CBMLBO! KPVSOBM! PG! TUPNBUPMPHZ · 2018-02-01 · temporomandibular joint disc, though such attempts are rare. This review points out the unique histological and biomechanical character

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BALKAN JOURNAL OF STOMATOLOGY ISSN 1107 - 1141 TUPNB

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Forthcoming Meeting

BALKAN STOMATOLOGICAL SOCIETY&

ROMANIAN DELEGATION OF THE BaSS

Invite you cordially to Bucharest on the occasion of the16th Congress of the Balkan Stomatological Society

President of the Congress Prof. Norina Forna

Dear colleagues and friends,

On behalf of the Local Organizing Committee, it gives me great pleasure to invite you for the 16th Balkan Stomatological Society Congress (BaSS) to be held in Bucharest, Romania, in April 2011. This event will be organized by the Romania delegation of the BaSS with the support of the Romanian Society of Oral Rehabilitation (ASSRO), The Romania Dental Council (CMDR) and Romanian Association of Oral Implantology and Biomaterials (SRIOB), under the theme “Update in dental medicine”.

The success of the Annual Bass Congress underlines the crucial role of the scientifi c programme’s quality. In recent years we have not only seen an increase in the number of participants but also the growth and development of what is now an outstanding scientifi c programme with continuing efforts to elevate Balkan stomatology to a higher level. This certainly could not have been achieved without your valuable support at the BASS congresses and for that we are extremely grateful.

As the Local Organizing Committee, we hope that, in addition to intellectual and professional growth, we can also provide you with a relaxing and enjoyable social experience. The capital of Romania is an attractive tourist destination, which offers the opportunity to all of you to pay a visit to the numerous historical sites and enjoy the extraordinary Romanian scenery.

We look forward to seeing you all in Bucharest.

Kind regards

Professor Norina Forna President of the Congress

Co-Organizer:Eurolink Medical CongressesStr. Carpaţi nr. 2, Iaşi, 700729, Româniaemail: [email protected]

16th Congress of the Balkan Stomatological Society28 April - 1 May, 2011, Bucharest, Romania

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ALBANIARuzhdie QAFMOLLA - Editor Address:Emil KUVARATI Dental University Clinic Besnik GAVAZI Tirana, Albania BOSNIA AND HERZEGOVINA Maida GANIBEGOVIĆ - Editor Address:Naida HADŽIABDIĆ Faculty of DentistryMihael STANOJEVIĆ Bolnička 4a 71000 Sarajevo, BIHBULGARIANikolai POPOV - Editor Address:Nikola ATANASSOV Faculty of DentistryNikolai SHARKOV G. Sofiiski str. 1 1431 Sofia, BulgariaFYROMJulijana GJORGOVA - Editor Address:Ana STAVREVSKA Faculty of DentistryLjuben GUGUČEVSKI Vodnjanska 17, Skopje Republika MakedonijaGREECEAnastasios MARKOPOULOS - Editor Address:Haralambos PETRIDIS Aristotle University Lambros ZOULOUMIS Dental School Thessaloniki, Greece

ROMANIAAlexandru-Andrei ILIESCU - Editor Address:Victor NAMIGEAN Faculty of Dentistry Cinel MALITA Calea Plevnei 19, sect. 1 70754 Bucuresti, Romania

SERBIAVojislav LEKOVIĆ - Editor Address:Slavoljub ŽIVKOVIĆ Faculty of Dentistry Zoran STAJČIĆ Dr Subotića 8 11000 Beograd, Serbia

TURKEYEnder KAZAZOGLU - Editor Address:Pinar KURSOGLU Yeditepe University Arzu CIVELEK Faculty of Dentistry Bagdat Cad. No 238 Göztepe 81006 Istanbul, TurkeyCYPRUSGeorge PANTELAS - Editor Address:Huseyn BIÇAK Gen. Hospital NicosiaAikaterine KOSTEA No 10 Pallados St. Nicosia, Cyprus

Editorial board

Editor-in-Chief Ljubomir TODOROVIĆ, DDS, MSc, PhD Faculty of Dentistry University of Belgrade Dr Subotića 8 11000 Belgrade Serbia

Council:President: Prof. P. KoidisPast President: Prof. A. IliescuPresident Elect: Prof. H. BostanciVice President: Prof. N. SharkovSecretary General: Prof. A.L. PissiotisTreasurer: Dr. E. HassapisEditor-in-Chief: Prof. Lj.Todorović

Members: R. Qafmolla P. Kongo M. Ganibegović S. Kostadinović A. Filchev D. Stancheva Zaburkova M. Carčev A. Minovska T. Lambrianidis S. Dalambiras

A. Adžić M. Djuričković N. Forna A. Bucur D. Stamenković M. Barjaktarević E. Kazazoglu M. Akkaya G. Pantelas S. Solyali

BALKAN STOMATOLOGICAL SOCIETYTUPNB

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The whole issue is available on-line at the web address of the BaSS (www.e-bass.org)

International Editorial (Advisory) Board Christoph HÄMMERLE - Switzerland George SANDOR - Canada Barrie KENNEY - USA Ario SANTINI - Great Britain Predrag Charles LEKIC - Canada Riita SUURONEN - Finland Kyösti OIKARINEN - Finland Michael WEINLAENDER - Austria

BALKAN JOURNAL OF STOMATOLOGY ISSN 1107 - 1141 STOMATOLOGIC

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RP I. Mistakidis The Prospect of Stem Cell-Based Regeneration of the TMJ Disc: 100 C. Cholopoulos Where Have We Gone So Far? I. Dimitrakopoulos

OP D. Veleska-Stevkovska Cytokines (IL-1, TNF-α, IL-6) and Oral Surgery Interventions 124

OP G. Cvijic Clinical Comparison between Implants with 133 L.L. Buarque e Silva 2 Chemically Different Surfaces Placed in the Maxilla W.A. Buarque e Silva Previously Treated with Bone Block Graft R. Mazzonetto F. Andrade e Silva OP T. Tunali Akbay Salivary Thromboplastic Activity May Indicate 141 M. Guvercin Wound Healing in Tooth Extraction O. Gonul A. Yarat S. Akyuz R. Pisiriciler K. Göker

OP M. Popovska Oral Findings in Anaemias 145 M. Petrovski A. Atanasovska-Stojanovska Z. Antovska J. Dzurcevski

CR R. Gozneli Allergic Sensitivity to Denture Base Materials: A Clinical Report 149 Y. Kulak Ozkan E. Kazazoglu CR N. Topouzelis Orthodontic and Orthognathic Surgical Treatment of 153 N. Lazaridis Severe Skeletal Class III with the Use of F. Tarawneh Resorbable Plates and Screws: A Case Report M. Lazaridou

Contents

VOLUME 14 NUMBER 3 NOVEMBER 2010 PAGES 97-160

SUMMARYConservative and surgical methods utilized so far to treat

temporomandibular disorders have demonstrated inconsistent results. Tissue engineering is a new, rapidly expanding, multi-disciplinary field aiming to generate tissue and organ substitutes in vitro. Stem cells have already shown the potential to form multiple tissue types. Thus, their ability could be incorporated in contemporary paradigms to reconstruct the temporomandibular joint disc, though such attempts are rare. This review points out the unique histological and biomechanical character of the disc, trying to delineate the basic parameters that need to be considered in this process. In fact, the histological character of the disc cannot be defined as hyaline cartilage but fibro-cartilage. Particularly, the type I fibrillar collagen prevalence and relatively low glucosamino-glycan content, along with their specific topographic arrangement and distribution, correspond to the cellular content and correlate to the biomechanical characteristics of the disc, which mirror its functional role. Here, the various biomaterials, biochemical and biomechanical stimuli utilized for the reconstruction of hyaline cartilage are explored, since the experience and knowledge from these research fronts offer a useful guideline for the reconstruction of the temporomandibular joint disc. The cross-talk between these advancing scientific fields, together with a deeper comprehension of the physiology and pathophysiology of the temporomandibular complex can act as a catalyst, elevating the prospects of the disc’s regeneration to new heights.

Keywords: Tissue Engineering; Temporomandibular Joint (TMJ); Temporomandibular Joint Disc; Mesenchymal Stem Cells; Cartilage

Ilias Mistakidis1, Christos Cholopoulos2, Ioannis Dimitrakopoulos3

1Post-graduate studentAristotle University of Thessaloniki, Dental SchoolDepartment of Basic Dental Sciences 2Private dental practitioner, Thessaloniki, Greece3Aristotle University of Thessaloniki, Dental School Oral-Maxillofacial Surgery DepartmentThessaloniki, Greece

REVIEW PAPER (RP)Balk J Stom, 2010; 14:100-123

BALKAN JOURNAL OF STOMATOLOGY ISSN 1107 - 1141

The Prospect of Stem Cell-Based Regeneration of the TMJ Disc: Where Have We Gone So Far?

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Introduction

The temporomandibular joint (TMJ), a diarthrodial, synovial joint, is the only joint contained in the craniofacial region. Its importance is highlighted by its involvement in the processes of mastication, ingestion and speech. The TMJ is basically unique in the sense that it is composed by 2 separate joints, located on either sides of the mandible, whose coordinated function is necessary to perform the opening and closure of the jaw. The joint of either side consists of the articulating surfaces of the temporomandibular (or glenoid) fossa and the head of the mandibular condyle, as well as an intervening articular disc. The disc together with the

synovial membrane serves to lubricate and enhance the congruity of the articular surfaces, stabilize the joint and absorb shock. The articular surfaces of the TMJ and the articular disc normally experience dynamic loading during physiological functions, such as chewing and speaking. Parafunctional habits, such as clenching, grinding or bruxism, apply static loading to the joint and are considered as causal factors of a number of disorders. Cartilage has a low cellular content and is in short supply of progenitor cells from the blood or the bone marrow since it lacks vasculature. As a result, it has a low self-repair capacity which hinders therapeutic approaches. Temporomandibular disorders (TMDs) are manifested with cervical pain, headaches, impaired movement,

clicking and popping of the jaw, all of which eventually interfere with the physiological function of the TMJ and negatively affect the quality of life.

The etiology and clinical management of TMDs are subjects of considerable controversy1. This broad category of diseases involves both TMJ and related musculoskeletal structures of the head and neck2,3. Epidemiological data show that TMDs affect roughly a quarter of the general population and 70% of them suffer from TMJ dysfunction4-6. Abnormalities of the TMJ may develop as a part of systemic disease as well. Osteoarthritis mainly affects older people and involves the erosion of the articulating surfaces of joints. It is mainly caused by excessive stress on joints over time, leading to the gradual degeneration of the cartilage matrix, while genetic disorders of the molecular components of the matrix may also predispose individuals to osteoarthritis. Rheumatoid arthritis is another systemic disease that attacks joints including the TMJ. Its cause remains unclear, though autoimmune, bacterial antigen and genetic mechanisms have been proposed7-9. At a local level, several causal factors such as occlusal dysfunction or stress related parafunctional habits have been explored and found to play a key role in the pathophysiology of TMDs. These conditions, in combination with genetic predispositions, hormonal and inflammatory mediators, may participate in the rise and development of TMDs.

Nearly 3-4% of the population seeks treatment for TMDs and interestingly, significantly more women are treated for TMDs than men, with a female to male patient prevalence reported between 3:1 to 8:14,10,11. Conditions requiring treatment include internal joint derangement, ankylosis, arthritic conditions, anterior disc displacement, traumatic injuries and tumours. With this vast array of abnormalities and related symptoms, dental practitioners and surgeons have encountered a great dilemma over treatment selection.

Treatment methods range from conservative, such as physical therapy (including self-care exercises12 and splints13) and medication, to various surgical procedures. The latter are nowadays considered as last options when non-surgical approaches fail to give satisfactory results14. Discectomy was initially performed to treat all kinds of disc derangement, regardless of their causality and severity15. Today, this procedure is used only in severe cases of TMDs to remove a damaged or dislocated disc and may involve the use of a disc replacement. Alternatively, as was first described by Farrar and McCarty5 in 1979, a surgical procedure called discoplasty is utilized for the repositioning of the disc, in a less aggressive approach. When removal of the disc does not succeed in relief of pain and recovery of functional joint mobility, the need of its replacement arises. This drove to the development of disc analogues from alloplastic materials, such as silicone rubber16 and Proplast/Teflon17. Though early reports showed promising

results18, both implant systems were found to undergo fragmentation gradually, due to their vulnerability to the repeated mechanical stresses and ultimately cause a foreign body reaction; this fact led to their withdrawal from the market19. Because of the disastrous results from the use of alloplastic materials, autogenous tissues, such as dermis20 and the temporalis muscle21 were used to replace the disc but showed inconsistent results. In an approach for the reconstruction of the total TMJ complex, 3 implant systems, approved by the FDA, are currently commercially available (Christensen TMJ Implant, W. Lorenz/Biomet TMJ Implant, Techmedica/TMJ Concepts TMJ Implant).

Given the marginal success displayed by the methods used so far for the treatment of TMDs, tissue engineering (TE) arises as the perfect candidate for the regeneration and replacement of a tissue that, as mentioned above, has limited ability for intrinsic repair. Tissue engineering can be defined as an interdisciplinary field that applies principles of engineering and life sciences (trying to understand the mechanisms of tissue growth) toward the development of biological substitutes that restore, maintain or improve functions of tissues or whole organs22. In early attempts, transplantation of in vitro expanded autologous chondrocytes into defect sites underneath membranes (ACT), was used to regenerate cartilage in major joints, such as the knee joint giving promising functional results23-25. The isolation of mesenchymal stem cells (MSCs) from multiple tissue sources26-30 and identification of their ability to differentiate into multiple mesodermal cell lines31-33 and form tissues of mesenchymal origin, combined with recent advances in biotechnology led to the evolution of TE approaches and raised therapeutic prospects (Fig. 1). Briefly, the field of TE comprises of 3 basic axes: the adequate cells, a biocompatible scaffold and the delivery of specific bioactive molecules. Contemporary concepts involve the use of cell lines capable to generate tissues with functional, biochemical and biomechanical properties similar to those of native tissues. In order to achieve such results, these cells must first be cultivated and conditioned with specific biological differentiating stimuli. As such, growth factors (GF) can not only induce differentiation of multipotent cells, but can also promote their proliferation and anabolic activity. The use of biomaterial scaffolds of synthetic or natural origin provides a hospitable environment for cellular attachment, proliferation and growth. Through cellular synthetic activity and concurrent scaffold degradation, the latter is eventually replaced by the newly formed tissue. The in vitro maturation process of the compound takes place in bioreactors, devices that provide the necessary biochemical and biomechanical micro-environmental features in a controlled manner. Alternatively and/or additionally, a period of in vivo maturation may be required. Scaffolds provide the means to deliver the cells to the site of the defect and ensure

Balk J Stom, Vol 14, 2010 Regeneration Possibilities of the TMJ Disc 101

102 Ilias Mistakidis et al. Balk J Stom, Vol 14, 2010

after transplantation that they stay in place long enough to achieve the desired repair. Furthermore, incorporated bioactive molecules trigger the chemotactic migration of host cells in a means to enhance in vivo maturation and integration of the construct.

In this review, an attempt is made to present the state of knowledge in the rapidly advancing field of TE on cartilage reconstruction. This presentation will focus on the prospect of reconstructing the TMJ disc, highlighting

its unique histological and biomechanical characteristics. Taking into consideration the fact that articular cartilage and the TMJ disc share several functional and histological similarities and given the scarcity of attempts to reconstruct the TMJ disc34-36, a brief presentation of cartilage engineering research is going to follow. Knowledge of the anatomical and histological features of the TMJ gives the theoretical background for understanding these efforts.

Figure 1. Title: Stem cell-based tissue engineering

Anatomy and Histology of the TMJ

The TMJ is a diarthrodial synovial joint composed of the condylar process of the mandible on one side, the glenoid fossa and articular eminence of the temporal bone on the other and an articular disc located in

between (Fig. 2). Both the surfaces of the condyle and glenoid fossa are covered with fibro-cartilage. The glenoid fossa is an oval depression in the temporal bone’s lower surface, anteriorly to acoustic meatus. The condyle is situated over the ascending mandibular ramus. A capsule encloses the joint and consists of


2 layers: an outer fibrous one and an inner synovial one. The latter contains the disc which together with its surrounding attachments divides the joint into superior and inferior joint spaces. Both cavities contain approximately one millilitre of synovial fluid each. The synovial membrane-disc complex serves to absorb shock, lubricate and improve the affinity of the joint surfaces. As the articular disc has little vasculature, the transfer of nutrients, oxygen and waste products occurs through the synovial fluid. The disc is biconcave in shape, with a thinner centre and a thicker periphery; it is subdivided into the anterior, intermediate and the posterior zones and additionally into lateral, central and medial zones. Dislocation of the disc is prevented by

its peripheral attachments: posterior to the retrodiscal tissue and the condyle, medially and laterally to the disc capsule and the medial and lateral poles of the condyle via triangular zones of connective tissue and anteriorly to the eminence, the head of the condyle and through a fibrous insertion to the lateral pterygoid muscle. The latter facilitates the disc’s movement forwards, following the rotation of the condyle during jaw opening, while the repositioning of the disc is attributed to the elastic recoil of the superior lamella of the retrodiscal tissue pad. The masseter and temporalis muscles also have fibrous connections to the disc. The capsule and to a lesser degree the disc are innervated by small unmyelinated nerve fibres of the trigeminal ganglion37.

Figure 2. Graphic representation of the TMJ complex: Temporomandibular joint disc, located between the glenoid fossa [F], the anterior eminence [E], the mandibular condyle [C], is subdivided into posterior [P], intermediate [IZ] and anterior [A] zones. Lateral pterygoid muscle [LP],

acoustic meatus [AM]

From a histological point, the TMJ condylar cartilage is different from other joints. There are 3 types of cartilage (hyaline, elastic and fibro-cartilage) present in adults, depending on function and location. The mandibular condyle’s articular cartilage is fibroelastic38, having characteristics similar to both hyaline and fibro-cartilage, mirroring the functional needs of the jaw movement. It consists mostly of type I fibrillar collagen unlike hyaline cartilage, which is made out of type II collagen39. Specifically, the cartilage of the condyle displays 4 distinct layers:

The superficial or fibrous articular zone, forming the articular surface, is composed of dense fibrous tissue. On the contrary, most joint surfaces consist of hyaline cartilage. Most fibres are collagenous and are arranged parallel to the surface, running anteroposteriorly through the centre of the condyle and circumferentially around the periphery. Elastin fibres exist as well but in a low

extent. Chondrocytes are scarce in this region and appear flattened and elongated;

The cell-rich or prechondroblastic layer is the site where proliferation occurs, providing a source of cells to adjacent layers. This layer has been reported to thin down or even disappear with age;

The chondroblastic or fibrocartilagenous zone. Here, the round chondrocyte-like cells are irregularly distributed unlike in growth plates of axial bones. This arrangement allows growth and remodelling of cartilage in all three dimensions40;

The endochondral ossification or hypertrophic zone, where hypertrophic chondrocytes, calcified cartilage blood and lymph vessels are observed.

The synovial membrane lining the inner surface of the fibrous joint capsule is composed entirely of loose connective tissue rich in type V collagen. Deep stromal cells and 2 types of lining cells: macrophage-like cells


(type A cells) and fibroblast-like cells (type B cells) populate the membrane41,42. Type A cells are arranged so, as for their apex to protrude into the synovial space and therefore seem to participate in the formation of the viscous synovial fluid and removal of waste degradation products. Efficient lubrication is considered a necessity for proper joint function and is attributed to surface-active phospholipids and hyaluronic acid43-45. Surface-active phospholipids cover the articulating surfaces while hyaluronic acid forms a mucus film separating them to prevent friction43,44. Phospholipids consist mainly of phosphatidylcholine and are subject to hydrolysis by phospholipase A2. Adherence of hyaluronic acid to phospholipid membranes (liposomes) was seen in vitro and protected those membranes from phospholipase A2 activity. This observation suggests that in situ hyaluronic acid probably plays a key role by adhering to the surface-active phospholipids and preventing their uncontrolled degradation from phospholipase A245.

The articular disc of the TMJ is a dense fibrous tissue and its histology has not been investigated thoroughly as for its cell distribution and extracellular matrix composition in humans46. Several animal models have been used instead, including mice37,47, rabbits48,49, bovines50 and porcine51. The porcine model is suggested to be the most suitable animal model for biomechanics research and tissue engineering studies. A study by Detamore et al52 on porcine TMJ discs investigated the cell types and distribution. Cells were classified either as fibroblasts (spindle-shaped with no discernable cell boundary) or chondrocyte-like (polygonal, in lacunae, therefore described as fibro-chondrocytes). Predominant in total cell count were fibroblasts (70% ±11%, mean ± SD). Cells were unevenly distributed through the anteroposterior and mediolateral axes. The anterior and posterior bands contained fewer cells than the intermediate zone, which in turn had fewer cells than the lateral and medial regions. This contradicts previous studies on rabbits53 and primates54 reporting that cells were more numerous in or near the anterior and posterior bands compared to the intermediate zone. The cell type regional distribution varied significantly; fibroblasts were predominant in the anterior and posterior bands. Fibro-chondrocytes showed higher relative numbers in the centre and through the superoinferior axis in the inferior layer of the disc. Regarding the ultra-structure of the cells, Detamore’s group reported that fibroblasts appeared to have large nuclei and few organelles, poorly developed smooth and rough endoplasmic reticulum (ER) and rarely observed Golgi apparatus and mitochondria. Chondroblast-like cells also had relatively large nuclei, small amounts of rough ER and Golgi apparatus, but often displayed several mitochondria, a sign of high metabolic activity.

The extracellular matrix (ECM) of the disc consists of tissue fluid (65-85%) and macromolecules which

mainly refer to collagen (85-90%) and proteoglycans (10-15%)55,56. Smooth muscle has also been detected, mainly in the anterior section of the disc, implying the presence of vasculature52. The primary glucosaminoglycan (GAG) was Chondroitin Sulphate followed by Dermatan sulphate. The high content of both GAGs (as components of the large proteoglycan, aggrecan) found in the intermediate and anterior parts of the disc51,55,57 corresponds well with the increased compressive stiffness of the area reported in other studies58,59 and is attributed to the abundance of fibro-chondrocytes in that region. Proteoglycans (PGs) attribute significant compressive strength, because they have the ability to trap amounts of water, which is rather incompressible, due to their highly anionic nature. Regarding the prevalence of type I collagen and noticeable amounts of elastin found in the central parts of both the anterior and posterior bands51, a correlation with the predominance of fibroblasts in those regions is indicated52. Furthermore, the same research group observed the collagen fibres’ arrangement in the disc using scanning electron microscopy and reported fibres running in a circular manner around the periphery and anteroposteriorly through the intermediate zone51. These characteristics are similar to those of condylar cartilage and demonstrate their close functional relation.

Concluding, the TMJ disc is both fibrous and cartilaginous in nature; its cellular and histological composition shares characteristics with articular cartilage, tendon and meniscus (Tab. 1).

Table 1. Summary of TMJ disc key characteristics:

• Predominance of fibrillar type I Collagen (not type II).• Collagen fibers arranged parallel to the surface, running

circularly around the periphery and anteroposteriorly through the intermediate zone.

• Main GAG is Chondroitin sulphate, total GAG content is lower than hyaline cartilage.

• High content of GAGs in anterior and intermediate regions attribute increased compressive stiffness.

• Main PG is aggrecan that consists of Chondroitin sulphate. • Uneven distribution of fibroblasts and fibrochondroblasts

through its zones.

Stem Cells

A stem cell (SC) is one that, through asymmetric mitotic division, is able to differentiate into several specialized cell lineages (multipotency), while retaining the potential for self-renewal. Such division results in 2 daughter cells, 1 of which follows the mothers’ genotype while the other (either by genetic or environmental influence) targets a specialized lineage60. Stem cells exist


at different hierarchical levels during the development of an organism. At one end of the spectrum are SCs that can give rise to all cell types in the body, which are called pluripotent. Other SCs may be partially committed and, thus, only retain ability to differentiate into a more restricted subset of different cell types; these are called multipotent. Sources of stem cells can be either embryonic or adult. Embryonic stem cells are obtained from the inner cell mass of blastocyst stage embryos61; however, their use has given rise to ethical considerations. Although adult stem cells can be found in children, adolescences and adults, and can be obtained from multiple tissues, the question “how age might affect their capacity?” is still unclear62.

In the early sixties Petrakova et al63 were the first to show that fragments of bone marrow had the potential to form osseous tissue when implanted ectopically. This was the first indication that bone marrow contains some kind of cells with osteogenic capacity. Isolation of such cells from bone marrow was performed by Fridenshtein64 in the late sixties, and they were described as “colony forming fibroblast-like cells”. Almost 2 decades later, Caplan65 gave them their current name (mesenchymal stem cells, MSCs) and identified their full potential to differentiate into multiple mesodermal cell lines, forming tissues of mesenchymal origin66. This revelation broke new ground for the prospect of tissue regeneration. Multiple sources of adult stem cells have been identified since then, including adipose tissue27,67, heart28, umbilical cord blood68, skeletal muscle69, dermis of skin30,70, the bulge region of the hair follicle71 as well as brain72,73. In the craniofacial region, stem cells can be isolated from orofacial bone77, dental pulp74,75 exfoliated deciduous teeth76, and periodontal ligament78. The 2 most extensively examined stem cell sources for their capacity to differentiate into various cell lineages are bone marrow derived stem cells (BMSCs) and adipose derived stem cells (ASCs).

BMSCs are considered the premier source for tissue engineering. They can be easily obtained from various locations79 and have the ability to form mesenchymal and connective tissues32 such as bone, cartilage, ligament, fat and muscle80-82. Moreover, BMSCs have been reported to differentiate into hepatic, cardiac, renal and neural cells33,83-85, although these pathways are not normally utilized to prove multipotency of isolated MSCs as will be described later in this paper. At this point, it should be noted that clonal studies showed that adhering cell populations isolated from bone marrow are functionally heterogeneous, containing undifferentiated stem progenitors and lineage restricted precursors86,87,95. This heterogeneity can partially explain the variations of the differentiation capacities observed in related studies composing a hurdle in their comparative evaluation.

Though being considered primarily as a metabolic reservoir, the adipose compartment appears to be a good and plentiful source of MSCs as first Zuk67 found. ASCs

can be readily obtained via lipoaspirate, a minimally invasive procedure, of great need nowadays. Several groups reported that ASCs undergo adipogenesis, chondrogenesis, osteogenesis, and myogenesis27,67,88,89, while others even suggest that ASCs are able to differentiate into non-mesodermal tissues, such as hepatic and neuronal-glial 90-92.

Even though there are no definite markers of MSCs, different methods have been used for their identification. One of the main characteristics of MSCs, still used in contemporary identification processes, is their ability to adhere to the plastic plate surfaces when maintained in standard culture conditions, as was first observed by Friedenstein64. Currently, more sophisticated procedures that researchers utilize include positive selection with microbeads combined with fluorescence-activated cell-sorting or magnetic-activated cell-sorting that enable the defined isolation and precise characterization of MSCs93,94. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy96 has listed the minimal criteria required for a cell to be regarded as an MSC, providing a general consensus (Fig. 3). The list includes several markers that cells should lack or exhibit additionally to the ability to differentiate to osteoblasts, adipocytes and chondroblasts in vitro. However, new ones are proposed over time.

Today, despite the fact that multiple sources of stem cells have been identified, certain drawbacks related to the harvesting process of BMSCs such as pain, stigma and low yield62 as well as the debate surrounding the use of embryonic stem cells led to the elevation of ASCs as an attractive option for a wide range of medical applications. Additionally, alternative orofacial sources of SCs and their capacities where also investigated. As mentioned, SCs can be isolated from the dental pulp of extracted humans 3rd molars74,75, exfoliated deciduous teeth76 and the periodontal ligament78. These sources present tempting alternatives through minimally invasive harvesting methods. Although small in number, dental pulp SCs (DPSCs) demonstrated higher proliferation rates compared to BMSCs in a study by Shi et al97. DPSCs can not only form dentin/pulp-like structures when implanted in immunodeficient mice98, but have also shown the ability to differentiate into neuron-like and glial-like cells (expressing nestin and glial-fibrillar acid protein)74. Furthermore, DPSCs are often mentioned in recent discussions about regenerative endodontics99,100. Accordingly, when SCs from exfoliated deciduous teeth were implanted in immunodeficient mice in a HA/TCP carrier, they formed dentin-like structures76. Periodontal ligament SCs (PDLSCs) generated cementum/PDL-like structures, containing a thin layer of cementum interfaced with dense collagen fibres similar to Sharpey’s fibres in a small animal in vivo model78. A recent review article by Huang et al101 discussed availability of dental derived stem cells in regenerative medicine.


Based on the great potential of tissue engineering and other stem cell based therapies, storage banks were established to isolate and freeze-store stem cells from multiple sources, such as extracted 3rd molars and the umbilical cord. These banks provide donors a prospect for stem cell-based therapies in the future if such need arises and given that stem cell science has evolved. However, there is the need to fully certify and standardize the procedures utilized for the isolation and particularly the maintenance of cells in a viable state.

But to what extent do stem cells, originating from different tissue, differ in their potential? Moreover, does their potential lie with their intrinsic features, or with the fact that stem cell populations might include lineage restricted precursors? A study by Im et al102 suggested that ASCs have inferior chondrogenic potential than BMSCs, a view supported by the work of Vidal et al103 and Kisiday et al104. In addition, a comparative study between orofacial and iliac crest derived MSCs reported that the

latter showed more proliferative and fewer senescence properties and different chondrogenic and adipogenic potentials77. In a comparative study by Wagner105 in 2005, however, no phenotypic differences between MSCs from bone marrow, adipose tissue and umbilical cord blood were observed. Thus, research must focus on identifying and comparing the distinct capacities of stem cells from different sources.

Scaffolds

In order to provide a hospitable 3D environment for cell attachment, proliferation and differentiation to form a tissue analogue, the use of biomaterial scaffolds was introduced in tissue engineering. Scaffolds must satisfy a number of requirements, primarily to ensure their safe usage and secondarily to fit the specific

Figure 3. Minimal criteria for defining multipotent mesenchymal stromal cells. MSCs must: (1) be plastic-adherent when maintained in standard culture conditions; (2) differentiate to osteoblasts, adipocytes and chondroblasts in vitro; (3) express CD105, CD73 and CD90, and lack expression of

CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules


called integrins. In physiological cell-ECM interactions, integrins play an important role in tissue homeostasis. Binding of ECM proteins to these receptors promotes focal adhesion. Additionally, this process leads to activation of intracellular signalling pathways that mediate changes in gene expression and thus affect cell proliferation, survival and differentiation117-120. Though results from their use are so far controversial121,122, integration of such signalling molecules in scaffolds is of great interest and strikes promise for the use in chondrogenic induction of MSCs.

For tissue engineering purposes, a wide variety of different scaffold materials exists, which can be natural, synthetic or composite. Natural scaffold biomaterials can be further classified to protein-base and polysaccharide-based, while synthetic materials can be subdivided into ceramics (hydroxyapatites or HA), calcium phosphates (CaPs) and synthetic polymers. In cartilage engineering, the main scaffold materials found in literature are summarized in table 2; so far however, none seems to possess the ideal characteristics to rise up as the perfect candidate. Particularly natural biomaterials could stimulate cell attachment, proliferation, differentiation and synthetic activity as they home in the cell’s natural environment. On the contrary, synthetic scaffold biomaterials offer significant advantages over the purely biological ones in terms of unlimited supply and manipulation of their mechanical properties, macro, micro and nano-topography and degradation behaviour.

Looking to the future, a minimally invasive surgical technique, such as an injection of a gel or paste material, seeded with MSCs or committed cells into a single or multi-tissue defect and shaped in situ seems ideal. In this direction several promising materials are being developed including, calcium phosphate cements123,124, in situ polymerizable and cross-linkable materials125,126, stimulus responsive systems127,128, and self-assembling materials129,130. A detailed review of injectable materials focusing on the regeneration of complex craniofacial tissues is available by Kretlow et al131.

characteristics of the tissue we intend to reconstruct. For example, scaffolds used for bone modelling must have comparable mechanical properties to bone, while for engineering of soft tissues (i.e. dermis, oral mucosa), a must softer scaffold is often required. It is essential that biocompatibility of the material and its subsequent degradation products is ensured106. Additionally, scaffolds must be biodegradable, with a controlled, predetermined degradation rate; after implantation, they must act as a temporary framework securing the initial stability and mechanical support of the construct itself, protecting the cells contained within, and surrounding tissues. Gradually they should give space to the newly formed tissue, allowing its ingrowth. Ideally scaffold materials should also demonstrate good handling characteristics (the ability to be shaped and fit into complex defects).

Moreover, scaffolds must have sufficient porosity to enable cellular colonization and tissue ingrowth (pore size is dependent to the specific cell type needs), as well as pore interconnectivity107-111 to facilitate the transport of nutrients and oxygen inwards, degradation and cellular metabolic by-products outwards. It is possible to modulate the pore topography and size to suit a particular cell type. Research has shown that pore shape can have a profound effect on both cell attachment and long term survival of cells112. Current developments in 3D computerized-tomography, 3D computer-aided design and rapid prototyping techniques are used for the design and engineering of customized cartilaginous implants made of synthetic polymers in defined pore architecture113,114. Porosity can, however, adversely affect important mechanical characteristics of the scaffold, thus precise computational design techniques are needed to predict and ultimately optimize the microstructure to achieve the desired balance106.

In an attempt to enhance the attachment of cells onto scaffolds, scientists have incorporated small peptide sequences (such as the RGD: Asp-Gly-Asp sequence115 and the IKVAV sequence116) in scaffold biomaterials. These peptides are recognized by cell surface receptors,

Table 2. Scaffold materials used for cartilage tissue engineering

Type Natural References Synthetic References

Material Agarose 132, 133, 134 PLA, poly(lactic acid 155, 156, 157

Alginate 133, 134, 135, 136 PGA, poly(glycolic acid) 158, 159, 160

Cellulose 137, 154 PLGA, poly(L-lactic-co-glycolic acid) 161, 162, 163

Chitosan 138, 139, 140 PEG, poly(ethylene-glycol) 164, 165, 166

Hyaluronic Acid 141, 142, 143, 144 PCL, poly(capro-lactone) 167, 168, 169, 170

Collagen 145, 146, 147, 156 POC, poly(1,8-octenadiol citrate) 171, 172

Fibrin 147, 148, 149, 150 PU, poly(urethane) 173, 174

Silk 151, 152, 153


technology should include developments of automated closed systems equipped with sophisticated tools, providing the means to both monitor physiochemical variables and enable adjustment of their values, thus allowing reproducibility and standardization of culture conditions. Aiming to predict the maturation level of the engineered construct advanced non-destructive analysis techniques (such as micro computerized tomography) could be integrated.

Mechanical stimulation seems to be a factor that greatly influences the developmental process. Research has shown induction of multipotent cells, seeded in engineered constructs subjected to physiological loads, towards various cell lines depending on the type of mechanical stimulation as it will be discussed later in this paper. Different stress protocols have been incorporated in bioreactor systems and applied to cell-loaded scaffolds. These include perfusion flow, shear, compression and tension among others. When these forces are applied in an advantageous combination with specific magnitudes and frequencies, they may not only enable controlled induction of differentiation but also enhance the quality of the generated construct. Schulz and Bader have recently reviewed different bioreactor systems used for the cultivation and stimulation of chondrocytes for the purposes of cartilage tissue engineering177.

Signalling Molecules

In the processes of tissue engineering, numerous hurdles need to be surpassed, as different approaches, referring to the selection of cell types, can be adopted. For example, when dealing with MSCs, apart from an appropriate 3D scaffold, the use of a series of signaling molecules is required for the in vitro proliferation, guided differentiation of the cells and the induction of neo-tissue formation. In vivo, MSCs’ differentiation is initiated through interactions of molecular signals, emitted from neighboring cells, transduced via extra or intra-cellular pathways and induced by cytokines, growth factors and ECM proteins (such as PGs and collagens), or through direct interaction with surface proteins of neighbouring cells. Trying to mimic the in vivo environment, researchers provide controlled delivery of stimulating factors to stem cells cultured in vitro, but furthermore have to ensure the long term maintenance of their phenotype. This last requirement also stands for the in vitro culturing of differentiated cells, such as autologous chondrocytes. A series of excellent reviews of the growth factors used for those purposes is available in the literature178-180 and a brief presentation of them follows. Among the growth factors investigated, some families of molecules have demonstrated a great impact on articular cartilage TE.

Bioreactors

Bioreactors can be defined as devices in which biological and/or biochemical processes are re-enacted under controlled conditions175, providing an environment that is advantageous for the creation of a desired product, weather it is wine or ECM as in cartilage tissue engineering. A number of parameters such as pH, temperature, pressure, nutrient supply, oxygen supply and waste removal should be carefully controlled and kept in optimal levels in the challenging attempt to mimic in vitro the dynamics of in vivo tissue growth. The in vitro generation of large three dimensional tissue analogues requires the development of sophisticated and complex systems that allow cells to locate them in a 3D scaffold, differentiate, proliferate and produce ECM, at the same time satisfying their physiochemical demands. The accurate simulation of the native physiological microenvironment will not only enhance the quality of the tissue engineered construct, but also hopefully ensure cell survival, tissue integration and proper function following implantation.

The simplest and most commonly used bioreactors are culture discs and flasks. They are easy to handle, affordable and through mechanisms of passive diffusion of nutrients and oxygen allow rapid multiplication of cells when cultured in monolayer. When multilayer approaches are adopted, transport of these substrates in and out of the cell containing microenvironment is mediated by inducing fluid flow, and/or passive diffusion along concentration gradients.

The advances in technology of bioreactor systems for the hosting of scaled-up scaffolds, trying to facilitate improved cell survival, reach new levels of complexity in tissue engineering techniques. Current standard culture conditions seem inadequate to distribute nutrients and oxygen throughout the construct and removal low molecular metabolites and waste products from it, which occurs mainly through diffusion phenomena from the center of the scaffold. This leads to migration of cells, initially located in the inner areas, to more superficial areas of the scaffold, where the nutrient concentration is higher. Eventually a steep fall in the cell density in the deeper zones of the construct is observed, affecting the homogeneity of the ECM and cell distribution in the final tissue equivalent176.

As summarized by Depprich et al175 in an excellent review, the concept of improving the bioreactor and scaffold design, in order to reduce diffusional limitations and ensure the viability of inner areas of the three dimensional construct, requires advances in both bioreactor and scaffold technology. From this point of view a dynamic laminar flow environment, achieved by perfusion directly through bioreactor inherent tubes and prefabricated interconnecting pores of the scaffold, would seem an ideal prospect175. Further advances in bioreactor


development and degeneration is provided by Chun et al199.

The Hypoxia induced factor (HIF) family consists of 3 subgroups (HIF-1a, HIF-2a and HIF-3a) of transcription factors capable of sensing and responding to alterations of the oxygen tension in the cell’s microenvironment. Recent studies show enhanced chondrogenic differentiation of rat MSCs, in hypoxic conditions, through up-regulation of the HIF-1a levels200. The importance of HIF-2a induction of the human articular chondrocytic phenotype in hypoxic condition has been suggested, whereas HIF-1a has been shown to be essential for growth arrest and survival of chondrocytes201. Another study showed that chondrocytes produce increased quantities of collagen type II in vitro, through HIF-1a202.

Other substances used for the induction of chondrogenesis include dexamethasone, β-glycerophosphate and ascorbic acid. Dexamethasone, a synthetic glycocorticoid, stimulating chondrogenesis by enhancing expression of cartilage extracellular matrix genes is used in standard cultivation media203. In animal studies, it is reported to be a potent stimulant of chondrogenesis204. In a study by Bean et al205, the use of 25 μg/ml concentration of ascorbic acid in culture media showed increased production of collagen by TMJ disc cells and is considered to be effective for the regeneration of the TMJ disc.

Even though extensive research is being conducted in the field of growth factors, the sometimes contradictory results for their potential role in in vitro chondrogenic differentiation of MSCs, and in vivo chondrogenic development, cartilage homeostasis and function, highlight the need for a better characterization of their signalling pathways. Currently, in the effort to simulate physiological in vivo processes of tissue development and maintenance through the series of interactions of biological factors, researchers seem to be trying to decode and reproduce an unknown tongue. In fact this is the richest and most complex language that exists, the one of nature. So far in this challenge, we have succeeded in identifying only a few words (could be GFs) and their likely meanings (could be their role), but are still unable to generate meaningful sentences. Thus, exploring the vocabulary and understanding the syntax, based on which these words are used (put in order), are essential to both understand and speak this language.

Accordingly, a profound understanding of the underlying biological and physiological background, accurately delineating the stages and defining the molecular markers involved in chondrogenic differentiation and development, can facilitate coordinated supply of growth and differentiation factors. Furthermore, sophisticated materials have to be developed to enable controlled spatial and dynamic release of these factors.

Alternative approaches for the delivery of GFs, involving gene therapy techniques, have also been investigated. The concept of gene therapy, as first

The transforming growth factor (TGF) superfamily, include 5 members of TGFs-β (1-5), the BMPs, activins and inhibins which interact with type I and II cell surface receptors and activate intracellular mechanisms. TGF-β1, 2 and 3 are predominantly produced in bone and cartilage and are considered to be potent stimulators of proteoglycan and type II collagen production and induce chondrogenic differentiation of MSCs in vitro181. It seems that TGF-β, as part of a serum-free differentiating medium, is one of the most widely used GFs and, in addition to promoting chondrogenesis, has also been shown to inhibit osteogenic and adipogenic differentiation32,182,183. BMPs compose a large subgroup of 20 polypeptides mainly investigated for their role in bone repair and seem to be key regulators of skeletal development. There is evidence indicating that BMP-2, -4, and -6 can stimulate chondrogenic differentiation of MSCs184 and synthesis of type II collagen and aggrecan by chondrocytes in vitro185. BMP-7 (osteogenic protein-1) was reported to enhance repair articular cartilage defect in animal models186, 187.

The fibroblast growth factor (FGF) family comprises 22 members that bind to one of 4 FGF receptors (FGFRs). The importance of FGFs in skeletal development is highlighted by number of dysplasias that have been attributed to specific mutations of genes encoding FGFRs. Research showed that FGF-18 stimulated cell proliferation, differentiation and cartilage ECM formation both in vitro and in vivo188,189. Expansion of MSCs in medium containing FGF-2 has also shown to promote chondrogenesis190-192. In a rabbit model, FGF-2 stimulated cartilage restoration in temporomandibular or articular cartilage defects193.

The insulin-like growth factor (IGF) family comprises of IGF-1, IGF-2, their respectful receptors (IGF1R, IGF2R) and several IGF-binding proteins and proteases that regulate their activity. Their anabolic role is supported by severe growth retardations observed in mice with IGF-1 mutations. Expression of IGF-1 and IGFR1 in adults occurs in chondrocytes, osteoblasts and osteoclasts194. Additionally, it was shown that IGF-1 induces proteoglycan synthesis, chondrocyte survival and proliferation195 and promotes articular cartilage regeneration196, a fact that supports its role as a mediator of cartilage growth and homeostasis. IGF-1 has also been shown to stimulate chondrogenic differentiation of MSCs197.

The Wingless (Wnt) family contains more than 20 members involved in skeletal development and the control chondrogenesis. Church et al198 investigated the role of Wnts in chondrogenic development and found that Wnt-5a, Wint-5b and Wnt-11 regulate chondrocyte proliferation and hypertrophic maturation in embryonic and post natal growth plates, while it seems that deregulation of Wnt signalling might lead to disease, in particular osteoarthritis. A recent review on the role of Wnts in cartilage


pellet cultures212. It seems that dynamic compression can further stimulate chondrocyte metabolism and enhance cartilage ECM production213-215. However, these specific effects are highly dependent on the magnitude and frequency of the applied stimuli. Furthermore, engineered tissues in different stages of development, progressing from scaffold occupied towards ECM occupied constructs, may require different mechanical stimuli. Osteogenesis, on the other hand, appears to be promoted by cyclic mechanical stretching216 and continuous perfusion217.

Articular cartilage experiences low oxygen concentrations due to its avascular nature. However, chondrocytes are adapted to these hypoxic conditions and produce ATP through anaerobic glycolysis mediated by the HIF-1a transcription factor218,219. Based on that fact, studies have investigated the effects of oxygen levels on cartilage regeneration in vitro. Reports have shown hypoxia to induce the chondrogenic differentiation of MSCs220,221 as well as the synthesis of ECM proteins in cultured chondrocytes222, while higher oxygen tension leads to osteogenic differentiation of MSCs223. Studies suggest that low oxygen levels inhibit the expression of type X collagen, a marker of hypertrophic chondrocytes leading to bone formation224. Recently, Wuertz et al225,226 showed that low pH inhibits the expression of aggrecan by MSCs, while in another study they reported that, when cultured in an environment containing low glucose, low pH and high osmolarity, MSCs displayed lower proliferation rates and lower expression of matrix genes compared to standard conditions.

As described previously, TGF-β, FGF-2 and IGF-1 are the most commonly used growth factors for chondrogenesis. However, in physiological tissues, none of these substances acts independently, they rather participate in an orchestrated sequence of interactions that mediate physiological function of tissues.

Consequently, researchers have attempted to deliver simultaneously various signalling molecules implicated in chondrogenesis. In particular, a combination of TGF-β3 and BMP-2 gave improved chondrogenic differentiation compared to either GF alone, or the combination of TGF-β3 with either BMP-4 or BMP-6184. Likewise the combination of TGF-β and application of cyclic mechanical load better promoted chondrogenesis of MSCs of either stimulant alone227. Indrawattana et al228 demonstrated how combinations such as TGF-β3/BMP-6 and TGF-β3/IGF-1 and the cycling of these combinations can induce chondrogenesis in MSCs. Additionally, a study by Chua et al229 demonstrated that the combination of IGF-1, bFGF and TGF-β2 increased cartilage-specific ECM expression and enhanced the histological features of the engineered cartilage. IGF-1 could be involved in synergism with TGF-β1, as the expression of chondrogenic-specific transcription factor SOX9 and production of type II collagen and cartilage specific PGs

described by Evans206, involves the delivery of a cDNA specific to a GF or signalling molecule into target cells which then produce that factor in vivo. Gene therapy can be performed directly, through delivery of transfection vectors in the targeted site or indirectly, where cells are transfected in vitro prior to their in situ implantation. The vectors can be either non-viral or viral which include lentiviruses, adenoviruses and adeno-associated viruses. Each approach displays different advantages and disadvantages. For example: the use of non-viral vectors is relatively simple but their expression is generally transient. On the other hand, the use of viral vectors results in prolonged transgene expression, but carries the risk of insertional mutagenesis. The incorporation of plasmid-DNA into scaffold materials is a similar idea that has also been explored in a number of studies207,208. Recently, the concept of gene delivery in cartilage repair has been reviewed by Steinert et al209.

Since our clinical experience with GF technology is relatively new, issues such as the long-term effects of implanting materials containing supra-physiological doses of GFs and the safety of gene therapy approaches, have to be addressed.

Research in Cartilage Tissue Engineering

In the literature, reports for reconstruction of the TMJ disc using stem cell-based TE approaches are scarce36. On the other hand, substantial work has focused on the TE of the hyaline cartilage, which is not surprising taking into consideration its significantly higher commercial interest. The experience and knowledge from the research in hyaline cartilage TE could provide an insight for attempts to reconstruct the TMJ disc, given the similarities of those tissues, while not overlooking their compositional and functional differences. Thus, a brief presentation of cartilage engineering will follow.

In vitro studies have attempted to determine the optimal mechanical and biochemical stimuli required for proliferation and differentiation of stem cells into the chondroblastic line, as well as for the maintenance of their phenotype and ECM production. As stated above, a number of different techniques can be used including: perfusion flow, cyclic strain and/or compression, bending, controlled pH, controlled oxygen levels and growth factor delivery. Elder210 demonstrated an enhanced chondrogenic differentiation under cyclic mechanic compression (9.25 KPa, 0.33 Hz, 2 h, for 3 days) of BMSCs in agarose gel. The same group reported that cyclic hydrostatic compression induced chondroinduction of MSCs211. Cyclic hydrodynamic stimulation has been shown to promote the chondrogenic commitment of hBMSCs in


by TGF-β, IGF-1 and FGF-2 has been shown to direct re-differentiation of de-differentiated cells238,239 and increase the cells’ proliferation rate238,240.

Stem cells were introduced early in the field of TE, as they possess the potential to differentiate into almost all types of cells. Multipotent stem cells, as mentioned before, can be obtained easily from various tissues. BMSCs have already demonstrated their tendency to undergo osteogenic and chondrogenic differentiation when cultured under certain conditions. However, it should be noted that adhering cell populations isolated from bone marrow are functionally heterogeneous, containing undifferentiated stem progenitors and lineage restricted precursors, with varying differentiation capacities86,87,95. Moreover, chondrogenic differentiation of MSCs in standard in vitro culture systems routinely results in expression of hypertrophic markers, such as type X collagen and alkaline phosphatise, which correlate with calcification and vascular invasion242. This undesirable phenomenon can be restrained by conditioning the cultured cells with biological, biochemical and biomechanical stimuli.

A vast number of animal studies have explored their ability to repair mainly osseous defects displaying controversial results. Bone marrow derived MSCs have been, combined with HA/TCP scaffolds to help build calvarial and alveolar bone defects in canines243,244 and mice245, seeded into hyaluronan-based polymers for reconstruction of orbital rim defects in pigs246. They have been also loaded onto gelatin sponges for repair of calvarial defects in mice247 and onto poly(caprolactone)(PCL)-based scaffolds to repair cranial defects in rabbits248. Adipose derived MSCs have been used in combination with gel foam scaffolds into rabbit calvarial defects249 and seeded onto PLGA scaffolds for rat critical-sized calvarial defects250, and showed moderate and significant results, respectively. In a worth mentioning human case by Warnke251, a gross and fully vascularized mandibular construct was created through the combined use of a custom designed titanium mesh scaffold, loaded with hydroxyapatite (HA) blocks and a bovine type I collagen coating containing BMP-7 and bone marrow aspirate. The compound was allowed to maturate in a prefabricated muscle flap and revascularized through vessels’ ingrowth from the thoracodorsal artery. After 7 weeks of maturation, the construct with its accompanying soft tissue envelope (including the adjacent vessel pedicle) was transplanted into the defect. The thoracodorsal artery and vein were anastomosed onto the external carotid artery and the cephalic vein, respectively. The patient’s ability to eat and speak improved greatly, but unfortunately the researchers were able to follow up only for 15 months when the patient passed away from cardiac arrest.

The repair of osteochondral defects has also been addressed through the use of MSCs in scaffold and

by stimulated MSCs were comparable to those of mature chondrocytes, as shown by Longobardi et al230. Addition of the parathyroid hormone PTHrP to TGF-β3-stimulated MSCs has been shown to inhibit the expression of type X collagen and suppress cells’ terminal differentiation231. At the same time, other reports show negative and no response to the use of the combinations IGF-1/FGF-2232 and IGF-1/TGF-β233, respectively.

Current experimental data give us a glimpse of the complexity of interactions involved in the processes of tissue growth and homeostasis; and as they emphasize the limited extent of our knowledge, they indicate a need for deeper understanding of the background of this field.

Discussion

To what extent can we really hope that TE techniques can result in a viable disc analogue? The idea of seeding multipotent cells in specially designed scaffolds with concurrent orchestrated delivery of biological factors, creating an environment for directed differentiation and tissue maturation for the reconstruction of a fully functional tissue, TMJ disc in this case, seems to be an ideal prospect. In this process, however, a number of issues still remain unsolved.

For example, which is the most favourable cell source? A cell source of chondrogenic and/or fibroblastic cells is needed. The most apparent choice would be native tissue cells, already possessing the tissue specific differentiated phenotype; however, in our case either physiological or degenerated disc cells are in sort supply. Furthermore, their harvesting process is associated with an additional surgical procedure and post-operative donor site morbidity. The use of mature chondrocytes from either non-load bearing or non-articular cartilage, such as nasal, rib and auricular cartilage, has also been proposed, an approach that reduces the site morbidity due to the lower levels of physical forces it experiences. An alternative choice could be tissue specific cells of xenogenic origin, which would be limitless in number and easy to obtain, but their use could potentially implicate the possibility of disease transmission and histocompatibility issues.

The major obstacles in the utilization of mature chondrocytes for TE purposes is their low expansion rates and de-differentiation leading to loss of function when cultivated in vitro234. The de-differentiation phenomenon is accompanied by a shift towards a fibroblast-like phenotype characterized by the adoption of a spindle-like shape and type I collagen production235. De-differentiation seems to be reversible when these cells are relocated in a 3D environment222,236,237. This observation points out significance of the 3D scaffold architecture in promoting the chondrocytic phenotype. Also, stimulation


maturation and conditioning in vitro should diminish the incidence of undesirable metaplasia, meaning mineralization of the chondral part towards bone. A number of TE studies for the mandibular condyle are available as the number of scientists in the field is growing exponentially262.

Nevertheless, these efforts did not incorporate current data regarding histological and biomechanical characteristics of the mandibular condyle’s cartilage. This is not surprising since our experience on TE is limited. Particularly, the cartilage of the mandibular condyle contains a heterogeneously distributed ECM. The type and arrangement of collagen fibres and the type and distribution of GAG-associated PGs through its regions mirrors the function of the TMJ. For the construction of a viable and fully functional mandibular condyle we should not only attempt to mimic the macroscopic gross anatomy and composition of the targeted tissue. The construct should also display the same detailed organization of its cellular and non-cellular components to meet the requirements of native tissue. So far, efforts proposed for engineering composite constructs are based on specific strategies for selection of scaffolds and cell sources. These include single homogenous and biphasic scaffolds in cell-free approaches or loaded with chondrogenic, osteogenic or multipotent cells263.

Issues similar to the ones of tissue engineered osteochondral constructs complicate the disc reconstruction. The TMJ disc histology has not been investigated thoroughly as for its cell distribution and extracellular matrix composition in humans46, but several animal models have been used, including mice37,47, rabbits48,49, bovines50 and porcine51. It has been suggested, based on a TE study where no differences were observed between results from porcine and human cells264, that the porcine model is suitable for characterization and TE studies of the TMJ disc. Furthermore, the first TMJ Bioengineering Conference held in May 2006 in Broomfield, Colorado263 proposed that, given the high cost of primate research and the similarity of the gross morphology of the constituents of the TMJ including the disc265-269, the porcine model is the most suitable animal model for biomechanics research and TE studies. Data obtained from the histological, biochemical and biomechanical analysis of TMJ discs of animal models, such as the porcine, show that the disc contains a vast population of fibroblasts and relatively less fibro-chondroblasts (chondrocyte-like cells); accordingly, it is composed mainly of type I collagen and its total elastin and GAG content gives it a unique character that falls in between hyaline cartilage and meniscus. The compressive and tensile properties, demonstrated by the disc, are attributed to its composition and beyond that, to the topographic arrangement of its macromolecules. The high GAG concentrations, located in the intermediate zone and anterior band of the disc51,57,270 seem to contribute

scaffoldless approaches. A combination of soluble scaffolds with MSCs have been injected in knees of rabbits and goats and showed controversial effects252-254. A biphasic PCL/TCP scaffold with MSCs was used in condylar defects of pigs255 and a PLGA/nano-hydroxyapatite (NHA) composite loaded with MSCs was used in rats’ knees256 to repair osteochondral defects; both gave promising results. Accordingly, Wakitani et al257-259 investigated the effect of transplantation of MSCs embedded in collagen gel for repair of human full-thickness articular cartilage defects with promising results. Taking into consideration that there is no single method regarded as a standard for isolation and expansion of MSCs, in field of TE; it is important to realize that these varying approaches make it difficult to directly compare experimental results.

These efforts for osteochondral tissue reconstruction could potentially play a key role in TMJ TE, especially when dealing with defects involving both condylar bone and articular cartilage. Furthermore, as pointed out during the TE session of the TMJ Bioengineering Conference, surgical implantation and attachment of a TMJ disc alone is considered difficult or even impossible in practice by most. Thus constructing and attaching an engineered disc/condyle composite would be the most rational approach for implanting an engineered disc. This approach would better fit cases where disc abnormalities are concurrent or induced by primary degenerations of the condyle. Alhadlaq and Mao260,261 succeeded in generating constructs with the shape and dimensions of the human mandibular condyle. They used rat MSCs, independently stimulated towards the chondrogenic and osteogenic lines, seeded onto a photo-polymerizable, 2-layered polyethylene-glycol diacrylate (PEGDA) scaffold and implanted into the dorsum of immunodeficient mice for 12-week maturation. Each layer of the construct demonstrated tissue distinctive microscopic characteristics. Infiltration between osseous and cartilaginous compartments was enhanced by increasing cell encapsulation density. However, integration of the condyle analogue to the ascending ramus and surrounding attachments still composes a hurdle.

Furthermore, a number of other issues need to be addressed in the challenge of implanting an engineered condyle. The tissue engineered condylar graft should be adequately vascularized whereas its attachments and mechanical properties should ensure the ability to withstand the early shear and torque stresses. Mechanical properties of the condylar construct depend highly on the level of in vitro maturation. Longer periods of maturation seem to lead to a better developed cartilaginous ECM, displaying better mechanical properties and enhanced success rates when subjected to physiological mechanical loads. However this prolonged maturation could negatively affect the construct’s capacity to integrate with the adjacent tissues. In addition, proper construct


found that the presence of succinic acid and lactic acid is a reliable marker of septic arthritis. Lavage of the upper compartment of the TMJ washes away degraded particles and inflammatory components, such as radical oxygen species (ROS), phospholipase A2286 and interleukins 1β and 6287 among others, concurrently decreasing the intra-articular pressure. So far, diagnostic criteria in various TMDs, through biochemical analysis of the synovial fluid, are insufficiently defined, leading to inefficient treatment. Progressive evaluative analysis of changes occurring in different developmental stages of the pathogenesis of TMDs can offer an insight on the ongoing pathogenetic mechanisms. This could facilitate a deeper understanding of the etiology of these pathoses and subsequently enable the development of new successful treatments.

The ultimate goal is reconstruction of a fully viable, rapidly integrating disc equivalent possessing the biomechanical and histological characteristics of a physiological human disc. In this direction, data derived from engineering research of relevant tissues (such as the knee joint) can be incorporated in TMJ disc TE paradigms. Concurrently scientists have to focus on the TMJ for a better comprehension of its physiology, pathophysiology and biomechanics. Advances in scientific fields, such as molecular and cellular biology, biomaterials and bioengineering, constitute a basis of collective wisdom. The cross-talk between specialties, forming interdisciplinary and multidisciplinary partnerships, will be the catalyst in moving the field of TE to new heights. Adult stem cells are a powerful tool for regenerative medical applications. TMJ tissue engineering is an opportunity that dentistry cannot afford to miss.

Abbreviations:TE: tissue engineeringTMJ: temporomandibular jointTMD: temporomandibular disorderMSC: mesenchymal stem cellBMSC: bone marrow derived stem cellASC: adipose derived stem cellDPSCs: dental pulp derived stem cellsPDLSCs: periodontal ligament derived stem cellsECM: extra-cellular matrixGAG: glucosaminoglycanPGs: proteoglycansGF: growth factor

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to the high compressive strength of these parts58,59; collagen fibres running circularly around the periphery and anteroposteriorly through the intermediate zone of the disc51 also correspond to the high tensile stiffness reported by a previous study271 and to the taut tension, applied through the disc’s anteroposterior axis by its anterior and posterior attachments. Another arising issue is a theory of a more mechanically demanding environment in the inferior joint space, which is supported by the increased degeneration of the inferior disc surface observed in TMJ disorders272-274 and the high relative number of fibro-chondrocytes found in the inferior layer, compared to the superior and middle layers of the disc52. However, it must be taken into consideration that though porcine TMJ discs are approved for characterization, some early research data for human discs275 show controversy regarding the GAG distribution. In addition, differences existing between functional parameters of the TMJ, such as the frequency of mastication, indicate that they are not completely identical. These facts ultimately lead to the conclusion that human discs need to be better investigated and characterized, while efforts to generate a disc construct in vitro for the purpose of realistically treating humans, must incorporate the compositional and architectural features found in the native tissue.

However, investigation of etiology of temporomandibular disorders’ pathogenesis is also of great importance, as a large fraction of TMD’s causes remain unclear. For example, why are more women treated for TMDs than men6,276? Is it a matter of oestrogen receptors277? Is it just a matter of transmission or perception of pain278? Better understanding of TMD pathogenesis will not only help the prevention and treatment of TMDs, but will also give significantly better prospect to TE disc therapies. In fact, if the underlying degenerative disorder remains uncured, how can we hope that a tissue engineered disc equivalent can function for a long time in a viable and normal state unlike the disc it replaced?

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Correspondence and request for offprints to:

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SUMMARYIntroduction During oral surgery, trauma causes a number of different

local and systemic alterations to the host. Objective Determining the cytokines IL-1α, IL-6, TNF-α; and specifying

the signs of possible traumatic damage in the course of standard oral surgical procedures -cumulated surgical trauma on both soft and bone tissue.

Material and Method Research was conducted on 20 patients at the Clinic of Oral Surgery, Faculty of Stomatology in Skopje, planned for oral surgery. Cytokines serum levels of the patients were registered before surgery, 24 hours after it, and on the seventh day.

Results and Discussion Post-operative values of the examined cytokines showed statistically significant differences. The data showed connection between the changes in TNF-α during oral surgery and age of patients. The subjects differed only with respect to the number of the osteotomed surfaces, which had a discrete impact on the analyzed system parameters (weak correlation). There was a statistically positive correlation between duration and complexity of surgery (degree of the surgical trauma) and the occurrence of post-operative complications.

Conclusion The rapid post operative increase of the levels of the IL-1α, TNF-α, IL-6, demonstrated and proved high sensitivity. Keywords: Cytokines; Surgical Trauma; Immunology; Immunosuppression; Oral Surgery

Daniela Veleska-Stevkovska

University of Skopje, Faculty of DentistryClinic for Oral SurgerySkopje, FYROM

ORIGINAL PAPERBalk J Stom, 2010; 14:124-132


Cytokines (IL-1, TNF-α, IL-6) and Oral Surgery Interventions

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Introduction

Cytokines are low-molecular regulatory proteins, or glycol-proteins, which impact the intensity and duration of immunological response by stimulating or inhibiting its activation, proliferation or differentiation of various cells. They are secreted by leukocytes or various other cells as a response to different stimuli. Their average molecular mass is 30 kD. They posses an α helix structure and bind to specific receptors on the membrane of the target cells, causing signal transduction (Fig. 1), which results in change of the genetic expression of the target cells.

There are 3 known cytokine actions: autocrine (the receptor belongs to the cell secreting the cytokine), paracrine (the cytokine binds to its receptor in the proximity of the cell which produced it) and endocrine (the receptor is located in the distant parts of the organism). Furthermore, there are a number of cytokine functions: redundant action (2 or more cytokines with

similar function), synergetic (the combined action of 2 cytokines is greater than the additive effect of individual cytokines) and antagonistic (opposite effect)1-5.

Types of cytokines: - IL-1 to IL-18- TNFα and β-TNF family- IFN α, β, γ - interferon family- Hematopoietic family, factors of colony stimulation

(GM-CSF, G-CSF, IL-2-7, IL-11-13, IL-15)- Growth transformation factors (TGF-α and β)- Migration inhibition factors (MIF) causes the inhibition

of macrophage migration - Chemokynes (IL-8) responsible for chemotaxis.

Types of receptors:- Immunoglobulin super-family of receptors- Class 1 (hematopoietic)- Class 2 (interferon)- TNF receptor family- chemokyne receptor family (e.g. IL-8).

Cytokines and Surgical TraumaAll harmful stimuli and mechanical damage

contribute to ”awakening” of the organism response, called the acute phase response, which aims to minimise the damage and initiate the healing process. Very often, this does not represent an optimal reaction, but a hypo- or hyper-reaction, even though most of the reactions are essentially beneficial to the host. The direct effect of anaesthesia of immunological cells, stress-induced hormonal changes, suppressor activation, occurrence of suppressive serum factors6 and changes in cytokine production also play a role in post-surgical immuno-suppression. Cytokine measurements are conducted clinically on peripheral blood, but the absence of detectable cytokines in peripheral blood, often does not exclude local production of cytokines in the injured or inflamed organ7.

IL-1 is produced by all cells with a nucleus (including monocytes, macrophages, B-lymphocytes), NK (natural killer) cells, fibroblasts and muscular cells. The molecular weight of IL-1 is 17,500D. The human IL-1 is represented by 2 molecular forms IL-1α and IL-1β. The potential and the biological effects are identical in both forms and they have similar affinities in binding to receptors8. Both forms are encoded by different genes with different amino acid sequences and are connected in 3-dimensional levels. Some cell types also produce a third gene, which encodes an IL-1 receptor antagonist protein (IL-1ra) and is a competitive inhibitor of IL-1 α and IL-1β. IL-1 indicates the production of other cytokines. Various tissues constantly produce IL-1, e.g. the skin contains significant levels of IL-1, as well as the sweat and urine. Unlike them, macrophages and other cells produce IL-1 only as a result of external stimuli, for example bacterial poly-saccharide (LPS). IL-1 causes

several measurable clinical and biological activities, such as induction of increased body temperature, activation of endothelial cells and neutrophils, expression of adhesive molecules, induction of hypotension and shock. Furthermore, it stimulates the production of other cytokines IL-2, 4, 6 and 8, GM-CSF and IFN-γ - its concentrations rapidly increase following trauma, including moderate surgical trauma9.

TNF is capable of acting independently or in conjunction with a wide range of other factors. It exists in 2 forms: TNF α and TNF β. TNF α is produced by activated macrophages, though much less by other cell types, while TNF β is primarily a product of activated T lymphocytes. TNF α and TNF β bind to same receptors. They both have a half-life of 15-18 minutes, yet indicating metabolic and hemodynamic changes. There is a presence of endogenous inhibitors (transmembrane soluble TNF receptors-STNFRs) which inhibit the potential unregulated TNF activity. TNF α has cytotoxic effects on endothelial cells, it increases the adhesion of neutrophils to endothelial cells through regulation of adhesive molecules10 and increases vascular permeability, both directly and indirectly, through activation of neutrophils. It helps the release of prostaglandin PGE2, it activates PAF (thrombocyte activating factor) and the coagulation process. TNF and IL-1 are the most important inducers of acute phase response.

IL-6 has a molecular mass of 26kD. Its main features include synergism with IL-1 and TNF for the purpose of co-stimulating immunological response and inducing production of acute phase proteins. It has a short life span of about 1 hour. Following the occurrence of a trauma, its values can be detected after 1 hour, the concentration peak is reached after 4-6 hours and it can be present for 10 days in circulation. This cytokine is constantly detected in plasma, which suggests its constant production. It has been ascertained that the intestinal source of IL-6 is the “background” of its detectable plasma levels. It is also created in phagocytes, the vascular endothelial cells and the fibroblasts. IL-6 enables B-cell replication, differentiation and immunoglobulin production. IL-6 and IL-1 have the role of mediators of the acute phase response (CRP, fibrinogen, haptoglobin, amyloid α, α-1-antitrypsin and complement activation-C3 and factor B). It possesses imunomodulatory characteristics, including initiating PMN (polymorphonuclear leukocytes)-mediated hyper-inflammation and, paradoxically, deferred immunosuppression of the host. IL-6 can also serve as an anti-inflammatory mediator through a sophisticated mechanism of releasing sTNFs (soluble TNF receptors) and IL-1ra, which leads to attenuation of the functional capabilities of TNF and IL-1. IL-1 does not regulate the main inflammatory regulators, such as PG, nitric oxide, matrix metaloproteases, nor does it include the synthesis of adhesive molecules involved in the inflammatory response, such as ICAM-111. IL-6 even inhibits several

Figure 1. Signal transduction

Balk J Stom, Vol 14, 2010 Cytokines and Oral Surgery 125

126 Daniela Veleska Stevkovska Balk J Stom, Vol 14, 2010

key inflammatory responses by impacting the secretion of PGE2, which is a powerful immunosuppressive mediator of T-cells and macrophages. On the other hand, PGE2, induces the release of IL-10, which is a powerful anti-inflammatory mediator deactivating monocytes. This serves to explain the fact why IL-6 protects the organism from mortality in the occurrence of septic or toxic shock, which has been demonstrated through the administering of neutralising anti-bodies against IL-6. However, the increased plasma concentrations have been registered in acute states, such as surgical interventions, burns and bacterial infections. IL-6 is closely associated with the events occurring in the post-operative period (elective surgery) or following trauma, with the increased levels of IL-6 48 hours post-operatively constituting an alarm of possible infective post-operative complication, present immunosuppression or representing a reaction of the organism to a hyper-inflammatory condition12. There is a significant correlation between the IL-6 values and age. Still, IL-6 remains the cytokine that is most constantly elevated or most easily detectible.

Cytokine Response to Surgical InterventionSurgical intervention and trauma induce a series

of inflammatory responses, such as elevation of body temperature, increased sedimentation, leukocytosis and increased levels of acute phase proteins. The most up-to-date research shows that these responses are mediated by cytokines: IL-1, TNF, IL-6. IL-6 response is detected 4-6 hours post-operatively and prior to the increase of circulating acute phase proteins. The response is not always fast, but it can be long-lasting.

There is a correlation between the cytokine levels and the degree of surgical trauma - more pronounced trauma leads to greater changes. Furthermore, the duration of surgery and anaesthesia determine the cytokine response. Shenkin et al13 detected the increased levels of IL-6 30 min. after the incision of skin. Cruickshank et al14 detected the increased levels of IL-6 during the first 2-4 hours postoperatively. The type of intervention is very significant with regards to the cytokine response; for example, abdominal interventions show a sharp rise in the cytokine response. IL-6 is the only cytokine that is constantly elevated following tissue trauma and there is a clear correlation between prolonged and excessive IL-6 elevations and morbidity and mortality. Understanding the cytokine patho-physiological response serves as the basis for intervention therapy aimed at modulating the response to surgical trauma.

According to Bone’s theory15, the cytokine response to trauma is divided into 3 phases:1. Occurrence of local production of cytokines as a

reaction to a local injury;2. Small portions of cytokines are present in the systemic

circulation, which helps to optimise the defence mechanism;

3. The circulating levels of cytokines are higher than those necessary for the reparatory period and negatively impact the organism with their action. During this phase, a systemic inflammatory response (SIRS) can also occur, which if left untreated can be fatal.

In accordance with this, lower levels of measurable cytokines in systemic circulation in the post-operative period should be a goal which is worth insisting on. Consequently, it is necessary for locally produced cytokines to be “kept” locally.

Angele and Chaudry16 point to the fact that bone damage in conjunction with soft tissue trauma produce protracted depression of immunological function, as opposed to soft tissue trauma without bone trauma. This data suggest possible additive effects of traumatic damage on the depression of immunological functions. In their study, the authors indicate possible patho-physiological mechanism of immunosuppression during surgical interventions: depression of HLA-DR (Human leukocyte antigen receptor) expression, reduction of antigen-presenting capacity of macrophages, reduction of metabolic capacity of the organism, disturbance of homeostasis between the pro-inflammatory and anti-inflammatory cytokines. NO and prostaglandins are responsible for the depression of the antigen-presenting cells. Possible defects in the T-cell proliferation and cytokine secretion lead to reduced secretion of IL-2, IFN-γ and TNF α.

Aims of the Study

Following the most up-to-date scientific and research trends, which focus on the close relation between surgical traumatic stress and its direct impact on the first and immediate non-specific response, for the purpose of insuring complication free post-operative period and consequent quick, complete and functional recovery, we have formulated the following objectives of this paper:1. Quantitative determination of the serum components

of cytokines and their qualitative activity in the peri-operative period;

2. Establishment of correlation between the degree of traumatic damage and the levels of the cytokines IL-1α, IL-6, TNF-α in the peri-operative period, taking into account the complexity of the performance, as well as the correlation between the time interval of duration of the intervention and activity of the examined immunological parameters. We aimed to determine a potential impact of sex and age on immunological activity in the peri-operative period, and also to determine a correlation between immunological behaviour and possible objective clinical complications in the post-operative period;


3. Standardisation of a position on the protocol regarding minimising traumatic stress, eliminating the cumulative impact of other stress factors of non-traumatic origin, as well as defining a protocol for medical therapy, including immunotherapy.

Material and Method

Patient Selection and Working ProtocolFor the purpose of implementing the defined test

goals, the study involved 20 patients from the Clinic for Oral Surgery at the Faculty of Stomatology in Skopje undergoing removal of impacted third molars, following a detailed anamnesis, extra-oral and intra-oral clinical examination and x-ray analysis. Furthermore, the age and sex of the patients were also taken into consideration. This study did not include patients which, prior to being treated, showed any clinical sign of infection or neoplasm, or had been surgically treated at least 3 months before the date of the intervention. Each intervention was characterized by its own quantitative and qualitative attributes, such as: type of intervention, duration, complexity of performance, as well as the degree of surgical trauma of the periosteum and the bone tissue (osteotomy).

Prior to surgery, then 24 hours after, as well as on the seventh day postoperatively, the patients were tested for cytokine serum levels (IL-1α, IL-6, TNF-α), which provided an overview of the activities of the individual immune system.

The samples of vein blood were taken in quantities of 10 ml. The taken blood samples were then kept on temperatures ranging from 2-60C up to 24 hours. Following this, they were centrifuged and stored at a temperature of -200C until the time of laboratory testing.

Correlation was made between their variations before and 24 hours after the intervention and the objective post-operative complications on the first day after the intervention, which were of general (body temperature) and local character, the presence of post-operative

pain, oedema, haematoma, post-operative infection, and functional disturbance (trismus). Postoperatively, during the first 24 hours, patients were not administered antibiotic and anti-inflammatory drugs due to the requirements of the study and the need for achieving authentic results. On the seventh post-operative day, sutures were removed.

Principle of determining the cytokine concentration - ELISA method

The laboratory research was carried out at the Institute of Human Immunology and Genetics at the Medical Faculty in Skopje. For the purpose of determining the cytokine levels, blood was taken by using venipuncture, than left to coagulate, after which the serum is separated by centrifuging (5 min. on 4000 rpm). If it was necessary to keep the sample for longer than 1 day, it was stored at a temperature of -200C, while if it was required to store the samples for a period longer than a week, they were kept at -800C.

The cytokine concentration in the sample was determined by ELISA (enzyme linked immunosorbent assay) method (Medgenix Diagnostics, Belgium). Today, there are various commercially available toolkits to determine the cytokine concentration, but they do not always provide approximately the same results. Immunological tests represent an alternative to biological methods and flow cytometry. They are simple to perform and results are obtained in a matter of hours (Fig. 2).

Statistical analysisThe statistical analysis was conducted with the

statistical program STATISTICA 7.

Results and Discussion

The variations of IL-1α, IL-6 and TNF-α are presented both in tables and on charts (Tabs. 1-6 and Figs. 3-5).

Figure 2. Laboratory equipment


Table 1. Variations of IL-1α

IL-1a N Mean Confidence -95.0% Confidence +95.0% Min Max Std.Dev.

IL-1a / before 20 0.58 0.39 0.77 0.23 1.59 0.41

IL-1a / 24 h 20 0.60 0.39 0.81 0.21 2.02 0.45

IL-1a / 7 day 20 0.57 0.38 0.76 0.23 1.58 0.41

Table 2. Differences in IL-1α

Parameter N T Z p-level p Sig. / N. Sig.

IL-1abefore / IL-1a 24 h 20 93.00 0.08 0.94 p>0.05 N.Sig.

IL-1a before/ IL-1a 7 day 20 42.5 1.32 0.19 p>0.05 N.Sig.

Table 3. The variations of IL-6

IL-6 N Mean Confidence -95.0% Confidence +95.0% Min Max Std.Dev.

IL-6 / before 20 14.47 4.36 24.58 0.92 67.70 21.59

IL-6 / 24 h 20 18.27 5.21 31.34 1.34 89.90 27.92

IL-6 / 7 day 20 14.53 4.44 26.62 0.94 67.90 21.57

Table 4. Differences in IL-6


IL-6 before / IL-6 24 h 20 24.00 3.02 0.002 p<0.01 Sig.

IL-6 before / IL-6 7 day 20 33.5 2.67 0.008 p<0.01 Sig.

Table 5. The variations of TNF-α.

TNF-a N Mean Confidence -95.0% Confidence +95.0% Min Max Std.Dev.

TNF-a/ before 20 4.81 2.14 7.47 0.17 17.79 5.69

TNF-a/24 h 20 3.98 1.94 6.03 0.18 13.20 4.37

TNF-a / 7 day 20 4.52 2.12 6.93 0.18 15.99 5.14

Table 6. Differences in TNF-α.


TNF-a before/ TNF-a 24 h 20 101.00 0.15 0.031 p<0.05 Sig.

TNF-a before / TNF-a 7 day 20 61.00 1.37 0.171 p>0.05 N.Sig.


IL-1The studies of the following authors point out the

post-operative increase of IL-1 levels: Mokart et al17, Aasen et al18, Huang Tsung-Jen et al19. On the other hand, Baigrie et al20, Grzelak et al21 state in respective studies that they haven’t detected any changes in the serum levels of this cytokine.

Data on possible immunosuppression is provided by the following authors: Angele and Faist22 and Angele and Chaudry16.

IL-6The post-operative increase of IL-6 values is reported

by the following authors: Prondzinsky et al23, Mokart et al17, Baigrie et al20, Kashiwabara and Miyashita24, Decker et al25, Maruszynsk and Pojda26, while Kudoh et al18 provided data on their reduction.

TNF-α It is the earliest and most powerful mediator of the

immune response, with a rapid and very short period of existence. Still, it can initiate metabolic and hemodynamic changes by activating cytokines, even though it has a half-life of 15-18 min. It reaches its peak in the plasma 90 minutes after stimulation. Thus, it is a highly sensitive parameter with rapid and sharp peaks immediately after the operative treatment.

Post-operative alterations, i.e. the increase or reduction of cytokine levels, depend significantly on the TNF gene polymorphism. Infections, severe injuries and surgical trauma are known to affect cellular immunity, which is considered to be responsible for secondary complications, such as sepsis or multiple system organ failure. “Ex vivo” detection of LPS (lipopolysaccharide)-stimulated cytokine response is widely used as an indicator of a compromised response of the host in cases of accidents or surgical trauma.

Great number of authors indicate the occurrence of the reduction of TNF-α levels after the surgical intervention16,18,27,28, which also coincides with our own results and which might refer to a possible immunosuppression of the organism as an inadequate response to stress. In our examined materials, 50% of the subjects registered post-operative reduction of TNF-α values, while the rest registered an increase of the same values.

CorrelationsWe have only included statistically significant

correlations. When examining the relation between the surgical traumatic stress and the values of systemic parameters, we determined a weak positive correlation. This indicates that subjects differ among themselves only in the number of osteotomed surfaces, which results in only a discreet impact on the analyzed systemic parameters (weak correlation). According to Bone15, the

Figure 3. Variations of IL-1α

Figure 4. The variations of IL-6

Figure 5. The variations of TNF-α


organism, in accordance with its individual capacities, succeeds in keeping the locally produced mediators locally. This represents a good outcome of the post-operative process. Another group of authors clearly define the difference of the effects on the immunological system in various types of tissues, as well as the quantitative level of the release of the mediators of inflammation in various types of tissue (bone tissue contains greater number of tissue damage mediators than soft tissues)

Whitman29 examined the impact of isolated tissue damage (soft tissue or bone tissue), and the combined tissue damages on the immunological system, coming to the conclusion that in cases of the combined tissue damages there is a significant depression of IL-2, IL-3 and of IL-1, IL-6 as compared to isolated tissue damage..

Heating of bone tissue during oral surgery changes qualitatively the monocyte-macrophage function and impacts hematopoesis, quantitatively and qualitatively30. These cells produce less acute phase proteins (APPs) C3 and transferin, but possess greater cyto-toxicity measured according to the release of 1-lactate dehydrogenase.

The examined correlation between the type of intervention (interventions in which only impacted teeth are extracted and interventions where other neighbouring teeth are extracted simultaneously with the impacted teeth) and IL-1 (24 hours after the intervention) for R = -0.47 demonstrates medium high negative correlation, which means that in instances of increasing surgical trauma during surgery (increasing the number of simultaneously extracted teeth) reduces the post-operative values of analyzed parameters, which indicates an increased probability of post-operative immunosuppression.

The examined correlation between the type of intervention and TNF- (24 hours after the intervention) for R = -0.54 demonstrates medium high negative correlation.

The differences between the analyzed parameters, in regards to distribution according to sex, for p > 0.05, are insignificant. Male sex hormones have immunosuppressive capacities, unlike female sex hormones, which possess immunoprotective characteristics23.

There is data in the relevant literature31 that IL-6 naturally increases with age and is related to heart disease, type 2 diabetes, osteoporosis and other age-related conditions. It has been ascertained that individuals subjected to longer periods of chronic stress continue to have higher levels of IL-6 even after the ceasing of activity of the chronic stressor. Furthermore, they show high level of vulnerability to viral infections, high blood pressure and slower healing of wounds.

State of mind affects one’s state of health (mind-body interaction). In the material we examined, we came across data that indicate relation between TNF-α changes during oral surgery and patient’s age. These are considered to be authentic results.

Conclusions

Surgical intervention causes immune and acute phase response, aseptic inflammatory reaction, which universally accompany all types of trauma.

The rapid post-operative rise of the levels of IL-1α, TNF-α and IL-6 is demonstrated and proven to be of high sensitivity, thus enabling us to highlight their responsibility in the systemic immunological response of the organism and determining their roles as adequate indicators of tissue trauma.

Oral surgery induces systemic effects! During oral surgery there is a cumulative effect of the peri-operative stress: surgical traumatic stress, psychological stress and anaesthetic stress.

We determined justification of the use of diagnostic tests to assess the levels of the aforementioned cytokines as orientation marks for the level of post-operative inflammation. They also represent efficient prognostic instruments for early diagnosis of post-operative infection. The knowledge of cytokine levels can give insight to clinical practitioners into the intracellular changes (intracellular environment), as well as provide guidance for further treatment.

Very high post-operative levels of all measured parameters and the maintenance of those high levels after the 4th day (hyper-reaction) may indicate potential complications, post-operative infection and development of septic shock. The condition has the same negative effects as immunosuppression (hypo-responsiveness).

Our research and a great number of studies confirm the thesis that the greater the degree of surgical trauma (soft tissue, periosteum, bone tissue) the greater the possibility of inadequate reaction of the organism, i.e. immunosuppression.

Local post-operative complications depend on the level of local cytokine release. Systemic cytokines and other analyzed parameters are responsible for the general post-operative immunological status of the organism.

The benefits of the minimal invasive surgery (such as minimal pain, quicker recovery and shorter hospitalization) can be connected to decreased cytokine production. Therefore, we are inclined to suggest choosing the methods of the modern minimal invasive surgery rather than conventional surgery in all the cases where applicable.

Individuals suffering from chronic stress show greater degree of immunosuppression during oral surgery, which is a result of exhausted mechanisms of adaptation to the acute stressor - the intervention. This implies the need to ask one more question during the anamnesis: “Are you under stress?” This could help to select patients for whom you could more easily foresee the possibility of an inadequate post-operative reaction.

In accordance with previously stated conclusions, we can underlie the importance of minimizing traumatic


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stress during oral surgery by favouring an atraumatic approach, minimizing pressures and vibrations during the intervention, lowering the temperature developed during the osteotomy (by using systems with continuous internal and external cooling, reducing the number of revolutions per minute of the instruments), using drills made of high quality materials and adequate sharpness.

Equally important is the elimination of stress factors of non-traumatic origin by using premedication (anxiolytics, analgesics), as well as psychological techniques and stress reduction strategies.

It is recommended to use antibiotic and anti-inflammatory prophylaxis during complicated, long-lasting and comprehensive interventions

Steroids, non-steroid anti-inflammatory agents, the cytokines IL-4, IL-10, IL-13 reduce the production of pro-inflammatory cytokines. The most up-to-date administration of these agents can be effective in attenuating post-traumatic and post-operative hyper-inflammation.

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7. Tonnesen E, Christensen VB, Toft P. The role of cytokines in cardiac surgery. Int J Cardiol, 1996; Suppl 53:1-10.

8. Dinarello CA. IL-1 and IL-1 antagonism. Blood, 1991; 12:404-409.

9. Grzelak I, Olszewski WL, Rowinski W. Blood mononuclear cell production of IL-1 and IL-2 following moderate surgical trauma. Eur Surg Res, 1989; 21:114-122.


30. Sternfild DC, Ogle CK. Thermal injury functionally alters bone marrow derived macrophages. J Burn Care Rehabilitation, 1997; 18(6):505.

31. Kiecolt-Glaser JK, Glaser R. Mind and Immunity. Mind/Body Medicine, 1993; pp.39-59.

Correspondence and request for offprints to:Daniela Veleska-Stevkovska Faculty of Dentistry, Clinic for Oral surgery Vodnjanska 17, 1000 Skopje FYR Mcedonia Email: [email protected]

27. Menger M, Vollmar B. Surgical trauma: hyperinflammation versus immunosuppression? Langenbeck’s Archives of Surgery, 2004; 389(6):475-484.

28. Kawasaki T, Ogata M, et al. Surgical Stress induces Endotoxin hyporesponsivness and early decrease of monocyte mCD14 and HLA-DR expression during surgery. Anesth Analg, 2001; 92:1322-1326.

29. Withmann MW. Severe depression of host immune functions following closed bone fracture, soft tissue trauma and hemorragic shock. Critical Care Medicine, 1998; 26:8.

SUMMARYAim. Evaluation of the survival rate and the level of bone resorption

(LBR) between the implants with SLA® (the control group) and SLActive® (the test group) surfaces, placed in the maxilla previously treated with bone block grafts.

Methods. 17 patients were treated with 20 implants (10 with SLA and 10 with SLActive surface). The abutments were tightened with 35 Ncm, after 12 weeks (the control group) and 6 weeks (the test group). Periapical radiographies were made and evaluated after implant placement, after abutment tightening, 1 and 3 months with crown in function.

Results. Survival rate in the control group was 100% comparing to 80% in the test group. The mean value of LBR was 0.192 mm in the control group comparing to 0.106 mm in the test group.

Conclusion. The test group had smaller survival rate, as well as bone resorption, 3 months after loading.Keywords: Implant; Bone Graft; Single Crown

Gojko Cvijic1, Lígia Luzia Buarque e Silva1, Wilkens Aurélio Buarque e Silva1, Renato Mazzonetto2, Frederico Andrade e Silva1

FOP/UNICAMP, São Paulo, Brazil1Dept. of Prosthodontic2Dept. of Maxillofacial Surgery

ORIGINAL PAPER (OP)Balk J Stom, 2010; 14:133-140


Clinical Comparison between Implants with 2 Chemically Different Surfaces, Placed in the MaxillaPreviously Treated with Bone Block Graft

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Introduction

In the last years the use of dental implants has given new solutions for prosthodontic rehabilitation. First cases began in seventies1. Initially, implants with machined surface were used; they would be placed in bone and covered with flap. Many researches indicated the use of rough surface with 1-stage implants2. On that way the bone-to-implant contact was enhanced, which diminished time of osseointegration, while surgical treatment needed only 1 surgery. Comparing bone resorption between 1- and 2-stage implants, the biggest resoption occurred in the first 12 months after prostohodontic treatment. According to Cecchinato et al3 bone resorption around 1- stage implants was 0.02 mm while it was 0.17 mm around 2-stage implants (standard deviation was 0.38 and 0.51 mm respectively).

In 1990 SLA® surface was introduced (Institute Straumann AG, Basel, Switzerland), which diminished the healing time to 6 (I, II and III bone type) and to 12 weeks (IV bone type)4. Principal characteristics of the implant surface are: topography, chemical composition, energy, and hidrofilicity. These characteristics influence on protein adsorption and relation between the implant

surface and bone cells. Hypothetically, high surface energy enlarges the hidrofillicity, what consequently enhances the reaction with biological environment5.

The first implant surface, chemically activated, was introduced in 2004 (SLActive®, Institute Straumann AG, Basel, Switzerland). SLActive surface has the same microstructure as SLA; however, it is chemically different. Zhao et al6 compared the chemically and physically differentiated surfaces and concluded that chemical treatment can enlarge surface energy; due to special way of fabrication, it is protected of contamination, and has faster histological answer. The researches used the disks with SLA surface, wetted with water in the atmosphere with nitrogen, without any contact with air. After gamma sterilization the implants were placed in physiological solution, protecting them of contact with carbon and hydro-carbon. According to Buser et al7 the concentration of carbon and hydro-carbon is smaller on SLActive surface (C, 18.4 ± 2.7 at%) then on SLA (C, 37.3 ± 3.3 at%). Kasemo and Lausmaa8 showed that SLActive surface has dynamic contact angle of 00 comparing to 139.90 on SLA surface. It confirms the hidrofilicity, with high free surface energy of the mentioned chemically treated surface. It is possible that

134 Gojko Cvijic et al. Balk J Stom, Vol 14, 2010

chemical characteristic causes faster bone answer after the placement of implant with SLActive surface.

Independently of the implant surface, edentulous region should have adequate bone width and height to receive the implants. Otherwise, in the atrophic region, bone block graft should be used for succeeding necessary bone quantity9. In the literature, there is a little data explaining prosthodontic rehabilitation with single crown on the implant with SLActive surface, placed in the region that previously had been treated with bone block graft. The aim of this paper was to compare: (1) Survival rate between implants with SLA and SLActive surfaces, placed in the maxilla previously treated with bone block grafts; and (2) Level of bone resorption (LBR) between implants with 2 chemically different surfaces.

Material and Method

Patient SelectionIn this study 20 Straumann implants were used,

placed in 17 patients chosen according to inclusion and exclusion criteria. Every single implant was placed in the region between central incisor and second premolar, and prosthodontically treated with single screwed crown. When possible, the patients received 2 implants with 2 chemically different surfaces, while patients with 1 missed tooth received randomly chosen implant (SLA or SLActive).

The scientific project received approval from Ethic Committee (FOP-UNICAMP, Piracicaba, Brazil) and all patients assigned the Term of collaboration and research.

Inclusion Criteria. The patients were older than 18 years. All of them had lack of bone width in the region between central incisor and second premolar, which was treated with bone block graft, taken from the chin. The mentioned area planned for treatment with dental implants had either 7 to 9 mm from mesial do distal side of neighboring teeth, indicated for 1 implant with platform 4.8 mm, or 14 to 18 mm planned for 2 implants. Apico-coronal space, necessary for adequate crown height, had at least 6 mm. After treatment with bone block graft, the bone had at least 6.1 mm of width, and 10 mm of height, indicating 8 mm implant’s length. The patients were instructed to maintain oral hygiene properly.

Exclusion Criteria. Smokers were excluded from this study, as well as pregnant or lactating females, patients with uncontrolled diabetes mellitus, alcohol or drug abusers, patients with a presence of residual roots, and patients with clinical signs of bruxism. Beside that, there were excluded patients with removable lower dentures, or patients with 3 or more teeth lost in the region between the upper central incisor and second premolar. Also, we did not treat patients with loss of bone height, or inadequate bone quantity in chin region.

ProcedureAll patients underwent initial periodontal therapy,

basical motivation, information about oral hygiene, scaling, and root planning of all teeth. Each bone block had approximately 10 mm of length, 6 mm of width, and 4 mm of thickness. The patients were divided in 2 groups. In the first group, 6 months after the grafting of recipients site, 10 implants with SLA surface were placed. The same procedure was done in the test group, however with 10 Straumann implants with SLActive surface. 10 weeks after the SLA implants placing, and 4 weeks in case of SLActive implants, prosthodontic treatment began; it was finished 12 and 6 weeks afterward, respectively.

Preoperative Treatment. Initially, on each patient periapical radiographs, orthopantomography, and tomo graphy were done. The bone width and height were evaluated using tomography on the recipient and donor sites. Before surgery, the patients were submitted to basic periodontal treatment, during 2 visits. Beside that, they got 4 mg of dexametason and 2 g of amoxicillin, 1 hour before the surgery.

Surgical Procedure. For intraoral cleansing 0.12% chlorhexidine was used, while 0.2% chlorhexidine was used for extraoral cleansing. After that, the access in the recipient edentulous region was done, while the defect was analyzed and measured, planning the size of the bone graft. The recipient region was perforated with drill to stimulate bleeding. In the donor site, in the region of the mandible symphysis, the bone block graft was harvested according to dimension confirmed in the recipient site. The donor site was sutured in 2-steps approach. After block adaptation, it was fixed with the screw (Fig. 1), the borders were rounded, and the gap was fulfilled with cancelous bone. The sutures were removed 7 days postoperatively.

Figure 1. Bone block graft

Balk J Stom, Vol 14, 2010 Implantation in the Previously Bone Grafted Maxilla 135

The same surgical procedure was done for SLA and SLActive implants.

Postoperative Period. Patients were advised to use paracetamol 750 mg, 4 times per day, during 2 days. 7 days postoperatively the sutures were removed, while patients maintained oral hygiene regime in the treated region with 0.12% chlorhexidine, 1 min, 3 times per day, during 2 weeks.

Prosthodontic Procedure. During the period of osseointegration, none of the implants had mobility, pain, infection or any transparency on radiography. The patients were evaluated 10 weeks after SLA implant placement and 4 weeks after SLActive implants. The healing caps were removed, the implants were cleaned with an air and washed, and impression was taken

Implant Placement. The implant placement was done according to the ITI protocol10. After intraoral and extraoral cleansing, horizontal and sulcular incisions were done, accessing the edentulous region treated with bone block graft. The screw was taken out, the bone ridge was scalloped, and implant site perforated with 600 rpm drill speed. The Straumann Implants, 4.1 x 8 mm SP were placed with contra angle (NSK®, 20:1, Japan) with minimum torque value of 20 Ncm. Bone around the surgical implant site, on the vestibular and palatal sides, had at least 1 mm and on mesial and distal sides from 1.5 to 3 mm (Fig. 2). All implants were placed with rough surface completely in bone. The implants were closed with healing caps according to gingival height.

with original components (Institut Straumann, Basel, Switzerland - Fig. 3). SynOcta® abutments were defined and screwed on the plaster model for each case, and single screwed crowns were done (Fig. 4). All abutments were tightened with 35 Ncm and all crowns with 15 Ncm after clinical probe, adaptation on the implant shoulder and radiographic confirmation (Figs. 5 and 6). The crowns were closed with gutta-percha and photo-polymerized resin, with adequate oclusal relation and adjustment (Fig. 7).

Figure 2. Implant site

Figure 3. Healing cap in position

Figure 4. Impression cap


Radiographic Evaluation. After implant placement, periapical radiographies were done in the determined periods, listed below:● T0 - after implant placement;● T1 - impression taking, after 4 (SLActive surface) to

10 (SLA surface) weeks;● T2 - definitive tightening of synOcta abutment;● T3 - 1 month after definitive crown fixation;● T4 - 3 months after definitive crown fixation.

All radiographies were taken with prefabricated positioners, with the cone perpendicular to the radiographic film. The measurements were done with digital caliper (Digimess® Instrumentos de precisão Ltda., Brazil - Fig. 8). The percentage of distortion was calculated based on knowing measures of implant (9.8 mm = 8 mm implant body + 1.8 mm of trans-gingival part). The measure from radiography was diminished for the real value of implant length. Then, the distance between the implant shoulder and the first bone-to-implant contact was measured. From this value was

Figure 5. SynOcta® abutment on the model

Figure 8. Radiographical evaluation with caliper

Figure 7. Metalo – ceramic crown definitely tightened on Straumann® implant

Figure 6. manual tightening in the mouth


diminished 1.8 mm (which corresponds to the implant neck) plus the value of distortion in millimeters.

Finally, from this value the percentage of distortion was diminished, showing the level of bone resorption (LBR). LBR was defined as a distance from a border between rough and machined part of implant, until first bone-to-implant contact. The LBR was measured and evaluated from mesial and distal side of implant for every single implant, in each group, during 5 periods planned for analysis. Also the mean values of LBR on both side of the implant were measured and compared, as well as the difference between LBR of 2o groups. Finally, was compared mean value between 2 groups in the following periods: T0-T1; T0-T2; T0-T3 and T0-T4.

Results

The control group (SLA implants) was treated with single screwed crowns 12 weeks after implant placement. The abutments were tightened with 35 Ncm torque value. The same procedure was done with the test group (SLActive implants), while osseointegration time was 6 weeks before definitive tightening of screwed crowns. In the text bellow, we detected the alterations of LBR in the control as well as in the test group.

LBR around SLA surfaceBone resorption basically began in the third period

of evaluation, with single crown in function. In table 1, the LBR was observed in 4 periods of evaluation. The smallest relation was in the period: implant placing - abutment tightening. In other periods higher bone resorption vslues were found, particularly 3 months after tightening.

Table 1. Comparison of LBR around SLA implants in various periods of evaluations

Period LBR ± SD

Implant placing - impression 0.033 ± 0.017

Implant placing - tightening 0.029 ± 0.024

Implant placing - 1 month after tightening 0.099 ± 0.044

Implant placing - 3 months after tightening 0.161 ± 0.043

LBR around SLActive surfaceAccording to evaluation of LBR around the SLActive

surface, it was relatively stable, particularly in the first periods of evaluation. The main alteration occurred after abutment tightening and with the crowns in function (Tab. 2). The LBR diminished in the second period, but increased later. In this group, 2 implants were lost.

Table 2. The LBR around the SLActive surface between different periods of evaluation

Group SLActive

Period LBR ± SD

Implant placing - impression 0.036 ± 0.015

Implant placing - tightening 0.024 ± 0.007

Implant placing - 1 month later 0.052 ± 0.008

Implant placing - 3 month later 0.081 ± 0.012

Comparison between 2 implant types In the control group, no implant was lost during

3 months of evaluation (Tab. 3). In the test group, 2 implants lost stability in the moment of torque application, and they were removed 3 weeks after. Accordinglyhe survival rate in the test group, 3 months after crown tightening, was 80%.

Table 3. Percentage of survival rate of SLA and SLActive implants

Implants SLA SLActive

No 10 10

Loss 0 2

Survival rate 100% 80%

The value of LBR on the mesial and distal side enlarged with the time. The highest level was 0.231 mm and 0.153 mm in SLA group 3 months after the crown was placed, while in the SLActive group it was 0.109 mm and 0.104 mm, respectively (Tab. 4).

Table 4. Comparison of mean value of LBR on both sides of SLA and SLActive implants

Period Implant placing Impression Tightening 1 Month 3 Months

Side Mesial Distal Mesial Distal Mesial Distal Mesial Distal Mesial Distal

SLA 0.054 0.008 0.096 0.032 0.089 0.106 0.138 0.122 0.231 0.153

SLActive 0.011 0.039 0.083 0.039 0.051 0.046 0.090 0.063 0.109 0.104


Table 5. Mean value of LBR in SLA and SLActive group

Period Implant placing Impression Tightening 1 Month 3 Months

SLA 0.031 0.064 0.097 0.130 0.192

SLActive 0.025 0.061 0.048 0.076 0.106

Table 6. The LBR in different periods of evaluation between two groups

Period SLA SLActive Difference (%)

Implant installation/impression 0.033 0.036 0.003 9.1Implant installation/ abutment tightening 0.029 0.024 0.005 17.24Implant instalation/1 month after tightening 0.099 0.052 0.047 47.47Implant instalation/3 months after tightening 0.161 0.081 0.080 49.69

% - The percentage of difference between 2 groups

Table 5 shows mean values of each period planned in this research. The biggest LBR was 0.192 mm in the SLA group, 3 months after definitive crown placing. In the group with SLActive implants, the biggest value was 0.106 mm, in the same period.

The difference of LBR was 9.1% smaller in the SLA group, but only in the first period (Tab. 6). In other periods, the difference was significantly higher, and group SLActive had smaller value. In the last period, the difference was almost 50% smaller in the SLActive group, comparing to SLA.

Discussion

The dental implants are, nowadays, one of the main solutions for rehabilitation of edentulous regions. Depending of implant type, bone characteristics, indication, and patient profile, time for osseintegration varies between 6 weeks to 6 months4,10. The patient’s necessities and possibilities of the modern implantology bring time reduction. As a possible solution to reduce healing time even more, chemical and physical treatment of implant surface is used.

Our study was realized with the aim to verify the time of osseointegration of relatively short implants, chemically and physically treated, installed in the maxilla previously treated with bone block graft. The implants were loaded with single screwed crowns.

Becktor JP et al11 compared implants installed in the normal alveolar ridge with implants in the region treated with bone block graft. They lost implants basically in the period after loading, what is in concordance with our research refereeing to SLActive implants. Finally, as we did not loose any of SLA implants, we suspect that other uncontrolled variables could influence the implant loss, like: some unexpected surgical act unperceived during the

block fixation or implant placement, or some biological occurrence relating to the block grafts or surgical site.

Raghoebar et al12 evaluated the implants placed in the maxilla previously treated with bone block grafts. The authors headlined the importance of primary and secondary stability. According to them, one of the most important factors for implant stability is the technique for implant placement, and the implant length. In our research, the implants had 8 mm, and 2 of them, with SLActive surface, were lost. The length of the implants in Raghoebar’s paper was bigger. However, if the basic motive for implant loss were length, we would expect the loss of SLA implants as well, simply because they had the same length (8 mm). Nevertheless, no one of them was lost. So, this could lead us to suppose that the implants of 8 mm have certain predictability, even in the grafted region.

Keller et al13 emphasized that if the time after bone block grafts is longer, or/and prosthodontic rehabilitation is delayed, it can cause inadequate biological answer. The period without functional loading and without the stimulus in the bone around the implants could cause additional bone resorption. Beside that, the real time for loading of implants placed in the region treated with bone block is unknown and varies from patient to patient. We believe that the reduced healing time defined for implants with SLActive surface did not influence the loss that had occurred. The healing time of 3 months, for SLA implants, apparently could be considered as adequate even in the grafted region. However, we can not forget that in our research we used only 10 implants with the mentioned type of surface. We strongly believe that longer period of evaluation, with bigger number of implants placed in same conditions, could bring clearer answers. The size of the bone block used in bone augmentation corresponded to the edentulous region, and was adapted on the well known way. However, it can be more than 1 variable, that wasn’t controlled appropriately simply because it was clinical trial with certain difficulties to control.


Even though, considering this possibility of uncontrolled variable, basically relating to bone quality as possible cause of implant loss, we must headline that the graft was taken out from the chin, considering this region as an excellent donor site, with favourable bone quality. We suppose that histological analysis of bone in the region of the contact with graft, or histological analysis of bone sample trephined from the implant site could bring some responds about implant loss.

According to Lioubavina-Hack et al14, the implant stability is one of key-factors for success with dental implants. When we evaluated implants installed in both groups we did not realize any type of movements. Then, according to Albrektsson et al15, the test of mobility is unique clinical procedure that can give the evidence if the implant has adequate implant stability. Considering this fact, we can exclude the inadequate implant stability as a potential factor for implant loss, because all of the implants had insertion torque higher then 20 Ncm. Beside that, they did not have any lateral movement after insertion in the bone, perceived by surgeon. If we analyze the necessary insertion torque, we can find that there is no clear consensus in the current literature. Some authors mentioned 20 Ncm as minimum torque value necessary for implant placement, while enlarging the primary stability the chance for growing of fiber tissue getting down. Nevertheless, we can’t simply subestimate evident implant loss in group with SLActive surface, perhaps due to unregistered stability failure. This can be the motive for eventual use of other ways to detect and define primary stability; much more precise measure of implant stability, perhaps, could help to predict possible loss, as well as to define exact time of implant osseointegration, and consequent loading.

So, we can share the opinion of Raghoebar et al12 who stated that criteria of determining the minimal primary stability for implants placed in the region treated with block graft must be established. If it is to be done, it would be feasible to eliminate this variable as a possible cause of implant loss. The loss of implant stability occurred in the moment of torque application 6 weeks after the placement in the grafted region. According to Oates et al16, the implant stability can vary during time due to the histological process that occurs in bone. That is why the time of osseointegration is not unique parameter that influences the survival rate and implant loss. Considering the mentioned problems and explanations, it seems that clearer consensus could clarify the doubts about implants with chemically treated implant surface placed in the grafted region of mandible and maxilla.

The implants that were used in our study had diameter of 4.1 mm and length of 8 mm. According to research parameters, the group with SLA surface had 100% of survival rate after prosthodontic rehabilitation, during 3 months of evaluation. The control group had 80% of survival rate. However, from biological point

of view, it is a question whether the loss of 2 from 10 placed implants can be the parameter that indicates a longer period of osseointegration for SLActive surface? Due to relatively small number of implants chemically treated, we can not give strong affirmation that the time for healing must be longer then 6 weeks. Certainly, other researches can answer with more precisely that question. If we consider only numerical results, they show relatively high survival rate for SLActive implants, similar to other researches cited above.

Zolner et al17 compared LBR around SLActive implants. The evaluation was done between 383 implants loaded immediately and others with early loading. The success rate was 98% in the group with immediate loading and 97% in the group with early loading. The mean LBR was 0.82 mm ± 0.89 mm and 0.56 mm ± 0.73 mm, respectively. In our research, the mean LBR was 0.106 mm with definitive crown 3 months in function (Tab. 5). This result shows good bone answer around 1-stage implants. Comparing the LBR between determined periods, it varied between 0.036 ± 0.015 mm and 0.081 ± 0.012 mm (Tab. 2).

Cochran et al18 explained the principal reasons for bone resorption around implants, headlined that surgerical trauma usually causes alterations in blood supply and consequently insufficient bone nutrition. After abutment tightening, the additional bone resorption is expectable because of functional loading that principally affects coronal part of bone around implants. Our results have shown that the value of LBR in SLA group was 0.033 mm, comparing to SLActive group, where the LBR was 0.036 mm (Tab. 6). With the prosthesis in function, the LBR continued to grow to 0.099 mm (SLA) and 0.052 mm (SLActive) after 1 month, and 0.161 mm (SLA) and 0.081 mm (SLActive) after 3 months. The difference between 2 groups was 49.69% smaller in group with SLActive implants, which can be explained with high surface energy of chemically treated implants19.

Schwarz et al20 detected quantitative alteration in bone around SLActive surface that enlarged in the region with bone dehiscence prepared with the purpose for this research. The results in our research have shown low degree of bone resorption in the case of SLA surface, as well as around SLActive surface. In the group with SLActive surface, in some patients, we detected the bone growth. These data waren’t statistically evaluated for the 2 groups that we had (SLA and SLActive) simply because of the fact that the number of implants was relatively small. However, if we had more SLActive and SLA implants, placed in same conditions, we could expect statistical significance. Nevertheless, the bone growth could be explained with high surface energy6,19,20. Finally, it is necessary to evaluate more implants in same conditions, using the same parameters, but some new parameters as well, that can bring better and clearer explanations.


9. Yerit KC, Posch M, Hainich S, Klug C, Wanschitz F, Wagner A, et al. Long term implant survival in the grafted maxilla: results of a 12-year retrospective study. Clin Oral Impl Res, 2004; 15:693-699.

10. Buser D, Von Arx T, ten Bruggenkate C, Weingart D. Basic surgical principles with ITI implants. Clin Oral Impl Res, 2000; 11(Suppl):59-68.

11. Becktor JP, Isaksson S, Sennerby L. Survival Analysis of Endosseus Implants in Grafted and Non-grafted Edentulous Maxillae. Int J Oral Maxillofac Implants, 2004; 19:107-115.

12. Raghoebar GM, Schoen P, Meijar HJA, Stellingsma K, Vissink A. Early loading of endosseus implants in the augmented maxilla: a 1-year prospective study. Clin Oral Impl Res, 2003; 14:697-702 .

13. Keller EE, Tolman DE, Eckert S. Surgical-Prosthodontic Reconstruction of Advanced Maxillary Bone Compromise with Autogenous Onlay Block Bone Graft and Osseointegrated Endosseus Implants: A 12 year study of 32 Consecutive Patients. Int J Oral Maxillofac Implants, 1999; 14:197-209.

14. Lioubavina-Hack N, Lang PN, Karring T. Significance of primary stability for osseointegration of oral implants. Clin Oral Impl Res, 2006; 17:244-250.

15. Albrektsson T, Johansson CB, Sennerby L. Biological aspects of implant dentistry: osseointegration. Periodontology 2000, 1994; 4:48-73.

16. Oates TW, Valderrama P, Bischof M, Nedir R, Jones A, Simpson J, Toutenburg H, Cochran DL. Enhanced implant stability with chemically modified SLA surface: A randomized pilot study. Int J Oral Maxillofac Implants, 2007; 22:755-760.

17. Zolner A, Ganeles J, Korostoff J, Guerra F, Kraft T, Bragger U. Immediate and early non oclusal loading of Straumann implants with a chemically modified surface (SLActive) in the posterior mandible and maxilla: ínterim results from a prospective multicenter randomized-controlled study. Clin Oral Impl Res, 2008; 19:442-450.

18. Cochran DL, Nummikoski PV, Higginbottom FL, Herman JS, Makins SR, Buser D. Evaluation of an endosseus titanium implant with a sandblasted and acid-etched surface in the canine mandible: radiographic results. Clin Oral Impl Res, 1996; 7:240-252.

19. Rupp F, Scheideler L, Olshanska N, de Wild M, Weiland M, Geis-Gerstorfer J. Enhancing surface free energy and hidrophilicity through chemical modification of microstructured titanium implant surfaces. J Biomed Mater Res, 2006; 76A:323-334.

20. Schwarz F, Sager M, Ferrari D, Herten M, Wieland M, Becker J. Bone regeneration in dehiscence-type defects at non-submerged and submerged chemically modified (SLActive) and conventional SLA titanium implants: an immunohistochemical study in dogs. J Clin Periodontol, 2008; 35:64-75.


Dr. Gojko CvijicR.Dom Pedro II, 627 PiracicabaCEP 13400-390São Paulo, BrazilE mail: [email protected]

Conclusion

According to our method and the results obtained in this research, we can conclude that:

Implants with SLActive surface, placed in the maxilla in the region previously treated with block graft, and loaded with single screwed crowns 6 weeks after, have survival rate 20% smaller comparing to implants with SLA surface, in evaluation during 3 months after loading;

The LBR around implants with SLActive surface was smaller then for implants with SLA surface.

Acknowledgement: The authors wish to express their gratitude to Straumann Brazil for providing the material used in this research, and to Departments of Prosthodontics and Maxillo-Facial-Surgery, UNICAMP, Piracicaba, Brazil, for their support.

References

1. Branemark P-I, Hansson B-O, Adel R, Breine U, Lindstrom J, Hallen O, Ohman A. Osseointegrated implants in the treatment of edentulous jaw. Experience of 10-year-period. Scand J Plastic Reconstr Surg Hand Surg, 1977; 11(Supll. 16).

2. Buser D, Schenk DRK, Steinmann S, Fiorellini JP, Fox CH, Stich H. Influence of surface characteristic on bone integration of titanium implants. A histometric study in miniature pigs. Journal of Biomedical Materials Research, 1991; 25:889-902.

3. Cecchinato D, Olson C, Lindhe J. Submerged or non-submerged healing of endosseus implants to be used in the rehabilitation of partially dentate patients. J Clin Periodontology, 2004; 31:299-308.

4. Cochran DL, Buser D, Tem Brugenkate C, Weingart D, Taylor T, Bernard J, Peters F, Simpson J. The use of reduce healing times on ITI implants with sand-blasted and acid-etched (SLA) surface: Early results from clinical trials on ITI SLA implants. Clin Oral Impl Res, 2002; 13:144-153.

5. Kilpadi DV, Lemons JE. Surface energy characterization of unalloyed titanium implants. J Biomed Mater Res, 1994; 28:1419-1425.

6. Zhao G, Schwartz Z, Wieland M Rupp F, Geis-Gerstofer J, Cochran DL, Boyan BDJ. High surface energy enhances cell response to titanium substrate microstructure. Biomed Mater Res, 2005; 74A:49-58.

7. Buser D, Broggini N, Wieland M, Schenk RK, Denzer AJ, Cochran DL, et al. Enhanced bone apposition to a chemically modified SLA titanium surface. J Dent Res, 2004; 83(7):529-533.

8. Kasemo B, Lausmaa J. Biomaterial and implant surface: on the role of cleanliness, contamination, and preparation procedure. J Biomed Mater Res, 1988; 22:145-158.

SUMMARYThe oral mucosa is susceptible to tissue injury from many causes,

including infection, autoimmune disorders, surgical and accidental trauma, and gingival and periodontal inflammation. However, little is known about the events that influence wound healing in the mouth. This study investigated the changes in salivary thromboplastic activity prior to and after primary tooth extraction. Cytological smears and biochemical tests were also used in this study.

Salivary pH and salivary flow rate did not significantly change after primary tooth extraction. Salivary thromboplastic activity was not significantly higher after extraction than before extraction (p=0.068). Epithelial cells significantly decreased and leukocyte cells significantly increased in saliva after primary tooth extraction (p<0.001). It is concluded that oral cavity may aid the process of haemostasis and, perhaps, wound healing in the extraction area by increasing salivary leukocyte cells and thromboplastic activity. Thromboplastic activity may be a novel indicator of wound healing completion.Keywords: Salivary cells; Salivary flow rate; Salivary pH; Salivary thromboplastic activity; Tooth extraction

T. Tunali Akbay1, M. Guvercin2, O. Gonul2, A. Yarat1, S. Akyuz3, R. Pisiriciler4, K. Göker2

Marmara University, Faculty of Dentistry, Istanbul, Turkey 1 Department of Biochemistry2 Department of Maxillofacial Surgery3 Department of Paediatric Dentistry4 Department of Histology and Embryology



Salivary Thromboplastic Activity May Indicate Wound Healing in Tooth Extraction

TUPNBUP

MP

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Introduction

Wound healing is a dynamic, multi-faceted physiological process that involves a wide range of biological mediators1. Although primary tooth extraction is a simple intervention, some complications can occur. The oral mucosa is susceptible to tissue injury from many causes, including infection, autoimmune disorders, surgical and accidental trauma, and gingival and periodontal inflammation; however, little is known about the events that influence w ound healing in the mouth15. Normally tooth extraction induces some changes in the oral cavity2. Saliva is important in maintaining oral health and much of this protective activity is mediated by the variety of different salivary proteins3.

Damaged tissue release a lipoprotein known as thromboplastin (tissue factor FIII) which activates the extrinsic coagulation pathway. Activated monocytes and endothelial cells also express this thromboplastin

in their surface and participate in the coagulation4. Thromboplastin initiates the coagulation system and is a component of cell membrane but not found active in blood5,6. Like body fluids, saliva has also been known to have thromboplastic activity7- 10. Thromboplastin in saliva is thought to supply the haemostasis when injury takes place in the mouth and it facilitates the barrier function of buccal mucosa7,11. Thromboplastin is related to cells and cell fragments in saliva12,13. In the adult vertebrate body, physical integrity is continuously maintained by a tissue repair mechanism consisting of the vascular endothelium, tissue factor and circulating Factors VII, IX and X that governs localized thrombin elevations. The cellular effects of these thrombin elevations explain all aspects of tissue repair and maintenance14.

It has been shown that oral cavity is also affected from the disturbances of the haemostatic system in which spontaneous bleeding of the dental tissues, pettechies of oral soft tissues and ecchymosis are common in

142 T. Tunali Akbay et al. Balk J Stom, Vol 14, 2010

routine oral examination15. Post operative examinations show that minor oral surgeries can also cause bleeding. Salivary thromboplastin may establish the haemostasis after oral trauma7,11. Thromboplastin also contributes to wound healing, inflammatory response, tumour growth, metastasis and angiogenesis16,17. The disturbance of haemostatic balance in the oral cavity is an indicator of an acquired or congenital defect of the coagulation system18.

There is no study in the literature involving the relationship between the wound healing after primary tooth extraction and salivary thromboplastic activity. In this study, for the first time, wound healing in oral cavity is followed by the salivary thromboplastic activity after primary tooth extraction. Salivary pH and salivary flow rate were also determined and saliva imprint samples were also evaluated cytologically.

Material and Methods

This study was performed on 25 healthy children, age ranging 7-12 years. Children were randomly selected from individuals who attended Marmara University, Faculty of Dentistry. Oral dental examinations and restorative treatments were conducted by a pedodontist. Among the group, 80% of the children had experienced dental extractions before. Children were sent to the Oral and Maxillofacial Surgery Department for tooth extraction due to caries and pulpal pathology. Children had standard breakfast 1 hour before tooth extraction. 3 primary canine, 1 primary incisor, and 21 primary molar teeth were extracted from 25 children. Bleeding stopped in 60 minute in all children. Informed consent was obtained from each subject’s family before saliva collection.

All saliva samples were collected in resting position in the surgery department. First, the subjects rinsed their mouths with distilled water 3 times. Non-stimulated mixed saliva samples were collected by spitting into a funnel prior to and 1 hour after primary tooth extraction. Collection time was recorded and saliva volume was measured by pipetting the saliva into a new tube. Then the tubes were stored at -200C. Salivary flow rate was calculated by dividing the saliva volume to collection

time. Saliva samples were analyzed for pH by using pH paper (Neutralit-pH 5.5-9.0; Merck).

Thromboplastic activity of saliva samples was evaluated according to Quick’s 1 stage method using normal plasma19. This was performed by mixing 0.1 ml of saliva with 0.1 ml of 0.02 M CaCl2, with the clotting reaction being started on addition of 0.1 ml of plasma. All reagents were in the reaction temperature (370C) before admixture. Since the clotting time is inversely proportional to the salivary thromboplastic activity (STA), the lengthening of the clotting time is a manifestation of the decreased STA.

For cytological examinations, saliva samples were smeared over a glass microscope slide and fixed with air. They were stained with Giemsa stain20. All slides were examined microscopically (x100) for the presence of epithelium, erythrocyte, leukocyte and yeast cell.

The results were evaluated using a Student t-test, Wilcoxon test, Pearson correlation analysis and Spearman correlation analysis using the Unistat 5.0 statistical package programme.

Results

Mean age and body mass indices of 25 children were 9.88±1.39, and 16.70±2.23, respectively. According to sex, no significant differences were found in all parameters.

Salivary pH values and salivary flow rates did not significantly change after primary tooth extraction (Table 1). Salivary thromboplastic activity was moderately higher after extraction than before extraction (p= 0.068).

No significant difference was found concerning yeast cells in saliva imprint samples of both groups (p>0.1). There were no erythrocyte cells in saliva imprint samples (Table 2). Epithelial cells significantly decreased and leukocyte cells significantly increased in saliva imprint samples after primary tooth extraction (p<0.001).

Significant negative correlation was found between salivary flow rate and STA before primary tooth extraction (r= -0.413; p=0.04).

Table 1. Comparison of salivary flow rate, pH and salivary thromboplastic activity (STA) prior to and after primary tooth extraction

Pre-extraction period(n=25)

Post-extraction period(n=25)

p(t-test)

Salivary flow rate (ml/min) 0.21 ± 0.11 0.21 ± 0.08 0.782Salivary pH 7.22 ± 0.45 7.34 ± 0.40 0.136STA (sec) 72.64 ± 27.59 60.08 ± 26.23 0.068

Values are given as mean standard deviation. Since the clotting time is inversely proportional to the thromboplastic activity, the lengthening of the clotting time is a manifestation of decreased thromboplastic activity.

Balk J Stom, Vol 14, 2010 Wound healing and thromboplastic activity 143

Pre-extraction period(n=25)

Post-extraction period(n=25) p (Wilcoxon)

Epithelial cells(1)(2)(3)

16%28%56%

64%36%0%

0.001

Leukocyte cells(0)(1)(2)(3)

56%32%4%8%

4%16%56%24%

0.001

Yeast cells(0)(1

68%32%

68%32%

1.000

Epithelial cells: (1) 0-15 cells (normal) (2) 16-30 cells (medium) (3) more than 30 cells (too much)Leukocyte cells: (0) nonexistent (1) 3-5 cells (2) 6-15 cells (3) more than 16 cellsYeast cells: (0) nonexistent (1) present

they are exposed to vein media or collagen 23-25. Clot formation and wound healing process following tooth extraction are initiated by means of saliva. In the present study, leukocyte cell count increased 1 hour after tooth extraction. There was no significant difference in STA between pre and post tooth extraction; this may be due to the decrease in epithelial cell count or inhibition of thromboplastic activity by specific inhibitor like tissue factor pathway inhibitor. Saliva, as well as blood coagulation system, can play preventive role in oral cavity damages. Thromboplastin also plays an important role in wound healing, inflammatory response, tumour growth, metastasis and angiogenesis16,17.

The relationship between salivary pH and salivary flow rate is well known12,13. Fluctuations in salivary pH may change the secretion of thromboplastin from the cells present in saliva. Salivary pH and STA can be affected by changes of salivary flow rate. In the present study, salivary pH and flow rate did not change after primary tooth extraction. No significant difference in STA between groups may indicate wound healing after tooth extraction .

The negative correlation between STA and salivary flow rate has been shown in healthy subjects10. In the present study, there was also negative correlation between STA and salivary flow rate (r= -0.413; p=0.04) before tooth extraction; interestingly this correlation was not seen after tooth extraction. This finding may support the secretion of thromboplastin from the leukocyte cells in 60 minutes following tooth extraction.

It might be concluded that oral cavity aids the process of haemostasis and, perhaps, wound healing in the extraction area by increasing salivary leukocyte cells and thromboplastic activity. STA may be a novel indicator of wound healing completion.

Table 2. Cytological examination of saliva samples

Discussion

Wound healing process seem to be strictly regulated by multiple growth factors and cytokines released at the wound site18. In this study, possible new marker - thromboplastin is monitored in saliva after tooth extraction. Alterations that disrupt controlled timely healing process would extend tissue damage and prolong repair18.

Whole saliva is the most frequently used type of saliva for the diagnosis of systemic diseases, since it is readily collected and contains serum constituents. These constituents are derived from the local vasculature of the salivary glands and reach the oral cavity via the flow of gingival fluid. Analysis of saliva may be useful for the diagnosis of hereditary disorders, autoimmune diseases, malignant and infectious diseases, and endocrine disorders, as well as in the assessment of therapeutic levels of drugs and the monitoring of illicit drug use21,22. Salivary thromboplastin may establish haemostasis following oral trauma and facilitate barrier functions of oral mucosa7,11. As STA has been measured by PT test, shortened clot formation time shows increased thromboplastic activity19. STA was moderately increased after a single tooth extraction. Our preliminary assays (data not shown) showed a significant increase in STA after 2 teeth extraction. According to this finding, the size of an extraction area and the quality of the extraction may affect the STA.

Thromboplastin is related to the cells and cell fragments of saliva, so that high cell content (epithelial and leukocyte cells) of saliva increases the STA7,10,11. Normally, leukocyte cells do not have thromboplastic activity; they can only secrete thromboplastin when

144 T. Tunali Akbay et al. Balk J Stom, Vol 14, 2010

References

1. McGrory K, Flaitz CM, Klein JR. Chemokine changes during ora l wound healing. Biochem Biophys Res Commun, 2004; 324:317-320.

2. Simon E, Matee M. Post-extraction complications seen at a referral dental clinic in Dar Es Salaam, Tanzania. Int Dent J, 2001; 51:273-276.

3. Kaufman E, Lamster IB. The Diagnostic Applications of Saliva - A Review. Crit Rev Oral Biol Med, 2002; 13:197-212.

4. Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol, 2007; 25:9-18.

5. Bachli E. History of tissue factor. Br J Haematol, 2000; 110:248-255.

6. Selighson U. Disseminated intravascular coagulation (DIC). In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn URI (eds.). Williams Hematology. USA: McGraw-Hill Companies Inc., 2001; pp 1677-1695.

7. Zacharski LR, Rosenstein R. Reduction of salivary tissue factor (thromboplastin) activity by warfarin therapy. Blood, 1979; 53:366-374.

8. Lwaleed BA, Francis JL, Chrisholm M. Urinary tissue factor levels in neoplastic disease. Ann Saudi Med, 2000; 20:197-201.

9. Tutuarima JA. Hische EAH, Trotsenburg L, Helm HJ. Thromboplastic activity of cerebrospinal fluid in neurological disease. Clin Chem, 1985; 31:99-100.

10. Yarat A, Tunali T, Pisiriciler R, Akyuz S, Ipbuker A, Emekli N. Salivary thromboplastic activity in diabetics and healthy controls. Clin Oral Invest, 2004; 8:36-39.

11. Belikov PP. Blood coagulation factors in saliva in injury of oral mucous membrane. Stomatologia (Mosk), 1971; 50:72-74.

12. Flink H, Tegelberg A, Lagerlof F. Influence of the time of measurement of unstimulated human whole saliva on the diagnosis of hyposalivation. Arch Oral Biol, 2005; 50:553-559.

13. Fenoll-Palomares C, Munoz Montagud JV, Sanchiz V, Herreros B, Hernandez V, Minguez M, Benages A. Unstimulated salivary flow rate, pH and buffer capacity of saliva in healthy volunteers. Rev Esp Enferm Dig, 2004; 96:773-783.

14. Coleman LS. A hypothesis: Factor VII governs clot formation, tissue repair and apoptosis. Med Hypoth, 2007, 69:903-907.

15. Whelton H. The anatomy and physiology of the salivary glands. In: Edgar WM, OiMullane DM (eds). Saliva and Oral Health. Great Britain: Thanet Press Lim, 1996; pp. 1-8.

16. Rickles FR, Shoji M, Abe K. The role of hemostatic system in tumor growth, metastasis and angiogenesis : tissue factor is a bifunctional molecule capable of inducing both fibrin deposition and angiogenesis and cancer. Int J Hematol, 2001; 73:145-150.

17. Rickles FR, Patierno S, Fernandez PM. Tissue factor, thrombin and cancer. Chest, 2003; 124:58-68.

18. Sindet-Pederson S. Haemostasis in oral surgery the possible pathogenetic implications of oral fibrinolysis on bleeding. Experimental and clinical studies of the haemostatic balance in the oral cavity, with particular reference to patients with acquired and congenital defects of the coagulation system. Dan Medl Bull, 1991; 38:427-443.

19. Ingram GI, Hills M. Reference method for the one-stage prothrombin-time test on human blood. Thromb Haemost, 1976; 36:237-238.

20. Atay Z, Topalidis T. Cytodiagnostik der Serösen Höhlen. Atlas und Lehrbuch. Hannover: Wolfgang Pabst Verlag, 1994; p 356.

21. Humphrey SP, Williamson RT. A review of saliva: Normal composition, flow and function. J Prosth Dent, 2001; 85:162-169.

22. Jansen Van Rensburg BG. Saliva. In: Oral Biology. Berlin: Quinstessence Publishing Co Inc, 1995; pp 469-478.

23. Butenas S, Bouchard BA, Brummel-Ziedins KE, Parhami-Seren B, Mann KG. Tissue factor activity in whole blood. Blood, 2005; 105:2764-2770.

24. Day SM, Reeve JL, Pedersen B, Farris DM, Myers DD, Im M. Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood, 2005; 105:192-198.

25. Roberts HR, Monroel DM, Hoffman M. Molecular biology and biochemistry of the coagulation factors and pathways of hemostasis. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn URI (eds.). Williams Hematology. USA: McGraw-Hill Companies Inc, 2001; p 1409-1425.


Aysen YaratMarmara University, Faculty of DentistryDepartment of BiochemistryGuzelbahce, Buyukciftlik sok. No. 634365 NisantasiIstanbul, TurkeyE-mails: [email protected] [email protected]

SUMMARYWith intention to contribute to clinical findings at children with

haematology disorders, we formed the aim of our project - to notice the oral signs and symptoms at patients diagnosed with anaemia and acute leucosis, depending of disease’s duration, in order to realize similarities and differences between them.

40 children at age of 1-12 year, hospitalized at the Paediatric Clinic, Department of Haematology and Oncology, were examined. Anaemia was diagnosed in 15, and acute leucosis in 25 patients. Clinical examination consisted of inspection and palpation. Subjective parameters (pain, burning, indefinite taste, xerostomia) and objective signs (oral mucosa colour, bleeding, mucosal and lip changes and loss of tongue papillae) were evidenced in all patients.

The results showed that patients with anaemia (the disease was diagnosed at least 15 days previously) had exceptionally pale oral mucosa, evident loss of tongue papillae, angular cheillitis, glossodynia and glossopirosis. If the duration of the disease was at least 2 years, glossodinia and glossopirosis were discreet and weakly expressed, and cheillitis sicca was objectively present. Subjects with acute leucosis had comparatively much more prominent signs: burning pain and swelling were evident and strong, mouth opening limited, and there was a diffuse erythema throughout the oral cavity (the oral cavity was coloured red). Gingivitis with spontaneous bleeding, cheillitis exfolliativa exudativa and pale face were also present regardless time when the disease was diagnosed.

It was concluded that differences in oral clinical signs were due to bone marrow insufficiency in patients with leucosis, which influences all blood cells, and is manifested by disruption local immunity, in comparison with primary hypo-oxygenation present in patients with anaemias.Keywords: Blood Disorders; Anaemia; Leucosis; Bleeding, oral

Mirjana Popovska1, Mihajlo Petrovski1, Aneta Atanasovska-Stojanovska1, Zorica Antovska2, Jordan Dzurcevski1

1 Faculty of DentistryUniversity Dental Clinical CentreDepartment of Oral Pathology and Periodontology Skopje, FYROM 2 Faculty of Medicine, Paediatric Clinic Department of Haematology and OncologySkopje, FYROM



Oral Findings in Anaemias

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Introduction

Almost all haematological disorders can cause different kinds of changes in the oral cavity. Anaemias are the most common among haematological disorders. Lesions in the oral cavity can be noticed soon after signs of anaemia are noticed and the disease diagnosed, at the same time when disease is diagnosed. However, oral lesions could be the primary clinical manifestation of the disorder. Accordingly, it can be said that the role of dentists might be very important concerning diagnostic, prognostic and therapeutic issues.

Anaemia is a clinical condition characterized by acquired or hereditary abnormality of erythrocytes, their precursors or theirs’ structural elements3. However, sometimes it could be a consequence or manifestation of other, non-haematological disorders in the organism. In general, anaemia could be defined as quantitative deficit of erythrocytes, and due to this deficit, the oxygenic capacity of the blood is diminished. The basic elements for diagnosis of anaemia are reduction of the haematocrit, haemoglobin or hem. The enormous number of different kinds of anaemias can be classified into 3 groups:

146 Bahar Sezer et al. Balk J Stom, Vol 14, 2010

microcyt, normocyt and macrocyt anaemias, with different clinical entities1,4,10.

According to the statistic data from different sources, it could be concluded that the most frequent from all anaemias is fero-deficit anaemia (according to the WHO data13, 2.3% of children between 9-11 years of age, and 5.7% of children between 2-4 years of age, have fero-deficit anaemia).

The aim of this investigation was to notice oral signs and symptoms in patients diagnosed to have anaemia depending on the moment of diagnosing the disease (within 15 days, 3 months and six months).

Material and Method

45 children 1-12 years of age, hospitalized at the Paediatric clinic, Department of Haematology and Oncology, and examined at the Department of Oral Pathology and Periodontology were included into the study.

Depending on the disease duration, all the observed patients were divided in 3 groups:• First group - patients with anaemia diagnosed within

15 days;• Second group - patients with anaemia diagnosed

within 3 months;• Third group - patients with anaemia diagnosed within

6 months.Before the oral clinical examination, medical and

dental histories were evaluated. We faced a problem of precise communication with children due to their average age, so it was difficult to interpret the subjective parameters correctly. Of course, some of our patients didn’t talk at all, and we could not receive any information concerning their subjective difficulties. In these cases we used information from their parents.

In all of our patients, oral clinical examination was performed. To notice the existence of some oral changes, all subjective symptoms and objective signs were noticed. Concerning dental history, special attention was directed to probable genetics anamnesis, existence of bad habits, socio-economical conditions, etc. Subjective parameters as pain, burning, indefinite taste, or xerostomia were evidenced at every single subject.

Oral clinical examination was performed by inspection and palpation. Parameters like oral mucosa colour, mucosa bleeding, oral mucosa and lips changes and loss of the tongue papillae or their hypertrophy, the existence of erosions or ulcers were noticed and compared with subjective symptoms.

Data are processed and presented in tables and graphics.

Results

Figure 1 points out the percentage of different kinds of anaemias in the examined children. It can be noticed that the most common type of anaemia was fero-deficit type (39%); pernicious anaemia and anaemias associated with some chronic disorder were present in 27% of cases. In small percent of patients a combination of fero-deficit and pernicious anaemia was noticed.

The results point out the existence of pale oral mucosa, evident loss of tongue papillae, angular cheillitis, glossodynia and glossopirosis in patients with anaemia diagnosed within 15 days prior to the examination. Glossopirosis was frequently common in subjects with anaemia diagnosed within 3 months prior to the examination, but it was generally moderate in comparison to patients from the first group. In patients from the third group, where anaemia was diagnosed within 6 months prior to the examination, oral symptoms were very light, some glossodynia and glossopirosis and discrete increasing of saliva production (Tab. 1).

It is evident from table 2 that in patients with anaemia, in the beginning of the disease, all clinical symptoms were moderate (change of oral mucosa colour, loss of tongue papillae and cheillitis angularis). These symptoms calmed with the evolution of the disorder and were discretely present later. Eritema, petechiae, echymosis or gingival bleeding (caused by gingival inflammation) were not noticed in any period of the disease in none of the examined subjects.

Figure 2 depicts the fact that subjective symptoms calmed proportionally with the duration of the disorder.

Figure 1. Percentage of different kinds of anaemia in the examined children

Table 1. Subjective symptoms in the oral cavity according to the

duration of anaemia

Subjective symptoms Duration of anaemia

Within 15 days

Within 3 m.

Within 6 m.

Oral pain +++Glossodynia and glossopirosis +++ ++ +Undefined taste + +Hypersalivation +Spontaneous bleedings

Balk J Stom, Vol 14, 2010 Oral Findings in Anaemias 147

Figure 2. Appearance of oral objective signs in patients with anaemia during the disease

Discussion

Anaemias are more common diseases in children. Generally, anaemias have benign nature, but in children population can cause serious difficulties due to many subjective and objective problems in different stages of evolution of the disease. Clinical manifestations of the disease vary depending on age, extent, severity, prior existence of other systemic disorders and other factors. Slight and moderate controlled anaemias are mainly asymptomatic4. Symptom of main importance is paleness of oral mucosa, which is important in diagnostic and prognostic view.

In this context Siljanovski10 notices that anaemic patients, regardless of the type of anaemia, beside general symptoms characteristic for the disease, have oral changes too. According to this author, the most common (30-45%) are tongue changes, such as atrophic glossitis, as well as angular stomatitits and Candidiasis. The presence of these oral symptoms Siljanovski has commonly noticed with general symptoms like fatigue, fainting, paraestesias and other. In patient with aplastic anaemia, presence of gingival hyperplasia is noticed, especially in patients aged 5-7 years. In clinical presentation of this type of anaemia, a catharal gingivitis with moderate bleeding is noticed, but mainly related to some underlining systemic

Table 2. Oral objective signs according to the duration of anaemia

Objective signsDuration of anaemia

Within 15 days

Within 3 months

Within 6 months

Pale mucosa ++ +Loss of tongue papillae ++ + +Angular cheilitis ++ + +Cheilitis sicca + + +EritemaPetechiae and echimosesgingivitis Gingival bleedings

disorder like neutropenia or leukaemia8. Apart from that, in some patients with anaemia, a specific oral aphthous changes are noticed. Epithelisation of these lesions shorts when patients undergo blood transfusion and better haemoglobin blood level9. By some authorities in oral pathology and medicine, pethechiae and echimosis together with gingival hyperplasia are the most common in patients with aplastic anaemia2. The same authors noticed a presence of spontaneous gingival bleedings and herpetic lesions.

Our research disclosed that in patients with anaemia the most frequent subjective symptoms were glossodinia, indefinite taste and hypersalivation. Thess symptoms are more prominent at the beginning of the disease, and continuously reduce. After 6 months, the most of the symptoms were discrete, although glossodinia still persisted. The most frequent objective symptom in our patient was angular cheilitis. The symptom was noticed in all patients regardless the disorders’ duration. Pale mucosa, loss of the tongue papillae and angular chalets were noticed in patients at the beginning of the disorder. Similar findings were noticed by other authors too3,10.

As anaemias are defined as absolute reducing of erythrocytes numbers, reducing of haemoglobin concentration, or reducing of haematocrit, we can speculate that subjective symptoms and objective signs are caused by the reduced oxidative blood ability, reducing of normal transportation of the needing quantity of oxygen from lungs to peripheral blood. All this changes lead to tissue and cell hypo-oxygenation, which is clinically noticed in the oral cavity and other organs.

Different findings in our patients in different stages of the disorder (within 15 days, 3 months, or 6 months) might be explained in similar way: the most prominent symptoms at the beginning of the anaemia, i.e. in the acute stage of the disease, are the consequence of the genuine mechanism of the disease; later on, within 3 and 6 months after the diagnosis had been made, probably as a consequence of the undertaken treatment and normalisation of the disturbed haematological processes, oral symptoms also diminish. Our findings of subjective and objective improving in clinical findings within 6 months support this assumption.

References

1. Anderson C, Aronson I, Jacobs P. Erythropoiesis: Erythrocyte deformability is reduced and fragility increased by iron deficiency. Haematology, 2000; 4(5):457-602.

2. Brenan MT, Sankar V, Baccaglini L, Pillermer SR, Kingman A, Nunez O, Young NS, Atkinson JC. Oral manifestation in patients with aplastic anaemia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2001; 92(5):503-507.

3. Datz M. Approach to the patients with anaemia. Anaemia Research Today, Vol. 1, 2004.

148 Bahar Sezer et al. Balk J Stom, Vol 14, 2010

4. Evans WW, Porgel MA, Regezi JA. Oral mucosal lesion associated with sickle cell disease. Oral Dis, 1996; 75(5):303-304.

5. Girgwood RH. Drug induced anaemias. Drugs, 1976; 11(5):394-404.

6. Killip S, Bennet JM, Chambers MD. Iron deficiency Anaemia. Am Fam Physician, 2007; 75(5):671-678.

7. Lu SY, Wu HC. Initial diagnosis of anaemia from sore mouth and improved classification of anaemia by MCV and RDW in 30 patients. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 98:679-685.

8. Lucker J, Scully C, Oahhill A. Gingival swelling as a manifestation of aplastic anaemia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1991; 71(1):55-56.

9. Otan F, Ackigoz G, Sakallioglu U, Ozkan B. Reccurent aphtous ulcers in Fanconi’s anemia: A case report. Int J Paediatr Dent, 2004; 14: 214-217.

10. Siljanovski N. Anemija poradi nedostig na železo (sideropenična ili hipohromna anemija). Hematologija - Interna medicina, 2004; pp 877-895.

11. Tekcicek M, Tavil B, Cakar A, Pinar A, Unal S, Gumruk F. Oral and dental findings in children with Fanon anemia. Pediatric Dent, 2007; 29(3):248-252.

12. Tarai H, Shimahara M. Atrophic tongue associated with Candida. J Oral Pathol Med, 2005; 37(4):397-400.

13. Yepes JF. Anemia day. J Can Dent Assoc, 2007; 116:44-49.14. Yokoh K, Kimura S, Wada K, Morita Y, Ozawa M,

Kobayashi Y, Horiuchi Y, Maruo N, KondoM. Aplastic anemia associated with minimal serum M-protein treated successfully with repeated bolus methylprednisolone therapy. Rinisho Ketueki, 1990; 31(4):506-510.


Mirjana PopovskaVodnjanska 17 1000Skopje, FYR MacedoniaE-mail: [email protected]

SUMMARYA 52 year old partially edentulous woman was suffering from sore

mouth and uncomfortable with her removable partial dentures. The patient has been investigated in case of denture stomatitis and allergic sensitivity to dental materials used for prosthodontic treatment. Before the prosthodontic treatment, allergic contact test (back-skin test) was carried out and it was determined that allergic reactions were seen to some dental metals. The treatment proposed was the preparation of a combined fixed and removable partial denture by using titanium-fused-porcelain crowns and titanium made removable partial denture with precious attachments.Keywords: Allergic Sensitivity; Dental Alloy; Back-skin Test

Rifat Gozneli1, Yasemin Kulak Ozkan1, Ender Kazazoglu2

1Marmara University, School of Dentistry, Department of Prosthodontics, Istanbul, Turkey2 Yeditepe University, School of Dentistry, Department of Prosthodontics, Istanbul, Turkey

CASE REPORT (CR)Balk J Stom, 2010; 14:149-152


Allergic Sensitivity to Denture Base Materials:A Clinical Report

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Introduction

There are too many different dental alloys at the dental market, and a multitude of dental restorative procedures are based on the use of these cast alloys. However, during recent years, case reports have been published indicating that oral tissue reactions may occur in contact with dental cast alloys1-3. Reports of the prevalence of adverse effects to all dental materials used in daily practice, based on literature surveys, show that the frequency was estimated to be generally low. Kallus and Mjör4, found 46 cases of allergic sensitivity to dental materials out of 13.325 patients. Hensten and Pettersen5, also found that the incidence of adverse effects was estimated to be 1:400 in prosthodontic patients. Garhammer et al3, found that 250 patients, who attributed symptoms to dental alloys, represent about 0.03% of population of the region of Eastern Bavaria. Also they found that 17 patients out of 250 persons showed positive allergic reactions to metal salts in the back-skin patch test.

Overwhelming evidence from many case reports (results from skin tests) have shown that allergy may be a cause for adverse reactions towards dental cast alloys. Metals like nickel, palladium and cobalt rank high in allergy hit list6. Nickel, for example, is considered one of the most common causes of allergic dermatitis and is responsible for more allergic reactions than all other metals combined7. The element palladium is a known sensitizer with its sensitization rate in the range of 2-18%8.

In this article, a patient’s allergic sensitivity to some dental base alloys and treatment procedure, similar to that reported by Bezzon7, is reported.

Case Report

A 52-year-old woman, suffering for an unsatisfactory maxillary removable partial denture made of metal alloy based acrylic resin, has applied to our clinic (Fig. 1). While reporting her history, it was the second prosthesis made in different dental offices in 1 year, but she couldn’t use, because of suffering from sore mouth and burning mouth (Fig. 2). After intraoral examination, it was found that her intraoral tissues in contact with removable denture were hyperemic (Fig. 3). Intraoral examination indicated denture stomatitis and allergic sensitivity to dental materials used for prosthodontic treatment.

Clinical examination revealed the need for rehabilitation of the maxillary and mandibular arches (Fig. 4). For esthetic and functional reasons, the treatment proposed preparation of a combined fixed and removable partial denture with precision attachments. To find the appropriate material, the patient agreed to be submitted to a sensitivity test for determination of possible compatible alternative alloys. However, firstly the adequate intraoral hygiene should eliminate the hygiene sourced inflammation and erythema.

150 Rifat Gozneli et al. Balk J Stom, Vol 14, 2010

Table 1. Allergic sensitivity test results (Dental screening series)

1. Methyl methacrylate 2. Triethyleneglycol dimethacrylate 3. Urethane dimethacrylate 4. Ethyleneglycol dimethacrylate 5. BIS-GMA 6. N,N-dimethyl-4-toluidine 7. 2-Hydroxy-4-methonoxy-benzophenone 8. 1,4-Butanediol dimethacrylate 9. BIS-MA10. Potassium dichromate XX11. Mercury12. Cobalt chloride13. 2-Hydroxyethyl methacrylate14. Goldsodiumthiosulphate15. Nickel sulfate XX16. Eugenol17. Colophony18. N-Ethyl-4-toluenesulfonamide19. Formaldehyde20. 4-Tolyldiethanolamine21. Copper Sulfate22. Methylhydroquionone23. Palladium chloride XX24. Aluminium choloride hexahydrade25. Camphoroquinone26. N,N-dimethylaminoethyl mathacrylate27. 1,6 -Hexanediol diacrylate28. 2(2-Hydroxy-5-methylphenyl) benzotriazol29. Tetrahydrofurfuryl methacrylate30. Tin

Figure 1. Extra-oral appearance before treatment

Figure 2. Intra-oral appearance before treatment

Figure 3. Maxilla before treatment

Figure 4. Mandible before treatment

Balk J Stom, Vol 14, 2010 Allergic Sensitivity to Denture Base Materials 151

Before the prosthodontic treatment, allergic sensi-tivity contact test (back-skin test) was carried out at the University of Istanbul, Faculty of Medicine, Allergy Department. Tissue reaction (eczema-like eruptions) occurred by alloys that contained Nickel, Chromium and Palladium at her back skin (Tab. 1). So the prosthodontic treatment should provide the esthetic, function and also wouldn’t cause any allergic reaction. Most of the alloys that are used for prosthodontic treatment, contain those metal ions. So, it was decided that titanium based removable partial denture and

porcelain-fused-titanium fixed dentures could be used for prosthodontic treatment.

All of the teeth were prepared shoulder-type margin. Maxillary arch was treated by combined full titanium crown, porcelain-fused-titanium fixed dentures and titanium based removable partial denture with precision attachments (Fig. 5). Also, mandibular arch was treated with shortened arch and porcelain-fused-titanium fixed dentures instead of a removable partial denture, to supply better esthetic and comfortable prosthesis (Fig. 6). The prosthesis has been giving a satisfactory performance to the patient for the past 2 years (Fig. 7).

Figure 6. Mandible after treatmentFigure 5. Maxilla after treatment

Figure 7. Extra-oral appearance after treatment

Discussion

The use of metal alloys in dentistry is a necessity. Usually, because of being cheap and easy to find, cobalt-

chromium and nickel-chromium metal alloys are used so often. However, sometimes allergic sensitivity to those materials can be seen1-8. For the prosthodontic treatment of our patient, gold may be thought as another treatment choice. But, although titanium isn’t a cheap material, gold is even more expensive. Another reason why we didn’t use gold was due to the fact that gold alloys contain different metal ions to supply different physical properties. For example, gold alloys that are used for removable dentures contain 0-5% of palladium in alloy to harden the gold8. As mentioned before, the patient had allergic sensitivity to nickel, chromium and palladium. Because of these reasons it was concluded to use titanium based removable partial denture and porcelain-fused-titanium fixed dentures.

References

1. Taylor TD, Morton TH Jr. Ulcerative lesions of the palate associated with removable partial denture castings. J Prosthet Dent, 1991; 66:213-221.

152 Rifat Gozneli et al. Balk J Stom, Vol 14, 2010

2. Wataha JC. Biocompatibility of dental casting alloys: A review. J Prosthet Dent, 1992; 83:223-234.

3. Garhammer P, Schmalz G, Hiller KA, Reitinger T, Stolz W. Patients with local adverse effects from dental alloys: frequency, complaints, symptoms, allergy. Clin Oral Invest, 2001; 5:240-249.

4. Kallus T, Mjör IA. Incidence of adverse effects of dental materials. Scand J Dent Res, 1991; 99:236-240.

5. Hensten-Pettersen A. Casting alloys: Side effects. Adv Dent Res, 1992; 6:38-43.

6. Schmalz G, Garhammer P. Biological interactions of dental cast alloys with oral tissues. Dent Mater, 2002; 18:396-406.

7. Bezzon OL. Allergic sensitivity to several base metals: A clinical report. J Prosthet Dent, 1993; 69:243-244.

8. Berzins DW, Kawashima I, Graves R, Sarkar NK. Electrochemical characteristics of high-Pd alloys in relation to Pd-allergy. Dent Mater, 2000; 16:266-273.

Correspondence and request for offprints to:Rifat GozneliMarmara Üniversitesi, Dişhekimliği Fak.Güzelbahçe, Büyük Çiftlik Sokak, No. 634365 Nişantaşı, Istanbul, TurkeyE-mail: [email protected]

SUMMARYThis paper presents a case of a female patient with severe skeletal

Class III due to pronounced mandibular macrognathia combined with maxillary hypoplasia. The patient was treated with 2-jaw osteotomy in conjunction with pre- and post-operative orthodontic treatment. Resorbable surgical plates and screws were used for the orthognathic surgery to avoid potential complications from the use of metal plates and screws, even though fixation provided by metal plates is stronger than that provided by resorbable plates.

5 years following treatment, there has been no relapse. Our conclusion is that the resorbable plates and screw provided sufficient fixation and stability with no complications. Keywords: Class ΙΙΙ; Mandibular Macrognathia; Maxillary Hypoplasia;

Resorbable Plates Resorbable Screws

Nikolaos Topouzelis1, Nikolaos Lazaridis2, Fadi Tarawneh1, Maria Lazaridou2

1Department of Orthodontics, Dental School of Aristotle University of Thessaloniki2Department of Oral and Maxillofacial Surgery, Aristotle University of Thessaloniki, Papanikolaou General Hospital, Exohi, Thessaloniki

CASE REPORT (CR)Balk j Stom, 2010; 14:153-160


Orthodontic and Orthognathic Surgical Treatment of Severe Skeletal Class III with the Use of Resorbable Plates and Screws: A Case Report

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Introduction

Treating skeletal asymmetry of the orofacial system involves cooperation of an oral and maxillofacial surgeon and an orthodontist, so that the preoperative orthodontic treatment, the orthognathic surgical correction, as well as the postoperative orthodontic treatment might lead to the best possible result. In severe skeletal Class III cases, treatment planning often includes bilateral Dal Pont1 sagittal osteotomy of the mandibular rami combined with simultaneous Le Fort I osteotomy performed in the maxilla2. This paper describes a case of severe mandibular macrognathia combined with maxillary hypoplasia. It was treated with bimaxillary osteotomy combined with pre- and post-operative orthodontic treatment.

In order to stabilize maxillary and mandibular segments, resorbable surgical plates and screws were used to avoid any potential complications that might appear due to the use of metal plates and screws.

Case Report

Diagnosis and EtiologyA 20-year old woman with maxillofacial Class III

sought treatment. The clinical and radiological findings confirmed a brachyfacial type without asymmetry in the transverse dimension, a concave profile of osseous and soft tissues, mandibular protrusion due to anterior position, forward rotation and increased corpus length of the mandible (Fig. 1). When the mandible opened, there was a slight forward excursion from the centric relation position to the maximum intercuspation position. The patient presented no dysfunction of the temporomandibular joint (TMJ).

The patient had an increased caries index, teeth No 15 and 46 were missing, and her periodontal state was good. Dental arches presented some crowding (mild on the mandible, < 2 mm; medium on the maxilla, >4 mm), with a rotation of teeth No 13, 33, 45, and 47, while there was spacing between 45 and 47.

154 Nikolaos Topouzelis et al. Balk J Stom, Vol 14, 2010

There was posterior, bilateral and anterior cross bite with negative overjet -2 mm, while overbite was +2 mm. There was dental compensation of the skeletal asymmetry: the upper incisors presented pronounced labial inclination and the lower incisors lingual inclination. The upper mid-dental line had shifted 2 mm to the right in relation to the facial midline (Fig. 2).

Radiological Findings Analysis of the lateral cephalometric X-ray (Fig. 3a)

confirmed a skeletal Class III with negative convexity

(Α^Na-Pog -8mm) of osseous and soft tissues, due to maxillary hypoplasia (A to Na^-3 mm) and mandibular protrusion (Po-Or/Na-Pog 95o). Mandibular protrusion was due to an excessively long corpus (Xi-Pm 85 mm), forward rotation (Pt-Gn/Na-Ba 98o) and anterior position of the TMJ (Po- PtV 38mm). The lower anterior facial height was short (ANS-Xi-Pm 44o). Incisors presented negative overjet equal to -2 mm and overbite equal to +2 mm. Upper incisors presented pronounced labial inclination (I/Po-Or 127o) and lower incisors pronounced lingual inclination (i/Go-Me 79o).

Figure 1. Pre-treatment extraoral photographs

Figure 2. Pre-treatment intraoral photographs

Balk J Stom, Vol 14, 2010 Orthognathic Surgery with Resorbable Plates and Screws 155

The panoramic X-ray confirmed periodontal lesions and apical lesion in tooth No 37 (Fig. 3b).

Preoperative Orthodontics Initially, caries was treated and tooth No 25 was

extracted (due to its bad prognosis), the aim being to relieve crowding in the upper dental arch. Preoperative orthodontic treatment ensued using fixed appliances (Fig. 4). This entailed: (a) dental decompensation, with correction of the labial inclination of upper incisors and lingual inclination of lower incisors; this worsened overjet to -7 mm. Decompensation was of the utmost significance for the pre-operative correction of dental arches in cases of skeletal Class ΙΙΙ; (b) resolving crowding, closing spacing between 45 and 47, levelling and alignment of teeth; (c) achievement of the best possible compatibility of the maxillary and mandibular dental arches, which can only be checked on casts. Preoperative orthodontic treatment lasted 15 months. 6 months before its completion, the third molars (18, 28 and 38) were extracted so as to create bone in the region of the extractions, on the one hand, and so as not to burden the patient with extractions intra-operatively, on the other, since the jaw incision goes through the region of the third molars. Tooth No 48 was not extracted, because tooth No 46 was missing and teeth 47 and 48 were moved medially.

The operation was then planned on the basis of the preoperative lateral cephalometric X-ray and the casts mounted on a semi-adaptable articulator; it was decided to shift the maxilla forward by 4 mm and rotate it to the left by 2 mm, using Le Fort I osteotomy, while the mandible would be shifted backward by 6 mm using sagittal osteotomy of its rami. At this stage, 2 surgical splints were prepared. The first one, the intermediary

Figure 3. Pre-treatment X-rays: a. cephalometric radiograph; b. panoramic radiograph

a

b

Figure 4. Preoperative orthodontics


splint, was constructed in the new position of the maxilla, while the mandible remained unaffected in the initial centric relation position; the second and final splint was constructed so as to contain the new position of the distal mandibular stump in relation to the new maxillary position and determine the ultimate desirable occlusion.

Surgery and Postoperative Orthodontics Bimaxillary osteotomy was performed (Fig. 5).

Initially, a Le Fort I osteotomy was performed to move the maxilla forward by 4 mm, after the pre-constructed intermediary occlusal splint had led to temporary maxillo-mandibular fixation and osteosynthesis of the osteotomised maxilla with resorbable surgical plates and screws (Fig. 6). When the intermediary occlusal splint was removed, bilateral sagittal osteotomy of the mandibular rami was performed. The distal segment was shifted backward by 6 mm and osteosynthesis of 3 mandibular segments was performed using lag screws after the desirable occlusion had been achieved through the pre-planned use of the final splint3-5 (Fig. 7). Maxillo-mandibular fixation lasted 6 weeks. The patient’s post-operative course was uneventful.

In order to correct any imperfections of dental occlusion in the best possible way, post-operative orthodontic treatment followed 6 weeks later (Fig. 8); this was mainly based

on maxillo-mandibular elastic traction forces and lasted 5 months. The patient’s fixed appliances were left for 10 months for retention purposes.

At the end of the treatment the periodontium was healthy and teeth responded normally to vitality tests. The sagittal relation of the upper and lower molars was Angle Class II and of the rest of the teeth Class Ι. Incisor overjet was positive at +2 mm and incisor overbite was about 1/3 of the height of mandibular teeth (Fig. 9). Cephalometric comparison of the initial, pre-operative and final cephalometric X-ray (Fig. 10) is presented in table 1. These were the following main changes: Facial angle changed from 95° to 91°; facial convexity improved from -8 mm to -0 mm; the maxilla was placed in a more anterior position, since the distance of point A from MacNamara line improved from -3 mm to the ideal of 0 mm6,7; mandibular corpus length was reduced from 85 mm to 80 mm and facial height was increased from 44 mm to 47 mm.

Overall, there was a significant improvement in the patient’s dental (Fig. 9) and skeletal relations, in her aesthetic appearance (Fig. 11), the function of her dental-jaw system, as well as her psychological state. 5 years following treatment, there has been no relapse and the patient’s aesthetic and functional improvement has been preserved to date.

Figure 5. Bimaxillary osteotomy

Figure 6. Resorbable surgical plates and screws


Figure 7. Final splint

Figure 8. Postoperative orthodontic treatment

Figure 9. Post-treatment intraoral photographs


a b

Figure10. Post-treatment X-rays: a. cephalometric radiograph; b. panoramic radiograph

Figure 11. Post-treatment extraoral photographs

Table 1. Cephalometric comparison between initial, preoperative and final cephalometric analysis

Measurment Normal Values Initial Preoperative FinalFacial Axis (Pt-Gn/Na-Ba) 90o 3 98 98 84Facial angle (Po-Or/Na-Pog) 87o 3 95 95 91Mandibular plane (Po-Or/Go-Me) 26o 4 25 25 27Lower facial height angle(ANS-Xi-Pm) 47o 4 44 44 47Mandibular arc angle (DC-Xi-Pm) 26o 4 26 27 22Convexity at point A (ΑNa-Pog) 2 mm 2 -8 mm -8 mm 0 mmPoint A to MacNamara line (A to Na) 0 mm 2 -3 mm -3 mm 0 mmMandibular Incisor inclination 22o 4 79 89 83Mandibular Incisor to APog 1 mm 2 -6 mm -8 mm 4 mmMaxillary Incisor inclination 112 o 6 127 121 123E line -2 mm 2 -4 -3 -4Mandibular body length (Xi-Pm) 66 mm 3 85 mm 85 mm 80 mmPorion position (Po-PtV) 39 m 2.2 38 mm 39 mm 39 mm


Discussion

Pre-operative orthodontic treatment of this case aimed at dental decompensation of the skeletal anomaly and at aligning the teeth so that, mainly, maxillary and mandibular incisors would be properly corrected on the dental arches in the anteroposterior and vertical levels. This approach is called the theory of “2 patients”; according to this, the upper and lower dental arches are treated independently, as if they belonged to 2 patients8. Correction of the inclination of maxillary and mandibular incisors, which compensates skeletal asymmetry, allows 5 times as much posterior surgical shifting of the distal mandibular segment as compared to its potential shift, if there was no decompensation9.

Initially, a Le Fort I osteotomy was performed, which was followed by osteosynthesis of the osteotomised maxilla using resorbable surgical plates and screws, with the help of the pre-constructed intermediary occlusion splint. Resorbable surgical screws were also used following bilateral sagittal osteotomy of the mandibular rami, aiming at the osteosynthesis of 3 mandibular segments. We decided to use resorbable surgical plates and screws for the fixation of the jaw segments, so as to avoid potential complications that might appear if metal plates and screws were used instead, such as teeth sensitivity and sinusitis if plates and screws were placed too close to them, irritation due to palpability of the plates, pain and the need to surgically remove such materials later10-12.

Although metal plates provide stronger fixation than that provided by resorbable ones, research has shown that postoperative results are stable regardless of whether metal or resorbable plates have been used, particularly in cases where surgical shifts do not exceed 5 mm and when Le Fort I osteotomy and/or sagittal osteotomy of the mandibular ramus are performed. It has also been reported that the increased rigidity of metal plates might cause excessive tension/stress to bone10-16, while absorbable materials allow faster occlusion and TMJ adaptation17. Turvey et al18 noticed increased maxillary mobility during the postoperative phase in cases where resorbable plates had been used, as compared to cases where metal ones had been used. This mobility was considered desirable because it facilitated postoperative adaptation of jaw segments and it did not inhibit bone healing. The same thing was also observed by Costa et al11 in their research.

Conclusions

The cooperation of both maxillofacial surgeons and orthodontists is very important in treating cases of skeletal asymmetries of the orofacial system in order to obtain predictable and best possible result. This cooperation

involves treatment planning and coordination throughout the whole treatment phase.

In carefully selected cases, resorbable surgical plates and screws can be used for fixation, having predictable and similar results to metallic surgical plates and screws and, at the same time, avoiding disadvantages and complaints that accompany the later. This was demonstrated in the present case report, where 5 years following treatment the outcome of the treatment was stable and free of complications and complaints.

References

1. Dal Pont G. Retromolar osteotomy for correction of prognathism. J Oral Surg, 1961; 19:42-47.

2. Balan E. Erfahrungen bei kombinierten Osteotomien im Ober- und Unterkiefer. Dtsch Z Mund Kiefer Gesichts Chir, 1989; 13:101-112. (in German)

3. Obwegeer H. Der offene Biss in chirurgischer Sicht. Schweiz Monatsschr Zahnheilkd, 1964; 74:668-687. (in German)

4. Obwegeer H. Surgical Correction of Small or Retrodisplaced Maxillae. The “Dish-face” Deformity. Plast Reconstr Surg, 1969; 43:351-365.

5. Obwegeer H. Die einzeitige Vorrbewegung des Oberkiefers und Ruckbewegung des Unterkiefers zur Korrektur der extremen “progenie”. Schweiz Monatsshr Zahnheilkd, 1970; 80:547-556. (in German)

6. McNamara J. A method of cephalometric evaluation. Am J Orthod, 1984; 86:449-469.

7. McNamara J, Ellis E. Cephalometric analysis of untreated adults with ideal facial and occlusal relationships. Int J Orthod Oral Surg, 1988; 3:221-231.

8. Jacobs J, Sinclair P. Principles of orthodontic me chanics in orthognathic surgery cases. Am J Orthod, 1983; 84:399-407.

9. Langlande M. L’orthodontie orthognathie de l’adulte. In: Langlande M. Therapeutique orthodontique. Paris : Maloine SA, 1986; pp 767-796. (in French)

10. Fedorowicz Z, Nasser M, Newton JT, et al. Resorbable versus titanium plates for orthognathic surgery. Cochrane Database Syst Rev, 2007; 18; CD006204. Review.

11. Costa F, Robiony M, Zorzan E, Zerman N, Politi M. Stability of skeletal Class III malocclusion after combined maxillary and mandibular procedures: titanium versus resorbable plates and screws for maxillary fixation. J Oral Maxillofac Surg, 2006; 64:642-651.

12. Eppley BL. Bioabsorbable plate and screw fixation in orthognathic surgery. J Craniofac Surg, 2007; 18:818-825.

13. Edwards RC, Kiely KD, Eppley BL. Fixation of bimaxillary osteotomies with resorbable plates and screws: experience in 20 consecutive cases. J Oral Maxillofac Surg, 2001; 59:271-276.

14. Ferretti C, Reyneke JP. Mandibular sagittal split osteotomies fixed with biodegradable or titanium screws: a prospective comparative study of postoperative stability. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2002; 93:534-537.

15. Laine P, Kontio R, Lindqvist C, Suuronen R. Are there any complications with bioabsorbable fixation devices? A 10


18. Turvey TA, Bell RB, Tejera TJ, Proffit WR. The use of self-reinforced biodegradable bone plates and screws in orthognathic surgery. J Oral Maxillofac Surg, 2002; 60:59-65.


Nikolaos Topouzelis Aristotle University of ThessalonikiSchool of Dentistry, Department of OrthodonticsGreeceGR-54124 Thessaloniki, GreeceE-mail: [email protected]

year review in orthognathic surgery. Int J Oral Maxillofac Surg, 2004; 33:240-244.

16. Cheung LK, Chow LK, Chiu WK. A randomized controlled

trial of resorbable versus titanium fixation for orthognathic

surgery. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2004; 98:386-397.

17. Landes CA, Ballon A. Skeletal stability in bimaxillary

orthognathic surgery: P(L/DL)LA-resorbable versus titanium

osteofixation. Plast Reconstr Surg, 2006; 118:703-721.

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