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Research ArticleSteam Sterilization of Equine Bone BlockMorphological and Collagen Analysis
R Lo Giudice 1 G Rizzo2 A Centofanti2 A Favaloro2 D Rizzo2
G Cervino2 R Squeri2 B G Costa2 V La Fauci2 and G Lo Giudice 2
1Department of Clinical and Experimental Medicine Messina University Italy2Department of Biomedical and Dental Sciences and Morphofunctional Imaging Messina University Italy
Correspondence should be addressed to R Lo Giudice rlogiudiceunimeit
Received 29 May 2018 Accepted 1 August 2018 Published 13 August 2018
Academic Editor Tolga Tozum
Copyright copy 2018 R Lo Giudice et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Introduction The use of equine bone blocks is widely reported for bone augmentation techniques The block must be shapedaccording to the form of the defect that should be regenerated The shaping could be performed by hand before or during thesurgery in a sterile ambient or using a CNC milling machine that could not be sterile The aim of our study was to evaluate if asteam sterilization could provide a medical grade sterilization of the blocks and to evaluate if bone microstructure and collagenstructures change after different steam sterilization protocols provided bymainstream autoclaveMaterials andMethod Two blocksof equine bone were divided into 16 samples 1 sample was used as control and not submitted to any treatment 15 samples wereinfected with a Streptococcus faecalis bacterial culture The samples were singularly packed randomly divided into 3 groups andsubmitted to autoclave sterilization on the same device The groups were submitted to a sterilization cycle (Gr A 121∘C 116 barfor 201015840 Gr B134∘C 216 bar for 41015840 Gr C 134∘C 216 bar for 330min) 2 samples for each group were evaluated for the sterility 3samples for each group were observed at SEM to notice the macro- and microstructure modification and to confocal microscopeto observe the collagen Results All samples were sterile The SEM evaluation showed in all groups a preserved morphologicalstructure Confocal microscope evaluation shows that the collagen structure appears to be more uniform and preserved in groupC Conclusion Data show that autoclave steam sterilization could be reliable to obtain sterilization of equine bone blocks
1 Introduction
Bone regeneration is a reliable technique when the bone vol-ume is not sufficient to provide a long-term stability ofimplant-supported prosthetic restorations and functional andaesthetical outcome It is also indicated in the posttraumaticand oncologic reconstructive protocols [1]
The autologous bone is still considered the gold standardharvested from extra or intraoral site [2ndash4]
The homologous fresh frozen bone has also been reportedas effective thanks to its osteoconductive and it is poten-tially osteoinductive properties linked to its matrix containsgrowth factors such as bone morphogenetic protein (BMP)and vascular endothelial growth factor (VEGF) [5 6]
However in bone regeneration procedures due to theincreased morbidity limited quantities available and the
necessity of a second surgical site are widely used xenogenicmaterials of bovine porcine or equine origins [7 8]
Xenografts thanks to their chemical-physical character-istics similar to those of the human bone show osteoconduc-tive properties [9 10]
Enzyme-deantigenic equine bone has been used success-fully in several fields of oral surgery and implantology [11]including periapical cyst-removal management periodontaldefect correction [12] horizontal and vertical atrophic ridgereconstruction [13ndash17] and sinus augmentation [18ndash22]
Equine-derived biomaterials may be preferred to those ofbovine or porcine origins for issues related to TransmissibleSpongiform Encephalopathies It is well known in fact thatrabbits dogs and horses are the only mammalian speciesreported to be resistant to infection from prion diseasesisolated from other species [23 24]
HindawiBioMed Research InternationalVolume 2018 Article ID 9853765 8 pageshttpsdoiorg10115520189853765
2 BioMed Research International
In equine bone an enzymatic process to preserve thenative conformation of type I collagen equine bone is usedwhile making the bone nonantigenic The bone collagenmatrix contains a high amount of growth factors such as IGF-II TGF-beta IGF-I PDGF bFGF BMPs and others [25] andthis implies that the demineralized bone matrix (DBM) isable to stimulate the production of new bone tissue [25] whengrafted in another species [26ndash28]
Regarding cellular behavior it has been found that whenosteoclasts were cultured on enzyme-deantigenic equinebone they adhered on this material in greater numbers andexerted a more intense degrading activity [29] compared toinorganic bovine bone [30] possibly because of the presenceof preserved collagen in the equine xenograft
In vitro studies showed the effects of preserved bonersquos col-lagen on biological mechanisms related to bone regenerationA randomized clinical trial found that enzyme-deantigenicequine bone led to a significantly greater amount of newlyformed bone and a lower residual biomaterial comparedwithinorganic bovine bone [31]
The bone substitutes may be used in different forms suchas block or particulate along with the application of a long-lasting membrane to prevent soft tissue cells from invadingthe regenerating site (GBR) [32]
It has been reported that a bone block is a reliable mate-rial for regenerative procedures with predictable long-termpositive results [33] The space maintaining characteristics ofthe blocks can overcome the poor three-dimensional stabilityof the particulate substitutes offering an adequate mechanicalsupport to the overlying tissue [16 34 35]
Allograft and xenograft bone substitutes underwent toprocedures that greatly lower the potential risk of transmis-sion of bacteria viruses and prions
Different sterilization techniques are used for autologoustissues including gamma irradiation [36] ethylene oxide gas[37] thermal treatment with moist heat [38] beta-propio-lactone [39] chemical processing [40] and antibiotic soaks[41]
In xenografts the microbial safety is generally achievedthrough gamma or electron-beam (e-beam) irradiation [42]E-beam irradiation may be preferred to gamma because themore precise dose control allows a shorter irradiation time[43 44]
In the common dental practice the autoclave is used toobtain a high-grade sterility of various kind of material [45ndash47]
The aim of our research was to evaluate if different auto-clave sterilization protocols may modify the macroscopicaland microscopical bone structure of an equine block and ifthis protocols will affect the collagen matrix
2 Materials and Method
Two equine bone blocks (10x20x5mm) with collagen (OXBlock Osteoxenon Bioteck Italy) were divided into 16samples of cubic shape (5x5x5mm) using a stainless steelbone cutter Lindemann bur (Komet Komet It Srl Italy)
21 SterilizationTest One samplewas used as control and notsubmitted to any treatment
15 samples were infected with a bacterial culture
The Streptococcus faecalis used for sterilization test waspreviously isolated from a pharyngeal buffer and incubatedunder standardized conditions for 24 h in sterile thioglycol-late broth at 37plusmn2 C
The pH-values of the broths were measured at the begin-ning and end of each incubation cycle
1ml of thioglycollate broth contained 1 times 105 CFUmlStreptococcus faecalis colonies
Each sample was contaminated by using sterile micro-pipettes with 05ml of thioglycollate broth
The 15 samples were randomly divided in 3 groups andsubmitted to autoclave sterilization in the same device (Euro-nda E9 Med Euronda Italy)
A groupwas submitted to a sterilization cycle at 121∘C 116bar for 20min and 15min of drying
B group was submitted to a sterilization cycle at 134∘C at216 bar for 4min of sterilization and 15min of drying
C group was submitted to a sterilization cycle at 134∘C at216 bar for 330min of sterilization and 5min of drying
Two samples for each group were evaluated for thesterility
1 sample per group was incubated under standardizedconditions for 24 h in thioglycollate broth at 37plusmn2∘C
1 sample per group was incubated under standardizedconditions for 24 h in normal saline solution at 37plusmn2∘C
To evaluate the sterilization effects 18120583l of thioglycollateand 18120583l of normal saline solution were collected andincubated for 24 h at 37plusmn2∘C in CLED agar and ESCULINAagar for the bacterial count
After 24 h of incubation the bacterial presence was eval-uated by a conventional biochemical test and colonies werecounted and interpreted as colony-forming units
22 Structural Evaluation After autoclave steam steriliza-tion the 9 samples that were not evaluated for sterility under-went to SEM (Phenom Pro 5 Phenom-World BV Eind-hoven Netherlands) observation to directly observe the mac-roscopical and microscopical structure
Each sample was fractured to observe an uncut surfaceAll 9 samples underwent to optical observation to evalu-
ate the macroscopical structural modificationThree randomly areas of each surface were observed at
the samemagnification and themicrostructural changes wererecorded
After the SEM observation the 12 samples underwent tothe confocal microscope observation to evaluate the collagenpresence The uncut and unfractured surface was observed
23 Scanning Electron Microscopy Protocol After fixation in2 glutaraldehyde specimens were dehydrated in ethanoland amyl acetate and then were dried at critical point ina Balzers critical point drier using liquid CO2 The bonefractured surfaces were mounted on stub and platinumcoated with a sputtering system ldquoPlasma Sciences CrC-100Turbo Pumpedrdquo and observed by Phenom G2 pro scanningelectron microscope
24 Confocal Microscope Protocol After fixation in 2 glu-taraldehyde and rinsing in phosphate buffer 013molL pH
BioMed Research International 3
Figure 1 Group A spherical and ovoid cavities delimitated by tra-beculation (42x)
73 specimens were decalcified in 413 Ethylenediaminete-traacetic acid pH 72 dehydrated in ethanol and embeddedin paraffin A Leica microtome (Leica RM2255 Leica LeicaBiosystems MI Italy) was used to obtain eight-micrometer-thick sections
The sections were treated with the following antibodiesmouse monoclonal anticollagen I (diluted 11000 Sigma-Aldrich) which were demonstrated with IgG-Texas Red con-jugated anti-rabbit (1100 dilution Jackson ImmunoResearchLaboratories West Grove PA USA) [48 49]
Negative controls were carried out by treating the sectionsonly with the secondary antibodyThe samples were observedwith the Zeiss LSM 510 confocal microscope (Zeiss LSM 510Carl Zeiss SpA MI Italy) The ldquodisplay profilerdquo function ofthe laser scanning microscope was used to show the intensityprofile across an image along a freely selectable line [50]
3 Results
31 Sterilization Test The sterility was achieved in all sam-ples considering a SAL (Sterility Assurance Level) of 10minus6
32 Scanning Electron Microscopy Evaluation Group A im-age shows a defined bone architecture with cavities of spheri-cal shape delimited by a dense trabeculation (42x) (Figure 1)
It is possible to observe in detail a trabecula which de-limits two cavities and it is possible to see the presence ofsome residues of different shapes and sizes Bonemorphologyappears to be well preserved (1100x) (Figure 2)
Group B and group C images show a cavity of ovoid andspherical shape delimitated by a dense trabeculation (40x)(Figures 3 and 4)
Increasing the magnification it is possible to observe atrabecula with smaller communicating cavities the trabeculasurface appears to be well preserved (2500x) (Figures 5 and6)
33 Confocal Microscope Evaluation Group A the imageshows that type 1 collagen of the sample presents a fluores-cence pattern that is located at the periphery of the trabecula
Figure 2 Group A an intact trabecula with some residues on it
Figure 3 Group B morphologically well-preserved communicantcavities of ovoid and spherical shape delimitated by a dense trabecu-lation (40x)
Figure 4 Group C morphologically well-preserved communicantcavities of ovoid and spherical shape delimitated by a dense trabecu-lation (40x)
4 BioMed Research International
Figure 5GroupB a trabeculawith sand-like residuals on it (2500x)
Figure 6GroupC awell-preserved trabeculawith smaller commu-nicating cavities the trabecula surface appears to be well preserved(420x)
continuously A feeble fluorescence can be observed at theedges of some gap (Figure 7)
Group B the images show that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula sometimes continuous and in-tense sometimes weak and widespread A feeble fluorescenceis observed at the edges This group appears to be similar togroup A (Figure 8)
Group C the image shows that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula in a continuous and uniformman-ner The entire trabecula has a weak diffuse fluorescence pat-tern with a circumferential course sometimes more markedaround the gaps (Figure 9)
Display profile shows the fluorescence intensity along aselected line
The fluorescent signal intensity appears more pro-nounced and uniform in group C when compared to groups
Figure 7 Group A a continuous fluorescence pattern of type 1collagen is noticeable on the peripheral surface of the trabecula Alight fluorescence is evident
Figure 8 Group B the fluorescence pattern of the type 1 collagenis located in the peripheral of the trabecula and appears variouslyintense
Figure 9 Group C the fluorescence pattern of type 1 collagenis evident on trabecula and appears continuous and uniform Thewhole trabecula shows a fluorescence patternmore marked near thelacuna
BioMed Research International 5
Group C
Group BGroup A
Figure 10 Display profile analysis
A and B Comparing group A and group B a greater signalintensity is evident in group A (Figure 10)
4 Discussion
The bone augmentation procedure is widely studied in litera-ture and commonly used in clinical practice and might bemandatory in different clinical situations in both medicineand dentistry [5]
Materials different for origin and composition were deep-ly studied by the scientific community and the differencesbetween the particulate form and the bone block are com-monly known
Several studies analyzed the employment of bone equine-derived block containing collagen for ridge augmentation[51ndash53] These researches revealed the advantageous capacityto maintain the dimensions of the augmented ridge Benicet al demonstrated that the GBR technique performed withequine block with collagen membranes (CM) resulted inthe most favorable outcomes compared to bovine block andbovine granulate with CM [35]
It is mandatory to shape the bone block according to thedefect that should be filled The shaping could be performedby the industry that according to a CBCT provided by theclinician could send a preshaped and sterilized block or
a handmade ldquoin-houserdquo shaping before or during the surgeryThe handmade shaping has however a lack of precisionrelated to the method of shaping To overcome this issue aCNC milling machine could be used This machine has amicrometer precision and in addition to a three-dimensionalreconstruction could provide the clinician a precise modelhowever due to its characteristic the sterility requirement isnot satisfying andmust be achieved after shaping using othertechniques [54]
Cusinato et al [55] demonstrated the efficacy of hydro-gen peroxide treatment and e-beam irradiation on viralclearance in equine-derived xenografts (pericardium mem-brane collagen membrane and cortical and cancellous bonegrafts) spiked with three human viruses (Coxsackie virus B1influenza virus typeAwith subtypeH1N1 and herpes simplexvirus type 1)
Our work shows how it is possible to obtain a satisfactorysterility of the graft using a traditional device as the autoclave
The microbiological analysis shows how in agreementwith the literature the sterility was achieved in all samples
Our research was also aimed at understanding if this pro-cedure might modify the bone morphology and the collagen
The eye observation of the samples shows a fairly dehy-drated and a slightly lighter white surface when compared tothe control sample
6 BioMed Research International
The SEM observations between the control sample andthe samples of the three groups underline how at differentmagnifications themacrostructure does not show substantialmodifications [56]
In all samples the bone structure appears to be wellpreserved and themorphology appears to be very similar It isevident how the high temperature did not determine evidentand significantmodifications of the bone sample architecture
The collagen matrix that is a particular characteristic ofthe equine bone graft and is preserved due to the impossibilityof this species to be infected with prions appears to bevariously maintained
The confocal microscope immunofluorescence analysisshows how group C (131 fast) has the best-preserved collagenstructure This observation is also confirmed by the displayprofile analysis which evaluating the fluorescence intensityunderlines how the fluorescent signal appears to bemore uni-form and intense when compared to the other groups Whencomparing A and B groups the first one shows a strongerand a uniform fluorescence
The temperatures reached in steam autoclave sterilizationappear to be respectful of the collagen fibril that remainsalways below the thermal transitions temperatures [57 58]
The collagen resistance to heat and pressure damage soappears to be time related When a steam sterilization is per-formed a 134∘C and 216 bar for 330min and 5min of dryingprotocol is suggested
From the analysis of our data autoclave steam steriliza-tion method could be a reliable way to obtain sterilization ofbone graft
Giving the possibility of using a nonsterile CNC millingmachine will open a new scenario of precision in bonegrafting letting the surgeon to obtain a perfectly shaped andsterilized graft wherever before the surgery
5 Conclusion
The autoclave sterilization appears to be a reliable way toobtain amedical grade sterilization of the equine bone blocksand it does not affect the macro- and the microstructure andthe collagen fiber
Further studies are necessary to evaluate if there are anychemical modification and to evaluate if there are changes inthe in vivo osteointegration process of the bone that under-went the steam sterilization protocol
Data Availability
The data (photos) used to support the findings of this studyare available from the corresponding author upon request
Conflicts of Interest
Thestudy did not receive any financial support and therewerenot any conflicts of interest
Authorsrsquo Contributions
All individuals listed as authors have contributed substan-tially to the design performance analysis and reporting of
the work No others contributors to the articles than theauthors accredited
References
[1] S S Jensen and H Terheyden ldquoBone augmentation proceduresin localized defects in the alveolar ridge clinical results withdifferent bone grafts and bone-substitute materialsrdquo The Inter-national Journal of Oral amp Maxillofacial Implants vol 24 pp218ndash236 2009
[2] G A Catone B L Reimer DMcNeir and R Ray ldquoTibial auto-genous cancellous bone as an alternative donor site in max-illofacial surgery A preliminary reportrdquo Journal of Oral andMaxillofacial Surgery vol 50 no 12 pp 1258ndash1263 1992
[3] M G Donovan N C Dickerson and J C Mitchell ldquoCalvarialBone Harvest and Grafting Techniques for Maxillary andMan-dibular Implant Surgeryrdquo Atlas of the Oral and MaxillofacialSurgery Clinics vol 2 no 2 pp 109ndash122 1994
[4] C M Misch ldquoBone augmentation of the atrophic posteriormandible for dental implants using rhBMP-2 and titaniummesh clinical technique and early resultsrdquo International Journalof Periodontics and Restorative Dentistry vol 31 no 6 pp 581ndash589 2011
[5] F S de Ponte G Cutroneo R Falzea et al ldquoHistochemical andmorphological aspects of fresh frozen bone A preliminarystudyrdquo European Journal of Histochemistry vol 60 no 4 pp 5ndash9 2016
[6] A S Herford M Cicciu L F Eftimie et al ldquorhBMP-2 appliedas support of distraction osteogenesis A split-mouth histolog-ical study over nonhuman primates mandiblesrdquo InternationalJournal of Clinical and Experimental Medicine vol 9 no 9 pp17187ndash17194 2016
[7] M Cicciu ldquoReal opportunity for the present and a forward stepfor the future of bone tissue engineeringrdquo The Journal ofCraniofacial Surgery vol 28 no 3 pp 592-593 2017
[8] M Cicciu G Cervino A S Herford et al ldquoFacial Bone Recon-struction Using Both Marine or Non-Marine Bone SubstitutesEvaluation of Current Outcomes in a Systematic LiteratureReviewrdquoMarine Drugs vol 16 no 1 2018
[9] M EspositoM G Grusovin P Coulthard andH VWorthing-ton ldquoThe efficacy of various bone augmentation procedures fordental implants a Cochrane systematic review of randomizedcontrolled clinical trialsrdquo The International Journal of Oral ampMaxillofacial Implants vol 21 no 5 pp 696ndash710 2006
[10] GOrsini A ScaranoM PiattelliM Piccirilli S Caputi andAPiattelli ldquoHistologic and ultrastructural analysis of regeneratedbone in maxillary sinus augmentation using a porcine bone-derived biomaterialrdquo Journal of Periodontology vol 77 no 12pp 1984ndash1990 2006
[11] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoDental Implants Placed in Periodontally InfectedSites in Humansrdquo Clinical Implant Dentistry and RelatedResearch vol 19 no 1 pp 131ndash139 2017
[12] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoImmediate Implant Placement in Sockets withAsymptomatic Apical Periodontitisrdquo Clinical Implant Dentistryand Related Research vol 19 no 1 pp 20ndash27 2017
[13] T Traini P Valentini G Iezzi and A Piattelli ldquoA histologic andhistomorphometric evaluation of anorganic bovine bone re-trieved 9 years after a sinus augmentation procedurerdquo Journalof Periodontology vol 78 no 5 pp 955ndash961 2007
BioMed Research International 7
[14] A Barone P Toti A Quaranta et al ldquoClinical and Histologicalchanges after ridge preservation with two xenografts prelimi-nary results from a multicentre randomized controlled clinicaltrialrdquo Journal of Clinical Periodontology vol 44 no 2 pp 204ndash214 2017
[15] P Felice L Piana L ChecchiV CorvinoU Nannmark andMPiattelli ldquoVertical ridge augmentation of an atrophic posteriormandible with an inlay technique and cancellous equine boneblock A case reportrdquo International Journal of Periodontics andRestorative Dentistry vol 33 no 2 pp 159ndash166 2013
[16] R Pistilli L Signorini A Pisacane G Lizio and P Felice ldquoCaseof severe bone atrophy of the posterior maxilla rehabilitatedwith blocks of equine origin bone Histological resultsrdquo ImplantDentistry vol 22 no 1 pp 8ndash15 2013
[17] N De Angelis and M Scivetti ldquoLateral ridge augmentationusing an equine flex bone block infused with recombinanthuman platelet-derived growth factor BB A clinical and histo-logic studyrdquo International Journal of Periodontics andRestorativeDentistry vol 31 no 4 pp 383ndash388 2011
[18] D A di Stefano L Artese G Iezzi et al ldquoAlveolar ridge regen-eration with equine spongy bone a clinical histological andimmunohistochemical case seriesrdquo Clinical Implant Dentistryand Related Research vol 11 no 2 pp 90ndash100 2009
[19] L Artese A Piattelli D ADi Stefano et al ldquoSinus liftwith auto-logous bone alone or in addition to equine bone an immuno-histochemical study in manrdquo Implant Dentistry vol 20 no 5pp 383ndash388 2011
[20] S Tete R Vinci V L Zizzari et al ldquoMaxillary sinus augmen-tation procedures through equine-derived biomaterial or cal-varia autologous bone Immunohistochemical evaluation ofOPGRANKL in humansrdquo European Journal of Histochemistryvol 57 no 1 pp 60ndash65 2013
[21] G LoGiudice G Iannello A Terranova R LoGiudice G Pan-taleo and M Cicciu ldquoTranscrestal sinus lift procedure ap-proaching atrophic maxillary ridge A 60-month clinical andradiological follow-up evaluationrdquo International Journal ofDen-tistry vol 2015 2015
[22] S Tete V L Zizzari R Vinci et al ldquoEquine and porcine bonesubstitutes in maxillary sinus augmentation A histologicaland immunohistochemical analysis of VEGF expressionrdquo TheJournal of Craniofacial Surgery vol 25 no 3 pp 835ndash839 2014
[23] J Talairach and PThournouxCo-Planar Stereotaxic Atlas of theHuman Brain Thieme Medical Publishers Stuttgart Germany1988
[24] J Zhang ldquoThe structural stability of wild-type horse prion pro-teinrdquo Journal of Biomolecular Structure and Dynamics vol 29no 2 pp 369ndash377 2011
[25] D J Pacaccio and S F Stern ldquoDemineralized bonematrix Basicscience and clinical applicationsrdquo Clinics in Podiatric Medicineand Surgery vol 22 no 4 pp 599ndash606 2005
[26] K D Chesmel J Branger H Wertheim and N ScarboroughldquoHealing response to various forms of human demineralizedbone matrix in athymic rat cranial defectsrdquo Journal of Oral andMaxillofacial Surgery vol 56 no 7 pp 857ndash865 1998
[27] S Guizzardi R Scandroglio M D Silvestre R Savini and ARuggeri ldquoImplants of heterologous demineralized bone matrixfor induction of posterior spinal fusion in ratsrdquoThe Spine Jour-nal vol 17 no 6 pp 701ndash707 1992
[28] M S Rafael V Laize and M L Cancela ldquoIdentification of Spa-rus aurata bone morphogenetic protein 2 Molecular cloninggene expression and in silico analysis of protein conservedfeatures in vertebratesrdquoBone vol 39 no 6 pp 1373ndash1381 2006
[29] V Perrotti B M Nicholls and A Piattelli ldquoHuman osteoclastformation and activity on an equine spongy bone substituterdquoClinical Oral Implants Research vol 20 no 1 pp 17ndash23 2009
[30] V Perrotti B M Nicholls M A Horton and A PiattellildquoHuman osteoclast formation and activity on a xenogenousbone mineralrdquo Journal of Biomedical Materials Research Part Avol 90 no 1 pp 238ndash246 2009
[31] D A L Di Stefano G Gastaldi R Vinci L Cinci L Pieri andE Gherlone ldquoHistomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in SinusAugmentation A Randomized Clinical Trial with 3-Year Fol-low-Uprdquo The International journal of oral amp maxillofacial im-plants vol 30 no 5 pp 1161ndash1167 2015
[32] I Elgali O Omar C Dahlin and P Thomsen ldquoGuided boneregeneration materials and biological mechanisms revisitedrdquoEuropean Journal of Oral Sciences vol 125 no 5 pp 315ndash3372017
[33] J Waasdorp and M A Reynolds ldquoAllogeneic bone onlay graftsfor alveolar ridge augmentation a systematic reviewrdquo TheInternational Journal of Oral amp Maxillofacial Implants vol 25no 3 pp 525ndash531 2010
[34] M Schlee and D Rothamel ldquoRidge augmentation using cus-tomized allogenic bone blocks Proof of concept and histologi-cal findingsrdquo Implant Dentistry vol 22 no 3 pp 212ndash218 2013
[35] G I Benic D S Thoma R E Jung et al ldquoGuided bone regen-eration with particulate vs block xenogenic bone substitutes apilot cone beam computed tomographic investigationrdquo ClinicalOral Implants Research vol 28 no 11 pp e262ndashe270 2017
[36] H Nguyen D A F Morgan and M R Forwood ldquoSterilizationof allograft bone Effects of gamma irradiation on allograftbiology and biomechanicsrdquo Cell and Tissue Banking vol 8 no2 pp 93ndash105 2007
[37] T Arizono Y Iwamoto K Okuyama and Y Sugioka ldquoEthyleneoxide sterilization of bone grafts Residual gas concentrationand fibroblast toxicityrdquo Acta Orthopaedica vol 65 no 6 pp640ndash642 1994
[38] C Hofmann T Von Garrel and L Gotzen ldquoBone bank man-agement using a thermal disinfection system (Lobator SD-1) Acritical analysisrdquo Unfallchirurg vol 99 no 7 pp 498ndash508 1996
[39] R G T Geesink ldquoOsteoconductive coatings for total joint ar-throplastyrdquo Clinical Orthopaedics and Related Research no 395pp 53ndash65 2002
[40] D J Prolo P W Pedrotti and D H White ldquoEthylene oxidesterilization of bone dura mater and fascia lata for humantransplantationrdquo Neurosurgery vol 6 no 5 pp 529ndash539 1980
[41] J A Tom and S A Rodeo ldquoSoft tissue allografts for knee recon-struction in sportsmedicinerdquoClinical Orthopaedics and RelatedResearch no 402 pp 135ndash156 2002
[42] Block SS Disinfection Sterilization and Preservation LippincottWilliams andWilkins Philadelphia Pa USA 4th edition 1991
[43] E J Mitchell A M Stawarz R Kayacan and C M RimnacldquoThe effect of gamma radiation sterilization on the fatigue crackpropagation resistance of human cortical bonerdquoThe Journal ofBone amp Joint Surgery vol 86 no 12 pp 2648ndash2657 2004
[44] M Silindir andYA Ozeray ldquoSterilizationmethods and the com-parison of E-beam sterilization with gamma radiation steril-izationrdquo Journal of Pharmaceutical Sciences vol 34 pp 43ndash532009
[45] L C Huesca-Espitia M Suvira K Rosenbeck et al ldquoEffects ofsteam autoclave treatment on Geobacillus stearothermophilussporesrdquo Journal of AppliedMicrobiology vol 121 no 5 pp 1300ndash1311 2016
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
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Hindawiwwwhindawicom Volume 2018
ChemistryAdvances in
Hindawiwwwhindawicom Volume 2018
Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
2 BioMed Research International
In equine bone an enzymatic process to preserve thenative conformation of type I collagen equine bone is usedwhile making the bone nonantigenic The bone collagenmatrix contains a high amount of growth factors such as IGF-II TGF-beta IGF-I PDGF bFGF BMPs and others [25] andthis implies that the demineralized bone matrix (DBM) isable to stimulate the production of new bone tissue [25] whengrafted in another species [26ndash28]
Regarding cellular behavior it has been found that whenosteoclasts were cultured on enzyme-deantigenic equinebone they adhered on this material in greater numbers andexerted a more intense degrading activity [29] compared toinorganic bovine bone [30] possibly because of the presenceof preserved collagen in the equine xenograft
In vitro studies showed the effects of preserved bonersquos col-lagen on biological mechanisms related to bone regenerationA randomized clinical trial found that enzyme-deantigenicequine bone led to a significantly greater amount of newlyformed bone and a lower residual biomaterial comparedwithinorganic bovine bone [31]
The bone substitutes may be used in different forms suchas block or particulate along with the application of a long-lasting membrane to prevent soft tissue cells from invadingthe regenerating site (GBR) [32]
It has been reported that a bone block is a reliable mate-rial for regenerative procedures with predictable long-termpositive results [33] The space maintaining characteristics ofthe blocks can overcome the poor three-dimensional stabilityof the particulate substitutes offering an adequate mechanicalsupport to the overlying tissue [16 34 35]
Allograft and xenograft bone substitutes underwent toprocedures that greatly lower the potential risk of transmis-sion of bacteria viruses and prions
Different sterilization techniques are used for autologoustissues including gamma irradiation [36] ethylene oxide gas[37] thermal treatment with moist heat [38] beta-propio-lactone [39] chemical processing [40] and antibiotic soaks[41]
In xenografts the microbial safety is generally achievedthrough gamma or electron-beam (e-beam) irradiation [42]E-beam irradiation may be preferred to gamma because themore precise dose control allows a shorter irradiation time[43 44]
In the common dental practice the autoclave is used toobtain a high-grade sterility of various kind of material [45ndash47]
The aim of our research was to evaluate if different auto-clave sterilization protocols may modify the macroscopicaland microscopical bone structure of an equine block and ifthis protocols will affect the collagen matrix
2 Materials and Method
Two equine bone blocks (10x20x5mm) with collagen (OXBlock Osteoxenon Bioteck Italy) were divided into 16samples of cubic shape (5x5x5mm) using a stainless steelbone cutter Lindemann bur (Komet Komet It Srl Italy)
21 SterilizationTest One samplewas used as control and notsubmitted to any treatment
15 samples were infected with a bacterial culture
The Streptococcus faecalis used for sterilization test waspreviously isolated from a pharyngeal buffer and incubatedunder standardized conditions for 24 h in sterile thioglycol-late broth at 37plusmn2 C
The pH-values of the broths were measured at the begin-ning and end of each incubation cycle
1ml of thioglycollate broth contained 1 times 105 CFUmlStreptococcus faecalis colonies
Each sample was contaminated by using sterile micro-pipettes with 05ml of thioglycollate broth
The 15 samples were randomly divided in 3 groups andsubmitted to autoclave sterilization in the same device (Euro-nda E9 Med Euronda Italy)
A groupwas submitted to a sterilization cycle at 121∘C 116bar for 20min and 15min of drying
B group was submitted to a sterilization cycle at 134∘C at216 bar for 4min of sterilization and 15min of drying
C group was submitted to a sterilization cycle at 134∘C at216 bar for 330min of sterilization and 5min of drying
Two samples for each group were evaluated for thesterility
1 sample per group was incubated under standardizedconditions for 24 h in thioglycollate broth at 37plusmn2∘C
1 sample per group was incubated under standardizedconditions for 24 h in normal saline solution at 37plusmn2∘C
To evaluate the sterilization effects 18120583l of thioglycollateand 18120583l of normal saline solution were collected andincubated for 24 h at 37plusmn2∘C in CLED agar and ESCULINAagar for the bacterial count
After 24 h of incubation the bacterial presence was eval-uated by a conventional biochemical test and colonies werecounted and interpreted as colony-forming units
22 Structural Evaluation After autoclave steam steriliza-tion the 9 samples that were not evaluated for sterility under-went to SEM (Phenom Pro 5 Phenom-World BV Eind-hoven Netherlands) observation to directly observe the mac-roscopical and microscopical structure
Each sample was fractured to observe an uncut surfaceAll 9 samples underwent to optical observation to evalu-
ate the macroscopical structural modificationThree randomly areas of each surface were observed at
the samemagnification and themicrostructural changes wererecorded
After the SEM observation the 12 samples underwent tothe confocal microscope observation to evaluate the collagenpresence The uncut and unfractured surface was observed
23 Scanning Electron Microscopy Protocol After fixation in2 glutaraldehyde specimens were dehydrated in ethanoland amyl acetate and then were dried at critical point ina Balzers critical point drier using liquid CO2 The bonefractured surfaces were mounted on stub and platinumcoated with a sputtering system ldquoPlasma Sciences CrC-100Turbo Pumpedrdquo and observed by Phenom G2 pro scanningelectron microscope
24 Confocal Microscope Protocol After fixation in 2 glu-taraldehyde and rinsing in phosphate buffer 013molL pH
BioMed Research International 3
Figure 1 Group A spherical and ovoid cavities delimitated by tra-beculation (42x)
73 specimens were decalcified in 413 Ethylenediaminete-traacetic acid pH 72 dehydrated in ethanol and embeddedin paraffin A Leica microtome (Leica RM2255 Leica LeicaBiosystems MI Italy) was used to obtain eight-micrometer-thick sections
The sections were treated with the following antibodiesmouse monoclonal anticollagen I (diluted 11000 Sigma-Aldrich) which were demonstrated with IgG-Texas Red con-jugated anti-rabbit (1100 dilution Jackson ImmunoResearchLaboratories West Grove PA USA) [48 49]
Negative controls were carried out by treating the sectionsonly with the secondary antibodyThe samples were observedwith the Zeiss LSM 510 confocal microscope (Zeiss LSM 510Carl Zeiss SpA MI Italy) The ldquodisplay profilerdquo function ofthe laser scanning microscope was used to show the intensityprofile across an image along a freely selectable line [50]
3 Results
31 Sterilization Test The sterility was achieved in all sam-ples considering a SAL (Sterility Assurance Level) of 10minus6
32 Scanning Electron Microscopy Evaluation Group A im-age shows a defined bone architecture with cavities of spheri-cal shape delimited by a dense trabeculation (42x) (Figure 1)
It is possible to observe in detail a trabecula which de-limits two cavities and it is possible to see the presence ofsome residues of different shapes and sizes Bonemorphologyappears to be well preserved (1100x) (Figure 2)
Group B and group C images show a cavity of ovoid andspherical shape delimitated by a dense trabeculation (40x)(Figures 3 and 4)
Increasing the magnification it is possible to observe atrabecula with smaller communicating cavities the trabeculasurface appears to be well preserved (2500x) (Figures 5 and6)
33 Confocal Microscope Evaluation Group A the imageshows that type 1 collagen of the sample presents a fluores-cence pattern that is located at the periphery of the trabecula
Figure 2 Group A an intact trabecula with some residues on it
Figure 3 Group B morphologically well-preserved communicantcavities of ovoid and spherical shape delimitated by a dense trabecu-lation (40x)
Figure 4 Group C morphologically well-preserved communicantcavities of ovoid and spherical shape delimitated by a dense trabecu-lation (40x)
4 BioMed Research International
Figure 5GroupB a trabeculawith sand-like residuals on it (2500x)
Figure 6GroupC awell-preserved trabeculawith smaller commu-nicating cavities the trabecula surface appears to be well preserved(420x)
continuously A feeble fluorescence can be observed at theedges of some gap (Figure 7)
Group B the images show that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula sometimes continuous and in-tense sometimes weak and widespread A feeble fluorescenceis observed at the edges This group appears to be similar togroup A (Figure 8)
Group C the image shows that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula in a continuous and uniformman-ner The entire trabecula has a weak diffuse fluorescence pat-tern with a circumferential course sometimes more markedaround the gaps (Figure 9)
Display profile shows the fluorescence intensity along aselected line
The fluorescent signal intensity appears more pro-nounced and uniform in group C when compared to groups
Figure 7 Group A a continuous fluorescence pattern of type 1collagen is noticeable on the peripheral surface of the trabecula Alight fluorescence is evident
Figure 8 Group B the fluorescence pattern of the type 1 collagenis located in the peripheral of the trabecula and appears variouslyintense
Figure 9 Group C the fluorescence pattern of type 1 collagenis evident on trabecula and appears continuous and uniform Thewhole trabecula shows a fluorescence patternmore marked near thelacuna
BioMed Research International 5
Group C
Group BGroup A
Figure 10 Display profile analysis
A and B Comparing group A and group B a greater signalintensity is evident in group A (Figure 10)
4 Discussion
The bone augmentation procedure is widely studied in litera-ture and commonly used in clinical practice and might bemandatory in different clinical situations in both medicineand dentistry [5]
Materials different for origin and composition were deep-ly studied by the scientific community and the differencesbetween the particulate form and the bone block are com-monly known
Several studies analyzed the employment of bone equine-derived block containing collagen for ridge augmentation[51ndash53] These researches revealed the advantageous capacityto maintain the dimensions of the augmented ridge Benicet al demonstrated that the GBR technique performed withequine block with collagen membranes (CM) resulted inthe most favorable outcomes compared to bovine block andbovine granulate with CM [35]
It is mandatory to shape the bone block according to thedefect that should be filled The shaping could be performedby the industry that according to a CBCT provided by theclinician could send a preshaped and sterilized block or
a handmade ldquoin-houserdquo shaping before or during the surgeryThe handmade shaping has however a lack of precisionrelated to the method of shaping To overcome this issue aCNC milling machine could be used This machine has amicrometer precision and in addition to a three-dimensionalreconstruction could provide the clinician a precise modelhowever due to its characteristic the sterility requirement isnot satisfying andmust be achieved after shaping using othertechniques [54]
Cusinato et al [55] demonstrated the efficacy of hydro-gen peroxide treatment and e-beam irradiation on viralclearance in equine-derived xenografts (pericardium mem-brane collagen membrane and cortical and cancellous bonegrafts) spiked with three human viruses (Coxsackie virus B1influenza virus typeAwith subtypeH1N1 and herpes simplexvirus type 1)
Our work shows how it is possible to obtain a satisfactorysterility of the graft using a traditional device as the autoclave
The microbiological analysis shows how in agreementwith the literature the sterility was achieved in all samples
Our research was also aimed at understanding if this pro-cedure might modify the bone morphology and the collagen
The eye observation of the samples shows a fairly dehy-drated and a slightly lighter white surface when compared tothe control sample
6 BioMed Research International
The SEM observations between the control sample andthe samples of the three groups underline how at differentmagnifications themacrostructure does not show substantialmodifications [56]
In all samples the bone structure appears to be wellpreserved and themorphology appears to be very similar It isevident how the high temperature did not determine evidentand significantmodifications of the bone sample architecture
The collagen matrix that is a particular characteristic ofthe equine bone graft and is preserved due to the impossibilityof this species to be infected with prions appears to bevariously maintained
The confocal microscope immunofluorescence analysisshows how group C (131 fast) has the best-preserved collagenstructure This observation is also confirmed by the displayprofile analysis which evaluating the fluorescence intensityunderlines how the fluorescent signal appears to bemore uni-form and intense when compared to the other groups Whencomparing A and B groups the first one shows a strongerand a uniform fluorescence
The temperatures reached in steam autoclave sterilizationappear to be respectful of the collagen fibril that remainsalways below the thermal transitions temperatures [57 58]
The collagen resistance to heat and pressure damage soappears to be time related When a steam sterilization is per-formed a 134∘C and 216 bar for 330min and 5min of dryingprotocol is suggested
From the analysis of our data autoclave steam steriliza-tion method could be a reliable way to obtain sterilization ofbone graft
Giving the possibility of using a nonsterile CNC millingmachine will open a new scenario of precision in bonegrafting letting the surgeon to obtain a perfectly shaped andsterilized graft wherever before the surgery
5 Conclusion
The autoclave sterilization appears to be a reliable way toobtain amedical grade sterilization of the equine bone blocksand it does not affect the macro- and the microstructure andthe collagen fiber
Further studies are necessary to evaluate if there are anychemical modification and to evaluate if there are changes inthe in vivo osteointegration process of the bone that under-went the steam sterilization protocol
Data Availability
The data (photos) used to support the findings of this studyare available from the corresponding author upon request
Conflicts of Interest
Thestudy did not receive any financial support and therewerenot any conflicts of interest
Authorsrsquo Contributions
All individuals listed as authors have contributed substan-tially to the design performance analysis and reporting of
the work No others contributors to the articles than theauthors accredited
References
[1] S S Jensen and H Terheyden ldquoBone augmentation proceduresin localized defects in the alveolar ridge clinical results withdifferent bone grafts and bone-substitute materialsrdquo The Inter-national Journal of Oral amp Maxillofacial Implants vol 24 pp218ndash236 2009
[2] G A Catone B L Reimer DMcNeir and R Ray ldquoTibial auto-genous cancellous bone as an alternative donor site in max-illofacial surgery A preliminary reportrdquo Journal of Oral andMaxillofacial Surgery vol 50 no 12 pp 1258ndash1263 1992
[3] M G Donovan N C Dickerson and J C Mitchell ldquoCalvarialBone Harvest and Grafting Techniques for Maxillary andMan-dibular Implant Surgeryrdquo Atlas of the Oral and MaxillofacialSurgery Clinics vol 2 no 2 pp 109ndash122 1994
[4] C M Misch ldquoBone augmentation of the atrophic posteriormandible for dental implants using rhBMP-2 and titaniummesh clinical technique and early resultsrdquo International Journalof Periodontics and Restorative Dentistry vol 31 no 6 pp 581ndash589 2011
[5] F S de Ponte G Cutroneo R Falzea et al ldquoHistochemical andmorphological aspects of fresh frozen bone A preliminarystudyrdquo European Journal of Histochemistry vol 60 no 4 pp 5ndash9 2016
[6] A S Herford M Cicciu L F Eftimie et al ldquorhBMP-2 appliedas support of distraction osteogenesis A split-mouth histolog-ical study over nonhuman primates mandiblesrdquo InternationalJournal of Clinical and Experimental Medicine vol 9 no 9 pp17187ndash17194 2016
[7] M Cicciu ldquoReal opportunity for the present and a forward stepfor the future of bone tissue engineeringrdquo The Journal ofCraniofacial Surgery vol 28 no 3 pp 592-593 2017
[8] M Cicciu G Cervino A S Herford et al ldquoFacial Bone Recon-struction Using Both Marine or Non-Marine Bone SubstitutesEvaluation of Current Outcomes in a Systematic LiteratureReviewrdquoMarine Drugs vol 16 no 1 2018
[9] M EspositoM G Grusovin P Coulthard andH VWorthing-ton ldquoThe efficacy of various bone augmentation procedures fordental implants a Cochrane systematic review of randomizedcontrolled clinical trialsrdquo The International Journal of Oral ampMaxillofacial Implants vol 21 no 5 pp 696ndash710 2006
[10] GOrsini A ScaranoM PiattelliM Piccirilli S Caputi andAPiattelli ldquoHistologic and ultrastructural analysis of regeneratedbone in maxillary sinus augmentation using a porcine bone-derived biomaterialrdquo Journal of Periodontology vol 77 no 12pp 1984ndash1990 2006
[11] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoDental Implants Placed in Periodontally InfectedSites in Humansrdquo Clinical Implant Dentistry and RelatedResearch vol 19 no 1 pp 131ndash139 2017
[12] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoImmediate Implant Placement in Sockets withAsymptomatic Apical Periodontitisrdquo Clinical Implant Dentistryand Related Research vol 19 no 1 pp 20ndash27 2017
[13] T Traini P Valentini G Iezzi and A Piattelli ldquoA histologic andhistomorphometric evaluation of anorganic bovine bone re-trieved 9 years after a sinus augmentation procedurerdquo Journalof Periodontology vol 78 no 5 pp 955ndash961 2007
BioMed Research International 7
[14] A Barone P Toti A Quaranta et al ldquoClinical and Histologicalchanges after ridge preservation with two xenografts prelimi-nary results from a multicentre randomized controlled clinicaltrialrdquo Journal of Clinical Periodontology vol 44 no 2 pp 204ndash214 2017
[15] P Felice L Piana L ChecchiV CorvinoU Nannmark andMPiattelli ldquoVertical ridge augmentation of an atrophic posteriormandible with an inlay technique and cancellous equine boneblock A case reportrdquo International Journal of Periodontics andRestorative Dentistry vol 33 no 2 pp 159ndash166 2013
[16] R Pistilli L Signorini A Pisacane G Lizio and P Felice ldquoCaseof severe bone atrophy of the posterior maxilla rehabilitatedwith blocks of equine origin bone Histological resultsrdquo ImplantDentistry vol 22 no 1 pp 8ndash15 2013
[17] N De Angelis and M Scivetti ldquoLateral ridge augmentationusing an equine flex bone block infused with recombinanthuman platelet-derived growth factor BB A clinical and histo-logic studyrdquo International Journal of Periodontics andRestorativeDentistry vol 31 no 4 pp 383ndash388 2011
[18] D A di Stefano L Artese G Iezzi et al ldquoAlveolar ridge regen-eration with equine spongy bone a clinical histological andimmunohistochemical case seriesrdquo Clinical Implant Dentistryand Related Research vol 11 no 2 pp 90ndash100 2009
[19] L Artese A Piattelli D ADi Stefano et al ldquoSinus liftwith auto-logous bone alone or in addition to equine bone an immuno-histochemical study in manrdquo Implant Dentistry vol 20 no 5pp 383ndash388 2011
[20] S Tete R Vinci V L Zizzari et al ldquoMaxillary sinus augmen-tation procedures through equine-derived biomaterial or cal-varia autologous bone Immunohistochemical evaluation ofOPGRANKL in humansrdquo European Journal of Histochemistryvol 57 no 1 pp 60ndash65 2013
[21] G LoGiudice G Iannello A Terranova R LoGiudice G Pan-taleo and M Cicciu ldquoTranscrestal sinus lift procedure ap-proaching atrophic maxillary ridge A 60-month clinical andradiological follow-up evaluationrdquo International Journal ofDen-tistry vol 2015 2015
[22] S Tete V L Zizzari R Vinci et al ldquoEquine and porcine bonesubstitutes in maxillary sinus augmentation A histologicaland immunohistochemical analysis of VEGF expressionrdquo TheJournal of Craniofacial Surgery vol 25 no 3 pp 835ndash839 2014
[23] J Talairach and PThournouxCo-Planar Stereotaxic Atlas of theHuman Brain Thieme Medical Publishers Stuttgart Germany1988
[24] J Zhang ldquoThe structural stability of wild-type horse prion pro-teinrdquo Journal of Biomolecular Structure and Dynamics vol 29no 2 pp 369ndash377 2011
[25] D J Pacaccio and S F Stern ldquoDemineralized bonematrix Basicscience and clinical applicationsrdquo Clinics in Podiatric Medicineand Surgery vol 22 no 4 pp 599ndash606 2005
[26] K D Chesmel J Branger H Wertheim and N ScarboroughldquoHealing response to various forms of human demineralizedbone matrix in athymic rat cranial defectsrdquo Journal of Oral andMaxillofacial Surgery vol 56 no 7 pp 857ndash865 1998
[27] S Guizzardi R Scandroglio M D Silvestre R Savini and ARuggeri ldquoImplants of heterologous demineralized bone matrixfor induction of posterior spinal fusion in ratsrdquoThe Spine Jour-nal vol 17 no 6 pp 701ndash707 1992
[28] M S Rafael V Laize and M L Cancela ldquoIdentification of Spa-rus aurata bone morphogenetic protein 2 Molecular cloninggene expression and in silico analysis of protein conservedfeatures in vertebratesrdquoBone vol 39 no 6 pp 1373ndash1381 2006
[29] V Perrotti B M Nicholls and A Piattelli ldquoHuman osteoclastformation and activity on an equine spongy bone substituterdquoClinical Oral Implants Research vol 20 no 1 pp 17ndash23 2009
[30] V Perrotti B M Nicholls M A Horton and A PiattellildquoHuman osteoclast formation and activity on a xenogenousbone mineralrdquo Journal of Biomedical Materials Research Part Avol 90 no 1 pp 238ndash246 2009
[31] D A L Di Stefano G Gastaldi R Vinci L Cinci L Pieri andE Gherlone ldquoHistomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in SinusAugmentation A Randomized Clinical Trial with 3-Year Fol-low-Uprdquo The International journal of oral amp maxillofacial im-plants vol 30 no 5 pp 1161ndash1167 2015
[32] I Elgali O Omar C Dahlin and P Thomsen ldquoGuided boneregeneration materials and biological mechanisms revisitedrdquoEuropean Journal of Oral Sciences vol 125 no 5 pp 315ndash3372017
[33] J Waasdorp and M A Reynolds ldquoAllogeneic bone onlay graftsfor alveolar ridge augmentation a systematic reviewrdquo TheInternational Journal of Oral amp Maxillofacial Implants vol 25no 3 pp 525ndash531 2010
[34] M Schlee and D Rothamel ldquoRidge augmentation using cus-tomized allogenic bone blocks Proof of concept and histologi-cal findingsrdquo Implant Dentistry vol 22 no 3 pp 212ndash218 2013
[35] G I Benic D S Thoma R E Jung et al ldquoGuided bone regen-eration with particulate vs block xenogenic bone substitutes apilot cone beam computed tomographic investigationrdquo ClinicalOral Implants Research vol 28 no 11 pp e262ndashe270 2017
[36] H Nguyen D A F Morgan and M R Forwood ldquoSterilizationof allograft bone Effects of gamma irradiation on allograftbiology and biomechanicsrdquo Cell and Tissue Banking vol 8 no2 pp 93ndash105 2007
[37] T Arizono Y Iwamoto K Okuyama and Y Sugioka ldquoEthyleneoxide sterilization of bone grafts Residual gas concentrationand fibroblast toxicityrdquo Acta Orthopaedica vol 65 no 6 pp640ndash642 1994
[38] C Hofmann T Von Garrel and L Gotzen ldquoBone bank man-agement using a thermal disinfection system (Lobator SD-1) Acritical analysisrdquo Unfallchirurg vol 99 no 7 pp 498ndash508 1996
[39] R G T Geesink ldquoOsteoconductive coatings for total joint ar-throplastyrdquo Clinical Orthopaedics and Related Research no 395pp 53ndash65 2002
[40] D J Prolo P W Pedrotti and D H White ldquoEthylene oxidesterilization of bone dura mater and fascia lata for humantransplantationrdquo Neurosurgery vol 6 no 5 pp 529ndash539 1980
[41] J A Tom and S A Rodeo ldquoSoft tissue allografts for knee recon-struction in sportsmedicinerdquoClinical Orthopaedics and RelatedResearch no 402 pp 135ndash156 2002
[42] Block SS Disinfection Sterilization and Preservation LippincottWilliams andWilkins Philadelphia Pa USA 4th edition 1991
[43] E J Mitchell A M Stawarz R Kayacan and C M RimnacldquoThe effect of gamma radiation sterilization on the fatigue crackpropagation resistance of human cortical bonerdquoThe Journal ofBone amp Joint Surgery vol 86 no 12 pp 2648ndash2657 2004
[44] M Silindir andYA Ozeray ldquoSterilizationmethods and the com-parison of E-beam sterilization with gamma radiation steril-izationrdquo Journal of Pharmaceutical Sciences vol 34 pp 43ndash532009
[45] L C Huesca-Espitia M Suvira K Rosenbeck et al ldquoEffects ofsteam autoclave treatment on Geobacillus stearothermophilussporesrdquo Journal of AppliedMicrobiology vol 121 no 5 pp 1300ndash1311 2016
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
CorrosionInternational Journal of
Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
Journal of
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Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
ScienticaHindawiwwwhindawicom Volume 2018
Polymer ScienceInternational Journal of
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Advances in Condensed Matter Physics
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Applied ChemistryJournal of
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The Scientific World Journal
Volume 2018
TribologyAdvances in
Hindawiwwwhindawicom Volume 2018
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Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
BioMed Research International 3
Figure 1 Group A spherical and ovoid cavities delimitated by tra-beculation (42x)
73 specimens were decalcified in 413 Ethylenediaminete-traacetic acid pH 72 dehydrated in ethanol and embeddedin paraffin A Leica microtome (Leica RM2255 Leica LeicaBiosystems MI Italy) was used to obtain eight-micrometer-thick sections
The sections were treated with the following antibodiesmouse monoclonal anticollagen I (diluted 11000 Sigma-Aldrich) which were demonstrated with IgG-Texas Red con-jugated anti-rabbit (1100 dilution Jackson ImmunoResearchLaboratories West Grove PA USA) [48 49]
Negative controls were carried out by treating the sectionsonly with the secondary antibodyThe samples were observedwith the Zeiss LSM 510 confocal microscope (Zeiss LSM 510Carl Zeiss SpA MI Italy) The ldquodisplay profilerdquo function ofthe laser scanning microscope was used to show the intensityprofile across an image along a freely selectable line [50]
3 Results
31 Sterilization Test The sterility was achieved in all sam-ples considering a SAL (Sterility Assurance Level) of 10minus6
32 Scanning Electron Microscopy Evaluation Group A im-age shows a defined bone architecture with cavities of spheri-cal shape delimited by a dense trabeculation (42x) (Figure 1)
It is possible to observe in detail a trabecula which de-limits two cavities and it is possible to see the presence ofsome residues of different shapes and sizes Bonemorphologyappears to be well preserved (1100x) (Figure 2)
Group B and group C images show a cavity of ovoid andspherical shape delimitated by a dense trabeculation (40x)(Figures 3 and 4)
Increasing the magnification it is possible to observe atrabecula with smaller communicating cavities the trabeculasurface appears to be well preserved (2500x) (Figures 5 and6)
33 Confocal Microscope Evaluation Group A the imageshows that type 1 collagen of the sample presents a fluores-cence pattern that is located at the periphery of the trabecula
Figure 2 Group A an intact trabecula with some residues on it
Figure 3 Group B morphologically well-preserved communicantcavities of ovoid and spherical shape delimitated by a dense trabecu-lation (40x)
Figure 4 Group C morphologically well-preserved communicantcavities of ovoid and spherical shape delimitated by a dense trabecu-lation (40x)
4 BioMed Research International
Figure 5GroupB a trabeculawith sand-like residuals on it (2500x)
Figure 6GroupC awell-preserved trabeculawith smaller commu-nicating cavities the trabecula surface appears to be well preserved(420x)
continuously A feeble fluorescence can be observed at theedges of some gap (Figure 7)
Group B the images show that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula sometimes continuous and in-tense sometimes weak and widespread A feeble fluorescenceis observed at the edges This group appears to be similar togroup A (Figure 8)
Group C the image shows that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula in a continuous and uniformman-ner The entire trabecula has a weak diffuse fluorescence pat-tern with a circumferential course sometimes more markedaround the gaps (Figure 9)
Display profile shows the fluorescence intensity along aselected line
The fluorescent signal intensity appears more pro-nounced and uniform in group C when compared to groups
Figure 7 Group A a continuous fluorescence pattern of type 1collagen is noticeable on the peripheral surface of the trabecula Alight fluorescence is evident
Figure 8 Group B the fluorescence pattern of the type 1 collagenis located in the peripheral of the trabecula and appears variouslyintense
Figure 9 Group C the fluorescence pattern of type 1 collagenis evident on trabecula and appears continuous and uniform Thewhole trabecula shows a fluorescence patternmore marked near thelacuna
BioMed Research International 5
Group C
Group BGroup A
Figure 10 Display profile analysis
A and B Comparing group A and group B a greater signalintensity is evident in group A (Figure 10)
4 Discussion
The bone augmentation procedure is widely studied in litera-ture and commonly used in clinical practice and might bemandatory in different clinical situations in both medicineand dentistry [5]
Materials different for origin and composition were deep-ly studied by the scientific community and the differencesbetween the particulate form and the bone block are com-monly known
Several studies analyzed the employment of bone equine-derived block containing collagen for ridge augmentation[51ndash53] These researches revealed the advantageous capacityto maintain the dimensions of the augmented ridge Benicet al demonstrated that the GBR technique performed withequine block with collagen membranes (CM) resulted inthe most favorable outcomes compared to bovine block andbovine granulate with CM [35]
It is mandatory to shape the bone block according to thedefect that should be filled The shaping could be performedby the industry that according to a CBCT provided by theclinician could send a preshaped and sterilized block or
a handmade ldquoin-houserdquo shaping before or during the surgeryThe handmade shaping has however a lack of precisionrelated to the method of shaping To overcome this issue aCNC milling machine could be used This machine has amicrometer precision and in addition to a three-dimensionalreconstruction could provide the clinician a precise modelhowever due to its characteristic the sterility requirement isnot satisfying andmust be achieved after shaping using othertechniques [54]
Cusinato et al [55] demonstrated the efficacy of hydro-gen peroxide treatment and e-beam irradiation on viralclearance in equine-derived xenografts (pericardium mem-brane collagen membrane and cortical and cancellous bonegrafts) spiked with three human viruses (Coxsackie virus B1influenza virus typeAwith subtypeH1N1 and herpes simplexvirus type 1)
Our work shows how it is possible to obtain a satisfactorysterility of the graft using a traditional device as the autoclave
The microbiological analysis shows how in agreementwith the literature the sterility was achieved in all samples
Our research was also aimed at understanding if this pro-cedure might modify the bone morphology and the collagen
The eye observation of the samples shows a fairly dehy-drated and a slightly lighter white surface when compared tothe control sample
6 BioMed Research International
The SEM observations between the control sample andthe samples of the three groups underline how at differentmagnifications themacrostructure does not show substantialmodifications [56]
In all samples the bone structure appears to be wellpreserved and themorphology appears to be very similar It isevident how the high temperature did not determine evidentand significantmodifications of the bone sample architecture
The collagen matrix that is a particular characteristic ofthe equine bone graft and is preserved due to the impossibilityof this species to be infected with prions appears to bevariously maintained
The confocal microscope immunofluorescence analysisshows how group C (131 fast) has the best-preserved collagenstructure This observation is also confirmed by the displayprofile analysis which evaluating the fluorescence intensityunderlines how the fluorescent signal appears to bemore uni-form and intense when compared to the other groups Whencomparing A and B groups the first one shows a strongerand a uniform fluorescence
The temperatures reached in steam autoclave sterilizationappear to be respectful of the collagen fibril that remainsalways below the thermal transitions temperatures [57 58]
The collagen resistance to heat and pressure damage soappears to be time related When a steam sterilization is per-formed a 134∘C and 216 bar for 330min and 5min of dryingprotocol is suggested
From the analysis of our data autoclave steam steriliza-tion method could be a reliable way to obtain sterilization ofbone graft
Giving the possibility of using a nonsterile CNC millingmachine will open a new scenario of precision in bonegrafting letting the surgeon to obtain a perfectly shaped andsterilized graft wherever before the surgery
5 Conclusion
The autoclave sterilization appears to be a reliable way toobtain amedical grade sterilization of the equine bone blocksand it does not affect the macro- and the microstructure andthe collagen fiber
Further studies are necessary to evaluate if there are anychemical modification and to evaluate if there are changes inthe in vivo osteointegration process of the bone that under-went the steam sterilization protocol
Data Availability
The data (photos) used to support the findings of this studyare available from the corresponding author upon request
Conflicts of Interest
Thestudy did not receive any financial support and therewerenot any conflicts of interest
Authorsrsquo Contributions
All individuals listed as authors have contributed substan-tially to the design performance analysis and reporting of
the work No others contributors to the articles than theauthors accredited
References
[1] S S Jensen and H Terheyden ldquoBone augmentation proceduresin localized defects in the alveolar ridge clinical results withdifferent bone grafts and bone-substitute materialsrdquo The Inter-national Journal of Oral amp Maxillofacial Implants vol 24 pp218ndash236 2009
[2] G A Catone B L Reimer DMcNeir and R Ray ldquoTibial auto-genous cancellous bone as an alternative donor site in max-illofacial surgery A preliminary reportrdquo Journal of Oral andMaxillofacial Surgery vol 50 no 12 pp 1258ndash1263 1992
[3] M G Donovan N C Dickerson and J C Mitchell ldquoCalvarialBone Harvest and Grafting Techniques for Maxillary andMan-dibular Implant Surgeryrdquo Atlas of the Oral and MaxillofacialSurgery Clinics vol 2 no 2 pp 109ndash122 1994
[4] C M Misch ldquoBone augmentation of the atrophic posteriormandible for dental implants using rhBMP-2 and titaniummesh clinical technique and early resultsrdquo International Journalof Periodontics and Restorative Dentistry vol 31 no 6 pp 581ndash589 2011
[5] F S de Ponte G Cutroneo R Falzea et al ldquoHistochemical andmorphological aspects of fresh frozen bone A preliminarystudyrdquo European Journal of Histochemistry vol 60 no 4 pp 5ndash9 2016
[6] A S Herford M Cicciu L F Eftimie et al ldquorhBMP-2 appliedas support of distraction osteogenesis A split-mouth histolog-ical study over nonhuman primates mandiblesrdquo InternationalJournal of Clinical and Experimental Medicine vol 9 no 9 pp17187ndash17194 2016
[7] M Cicciu ldquoReal opportunity for the present and a forward stepfor the future of bone tissue engineeringrdquo The Journal ofCraniofacial Surgery vol 28 no 3 pp 592-593 2017
[8] M Cicciu G Cervino A S Herford et al ldquoFacial Bone Recon-struction Using Both Marine or Non-Marine Bone SubstitutesEvaluation of Current Outcomes in a Systematic LiteratureReviewrdquoMarine Drugs vol 16 no 1 2018
[9] M EspositoM G Grusovin P Coulthard andH VWorthing-ton ldquoThe efficacy of various bone augmentation procedures fordental implants a Cochrane systematic review of randomizedcontrolled clinical trialsrdquo The International Journal of Oral ampMaxillofacial Implants vol 21 no 5 pp 696ndash710 2006
[10] GOrsini A ScaranoM PiattelliM Piccirilli S Caputi andAPiattelli ldquoHistologic and ultrastructural analysis of regeneratedbone in maxillary sinus augmentation using a porcine bone-derived biomaterialrdquo Journal of Periodontology vol 77 no 12pp 1984ndash1990 2006
[11] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoDental Implants Placed in Periodontally InfectedSites in Humansrdquo Clinical Implant Dentistry and RelatedResearch vol 19 no 1 pp 131ndash139 2017
[12] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoImmediate Implant Placement in Sockets withAsymptomatic Apical Periodontitisrdquo Clinical Implant Dentistryand Related Research vol 19 no 1 pp 20ndash27 2017
[13] T Traini P Valentini G Iezzi and A Piattelli ldquoA histologic andhistomorphometric evaluation of anorganic bovine bone re-trieved 9 years after a sinus augmentation procedurerdquo Journalof Periodontology vol 78 no 5 pp 955ndash961 2007
BioMed Research International 7
[14] A Barone P Toti A Quaranta et al ldquoClinical and Histologicalchanges after ridge preservation with two xenografts prelimi-nary results from a multicentre randomized controlled clinicaltrialrdquo Journal of Clinical Periodontology vol 44 no 2 pp 204ndash214 2017
[15] P Felice L Piana L ChecchiV CorvinoU Nannmark andMPiattelli ldquoVertical ridge augmentation of an atrophic posteriormandible with an inlay technique and cancellous equine boneblock A case reportrdquo International Journal of Periodontics andRestorative Dentistry vol 33 no 2 pp 159ndash166 2013
[16] R Pistilli L Signorini A Pisacane G Lizio and P Felice ldquoCaseof severe bone atrophy of the posterior maxilla rehabilitatedwith blocks of equine origin bone Histological resultsrdquo ImplantDentistry vol 22 no 1 pp 8ndash15 2013
[17] N De Angelis and M Scivetti ldquoLateral ridge augmentationusing an equine flex bone block infused with recombinanthuman platelet-derived growth factor BB A clinical and histo-logic studyrdquo International Journal of Periodontics andRestorativeDentistry vol 31 no 4 pp 383ndash388 2011
[18] D A di Stefano L Artese G Iezzi et al ldquoAlveolar ridge regen-eration with equine spongy bone a clinical histological andimmunohistochemical case seriesrdquo Clinical Implant Dentistryand Related Research vol 11 no 2 pp 90ndash100 2009
[19] L Artese A Piattelli D ADi Stefano et al ldquoSinus liftwith auto-logous bone alone or in addition to equine bone an immuno-histochemical study in manrdquo Implant Dentistry vol 20 no 5pp 383ndash388 2011
[20] S Tete R Vinci V L Zizzari et al ldquoMaxillary sinus augmen-tation procedures through equine-derived biomaterial or cal-varia autologous bone Immunohistochemical evaluation ofOPGRANKL in humansrdquo European Journal of Histochemistryvol 57 no 1 pp 60ndash65 2013
[21] G LoGiudice G Iannello A Terranova R LoGiudice G Pan-taleo and M Cicciu ldquoTranscrestal sinus lift procedure ap-proaching atrophic maxillary ridge A 60-month clinical andradiological follow-up evaluationrdquo International Journal ofDen-tistry vol 2015 2015
[22] S Tete V L Zizzari R Vinci et al ldquoEquine and porcine bonesubstitutes in maxillary sinus augmentation A histologicaland immunohistochemical analysis of VEGF expressionrdquo TheJournal of Craniofacial Surgery vol 25 no 3 pp 835ndash839 2014
[23] J Talairach and PThournouxCo-Planar Stereotaxic Atlas of theHuman Brain Thieme Medical Publishers Stuttgart Germany1988
[24] J Zhang ldquoThe structural stability of wild-type horse prion pro-teinrdquo Journal of Biomolecular Structure and Dynamics vol 29no 2 pp 369ndash377 2011
[25] D J Pacaccio and S F Stern ldquoDemineralized bonematrix Basicscience and clinical applicationsrdquo Clinics in Podiatric Medicineand Surgery vol 22 no 4 pp 599ndash606 2005
[26] K D Chesmel J Branger H Wertheim and N ScarboroughldquoHealing response to various forms of human demineralizedbone matrix in athymic rat cranial defectsrdquo Journal of Oral andMaxillofacial Surgery vol 56 no 7 pp 857ndash865 1998
[27] S Guizzardi R Scandroglio M D Silvestre R Savini and ARuggeri ldquoImplants of heterologous demineralized bone matrixfor induction of posterior spinal fusion in ratsrdquoThe Spine Jour-nal vol 17 no 6 pp 701ndash707 1992
[28] M S Rafael V Laize and M L Cancela ldquoIdentification of Spa-rus aurata bone morphogenetic protein 2 Molecular cloninggene expression and in silico analysis of protein conservedfeatures in vertebratesrdquoBone vol 39 no 6 pp 1373ndash1381 2006
[29] V Perrotti B M Nicholls and A Piattelli ldquoHuman osteoclastformation and activity on an equine spongy bone substituterdquoClinical Oral Implants Research vol 20 no 1 pp 17ndash23 2009
[30] V Perrotti B M Nicholls M A Horton and A PiattellildquoHuman osteoclast formation and activity on a xenogenousbone mineralrdquo Journal of Biomedical Materials Research Part Avol 90 no 1 pp 238ndash246 2009
[31] D A L Di Stefano G Gastaldi R Vinci L Cinci L Pieri andE Gherlone ldquoHistomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in SinusAugmentation A Randomized Clinical Trial with 3-Year Fol-low-Uprdquo The International journal of oral amp maxillofacial im-plants vol 30 no 5 pp 1161ndash1167 2015
[32] I Elgali O Omar C Dahlin and P Thomsen ldquoGuided boneregeneration materials and biological mechanisms revisitedrdquoEuropean Journal of Oral Sciences vol 125 no 5 pp 315ndash3372017
[33] J Waasdorp and M A Reynolds ldquoAllogeneic bone onlay graftsfor alveolar ridge augmentation a systematic reviewrdquo TheInternational Journal of Oral amp Maxillofacial Implants vol 25no 3 pp 525ndash531 2010
[34] M Schlee and D Rothamel ldquoRidge augmentation using cus-tomized allogenic bone blocks Proof of concept and histologi-cal findingsrdquo Implant Dentistry vol 22 no 3 pp 212ndash218 2013
[35] G I Benic D S Thoma R E Jung et al ldquoGuided bone regen-eration with particulate vs block xenogenic bone substitutes apilot cone beam computed tomographic investigationrdquo ClinicalOral Implants Research vol 28 no 11 pp e262ndashe270 2017
[36] H Nguyen D A F Morgan and M R Forwood ldquoSterilizationof allograft bone Effects of gamma irradiation on allograftbiology and biomechanicsrdquo Cell and Tissue Banking vol 8 no2 pp 93ndash105 2007
[37] T Arizono Y Iwamoto K Okuyama and Y Sugioka ldquoEthyleneoxide sterilization of bone grafts Residual gas concentrationand fibroblast toxicityrdquo Acta Orthopaedica vol 65 no 6 pp640ndash642 1994
[38] C Hofmann T Von Garrel and L Gotzen ldquoBone bank man-agement using a thermal disinfection system (Lobator SD-1) Acritical analysisrdquo Unfallchirurg vol 99 no 7 pp 498ndash508 1996
[39] R G T Geesink ldquoOsteoconductive coatings for total joint ar-throplastyrdquo Clinical Orthopaedics and Related Research no 395pp 53ndash65 2002
[40] D J Prolo P W Pedrotti and D H White ldquoEthylene oxidesterilization of bone dura mater and fascia lata for humantransplantationrdquo Neurosurgery vol 6 no 5 pp 529ndash539 1980
[41] J A Tom and S A Rodeo ldquoSoft tissue allografts for knee recon-struction in sportsmedicinerdquoClinical Orthopaedics and RelatedResearch no 402 pp 135ndash156 2002
[42] Block SS Disinfection Sterilization and Preservation LippincottWilliams andWilkins Philadelphia Pa USA 4th edition 1991
[43] E J Mitchell A M Stawarz R Kayacan and C M RimnacldquoThe effect of gamma radiation sterilization on the fatigue crackpropagation resistance of human cortical bonerdquoThe Journal ofBone amp Joint Surgery vol 86 no 12 pp 2648ndash2657 2004
[44] M Silindir andYA Ozeray ldquoSterilizationmethods and the com-parison of E-beam sterilization with gamma radiation steril-izationrdquo Journal of Pharmaceutical Sciences vol 34 pp 43ndash532009
[45] L C Huesca-Espitia M Suvira K Rosenbeck et al ldquoEffects ofsteam autoclave treatment on Geobacillus stearothermophilussporesrdquo Journal of AppliedMicrobiology vol 121 no 5 pp 1300ndash1311 2016
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
CorrosionInternational Journal of
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Hindawiwwwhindawicom Volume 2018
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Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
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Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
4 BioMed Research International
Figure 5GroupB a trabeculawith sand-like residuals on it (2500x)
Figure 6GroupC awell-preserved trabeculawith smaller commu-nicating cavities the trabecula surface appears to be well preserved(420x)
continuously A feeble fluorescence can be observed at theedges of some gap (Figure 7)
Group B the images show that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula sometimes continuous and in-tense sometimes weak and widespread A feeble fluorescenceis observed at the edges This group appears to be similar togroup A (Figure 8)
Group C the image shows that type 1 collagen of thesample presents a fluorescence pattern that is located at theperiphery of the trabecula in a continuous and uniformman-ner The entire trabecula has a weak diffuse fluorescence pat-tern with a circumferential course sometimes more markedaround the gaps (Figure 9)
Display profile shows the fluorescence intensity along aselected line
The fluorescent signal intensity appears more pro-nounced and uniform in group C when compared to groups
Figure 7 Group A a continuous fluorescence pattern of type 1collagen is noticeable on the peripheral surface of the trabecula Alight fluorescence is evident
Figure 8 Group B the fluorescence pattern of the type 1 collagenis located in the peripheral of the trabecula and appears variouslyintense
Figure 9 Group C the fluorescence pattern of type 1 collagenis evident on trabecula and appears continuous and uniform Thewhole trabecula shows a fluorescence patternmore marked near thelacuna
BioMed Research International 5
Group C
Group BGroup A
Figure 10 Display profile analysis
A and B Comparing group A and group B a greater signalintensity is evident in group A (Figure 10)
4 Discussion
The bone augmentation procedure is widely studied in litera-ture and commonly used in clinical practice and might bemandatory in different clinical situations in both medicineand dentistry [5]
Materials different for origin and composition were deep-ly studied by the scientific community and the differencesbetween the particulate form and the bone block are com-monly known
Several studies analyzed the employment of bone equine-derived block containing collagen for ridge augmentation[51ndash53] These researches revealed the advantageous capacityto maintain the dimensions of the augmented ridge Benicet al demonstrated that the GBR technique performed withequine block with collagen membranes (CM) resulted inthe most favorable outcomes compared to bovine block andbovine granulate with CM [35]
It is mandatory to shape the bone block according to thedefect that should be filled The shaping could be performedby the industry that according to a CBCT provided by theclinician could send a preshaped and sterilized block or
a handmade ldquoin-houserdquo shaping before or during the surgeryThe handmade shaping has however a lack of precisionrelated to the method of shaping To overcome this issue aCNC milling machine could be used This machine has amicrometer precision and in addition to a three-dimensionalreconstruction could provide the clinician a precise modelhowever due to its characteristic the sterility requirement isnot satisfying andmust be achieved after shaping using othertechniques [54]
Cusinato et al [55] demonstrated the efficacy of hydro-gen peroxide treatment and e-beam irradiation on viralclearance in equine-derived xenografts (pericardium mem-brane collagen membrane and cortical and cancellous bonegrafts) spiked with three human viruses (Coxsackie virus B1influenza virus typeAwith subtypeH1N1 and herpes simplexvirus type 1)
Our work shows how it is possible to obtain a satisfactorysterility of the graft using a traditional device as the autoclave
The microbiological analysis shows how in agreementwith the literature the sterility was achieved in all samples
Our research was also aimed at understanding if this pro-cedure might modify the bone morphology and the collagen
The eye observation of the samples shows a fairly dehy-drated and a slightly lighter white surface when compared tothe control sample
6 BioMed Research International
The SEM observations between the control sample andthe samples of the three groups underline how at differentmagnifications themacrostructure does not show substantialmodifications [56]
In all samples the bone structure appears to be wellpreserved and themorphology appears to be very similar It isevident how the high temperature did not determine evidentand significantmodifications of the bone sample architecture
The collagen matrix that is a particular characteristic ofthe equine bone graft and is preserved due to the impossibilityof this species to be infected with prions appears to bevariously maintained
The confocal microscope immunofluorescence analysisshows how group C (131 fast) has the best-preserved collagenstructure This observation is also confirmed by the displayprofile analysis which evaluating the fluorescence intensityunderlines how the fluorescent signal appears to bemore uni-form and intense when compared to the other groups Whencomparing A and B groups the first one shows a strongerand a uniform fluorescence
The temperatures reached in steam autoclave sterilizationappear to be respectful of the collagen fibril that remainsalways below the thermal transitions temperatures [57 58]
The collagen resistance to heat and pressure damage soappears to be time related When a steam sterilization is per-formed a 134∘C and 216 bar for 330min and 5min of dryingprotocol is suggested
From the analysis of our data autoclave steam steriliza-tion method could be a reliable way to obtain sterilization ofbone graft
Giving the possibility of using a nonsterile CNC millingmachine will open a new scenario of precision in bonegrafting letting the surgeon to obtain a perfectly shaped andsterilized graft wherever before the surgery
5 Conclusion
The autoclave sterilization appears to be a reliable way toobtain amedical grade sterilization of the equine bone blocksand it does not affect the macro- and the microstructure andthe collagen fiber
Further studies are necessary to evaluate if there are anychemical modification and to evaluate if there are changes inthe in vivo osteointegration process of the bone that under-went the steam sterilization protocol
Data Availability
The data (photos) used to support the findings of this studyare available from the corresponding author upon request
Conflicts of Interest
Thestudy did not receive any financial support and therewerenot any conflicts of interest
Authorsrsquo Contributions
All individuals listed as authors have contributed substan-tially to the design performance analysis and reporting of
the work No others contributors to the articles than theauthors accredited
References
[1] S S Jensen and H Terheyden ldquoBone augmentation proceduresin localized defects in the alveolar ridge clinical results withdifferent bone grafts and bone-substitute materialsrdquo The Inter-national Journal of Oral amp Maxillofacial Implants vol 24 pp218ndash236 2009
[2] G A Catone B L Reimer DMcNeir and R Ray ldquoTibial auto-genous cancellous bone as an alternative donor site in max-illofacial surgery A preliminary reportrdquo Journal of Oral andMaxillofacial Surgery vol 50 no 12 pp 1258ndash1263 1992
[3] M G Donovan N C Dickerson and J C Mitchell ldquoCalvarialBone Harvest and Grafting Techniques for Maxillary andMan-dibular Implant Surgeryrdquo Atlas of the Oral and MaxillofacialSurgery Clinics vol 2 no 2 pp 109ndash122 1994
[4] C M Misch ldquoBone augmentation of the atrophic posteriormandible for dental implants using rhBMP-2 and titaniummesh clinical technique and early resultsrdquo International Journalof Periodontics and Restorative Dentistry vol 31 no 6 pp 581ndash589 2011
[5] F S de Ponte G Cutroneo R Falzea et al ldquoHistochemical andmorphological aspects of fresh frozen bone A preliminarystudyrdquo European Journal of Histochemistry vol 60 no 4 pp 5ndash9 2016
[6] A S Herford M Cicciu L F Eftimie et al ldquorhBMP-2 appliedas support of distraction osteogenesis A split-mouth histolog-ical study over nonhuman primates mandiblesrdquo InternationalJournal of Clinical and Experimental Medicine vol 9 no 9 pp17187ndash17194 2016
[7] M Cicciu ldquoReal opportunity for the present and a forward stepfor the future of bone tissue engineeringrdquo The Journal ofCraniofacial Surgery vol 28 no 3 pp 592-593 2017
[8] M Cicciu G Cervino A S Herford et al ldquoFacial Bone Recon-struction Using Both Marine or Non-Marine Bone SubstitutesEvaluation of Current Outcomes in a Systematic LiteratureReviewrdquoMarine Drugs vol 16 no 1 2018
[9] M EspositoM G Grusovin P Coulthard andH VWorthing-ton ldquoThe efficacy of various bone augmentation procedures fordental implants a Cochrane systematic review of randomizedcontrolled clinical trialsrdquo The International Journal of Oral ampMaxillofacial Implants vol 21 no 5 pp 696ndash710 2006
[10] GOrsini A ScaranoM PiattelliM Piccirilli S Caputi andAPiattelli ldquoHistologic and ultrastructural analysis of regeneratedbone in maxillary sinus augmentation using a porcine bone-derived biomaterialrdquo Journal of Periodontology vol 77 no 12pp 1984ndash1990 2006
[11] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoDental Implants Placed in Periodontally InfectedSites in Humansrdquo Clinical Implant Dentistry and RelatedResearch vol 19 no 1 pp 131ndash139 2017
[12] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoImmediate Implant Placement in Sockets withAsymptomatic Apical Periodontitisrdquo Clinical Implant Dentistryand Related Research vol 19 no 1 pp 20ndash27 2017
[13] T Traini P Valentini G Iezzi and A Piattelli ldquoA histologic andhistomorphometric evaluation of anorganic bovine bone re-trieved 9 years after a sinus augmentation procedurerdquo Journalof Periodontology vol 78 no 5 pp 955ndash961 2007
BioMed Research International 7
[14] A Barone P Toti A Quaranta et al ldquoClinical and Histologicalchanges after ridge preservation with two xenografts prelimi-nary results from a multicentre randomized controlled clinicaltrialrdquo Journal of Clinical Periodontology vol 44 no 2 pp 204ndash214 2017
[15] P Felice L Piana L ChecchiV CorvinoU Nannmark andMPiattelli ldquoVertical ridge augmentation of an atrophic posteriormandible with an inlay technique and cancellous equine boneblock A case reportrdquo International Journal of Periodontics andRestorative Dentistry vol 33 no 2 pp 159ndash166 2013
[16] R Pistilli L Signorini A Pisacane G Lizio and P Felice ldquoCaseof severe bone atrophy of the posterior maxilla rehabilitatedwith blocks of equine origin bone Histological resultsrdquo ImplantDentistry vol 22 no 1 pp 8ndash15 2013
[17] N De Angelis and M Scivetti ldquoLateral ridge augmentationusing an equine flex bone block infused with recombinanthuman platelet-derived growth factor BB A clinical and histo-logic studyrdquo International Journal of Periodontics andRestorativeDentistry vol 31 no 4 pp 383ndash388 2011
[18] D A di Stefano L Artese G Iezzi et al ldquoAlveolar ridge regen-eration with equine spongy bone a clinical histological andimmunohistochemical case seriesrdquo Clinical Implant Dentistryand Related Research vol 11 no 2 pp 90ndash100 2009
[19] L Artese A Piattelli D ADi Stefano et al ldquoSinus liftwith auto-logous bone alone or in addition to equine bone an immuno-histochemical study in manrdquo Implant Dentistry vol 20 no 5pp 383ndash388 2011
[20] S Tete R Vinci V L Zizzari et al ldquoMaxillary sinus augmen-tation procedures through equine-derived biomaterial or cal-varia autologous bone Immunohistochemical evaluation ofOPGRANKL in humansrdquo European Journal of Histochemistryvol 57 no 1 pp 60ndash65 2013
[21] G LoGiudice G Iannello A Terranova R LoGiudice G Pan-taleo and M Cicciu ldquoTranscrestal sinus lift procedure ap-proaching atrophic maxillary ridge A 60-month clinical andradiological follow-up evaluationrdquo International Journal ofDen-tistry vol 2015 2015
[22] S Tete V L Zizzari R Vinci et al ldquoEquine and porcine bonesubstitutes in maxillary sinus augmentation A histologicaland immunohistochemical analysis of VEGF expressionrdquo TheJournal of Craniofacial Surgery vol 25 no 3 pp 835ndash839 2014
[23] J Talairach and PThournouxCo-Planar Stereotaxic Atlas of theHuman Brain Thieme Medical Publishers Stuttgart Germany1988
[24] J Zhang ldquoThe structural stability of wild-type horse prion pro-teinrdquo Journal of Biomolecular Structure and Dynamics vol 29no 2 pp 369ndash377 2011
[25] D J Pacaccio and S F Stern ldquoDemineralized bonematrix Basicscience and clinical applicationsrdquo Clinics in Podiatric Medicineand Surgery vol 22 no 4 pp 599ndash606 2005
[26] K D Chesmel J Branger H Wertheim and N ScarboroughldquoHealing response to various forms of human demineralizedbone matrix in athymic rat cranial defectsrdquo Journal of Oral andMaxillofacial Surgery vol 56 no 7 pp 857ndash865 1998
[27] S Guizzardi R Scandroglio M D Silvestre R Savini and ARuggeri ldquoImplants of heterologous demineralized bone matrixfor induction of posterior spinal fusion in ratsrdquoThe Spine Jour-nal vol 17 no 6 pp 701ndash707 1992
[28] M S Rafael V Laize and M L Cancela ldquoIdentification of Spa-rus aurata bone morphogenetic protein 2 Molecular cloninggene expression and in silico analysis of protein conservedfeatures in vertebratesrdquoBone vol 39 no 6 pp 1373ndash1381 2006
[29] V Perrotti B M Nicholls and A Piattelli ldquoHuman osteoclastformation and activity on an equine spongy bone substituterdquoClinical Oral Implants Research vol 20 no 1 pp 17ndash23 2009
[30] V Perrotti B M Nicholls M A Horton and A PiattellildquoHuman osteoclast formation and activity on a xenogenousbone mineralrdquo Journal of Biomedical Materials Research Part Avol 90 no 1 pp 238ndash246 2009
[31] D A L Di Stefano G Gastaldi R Vinci L Cinci L Pieri andE Gherlone ldquoHistomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in SinusAugmentation A Randomized Clinical Trial with 3-Year Fol-low-Uprdquo The International journal of oral amp maxillofacial im-plants vol 30 no 5 pp 1161ndash1167 2015
[32] I Elgali O Omar C Dahlin and P Thomsen ldquoGuided boneregeneration materials and biological mechanisms revisitedrdquoEuropean Journal of Oral Sciences vol 125 no 5 pp 315ndash3372017
[33] J Waasdorp and M A Reynolds ldquoAllogeneic bone onlay graftsfor alveolar ridge augmentation a systematic reviewrdquo TheInternational Journal of Oral amp Maxillofacial Implants vol 25no 3 pp 525ndash531 2010
[34] M Schlee and D Rothamel ldquoRidge augmentation using cus-tomized allogenic bone blocks Proof of concept and histologi-cal findingsrdquo Implant Dentistry vol 22 no 3 pp 212ndash218 2013
[35] G I Benic D S Thoma R E Jung et al ldquoGuided bone regen-eration with particulate vs block xenogenic bone substitutes apilot cone beam computed tomographic investigationrdquo ClinicalOral Implants Research vol 28 no 11 pp e262ndashe270 2017
[36] H Nguyen D A F Morgan and M R Forwood ldquoSterilizationof allograft bone Effects of gamma irradiation on allograftbiology and biomechanicsrdquo Cell and Tissue Banking vol 8 no2 pp 93ndash105 2007
[37] T Arizono Y Iwamoto K Okuyama and Y Sugioka ldquoEthyleneoxide sterilization of bone grafts Residual gas concentrationand fibroblast toxicityrdquo Acta Orthopaedica vol 65 no 6 pp640ndash642 1994
[38] C Hofmann T Von Garrel and L Gotzen ldquoBone bank man-agement using a thermal disinfection system (Lobator SD-1) Acritical analysisrdquo Unfallchirurg vol 99 no 7 pp 498ndash508 1996
[39] R G T Geesink ldquoOsteoconductive coatings for total joint ar-throplastyrdquo Clinical Orthopaedics and Related Research no 395pp 53ndash65 2002
[40] D J Prolo P W Pedrotti and D H White ldquoEthylene oxidesterilization of bone dura mater and fascia lata for humantransplantationrdquo Neurosurgery vol 6 no 5 pp 529ndash539 1980
[41] J A Tom and S A Rodeo ldquoSoft tissue allografts for knee recon-struction in sportsmedicinerdquoClinical Orthopaedics and RelatedResearch no 402 pp 135ndash156 2002
[42] Block SS Disinfection Sterilization and Preservation LippincottWilliams andWilkins Philadelphia Pa USA 4th edition 1991
[43] E J Mitchell A M Stawarz R Kayacan and C M RimnacldquoThe effect of gamma radiation sterilization on the fatigue crackpropagation resistance of human cortical bonerdquoThe Journal ofBone amp Joint Surgery vol 86 no 12 pp 2648ndash2657 2004
[44] M Silindir andYA Ozeray ldquoSterilizationmethods and the com-parison of E-beam sterilization with gamma radiation steril-izationrdquo Journal of Pharmaceutical Sciences vol 34 pp 43ndash532009
[45] L C Huesca-Espitia M Suvira K Rosenbeck et al ldquoEffects ofsteam autoclave treatment on Geobacillus stearothermophilussporesrdquo Journal of AppliedMicrobiology vol 121 no 5 pp 1300ndash1311 2016
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
CorrosionInternational Journal of
Hindawiwwwhindawicom Volume 2018
Advances in
Materials Science and EngineeringHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of
Chemistry
Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
ScienticaHindawiwwwhindawicom Volume 2018
Polymer ScienceInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Advances in Condensed Matter Physics
Hindawiwwwhindawicom Volume 2018
International Journal of
BiomaterialsHindawiwwwhindawicom
Journal ofEngineeringVolume 2018
Applied ChemistryJournal of
Hindawiwwwhindawicom Volume 2018
NanotechnologyHindawiwwwhindawicom Volume 2018
Journal of
Hindawiwwwhindawicom Volume 2018
High Energy PhysicsAdvances in
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
TribologyAdvances in
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
ChemistryAdvances in
Hindawiwwwhindawicom Volume 2018
Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
BioMed Research International 5
Group C
Group BGroup A
Figure 10 Display profile analysis
A and B Comparing group A and group B a greater signalintensity is evident in group A (Figure 10)
4 Discussion
The bone augmentation procedure is widely studied in litera-ture and commonly used in clinical practice and might bemandatory in different clinical situations in both medicineand dentistry [5]
Materials different for origin and composition were deep-ly studied by the scientific community and the differencesbetween the particulate form and the bone block are com-monly known
Several studies analyzed the employment of bone equine-derived block containing collagen for ridge augmentation[51ndash53] These researches revealed the advantageous capacityto maintain the dimensions of the augmented ridge Benicet al demonstrated that the GBR technique performed withequine block with collagen membranes (CM) resulted inthe most favorable outcomes compared to bovine block andbovine granulate with CM [35]
It is mandatory to shape the bone block according to thedefect that should be filled The shaping could be performedby the industry that according to a CBCT provided by theclinician could send a preshaped and sterilized block or
a handmade ldquoin-houserdquo shaping before or during the surgeryThe handmade shaping has however a lack of precisionrelated to the method of shaping To overcome this issue aCNC milling machine could be used This machine has amicrometer precision and in addition to a three-dimensionalreconstruction could provide the clinician a precise modelhowever due to its characteristic the sterility requirement isnot satisfying andmust be achieved after shaping using othertechniques [54]
Cusinato et al [55] demonstrated the efficacy of hydro-gen peroxide treatment and e-beam irradiation on viralclearance in equine-derived xenografts (pericardium mem-brane collagen membrane and cortical and cancellous bonegrafts) spiked with three human viruses (Coxsackie virus B1influenza virus typeAwith subtypeH1N1 and herpes simplexvirus type 1)
Our work shows how it is possible to obtain a satisfactorysterility of the graft using a traditional device as the autoclave
The microbiological analysis shows how in agreementwith the literature the sterility was achieved in all samples
Our research was also aimed at understanding if this pro-cedure might modify the bone morphology and the collagen
The eye observation of the samples shows a fairly dehy-drated and a slightly lighter white surface when compared tothe control sample
6 BioMed Research International
The SEM observations between the control sample andthe samples of the three groups underline how at differentmagnifications themacrostructure does not show substantialmodifications [56]
In all samples the bone structure appears to be wellpreserved and themorphology appears to be very similar It isevident how the high temperature did not determine evidentand significantmodifications of the bone sample architecture
The collagen matrix that is a particular characteristic ofthe equine bone graft and is preserved due to the impossibilityof this species to be infected with prions appears to bevariously maintained
The confocal microscope immunofluorescence analysisshows how group C (131 fast) has the best-preserved collagenstructure This observation is also confirmed by the displayprofile analysis which evaluating the fluorescence intensityunderlines how the fluorescent signal appears to bemore uni-form and intense when compared to the other groups Whencomparing A and B groups the first one shows a strongerand a uniform fluorescence
The temperatures reached in steam autoclave sterilizationappear to be respectful of the collagen fibril that remainsalways below the thermal transitions temperatures [57 58]
The collagen resistance to heat and pressure damage soappears to be time related When a steam sterilization is per-formed a 134∘C and 216 bar for 330min and 5min of dryingprotocol is suggested
From the analysis of our data autoclave steam steriliza-tion method could be a reliable way to obtain sterilization ofbone graft
Giving the possibility of using a nonsterile CNC millingmachine will open a new scenario of precision in bonegrafting letting the surgeon to obtain a perfectly shaped andsterilized graft wherever before the surgery
5 Conclusion
The autoclave sterilization appears to be a reliable way toobtain amedical grade sterilization of the equine bone blocksand it does not affect the macro- and the microstructure andthe collagen fiber
Further studies are necessary to evaluate if there are anychemical modification and to evaluate if there are changes inthe in vivo osteointegration process of the bone that under-went the steam sterilization protocol
Data Availability
The data (photos) used to support the findings of this studyare available from the corresponding author upon request
Conflicts of Interest
Thestudy did not receive any financial support and therewerenot any conflicts of interest
Authorsrsquo Contributions
All individuals listed as authors have contributed substan-tially to the design performance analysis and reporting of
the work No others contributors to the articles than theauthors accredited
References
[1] S S Jensen and H Terheyden ldquoBone augmentation proceduresin localized defects in the alveolar ridge clinical results withdifferent bone grafts and bone-substitute materialsrdquo The Inter-national Journal of Oral amp Maxillofacial Implants vol 24 pp218ndash236 2009
[2] G A Catone B L Reimer DMcNeir and R Ray ldquoTibial auto-genous cancellous bone as an alternative donor site in max-illofacial surgery A preliminary reportrdquo Journal of Oral andMaxillofacial Surgery vol 50 no 12 pp 1258ndash1263 1992
[3] M G Donovan N C Dickerson and J C Mitchell ldquoCalvarialBone Harvest and Grafting Techniques for Maxillary andMan-dibular Implant Surgeryrdquo Atlas of the Oral and MaxillofacialSurgery Clinics vol 2 no 2 pp 109ndash122 1994
[4] C M Misch ldquoBone augmentation of the atrophic posteriormandible for dental implants using rhBMP-2 and titaniummesh clinical technique and early resultsrdquo International Journalof Periodontics and Restorative Dentistry vol 31 no 6 pp 581ndash589 2011
[5] F S de Ponte G Cutroneo R Falzea et al ldquoHistochemical andmorphological aspects of fresh frozen bone A preliminarystudyrdquo European Journal of Histochemistry vol 60 no 4 pp 5ndash9 2016
[6] A S Herford M Cicciu L F Eftimie et al ldquorhBMP-2 appliedas support of distraction osteogenesis A split-mouth histolog-ical study over nonhuman primates mandiblesrdquo InternationalJournal of Clinical and Experimental Medicine vol 9 no 9 pp17187ndash17194 2016
[7] M Cicciu ldquoReal opportunity for the present and a forward stepfor the future of bone tissue engineeringrdquo The Journal ofCraniofacial Surgery vol 28 no 3 pp 592-593 2017
[8] M Cicciu G Cervino A S Herford et al ldquoFacial Bone Recon-struction Using Both Marine or Non-Marine Bone SubstitutesEvaluation of Current Outcomes in a Systematic LiteratureReviewrdquoMarine Drugs vol 16 no 1 2018
[9] M EspositoM G Grusovin P Coulthard andH VWorthing-ton ldquoThe efficacy of various bone augmentation procedures fordental implants a Cochrane systematic review of randomizedcontrolled clinical trialsrdquo The International Journal of Oral ampMaxillofacial Implants vol 21 no 5 pp 696ndash710 2006
[10] GOrsini A ScaranoM PiattelliM Piccirilli S Caputi andAPiattelli ldquoHistologic and ultrastructural analysis of regeneratedbone in maxillary sinus augmentation using a porcine bone-derived biomaterialrdquo Journal of Periodontology vol 77 no 12pp 1984ndash1990 2006
[11] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoDental Implants Placed in Periodontally InfectedSites in Humansrdquo Clinical Implant Dentistry and RelatedResearch vol 19 no 1 pp 131ndash139 2017
[12] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoImmediate Implant Placement in Sockets withAsymptomatic Apical Periodontitisrdquo Clinical Implant Dentistryand Related Research vol 19 no 1 pp 20ndash27 2017
[13] T Traini P Valentini G Iezzi and A Piattelli ldquoA histologic andhistomorphometric evaluation of anorganic bovine bone re-trieved 9 years after a sinus augmentation procedurerdquo Journalof Periodontology vol 78 no 5 pp 955ndash961 2007
BioMed Research International 7
[14] A Barone P Toti A Quaranta et al ldquoClinical and Histologicalchanges after ridge preservation with two xenografts prelimi-nary results from a multicentre randomized controlled clinicaltrialrdquo Journal of Clinical Periodontology vol 44 no 2 pp 204ndash214 2017
[15] P Felice L Piana L ChecchiV CorvinoU Nannmark andMPiattelli ldquoVertical ridge augmentation of an atrophic posteriormandible with an inlay technique and cancellous equine boneblock A case reportrdquo International Journal of Periodontics andRestorative Dentistry vol 33 no 2 pp 159ndash166 2013
[16] R Pistilli L Signorini A Pisacane G Lizio and P Felice ldquoCaseof severe bone atrophy of the posterior maxilla rehabilitatedwith blocks of equine origin bone Histological resultsrdquo ImplantDentistry vol 22 no 1 pp 8ndash15 2013
[17] N De Angelis and M Scivetti ldquoLateral ridge augmentationusing an equine flex bone block infused with recombinanthuman platelet-derived growth factor BB A clinical and histo-logic studyrdquo International Journal of Periodontics andRestorativeDentistry vol 31 no 4 pp 383ndash388 2011
[18] D A di Stefano L Artese G Iezzi et al ldquoAlveolar ridge regen-eration with equine spongy bone a clinical histological andimmunohistochemical case seriesrdquo Clinical Implant Dentistryand Related Research vol 11 no 2 pp 90ndash100 2009
[19] L Artese A Piattelli D ADi Stefano et al ldquoSinus liftwith auto-logous bone alone or in addition to equine bone an immuno-histochemical study in manrdquo Implant Dentistry vol 20 no 5pp 383ndash388 2011
[20] S Tete R Vinci V L Zizzari et al ldquoMaxillary sinus augmen-tation procedures through equine-derived biomaterial or cal-varia autologous bone Immunohistochemical evaluation ofOPGRANKL in humansrdquo European Journal of Histochemistryvol 57 no 1 pp 60ndash65 2013
[21] G LoGiudice G Iannello A Terranova R LoGiudice G Pan-taleo and M Cicciu ldquoTranscrestal sinus lift procedure ap-proaching atrophic maxillary ridge A 60-month clinical andradiological follow-up evaluationrdquo International Journal ofDen-tistry vol 2015 2015
[22] S Tete V L Zizzari R Vinci et al ldquoEquine and porcine bonesubstitutes in maxillary sinus augmentation A histologicaland immunohistochemical analysis of VEGF expressionrdquo TheJournal of Craniofacial Surgery vol 25 no 3 pp 835ndash839 2014
[23] J Talairach and PThournouxCo-Planar Stereotaxic Atlas of theHuman Brain Thieme Medical Publishers Stuttgart Germany1988
[24] J Zhang ldquoThe structural stability of wild-type horse prion pro-teinrdquo Journal of Biomolecular Structure and Dynamics vol 29no 2 pp 369ndash377 2011
[25] D J Pacaccio and S F Stern ldquoDemineralized bonematrix Basicscience and clinical applicationsrdquo Clinics in Podiatric Medicineand Surgery vol 22 no 4 pp 599ndash606 2005
[26] K D Chesmel J Branger H Wertheim and N ScarboroughldquoHealing response to various forms of human demineralizedbone matrix in athymic rat cranial defectsrdquo Journal of Oral andMaxillofacial Surgery vol 56 no 7 pp 857ndash865 1998
[27] S Guizzardi R Scandroglio M D Silvestre R Savini and ARuggeri ldquoImplants of heterologous demineralized bone matrixfor induction of posterior spinal fusion in ratsrdquoThe Spine Jour-nal vol 17 no 6 pp 701ndash707 1992
[28] M S Rafael V Laize and M L Cancela ldquoIdentification of Spa-rus aurata bone morphogenetic protein 2 Molecular cloninggene expression and in silico analysis of protein conservedfeatures in vertebratesrdquoBone vol 39 no 6 pp 1373ndash1381 2006
[29] V Perrotti B M Nicholls and A Piattelli ldquoHuman osteoclastformation and activity on an equine spongy bone substituterdquoClinical Oral Implants Research vol 20 no 1 pp 17ndash23 2009
[30] V Perrotti B M Nicholls M A Horton and A PiattellildquoHuman osteoclast formation and activity on a xenogenousbone mineralrdquo Journal of Biomedical Materials Research Part Avol 90 no 1 pp 238ndash246 2009
[31] D A L Di Stefano G Gastaldi R Vinci L Cinci L Pieri andE Gherlone ldquoHistomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in SinusAugmentation A Randomized Clinical Trial with 3-Year Fol-low-Uprdquo The International journal of oral amp maxillofacial im-plants vol 30 no 5 pp 1161ndash1167 2015
[32] I Elgali O Omar C Dahlin and P Thomsen ldquoGuided boneregeneration materials and biological mechanisms revisitedrdquoEuropean Journal of Oral Sciences vol 125 no 5 pp 315ndash3372017
[33] J Waasdorp and M A Reynolds ldquoAllogeneic bone onlay graftsfor alveolar ridge augmentation a systematic reviewrdquo TheInternational Journal of Oral amp Maxillofacial Implants vol 25no 3 pp 525ndash531 2010
[34] M Schlee and D Rothamel ldquoRidge augmentation using cus-tomized allogenic bone blocks Proof of concept and histologi-cal findingsrdquo Implant Dentistry vol 22 no 3 pp 212ndash218 2013
[35] G I Benic D S Thoma R E Jung et al ldquoGuided bone regen-eration with particulate vs block xenogenic bone substitutes apilot cone beam computed tomographic investigationrdquo ClinicalOral Implants Research vol 28 no 11 pp e262ndashe270 2017
[36] H Nguyen D A F Morgan and M R Forwood ldquoSterilizationof allograft bone Effects of gamma irradiation on allograftbiology and biomechanicsrdquo Cell and Tissue Banking vol 8 no2 pp 93ndash105 2007
[37] T Arizono Y Iwamoto K Okuyama and Y Sugioka ldquoEthyleneoxide sterilization of bone grafts Residual gas concentrationand fibroblast toxicityrdquo Acta Orthopaedica vol 65 no 6 pp640ndash642 1994
[38] C Hofmann T Von Garrel and L Gotzen ldquoBone bank man-agement using a thermal disinfection system (Lobator SD-1) Acritical analysisrdquo Unfallchirurg vol 99 no 7 pp 498ndash508 1996
[39] R G T Geesink ldquoOsteoconductive coatings for total joint ar-throplastyrdquo Clinical Orthopaedics and Related Research no 395pp 53ndash65 2002
[40] D J Prolo P W Pedrotti and D H White ldquoEthylene oxidesterilization of bone dura mater and fascia lata for humantransplantationrdquo Neurosurgery vol 6 no 5 pp 529ndash539 1980
[41] J A Tom and S A Rodeo ldquoSoft tissue allografts for knee recon-struction in sportsmedicinerdquoClinical Orthopaedics and RelatedResearch no 402 pp 135ndash156 2002
[42] Block SS Disinfection Sterilization and Preservation LippincottWilliams andWilkins Philadelphia Pa USA 4th edition 1991
[43] E J Mitchell A M Stawarz R Kayacan and C M RimnacldquoThe effect of gamma radiation sterilization on the fatigue crackpropagation resistance of human cortical bonerdquoThe Journal ofBone amp Joint Surgery vol 86 no 12 pp 2648ndash2657 2004
[44] M Silindir andYA Ozeray ldquoSterilizationmethods and the com-parison of E-beam sterilization with gamma radiation steril-izationrdquo Journal of Pharmaceutical Sciences vol 34 pp 43ndash532009
[45] L C Huesca-Espitia M Suvira K Rosenbeck et al ldquoEffects ofsteam autoclave treatment on Geobacillus stearothermophilussporesrdquo Journal of AppliedMicrobiology vol 121 no 5 pp 1300ndash1311 2016
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
CorrosionInternational Journal of
Hindawiwwwhindawicom Volume 2018
Advances in
Materials Science and EngineeringHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of
Chemistry
Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
ScienticaHindawiwwwhindawicom Volume 2018
Polymer ScienceInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Advances in Condensed Matter Physics
Hindawiwwwhindawicom Volume 2018
International Journal of
BiomaterialsHindawiwwwhindawicom
Journal ofEngineeringVolume 2018
Applied ChemistryJournal of
Hindawiwwwhindawicom Volume 2018
NanotechnologyHindawiwwwhindawicom Volume 2018
Journal of
Hindawiwwwhindawicom Volume 2018
High Energy PhysicsAdvances in
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
TribologyAdvances in
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
ChemistryAdvances in
Hindawiwwwhindawicom Volume 2018
Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
6 BioMed Research International
The SEM observations between the control sample andthe samples of the three groups underline how at differentmagnifications themacrostructure does not show substantialmodifications [56]
In all samples the bone structure appears to be wellpreserved and themorphology appears to be very similar It isevident how the high temperature did not determine evidentand significantmodifications of the bone sample architecture
The collagen matrix that is a particular characteristic ofthe equine bone graft and is preserved due to the impossibilityof this species to be infected with prions appears to bevariously maintained
The confocal microscope immunofluorescence analysisshows how group C (131 fast) has the best-preserved collagenstructure This observation is also confirmed by the displayprofile analysis which evaluating the fluorescence intensityunderlines how the fluorescent signal appears to bemore uni-form and intense when compared to the other groups Whencomparing A and B groups the first one shows a strongerand a uniform fluorescence
The temperatures reached in steam autoclave sterilizationappear to be respectful of the collagen fibril that remainsalways below the thermal transitions temperatures [57 58]
The collagen resistance to heat and pressure damage soappears to be time related When a steam sterilization is per-formed a 134∘C and 216 bar for 330min and 5min of dryingprotocol is suggested
From the analysis of our data autoclave steam steriliza-tion method could be a reliable way to obtain sterilization ofbone graft
Giving the possibility of using a nonsterile CNC millingmachine will open a new scenario of precision in bonegrafting letting the surgeon to obtain a perfectly shaped andsterilized graft wherever before the surgery
5 Conclusion
The autoclave sterilization appears to be a reliable way toobtain amedical grade sterilization of the equine bone blocksand it does not affect the macro- and the microstructure andthe collagen fiber
Further studies are necessary to evaluate if there are anychemical modification and to evaluate if there are changes inthe in vivo osteointegration process of the bone that under-went the steam sterilization protocol
Data Availability
The data (photos) used to support the findings of this studyare available from the corresponding author upon request
Conflicts of Interest
Thestudy did not receive any financial support and therewerenot any conflicts of interest
Authorsrsquo Contributions
All individuals listed as authors have contributed substan-tially to the design performance analysis and reporting of
the work No others contributors to the articles than theauthors accredited
References
[1] S S Jensen and H Terheyden ldquoBone augmentation proceduresin localized defects in the alveolar ridge clinical results withdifferent bone grafts and bone-substitute materialsrdquo The Inter-national Journal of Oral amp Maxillofacial Implants vol 24 pp218ndash236 2009
[2] G A Catone B L Reimer DMcNeir and R Ray ldquoTibial auto-genous cancellous bone as an alternative donor site in max-illofacial surgery A preliminary reportrdquo Journal of Oral andMaxillofacial Surgery vol 50 no 12 pp 1258ndash1263 1992
[3] M G Donovan N C Dickerson and J C Mitchell ldquoCalvarialBone Harvest and Grafting Techniques for Maxillary andMan-dibular Implant Surgeryrdquo Atlas of the Oral and MaxillofacialSurgery Clinics vol 2 no 2 pp 109ndash122 1994
[4] C M Misch ldquoBone augmentation of the atrophic posteriormandible for dental implants using rhBMP-2 and titaniummesh clinical technique and early resultsrdquo International Journalof Periodontics and Restorative Dentistry vol 31 no 6 pp 581ndash589 2011
[5] F S de Ponte G Cutroneo R Falzea et al ldquoHistochemical andmorphological aspects of fresh frozen bone A preliminarystudyrdquo European Journal of Histochemistry vol 60 no 4 pp 5ndash9 2016
[6] A S Herford M Cicciu L F Eftimie et al ldquorhBMP-2 appliedas support of distraction osteogenesis A split-mouth histolog-ical study over nonhuman primates mandiblesrdquo InternationalJournal of Clinical and Experimental Medicine vol 9 no 9 pp17187ndash17194 2016
[7] M Cicciu ldquoReal opportunity for the present and a forward stepfor the future of bone tissue engineeringrdquo The Journal ofCraniofacial Surgery vol 28 no 3 pp 592-593 2017
[8] M Cicciu G Cervino A S Herford et al ldquoFacial Bone Recon-struction Using Both Marine or Non-Marine Bone SubstitutesEvaluation of Current Outcomes in a Systematic LiteratureReviewrdquoMarine Drugs vol 16 no 1 2018
[9] M EspositoM G Grusovin P Coulthard andH VWorthing-ton ldquoThe efficacy of various bone augmentation procedures fordental implants a Cochrane systematic review of randomizedcontrolled clinical trialsrdquo The International Journal of Oral ampMaxillofacial Implants vol 21 no 5 pp 696ndash710 2006
[10] GOrsini A ScaranoM PiattelliM Piccirilli S Caputi andAPiattelli ldquoHistologic and ultrastructural analysis of regeneratedbone in maxillary sinus augmentation using a porcine bone-derived biomaterialrdquo Journal of Periodontology vol 77 no 12pp 1984ndash1990 2006
[11] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoDental Implants Placed in Periodontally InfectedSites in Humansrdquo Clinical Implant Dentistry and RelatedResearch vol 19 no 1 pp 131ndash139 2017
[12] R Crespi P Cappare G Crespi G LoGiudice G Gastaldi andE Gherlone ldquoImmediate Implant Placement in Sockets withAsymptomatic Apical Periodontitisrdquo Clinical Implant Dentistryand Related Research vol 19 no 1 pp 20ndash27 2017
[13] T Traini P Valentini G Iezzi and A Piattelli ldquoA histologic andhistomorphometric evaluation of anorganic bovine bone re-trieved 9 years after a sinus augmentation procedurerdquo Journalof Periodontology vol 78 no 5 pp 955ndash961 2007
BioMed Research International 7
[14] A Barone P Toti A Quaranta et al ldquoClinical and Histologicalchanges after ridge preservation with two xenografts prelimi-nary results from a multicentre randomized controlled clinicaltrialrdquo Journal of Clinical Periodontology vol 44 no 2 pp 204ndash214 2017
[15] P Felice L Piana L ChecchiV CorvinoU Nannmark andMPiattelli ldquoVertical ridge augmentation of an atrophic posteriormandible with an inlay technique and cancellous equine boneblock A case reportrdquo International Journal of Periodontics andRestorative Dentistry vol 33 no 2 pp 159ndash166 2013
[16] R Pistilli L Signorini A Pisacane G Lizio and P Felice ldquoCaseof severe bone atrophy of the posterior maxilla rehabilitatedwith blocks of equine origin bone Histological resultsrdquo ImplantDentistry vol 22 no 1 pp 8ndash15 2013
[17] N De Angelis and M Scivetti ldquoLateral ridge augmentationusing an equine flex bone block infused with recombinanthuman platelet-derived growth factor BB A clinical and histo-logic studyrdquo International Journal of Periodontics andRestorativeDentistry vol 31 no 4 pp 383ndash388 2011
[18] D A di Stefano L Artese G Iezzi et al ldquoAlveolar ridge regen-eration with equine spongy bone a clinical histological andimmunohistochemical case seriesrdquo Clinical Implant Dentistryand Related Research vol 11 no 2 pp 90ndash100 2009
[19] L Artese A Piattelli D ADi Stefano et al ldquoSinus liftwith auto-logous bone alone or in addition to equine bone an immuno-histochemical study in manrdquo Implant Dentistry vol 20 no 5pp 383ndash388 2011
[20] S Tete R Vinci V L Zizzari et al ldquoMaxillary sinus augmen-tation procedures through equine-derived biomaterial or cal-varia autologous bone Immunohistochemical evaluation ofOPGRANKL in humansrdquo European Journal of Histochemistryvol 57 no 1 pp 60ndash65 2013
[21] G LoGiudice G Iannello A Terranova R LoGiudice G Pan-taleo and M Cicciu ldquoTranscrestal sinus lift procedure ap-proaching atrophic maxillary ridge A 60-month clinical andradiological follow-up evaluationrdquo International Journal ofDen-tistry vol 2015 2015
[22] S Tete V L Zizzari R Vinci et al ldquoEquine and porcine bonesubstitutes in maxillary sinus augmentation A histologicaland immunohistochemical analysis of VEGF expressionrdquo TheJournal of Craniofacial Surgery vol 25 no 3 pp 835ndash839 2014
[23] J Talairach and PThournouxCo-Planar Stereotaxic Atlas of theHuman Brain Thieme Medical Publishers Stuttgart Germany1988
[24] J Zhang ldquoThe structural stability of wild-type horse prion pro-teinrdquo Journal of Biomolecular Structure and Dynamics vol 29no 2 pp 369ndash377 2011
[25] D J Pacaccio and S F Stern ldquoDemineralized bonematrix Basicscience and clinical applicationsrdquo Clinics in Podiatric Medicineand Surgery vol 22 no 4 pp 599ndash606 2005
[26] K D Chesmel J Branger H Wertheim and N ScarboroughldquoHealing response to various forms of human demineralizedbone matrix in athymic rat cranial defectsrdquo Journal of Oral andMaxillofacial Surgery vol 56 no 7 pp 857ndash865 1998
[27] S Guizzardi R Scandroglio M D Silvestre R Savini and ARuggeri ldquoImplants of heterologous demineralized bone matrixfor induction of posterior spinal fusion in ratsrdquoThe Spine Jour-nal vol 17 no 6 pp 701ndash707 1992
[28] M S Rafael V Laize and M L Cancela ldquoIdentification of Spa-rus aurata bone morphogenetic protein 2 Molecular cloninggene expression and in silico analysis of protein conservedfeatures in vertebratesrdquoBone vol 39 no 6 pp 1373ndash1381 2006
[29] V Perrotti B M Nicholls and A Piattelli ldquoHuman osteoclastformation and activity on an equine spongy bone substituterdquoClinical Oral Implants Research vol 20 no 1 pp 17ndash23 2009
[30] V Perrotti B M Nicholls M A Horton and A PiattellildquoHuman osteoclast formation and activity on a xenogenousbone mineralrdquo Journal of Biomedical Materials Research Part Avol 90 no 1 pp 238ndash246 2009
[31] D A L Di Stefano G Gastaldi R Vinci L Cinci L Pieri andE Gherlone ldquoHistomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in SinusAugmentation A Randomized Clinical Trial with 3-Year Fol-low-Uprdquo The International journal of oral amp maxillofacial im-plants vol 30 no 5 pp 1161ndash1167 2015
[32] I Elgali O Omar C Dahlin and P Thomsen ldquoGuided boneregeneration materials and biological mechanisms revisitedrdquoEuropean Journal of Oral Sciences vol 125 no 5 pp 315ndash3372017
[33] J Waasdorp and M A Reynolds ldquoAllogeneic bone onlay graftsfor alveolar ridge augmentation a systematic reviewrdquo TheInternational Journal of Oral amp Maxillofacial Implants vol 25no 3 pp 525ndash531 2010
[34] M Schlee and D Rothamel ldquoRidge augmentation using cus-tomized allogenic bone blocks Proof of concept and histologi-cal findingsrdquo Implant Dentistry vol 22 no 3 pp 212ndash218 2013
[35] G I Benic D S Thoma R E Jung et al ldquoGuided bone regen-eration with particulate vs block xenogenic bone substitutes apilot cone beam computed tomographic investigationrdquo ClinicalOral Implants Research vol 28 no 11 pp e262ndashe270 2017
[36] H Nguyen D A F Morgan and M R Forwood ldquoSterilizationof allograft bone Effects of gamma irradiation on allograftbiology and biomechanicsrdquo Cell and Tissue Banking vol 8 no2 pp 93ndash105 2007
[37] T Arizono Y Iwamoto K Okuyama and Y Sugioka ldquoEthyleneoxide sterilization of bone grafts Residual gas concentrationand fibroblast toxicityrdquo Acta Orthopaedica vol 65 no 6 pp640ndash642 1994
[38] C Hofmann T Von Garrel and L Gotzen ldquoBone bank man-agement using a thermal disinfection system (Lobator SD-1) Acritical analysisrdquo Unfallchirurg vol 99 no 7 pp 498ndash508 1996
[39] R G T Geesink ldquoOsteoconductive coatings for total joint ar-throplastyrdquo Clinical Orthopaedics and Related Research no 395pp 53ndash65 2002
[40] D J Prolo P W Pedrotti and D H White ldquoEthylene oxidesterilization of bone dura mater and fascia lata for humantransplantationrdquo Neurosurgery vol 6 no 5 pp 529ndash539 1980
[41] J A Tom and S A Rodeo ldquoSoft tissue allografts for knee recon-struction in sportsmedicinerdquoClinical Orthopaedics and RelatedResearch no 402 pp 135ndash156 2002
[42] Block SS Disinfection Sterilization and Preservation LippincottWilliams andWilkins Philadelphia Pa USA 4th edition 1991
[43] E J Mitchell A M Stawarz R Kayacan and C M RimnacldquoThe effect of gamma radiation sterilization on the fatigue crackpropagation resistance of human cortical bonerdquoThe Journal ofBone amp Joint Surgery vol 86 no 12 pp 2648ndash2657 2004
[44] M Silindir andYA Ozeray ldquoSterilizationmethods and the com-parison of E-beam sterilization with gamma radiation steril-izationrdquo Journal of Pharmaceutical Sciences vol 34 pp 43ndash532009
[45] L C Huesca-Espitia M Suvira K Rosenbeck et al ldquoEffects ofsteam autoclave treatment on Geobacillus stearothermophilussporesrdquo Journal of AppliedMicrobiology vol 121 no 5 pp 1300ndash1311 2016
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
CorrosionInternational Journal of
Hindawiwwwhindawicom Volume 2018
Advances in
Materials Science and EngineeringHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of
Chemistry
Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
ScienticaHindawiwwwhindawicom Volume 2018
Polymer ScienceInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Advances in Condensed Matter Physics
Hindawiwwwhindawicom Volume 2018
International Journal of
BiomaterialsHindawiwwwhindawicom
Journal ofEngineeringVolume 2018
Applied ChemistryJournal of
Hindawiwwwhindawicom Volume 2018
NanotechnologyHindawiwwwhindawicom Volume 2018
Journal of
Hindawiwwwhindawicom Volume 2018
High Energy PhysicsAdvances in
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
TribologyAdvances in
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
ChemistryAdvances in
Hindawiwwwhindawicom Volume 2018
Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
BioMed Research International 7
[14] A Barone P Toti A Quaranta et al ldquoClinical and Histologicalchanges after ridge preservation with two xenografts prelimi-nary results from a multicentre randomized controlled clinicaltrialrdquo Journal of Clinical Periodontology vol 44 no 2 pp 204ndash214 2017
[15] P Felice L Piana L ChecchiV CorvinoU Nannmark andMPiattelli ldquoVertical ridge augmentation of an atrophic posteriormandible with an inlay technique and cancellous equine boneblock A case reportrdquo International Journal of Periodontics andRestorative Dentistry vol 33 no 2 pp 159ndash166 2013
[16] R Pistilli L Signorini A Pisacane G Lizio and P Felice ldquoCaseof severe bone atrophy of the posterior maxilla rehabilitatedwith blocks of equine origin bone Histological resultsrdquo ImplantDentistry vol 22 no 1 pp 8ndash15 2013
[17] N De Angelis and M Scivetti ldquoLateral ridge augmentationusing an equine flex bone block infused with recombinanthuman platelet-derived growth factor BB A clinical and histo-logic studyrdquo International Journal of Periodontics andRestorativeDentistry vol 31 no 4 pp 383ndash388 2011
[18] D A di Stefano L Artese G Iezzi et al ldquoAlveolar ridge regen-eration with equine spongy bone a clinical histological andimmunohistochemical case seriesrdquo Clinical Implant Dentistryand Related Research vol 11 no 2 pp 90ndash100 2009
[19] L Artese A Piattelli D ADi Stefano et al ldquoSinus liftwith auto-logous bone alone or in addition to equine bone an immuno-histochemical study in manrdquo Implant Dentistry vol 20 no 5pp 383ndash388 2011
[20] S Tete R Vinci V L Zizzari et al ldquoMaxillary sinus augmen-tation procedures through equine-derived biomaterial or cal-varia autologous bone Immunohistochemical evaluation ofOPGRANKL in humansrdquo European Journal of Histochemistryvol 57 no 1 pp 60ndash65 2013
[21] G LoGiudice G Iannello A Terranova R LoGiudice G Pan-taleo and M Cicciu ldquoTranscrestal sinus lift procedure ap-proaching atrophic maxillary ridge A 60-month clinical andradiological follow-up evaluationrdquo International Journal ofDen-tistry vol 2015 2015
[22] S Tete V L Zizzari R Vinci et al ldquoEquine and porcine bonesubstitutes in maxillary sinus augmentation A histologicaland immunohistochemical analysis of VEGF expressionrdquo TheJournal of Craniofacial Surgery vol 25 no 3 pp 835ndash839 2014
[23] J Talairach and PThournouxCo-Planar Stereotaxic Atlas of theHuman Brain Thieme Medical Publishers Stuttgart Germany1988
[24] J Zhang ldquoThe structural stability of wild-type horse prion pro-teinrdquo Journal of Biomolecular Structure and Dynamics vol 29no 2 pp 369ndash377 2011
[25] D J Pacaccio and S F Stern ldquoDemineralized bonematrix Basicscience and clinical applicationsrdquo Clinics in Podiatric Medicineand Surgery vol 22 no 4 pp 599ndash606 2005
[26] K D Chesmel J Branger H Wertheim and N ScarboroughldquoHealing response to various forms of human demineralizedbone matrix in athymic rat cranial defectsrdquo Journal of Oral andMaxillofacial Surgery vol 56 no 7 pp 857ndash865 1998
[27] S Guizzardi R Scandroglio M D Silvestre R Savini and ARuggeri ldquoImplants of heterologous demineralized bone matrixfor induction of posterior spinal fusion in ratsrdquoThe Spine Jour-nal vol 17 no 6 pp 701ndash707 1992
[28] M S Rafael V Laize and M L Cancela ldquoIdentification of Spa-rus aurata bone morphogenetic protein 2 Molecular cloninggene expression and in silico analysis of protein conservedfeatures in vertebratesrdquoBone vol 39 no 6 pp 1373ndash1381 2006
[29] V Perrotti B M Nicholls and A Piattelli ldquoHuman osteoclastformation and activity on an equine spongy bone substituterdquoClinical Oral Implants Research vol 20 no 1 pp 17ndash23 2009
[30] V Perrotti B M Nicholls M A Horton and A PiattellildquoHuman osteoclast formation and activity on a xenogenousbone mineralrdquo Journal of Biomedical Materials Research Part Avol 90 no 1 pp 238ndash246 2009
[31] D A L Di Stefano G Gastaldi R Vinci L Cinci L Pieri andE Gherlone ldquoHistomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in SinusAugmentation A Randomized Clinical Trial with 3-Year Fol-low-Uprdquo The International journal of oral amp maxillofacial im-plants vol 30 no 5 pp 1161ndash1167 2015
[32] I Elgali O Omar C Dahlin and P Thomsen ldquoGuided boneregeneration materials and biological mechanisms revisitedrdquoEuropean Journal of Oral Sciences vol 125 no 5 pp 315ndash3372017
[33] J Waasdorp and M A Reynolds ldquoAllogeneic bone onlay graftsfor alveolar ridge augmentation a systematic reviewrdquo TheInternational Journal of Oral amp Maxillofacial Implants vol 25no 3 pp 525ndash531 2010
[34] M Schlee and D Rothamel ldquoRidge augmentation using cus-tomized allogenic bone blocks Proof of concept and histologi-cal findingsrdquo Implant Dentistry vol 22 no 3 pp 212ndash218 2013
[35] G I Benic D S Thoma R E Jung et al ldquoGuided bone regen-eration with particulate vs block xenogenic bone substitutes apilot cone beam computed tomographic investigationrdquo ClinicalOral Implants Research vol 28 no 11 pp e262ndashe270 2017
[36] H Nguyen D A F Morgan and M R Forwood ldquoSterilizationof allograft bone Effects of gamma irradiation on allograftbiology and biomechanicsrdquo Cell and Tissue Banking vol 8 no2 pp 93ndash105 2007
[37] T Arizono Y Iwamoto K Okuyama and Y Sugioka ldquoEthyleneoxide sterilization of bone grafts Residual gas concentrationand fibroblast toxicityrdquo Acta Orthopaedica vol 65 no 6 pp640ndash642 1994
[38] C Hofmann T Von Garrel and L Gotzen ldquoBone bank man-agement using a thermal disinfection system (Lobator SD-1) Acritical analysisrdquo Unfallchirurg vol 99 no 7 pp 498ndash508 1996
[39] R G T Geesink ldquoOsteoconductive coatings for total joint ar-throplastyrdquo Clinical Orthopaedics and Related Research no 395pp 53ndash65 2002
[40] D J Prolo P W Pedrotti and D H White ldquoEthylene oxidesterilization of bone dura mater and fascia lata for humantransplantationrdquo Neurosurgery vol 6 no 5 pp 529ndash539 1980
[41] J A Tom and S A Rodeo ldquoSoft tissue allografts for knee recon-struction in sportsmedicinerdquoClinical Orthopaedics and RelatedResearch no 402 pp 135ndash156 2002
[42] Block SS Disinfection Sterilization and Preservation LippincottWilliams andWilkins Philadelphia Pa USA 4th edition 1991
[43] E J Mitchell A M Stawarz R Kayacan and C M RimnacldquoThe effect of gamma radiation sterilization on the fatigue crackpropagation resistance of human cortical bonerdquoThe Journal ofBone amp Joint Surgery vol 86 no 12 pp 2648ndash2657 2004
[44] M Silindir andYA Ozeray ldquoSterilizationmethods and the com-parison of E-beam sterilization with gamma radiation steril-izationrdquo Journal of Pharmaceutical Sciences vol 34 pp 43ndash532009
[45] L C Huesca-Espitia M Suvira K Rosenbeck et al ldquoEffects ofsteam autoclave treatment on Geobacillus stearothermophilussporesrdquo Journal of AppliedMicrobiology vol 121 no 5 pp 1300ndash1311 2016
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
CorrosionInternational Journal of
Hindawiwwwhindawicom Volume 2018
Advances in
Materials Science and EngineeringHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of
Chemistry
Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
ScienticaHindawiwwwhindawicom Volume 2018
Polymer ScienceInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Advances in Condensed Matter Physics
Hindawiwwwhindawicom Volume 2018
International Journal of
BiomaterialsHindawiwwwhindawicom
Journal ofEngineeringVolume 2018
Applied ChemistryJournal of
Hindawiwwwhindawicom Volume 2018
NanotechnologyHindawiwwwhindawicom Volume 2018
Journal of
Hindawiwwwhindawicom Volume 2018
High Energy PhysicsAdvances in
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
TribologyAdvances in
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
ChemistryAdvances in
Hindawiwwwhindawicom Volume 2018
Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
8 BioMed Research International
[46] B T Garibaldi M Reimers N Ernst et al ldquoValidation of auto-clave protocols for successful decontamination of category amedical waste generated fromcare of patientswith serious com-municable diseasesrdquo Journal of ClinicalMicrobiology vol 55 no2 pp 545ndash551 2017
[47] S Akyalcin H P McIver J D English J C Ontiveros and RL Gallerano ldquoEffects of repeated sterilization cycles on primarystability of orthodontic mini-screwsrdquo The Angle Orthodontistvol 83 no 4 pp 674ndash679 2013
[48] D diMauro RGaeta A Arco et al ldquoDistribution of costamericproteins in normal human ventricular and atrial cardiac mus-clerdquo Folia Histochemica et Cytobiologica vol 47 no 4 pp 605ndash608 2009
[49] F Osculati M Bentivoglio M Castellucci S Cinti C Zanca-naro and A Sbarbati ldquoThe solitary chemosensory cells and thediffuse chemosensory system of the airwayrdquo European Journalof Histochemistry vol 51 supplement 1 pp 65ndash72 2007
[50] G Anastasi G Cutroneo G Santoro et al ldquoIntegrins muscleagrin and sarcoglycans during muscular inactivity conditionsAn immunohistochemical studyrdquo European Journal of Histo-chemistry vol 50 no 4 pp 327ndash336 2006
[51] A Barone P Toti G-B Menchini-Fabris P Felice S Mar-chionni andU Covani ldquoEarly volumetric changes after verticalaugmentation of the atrophic posterior mandible with inter-positional block graft versus onlay bone graft A retrospectiveradiological studyrdquo Journal of Cranio-Maxillo-Facial Surgeryvol 45 no 9 pp 1438ndash1447 2017
[52] M Simion M Nevins I Rocchietta et al ldquoVertical ridge aug-mentation using an equine block infused with recombinanthuman platelet-derived growth factor-BB a histologic study in acanine modelrdquo International Journal of Periodontics and Re-storative Dentistry vol 29 no 3 pp 245ndash255 2009
[53] F Schwarz D Ferrari E Balic D Buser J Becker andM SagerldquoLateral ridge augmentation using equine- and bovine-derivedcancellous bone blocks a feasibility study in dogsrdquoClinical OralImplants Research vol 21 no 9 pp 904ndash912 2010
[54] F Mangano A Macchi J A Shibli et al ldquoMaxillary ridge aug-mentation with custom-made CADCAM scaffolds A 1-yearprospective study on 10 patientsrdquo Journal of Oral Implantologyvol 40 no 5 pp 561ndash569 2014
[55] R CusinatoM Pacenti T Martello P FattoriMMorroni andG Palu ldquoEffectiveness of hydrogen peroxide and electron-beamirradiation treatment for removal and inactivation of viruses inequine-derived xenograftsrdquo Journal of Virological Methods vol232 pp 39ndash46 2016
[56] M A Rosa P Gugliandolo A Favaloro et al ldquoMorpho-struc-tural alterations of sub-chondral bone tissue in patients withosteoarthritis A scanning electron microscopy studyrdquo ItalianJournal of Anatomy and Embryology vol 120 no 1 pp 71ndash812015
[57] L Bozec and M Odlyha ldquoThermal denaturation studies of col-lagen by microthermal analysis and atomic force microscopyrdquoBiophysical Journal vol 101 no 1 pp 228ndash236 2011
[58] N A ValenteA CalascibettaG Patianna TMangMHattonand S Andreana ldquoThermodynamic effects of 3 different diodelasers on an implant-bone interface An ex-vivo study with re-view of the literaturerdquo Journal of Oral Implantology vol 43 no2 pp 94ndash99 2017
CorrosionInternational Journal of
Hindawiwwwhindawicom Volume 2018
Advances in
Materials Science and EngineeringHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of
Chemistry
Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
ScienticaHindawiwwwhindawicom Volume 2018
Polymer ScienceInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Advances in Condensed Matter Physics
Hindawiwwwhindawicom Volume 2018
International Journal of
BiomaterialsHindawiwwwhindawicom
Journal ofEngineeringVolume 2018
Applied ChemistryJournal of
Hindawiwwwhindawicom Volume 2018
NanotechnologyHindawiwwwhindawicom Volume 2018
Journal of
Hindawiwwwhindawicom Volume 2018
High Energy PhysicsAdvances in
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
TribologyAdvances in
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
ChemistryAdvances in
Hindawiwwwhindawicom Volume 2018
Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
CorrosionInternational Journal of
Hindawiwwwhindawicom Volume 2018
Advances in
Materials Science and EngineeringHindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Journal of
Chemistry
Analytical ChemistryInternational Journal of
Hindawiwwwhindawicom Volume 2018
ScienticaHindawiwwwhindawicom Volume 2018
Polymer ScienceInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Advances in Condensed Matter Physics
Hindawiwwwhindawicom Volume 2018
International Journal of
BiomaterialsHindawiwwwhindawicom
Journal ofEngineeringVolume 2018
Applied ChemistryJournal of
Hindawiwwwhindawicom Volume 2018
NanotechnologyHindawiwwwhindawicom Volume 2018
Journal of
Hindawiwwwhindawicom Volume 2018
High Energy PhysicsAdvances in
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
TribologyAdvances in
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
ChemistryAdvances in
Hindawiwwwhindawicom Volume 2018
Advances inPhysical Chemistry
Hindawiwwwhindawicom Volume 2018
BioMed Research InternationalMaterials
Journal of
Hindawiwwwhindawicom Volume 2018
Na
nom
ate
ria
ls
Hindawiwwwhindawicom Volume 2018
Journal ofNanomaterials
Submit your manuscripts atwwwhindawicom
