Cell Attachment Properties of Portland Cement–based Endodontic Materials: Biological and...

Review Article

Cell Attachment Properties of Portland Cement–basedEndodontic Materials: Biological and MethodologicalConsiderationsHany Mohamed Aly Ahmed, BDS, HDD (Endo),*Norhayati Luddin, BDS, Grad Dip Clin Dent, D Clin Dent, FRACDS,*Thirumulu Ponnuraj Kannan, BSc, MSc, PhD,*† Khairani Idah Mokhtar, BSc, MSc, PhD,*and Azlina Ahmad, BSc, MSc, PhD*†

Abstract

From the *School of Dental Sciences and †Human Genome CenAddress requests for reprints to Dr Hany Mohamed Aly Ahmed,

Kerian, 16150 Kelantan, Malaysia. E-mail address: hany_endodonti0099-2399/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2014.06.013

JOE — Volume 40, Number 10, October 2014

Key WordsCell attachment, mineral trioxide aggregate, Portland cement, scanning electron micro-scope

In vitro cytotoxicity tests are essential stages of the biocompatibility screening process,with different assays being used to assess the effects of a biomaterial on cell growth,cell membrane integrity, enzyme activity, or genetic effects (1). Cell adhesion is the firststep necessary before cells can proliferate, differentiate, and produce an extracellularmineralized matrix on a substrate (2). Thus, cell attachment onto endodontic bioma-terials is generally agreed to be a valid criterion for the evaluation of their biologicalproperties (3, 4). The scanning electron microscope (SEM) provides importantinformation in establishing biocompatibility by aiding in the observation of cellmorphology and material-cell interactions (5, 6).

Portland cement (PC)–based endodontic materials such as mineral trioxideaggregate (MTA) continue to be the subject of active research since the early 1990s(7). Research has focused on the biosynthetic activity of pulp and periodontal cellswhen applied in contact with the biologically active MTA and other PC-based formula-tions (7–9). Cellular interactions and the development of cytoplasmic processes alongthe crystalline structures of PC-based endodontic materials that are able to regulateosteogenic/dentinogenic events are of particular concern (6, 8, 10). Hence, thisreview was undertaken to discuss the cell attachment properties of commercial andexperimental PC-based materials on different cell cultures by using SEM.

Review QuestionsThe following questions are addressed:

1. What are the methodological procedures used for examining the cell attachmentproperties of PC-based formulations in vitro by using SEM, and which is themost relevant protocol that would simulate the clinical situation?

2. How can researchers overcome the technical challenges faced while examining sam-ples of PC-based formulations under SEM?

3. Is there any detectable variation in the cell attachment properties of PC-based for-mulations when applied with different cell cultures?

4. To what extent are SEM observations accurate? Is it necessary to substantiate SEMfindings with other examination tools?

tre, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia.Department of Restorative Dentistry, School of Dental Sciences, Universiti Sains Malaysia, Kubangst@hotmail.com

Properties of Portland Cement–based Materials 1517

Review Article

MethodsLiterature Search Methodology

A PubMed electronic search (http://www.ncbi.nlm.nih.gov/pubmed) was conducted, spanning the period from January 1993 toOctober 2013, to identify the available investigations on the cell attach-ment properties of PC-based endodontic materials written in the Englishlanguage. The following key words were used in the search: ‘‘Portlandcement’’ AND ‘‘scanning electron microscope’’ OR ‘‘cell attachment’’,‘‘Mineral trioxide aggregate’’ AND ‘‘scanning electron microscope’’OR ‘‘cell attachment’’. After deleting duplicate articles, the cross cita-tions of the selected articles were identified, and the data were thenanalyzed. Table 1 (1–5, 9–26) shows the retrieved studies andsummarizes the methodological procedures used for SEM analysis.

Preparation of SamplesPC-based samples are usually examined after mixing and letting

them set for a given period of time (1–5, 9–11, 13, 15–20, 22–24,26). Samples on coverslips or disks embedded in well-plates are themost common preparation procedure (Table 1); nevertheless, exam-ining the set samples after application in root slices or sterile acrylicmolds is another valid alternative, where cells can be viewed on thetop of the material and at the interface (1) (Fig. 1A–C). The latter wouldprovide better simulation to the clinical application of PC-based mate-rials, where they are frequently applied in prepared root-end/furcation/side cavities. In addition, coverslips may not withstand the rigors ofdehydration, drying, and metal coating, and some samples may becomelost (27).

The cell attachment properties of MTA have also been examined inthe freshly mixed formulation (12, 14, 16). Such formulation wouldrelease a considerable amount of chemical by-products that are toxicto the cells in culture. However, in the clinical situation, these by-products are likely to be diluted in the interstitial tissue fluids andare eliminated through the vasculature, a biological response that islacking in the tissue culture (28). Therefore, using set samples forSEM evaluation seems to be more suitable.

Sterilizing the samples before application into a given biologicalexamination is preferred to avoid contamination (17, 26, 29).Ultraviolet radiation with or without immersion in 70% ethanol is themost common procedure (2, 5, 15, 17, 23–25). However, otherstudies have used chemical disinfection such as penicillin/streptomycin followed by washing with phosphate-buffered saline (9,19), or immersion in ethanol followed by drying (13). The use ofgamma irradiation and ethylene oxide gas has also been reported(21, 26). Comparative studies are warranted to determine the mosteffective yet inert sterilization method.

The repeated immersion of study samples in culture medium ordistilled water for a given time before application of the cell culturehas been reported (3, 4, 17, 22, 23, 25, 26). This step aims toensure removal of potentially toxic by-products that may affect the adhe-sion of cultured cells other than the surface of the material (4, 25). Itcould be argued that removal of such elutes from the cell culturemedium may overestimate the biological profile of the material,especially that such samples usually are immersed after being set (4,17, 22, 23, 25, 26), which is less toxic than their clinical applicationin the freshly mixed formulation. Notably, adsorption of serumprotein on the surface of study samples immersed in serumcontaining culture medium may also improve cell adhesion (2). Thismight explain why this immersion procedure was not performed inmany studies (1, 2, 5, 10–16, 18–21, 24). It is worth noting thatSEM may provide additional information by examining the effect ofsuch elutes on the attachment and spreading of cells at areas around

1518 Ahmed et al.

the material. Toxic by-products, eluted from set samples, may affectthe attachment and spreading of cells applied on a biocompatible sub-strate other than the surface of the test material (Fig. 1D–G).

Application of Cell CulturesAdhesion of mammalian cells to dental biomaterials is an area of

active research. The literature shows that human osteosarcoma celllines (MG-63 and Saos-2) are most commonly used for cell attachmentevaluation of PC-based materials (Table 1). Both osteosarcoma celllines possess several osteoblastic features (30, 31); however, theyexhibit abnormal molecular and cellular functions because of theirchromosomal alterations (32). As such, these cell lines may not beideally suited for studying all aspects of osteoblast function (14, 31,32). Other primary cell cultures and cell lines, such as humanfibroblasts from the gingiva and periodontal ligament, murinecementoblasts, and dental pulp stem cells, have also been examined(Table 1).

The application of a given cell culture onto study samples is a rela-tively simple procedure. Preparing the samples onto sterile coverslipsor in transparent acrylic molds would help the researcher to observethe cellular responses at areas around the interface via an inverted mi-croscope before being processed for SEM (Fig. 1A–G). Observing thecytotoxic effects of the material at this stage is beneficial becausedead cells cannot be detected via SEM because they usually detachand become washed during evacuation of the medium, rinsing, andadding of the fixative agent (Fig. 1D–G).

Methods of ProcessingExcept for 2 studies (14, 24), all investigations listed in Table 1

performed the fixation step via immersion in 2.5% glutaraldehyde so-lution. Cacodylate buffer has been used as an additive to avoid reactionof the medium with the released calcium hydroxide (2, 3, 15–17). Onestudy used Karnovsky’s fixative solution (1% paraformaldehyde, 1.5%glutaraldehyde, and 0.1 mol/L sodium cacodylate buffer, pH 7.2–7.4) (9). Despite being an expensive and severely toxic material(33), the application of osmium tetroxide for further fixation hasalso been reported (3–5, 12, 13, 19, 21, 25).

Dehydration in a series of increasing concentrations of ethanol is acommon procedure before mounting the samples on stubs and coatingwith gold or gold/palladium. If an acrylic mold is used, immersing thesamples at higher concentrations of ethanol (>70%) for a shorttime (3–5 minutes each) is preferred to prevent cracking of theacrylic mold. Critical point dryness has been used as the final stepbefore mounting the samples (1, 4, 5, 11–16, 20, 23).Hexamethyldisilazane has also been used as a time-saving, inexpensivealternative to critical point drying (2, 3, 17, 21, 34).

Camilleri et al (15) compared the different processing methods ofMTA samples and found that critical point dryingmay affect the material.The calcium hydroxide produced during setting may react on contactwith CO2 to produce a calcium carbonate precipitate on the specimens,which may affect the field viewed under the SEM (15). The use of hex-amethyldisilazane is a reasonable substitute for critical point drying thatmay allow better cell preservation (15). However, this technique canstill produce surface carbonation of the materials (15). Drying the sam-ples in dry air may also show satisfactory results (9) (Fig. 1A–G).

The formation of different shapes and sizes of calcium compoundcrystals (mainly composed of calcium carbonate or calcium phos-phate) has been reported (4, 35, 36). The same crystals can also beobserved when a setting accelerator, such as calcium chloridedihydrate (CaCl2$2H2O), is added to either white MTA (WMTA)(Fig. 1H and I) or PC (5), which may show the presence of sulfur in

TABLE 1. Summary of Studies on Cell Attachment Properties of PC-based Materials by Using SEM

Author/s Year Test material(s)/cell type Methodology Results

Koh et al (11) 1997 GMTA (LLU)/human MG-63 cell line Material was placed in a Petridish having the cell line.

Material favored cell attachment.

Koh et al (12) 1998 GMTA (LLU)/human MG-63 cell line Material on coverslips wasplaced in a Petri dish havingthe cell line.

Cells appeared flat and adhered to the material.

Mitchell et al (13) 1999 GMTA (LLU)/human MG-63 cell line Materials on coverslips wereplaced in Petri dishes havingthe cell line.

Cells appeared flat and adhered to the material.

Zhu et al (3) 2000 GMTA (LLU)/human Saos-2 cell line Material disks were placed in thebottom of 4-well plates.

Cells showed favorable attachment andspreading on surface of MTA.

Abdullah et al (5) 2002 GMTA (LLU) and PC and PC +CaCl2/Saos-2 cell line

Materials on coverslips were placedin 16-well plates having the cell line.

Cells showed favorable attachment andspreading on surface of GMTA and PCand PC + CaCl2

P�erez et al (14) 2003 GMTA (PRO) and WMTA/ratprimary osteoblasts and MG-63cell line

Material pellets were placed in thebottom of 8-well slides.

Number of cells increased in all samples,except for WMTA where no osteoblastswere visible on top of WMTA by end ofday 13.

Thomson et al (2) 2003 MTA/primary murine cementoblasts(OCCM.30)

Material disks were placed in the bottomof 12-well culture plate, and cells werethen seeded.

Camilleri et al (15) 2004 GMTA (PRO) and WMTA (PRO)/Saos-2 cell line

Materials on coverslips were placedin tissue culture dishes having thecell line.

All samples cured for 1 day showed confluentcell monolayer after 5 and 7 days. Materialscured for 28 days showed incomplete cellconfluence after 1 and 5 days.

Balto (16) 2004 MTA (PRO)/human periodontalligament fibroblasts

Root slices filled with the material. Set MTA favored cell attachment. Freshlyprepared MTA did not favor cell attachment.

Nakayama et al (10) 2005 MTA (PRO)/primary rat bonemarrow osteoblast cells

Three dishes of each material were used,and cells were seeded on each material.

Al-Rabeah et al (17) 2006 GMTA (PRO) and WMTA (PRO)and MTA mixed with localanesthetic solution/primaryhuman alveolar bone cells

Cells were seeded onto multiple MTA disksin 24-well culture plates.

Cell attachment with all groups was comparablypropagated for 14 days.

Min et al (18) 2007 PC/primary human pulp cells Cells were seeded onto PC disks in 12-wellculture plates.

Material favored cell attachment and spreading.

Trubiani et al (19) 2007 MTA/primary human dentalpulp stem cells

MTA was seeded into the cell pellet. Material favored cell attachment and spreading.

Gandofli et al (9) 2008 G1: powder: white PC, bismuth oxide,CaCl2, calcium sulfate emihydrate.Liquid: Alfacaine SP.

G2: powder: white PC, bismuth oxide,CaCl2, calcium sulfate emihydrateand phyllosilicate. Liquid: Alfacaine SP

G3: powder: white PC, bismuth oxide, CaCl2,calcium sulfate emihydrate, sodium fluorideand phyllosilicate. Liquid: Alfacaine SP.

G4: WMTA (PRO)/Saos-2 cell line

Cells were seeded onto materialdisks.

All materials favored cell attachment and spreading.

Min et al (20) 2009 PC and PC + Bi2O3/primary dental pulp cellculture

Material disks were placed in the bottomof 12-well culture plate, and cells werethen seeded.

All materials favored cell attachment and spreading.

(Continued )

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JOE—

Volume40,

ber10,

October

2014Properties

ofPortland

Cement–

basedMaterials

TABLE1.

(Continued)

Author/s

material(s)/celltype

Methodology

Results

Raldietal(21)

(ANG)/humangingivalfibroblast

cellline

Materialwasco

ndensedinto

standardized

rootcavities,andcellswere

appliedonto

thesamples.

allowedcelladhesion,despitebeing

lowerthanirradiatedgroups(Er:YAGlaser

andhigh-powerdiodelaser).

Maetal(4)

(PRO)/primary

humangingival

fibroblasts

Cellswere

seededonto

materialdisks.

Allmaterialsfavo

redcellattach

mentand

spreading.

Al-Hiyasatetal(1)

(PRO)/balb/C

3T3mouse

fibrobast

cellline

Materialwasco

ndensedinto

standardizedrootcavities,and

cellswere

appliedonto

thesamples.

Materialfavo

redcellattach

mentandspreading.

Asgary

etal(22)

(PRO)/humangingivalfibroblasts

Cellswere

seededonto

materialdisks.

Materialfavo

redcellattach

mentandspreading.

Trichaiyaponetal(23)

(PRO)/primary

humanperiodontal

ligamentcells

Materialdiskswere

elutedfor4days;

thencellswere

seeded.

Materialfavo

redcellattach

mentandspreading.

G€ uve

netal(24)

(ANG-Fillapex)/humantooth

Materialswere

appliedonco

verslips,

andcellswere

thenseeded.

Materialdid

notfavo

rcellattach

Zhouetal(25)

(PRO)/primary

humangingival

fibroblasts

Cellswere

seededonto

materialdisks.

Materialfavo

redcellattach

mentandspreading

after3and7days

lture.

Kangetal(26)

(PRO),W

(PRO)+0.1%

citric

acid,W

(PRO)+10%

calcium

chloride,WMTA

(PRO)+

calcium

lactate

MG-63cellline

Materialdiskswere

immersedin

preparedmedium

for3days;

thencellswere

seeded.

WithexceptionofMTA

+calcium

chloride,all

materialsdemonstratedgoodcellattach

properties.

ANG,Angelus;LLU,LomaLindaUniversity;PRO,ProRoot.

Review Article

1520 Ahmed et al.

their chemical composition (Fig. 1J and K). Notably, such crystals usu-ally do not prevent cell adhesion and spreading (4).

On the basis of the discussion above, it can be concluded that themethodological procedures used for preparing PC-based samplestogether with subsequent interpretation under SEM are challenging.This issue persists because of the processing remnants produced bychemical reactions between a bioactive PC-based material, which re-leases cationic components and triggers its precipitation on the surfaceand in the surrounding fluid (37), and reagents used during processing(15). These interactions would complicate the interpretation of cellmorphology, especially small and rounded cells with few cytoplasmicprocesses, which are a common landmark for samples examined within24 hours. Applying the cell culture onto the cement/mold assembly givesthe opportunity to trace the attachment of cells from the material/moldinterface inwards. Examining the attached cells after >2–3 days is rela-tively easier, although the medium should be regularly changed if thesamples are scheduled for examinations at longer time intervals (17).This is because cell death might occur as a result of loss of nutrientsin the culture medium rather than toxicity of the material (13).

Methods for EvaluationAdhesion and spreading of cells on amaterial surface are the initial

stages of normal cellular function (22). Rajaraman et al (38) outlinedthe process of adhesion and spreading in 4 events: (1) attachment ofcells at point of contact with the substratum, (2) centrifugal growthof filopodia (a cylindrical or conical process that is 10–20 mmlong), (3) cytoplasmic webbing, and (4) flattening of the centralmass. The cytoplasmic surface extensions formed by cultured cellscan be filopodia, microvilli (a process of 0.1–0.2mmdiameter), lamel-lipodia (flattened extension usually 0.1–0.5 mm thick), or blebs(spherical or hemispherical extension and usually 1–2 mm in diam-eter) (1, 16). The persistence of rounded cells with little or nospreading and vacuolization of the cytoplasm indicate that the surfaceof the material may be toxic (3, 16, 22) (Fig. 1D–G).

Researchers usually interpret the SEM images via the above-mentioned criteria. Some authors have also presented scoring systemsfor numeric evaluation (Table 2). Those systems describe cellmorphology by using certain ranks to provide a distinct landmark be-tween cellular responses toward a given biomaterial, and this could beuseful. However, the situation is different with PC-basedmaterials, espe-cially if the scoring system depends on percentages of alive-dying cells(12) or percentages of areas covered by the cells (13). The extent of cellcoverage may not be an ideal marker for cell-material interactionbecause the quantity of cell growth and its spread mainly depends ontime of incubation (5). In addition, the formation of calcium compoundcrystals may obscure an accurate numeric evaluation of cells attachedonto the surface.

It seems that slight or moderate differences between the cytotoxicprofiles of different biomaterials usually cannot be detected via SEM,and further comparative studies that use other assays are essential. Thatis why some researchers performed additional investigations, such asevaluation of the proliferation rate and cell viability via colorimetric assaysby using tetrazolium salt or alamar blue, to provide an accurate numericcomparison between groups, together with SEM (9, 10, 23).

Attachment of Cells onto PC-based Endodontic MaterialsMTA and PC. The data available on gray MTA (GMTA) and WMTAdemonstrate that the cell attachment properties are favorable whenthe samples are examined after setting (1–5, 10, 11, 13, 16, 21–23,25). However, at the freshly mixed formulation, the results arecontradictory. Koh et al (12) found that freshly mixed samples of

Figure 1. (A) Illustrated diagram showing evaluation of cell attachment properties onto the material and at area of interface by using a mold/cement assembly.Before application of culture medium, the cells in culture are applied onto the sample and left for 30 minutes to attach. Medium is added at the side of the well untilthe sample is completely covered by the medium. After scheduled incubation time, the cells are fixed, and the sample is dried. (B) The gold-coated mold/cementassembly is placed in a 6-well plate after dryness. (C) SEM images showing human periodontal ligament fibroblasts (HPLFs) attaching on top of PC-based material(Malaysian white PC + 10% wt CaCl2$2H2O) after 72 hours of incubation. (D) Image taken via inverted microscope shows HPLFs cultured over WMTA/transparentacrylic mold assembly and incubated for 24 hours. Cells at the area around the interface are spindle in shape (favorable attachment [white arrows]). (E) SEMimage of same sample after processing (sample was immersed in 2.5% glutaraldehyde for 2 hours, followed by immersion in ethanol at increasing concentrationsuntil 100%, drying in air, and then coating with gold). (F and G) Same procedure is applied on sample of amalgam. Both images show unfavorable attachment ofHPLFs after 24 hours (white arrows). (F) Movement of dead ‘‘floating’’ cells (white arrows) can be identified while shaking the 6-well plate gently. (H and I) SEMimage showing calcium compound crystals formed on sample of accelerated WMTA (WMTA + 10% wt CaCl2$2H2O). Calcium, oxygen, and carbon were detectedafter energy dispersive x-ray microanalysis. (J and K) In addition to said elements, sulphur can be identified in an accelerated PC formulation. Both samples wereimmersed in a prepared medium (Stromal Cell Growth Medium; Lonza, Walkersville, MD) seeded with HPLFs for 72 hours.

Review Article

GMTA favored the attachment of human osteosarcoma (MG-63) cellline. Conversely, SEM observations were not favorable when the sameformulation was examined with human periodontal ligament fibroblasts(16).

Perez et al (14) compared the cell attachment properties of GMTAwith those of WMTA on human MG-63 osteosarcoma cells and primary

rat osteoblasts at 3 time intervals. Their results revealed that MG-63 cellswere able to attach and spread over the surface of GMTA andWMTA simi-larly at all time intervals. However, osteoblasts responded differently, andin contrast to GMTA, WMTA failed to maintain a favorable attachment forlonger periods (by the end of day 13). The authors attributed their find-ings to differences in chemical composition and surface topography

TABLE 2. Summary of Scoring Systems Used for Evaluation of Cell Attachment Properties of PC-based Materials by Using SEM

Authors Year Scoring systems

Koh et al (12) 1998 (1) Cells flat (<10% cells showed shrinkage).(2) Cells partly rounded (<50% cells showed shrinkage).(3) Cell rounded (<90% cells showed shrinkage).(4) Cell death (<10% of cells still alive).

Mitchell et al (13) 1999 (0) Cells separated from material by a cell-free zone.(1) Cells growing up to but not onto material.(2) Healthy, fattened cells in intimate contact with material but covering#10% of the surface.(3) Healthy, fattened cells in intimate contact with material and covering 10%–50% of the surface.(4) Healthy, fattened cells in intimate contact with material and covering >50% of the surface. Mode scores

were calculated.Abdullah et al (5) 2002 (0) No surviving cell.

(1) Cells appear rounded, less flattened with fewer cytoplasmic extensions.(2) Cells appear flat, propagated on material, and exhibited intact, well-defined morphology with cytoplasmic

extension.(n) Not able to evaluate surface. Samples that were damaged and deemed unsuitable for evaluation were

excluded from the study and denoted as ’’n’’.Camilleri et al (15) 2004 (0) No cells.

(1) Occasional round cells.(2) Sparse flattened out cells.(3) Substantial cell growth, mostly flat cells with a few rounded cells.(4) Confluent.

Asgary et al (22) 2012 (1) Healthy cells were described as being flat, propagating over and adhering to surface of test biomaterials.(0) Less healthy or dying cells appeared round, had fewer cytoplasmic extensions, and were detached from the

surface.

Review Article

between both types of MTA. On the contrary, Camilleri et al (15) did notobserve much difference in the attachment of Saos-2 cells on both ma-terials after incubation for 7 days. Another study found that both mate-rials similarly favored the attachment and spreading of human alveolarbone cells by means of a matrix-like overlay, even after 14 days (17).

From the results of these investigations, it can be concluded thatdifferent cell lines and primary cell cultures may react differentlywhen applied in contact to freshly mixed and set GMTA and WMTA.

Few studies investigated the cell attachment properties of PCwithout additives. Abdullah et al (5) and Min et al (18) examined theattachment of SaOS-2 cells and human pulp cells on PC manufacturedin the United Kingdom and Korea, respectively. The results demon-strated the favorable attachment and spread of cells across the substratewith numerous cytoplasmic extensions. Similar results have also beenreported (20).

Modified Formulations of MTA and PC. MTA has numerousdesirable properties; however, it has some shortcomings such as pro-longed setting time and difficult handling characteristics (39, 40). Anumber of potential additives have been examined to overcome theselimitations. Similar combinations have been investigated with PC inaddition to radiopacifiers.

One study examined a local anesthetic solution (lidocaine HCl andepinephrine) as a substitute for the liquid provided by the manufacturerfor the powder of MTA and found that the cell attachment propertieswere similar for both formulations (17). A recent investigation hascompared 3 different combinations of accelerated MTA (citric acid, cal-cium chloride (CaCl2), and calcium lactate gluconate) (26). SEM ob-servations on MG-63 osteosarcoma cells have demonstrated favorableattachment onto the surfaces of MTA mixed with citric acid or calciumlactate gluconate, whereas MTA mixed with CaCl2 revealed only a smallnumber of round cells, which may be attributed to factors such as sur-face roughness. Despite the unfavorable response of MG-63 cells, itdoes seem appropriate to state that this combination is not suitable

1522 Ahmed et al.

for clinical application because cell types that would be in contactwith this combination in the clinical situation may react differently (7).

G€uven et al (24) examined the cell attachment properties of a newMTA-based root canal material (MTA and salicylate resin [Fillapex;Angelus, Londrina, Brazil]) on human tooth germ stem cells. The re-sults have demonstrated severe cytotoxic profile mainly because ofthe resin component. The cells appeared round at the first day, andat day 14, the cells became few and maintained the same roundedmorphology, indicating the toxic potential of the material.

A number of PC formulations have been examined as potential sub-stitutes toMTA. Abdullah et al (5) examined 2 percentages of CaCl2 (10%and 15%) mixed with PC and compared these with GMTA. The resultsrevealed that Saso-2 cells in contact with all test materials appearedflat and adhered well onto material surfaces at all time intervals. Anotherstudy introduced 3 innovative radiopaque PCs mixed with Alfacaine SP(articaine and epinephrine; SPAD, Dentsply, Rome Italy) (9). All exper-imental cements supported cellular growth, and numerous cellular ex-tensions and anchoring processes interacted with the cement surfacesand with adjacent cells. Min et al (20) reported similar findings withPC mixed with bismuth oxide and sterile distilled water.

Relevance of SEM in Determining the Biological Profileof PC-based Materials

The advantageous clinical outcomes of MTA reveal that the above-mentioned SEM findings are relevant. However, SEM mainly provides aqualitative analysis. Other examination tools such as confocal micro-scopy have proved similar results (41) and were able to prove that dif-ferences in chemical composition (or proportions of the same chemicalcomponents) between MTA brands (ProRoot and Angelus) may affectcell adhesion (42). Some researchers have compared the cell attach-ment properties of MTA with other root-end filling materials by usingquantitative attachment assays (quantification by a fluorescent dye)

Review Article

ConclusionsSEM observations demonstrate that commercial MTA formulations

show favorable cell attachment properties, which is consistent with theirsuccessful clinical outcomes. The favorable cell attachment propertiesof PC and its modified formulations support its potential use as a sub-stitute for MTA. However, researchers should carefully select cell typesfor their SEM investigations that would be in contact with the proposedPC-based combinations in the clinical situation.

Despite being a technical challenge, SEM provides useful informa-tion on the cell attachment properties of PC-based materials and theircytotoxic effects at areas around the interface. Other assays for cellproliferation and viability are essential to come up with an accuratein vitro biological profile of any given PC-based formulation.

AcknowledgmentsThe authors express their gratitude to Mr Mohd Yusuf Soon

Abdullah (Craniofacial Science laboratory, School of Dental Sci-ences), Miss Jamilah Afandi, Mr Johary Othman (Microscopy unit,School of Biological Sciences), and Mr Nik Fakuruddin (SEM unit,School of Health Sciences), Universiti Sains Malaysia (USM) fortheir technical support.

This study was supported by the USM Research University grantnumber 1001/PPSG/813056.

The authors deny any conflicts of interest related to this study.

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