Int. J. Oral Maxillofac. Surg. 2012; xxx: xxx–xxxmed.mui.ac.ir/clinical/stemcells/Dr.Shahnaseri.pdftreated with the appropriate indu-the factors. Cowan et al.11 demonstrated coated

YIJOM-2530; No of Pages 7

Research Paper

Cleft Lip and Palate

Int. J. Oral Maxillofac. Surg. 2012; xxx: xxx–xxxhttp://dx.doi.org/10.1016/j.ijom.2012.10.012, available online at http://www.sciencedirect.com

A comparison of tissue-engineered bone from adipose-derived stem cell withautogenous bone repair inmaxillary alveolar cleft model indogsN. Pourebrahim, B. Hashemibeni, S. Shahnaseri, N. Torabinia, B. Mousavi, S. Adibi,F. Heidari, M. Jafary Alavi: A comparison of tissue-engineered bone from adipose-derived stem cell with autogenous bone repair in maxillary alveolar cleft model in dogs.Int. J. Oral Maxillofac. Surg. 2012; xxx: xxx–xxx. # 2012 International Association ofOral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Abstract. This study was designed to compare bone regeneration of tissue-engineeredbone from adipose-derived stem cell and autogenous bone graft in a caninemaxillary alveolar cleft model. In this prospective clinical trial, mesenchymal stemcells (MSCs) were isolated from subcutaneous canine adipose tissue.Undifferentiated cells were incubated with a 3 mm � 3 mm � 3 mmhydroxyapatite/beta-tricalcium phosphate scaffold, in specific osteogenic mediumfor 21 days. Four mongrel dogs were prepared by removal of two of the threeincisors bilaterally and a 15 mm defect in bone was created from crest to nasal floor.After healing, repair was followed by a tissue engineered bone graft from adipose-derived stem cells on one side and corticocancellous tibial auto graft on the otherside. Bone regeneration was evaluated by histomorphometry on days 15 and 60 afterimplantation. The data were analysed with descriptive and t test methods (a = 0.05).Bone formation on the autograft sides was higher than on the stem cell sides at 15and 60 days, 45% and 96% versus 5% and 70%, respectively. Differences betweenthe two groups at 15 and 60 days were significant (p = 0.004 and 0.001,respectively). Although autograft is still the gold standard for bone regeneration,tissue engineered bone may provide an acceptable alternative.

Please cite this article in press as: Pourebrahim N, et al. A comparison of tissue-engineered bone fro

bone repair in maxillary alveolar cleft model in dogs, Int J Oral Maxillofac Surg (2012), http:/

0901-5027/000001+07 $36.00/0 # 2012 International Association of Oral and Maxillofacial Surge

N. Pourebrahim1, B. Hashemibeni2,S. Shahnaseri3, N. Torabinia4,B. Mousavi5, S. Adibi6, F. Heidari7,M. Jafary Alavi8

1Department of Maxillofacial Surgery, AzahraHospital, Isfahan University of MedicalSciences, Isfahan, Iran; 2AnatomicalSciences and Molecular Biology, IsfahanUniversity of Medical Sciences, Isfahan, Iran;3Department of Maxillofacial Surgery, AzahraHospital and Torabinejad Reasearch Center,Isfahan University of Medical Sciences,Isfahan, Iran; 4Oral and MaxillofacialPathology, Department of Oral andMaxillofacial Pathology, Isfahan University ofDental Sciences, Isfahan, Iran; 5TorabinejadResearch Center, Isfahan University of DentalSciences, Isfahan, Iran; 6Veterinary Surgeon,Private Practice, Isfahan, Iran; 7TorabinejadResearch center, Isfahan University ofmedical Sciences, Isfahan, Iran; 8Departmentof Biology, Isfahan University of MedicalSciences, Isfahan, Iran

Keywords: adipose-derived stem cell (ADSC);HA/TCP; alveolar cleft; tissue engineering ofbone; dogs; histomorphometry.

Accepted for publication 10 October 2012

Repair of bony defects remains a challen-ging part of many reconstructive proce-dures. The use of autogenous bone is thegold standard for grafting bone defects.

The reconstruction of alveolar cleftdefects is well established, with the mostwidely accepted approach being second-ary alveolar cleft osteoplasty in the mixed

dentition phase.1 In conventional meth-ods, autogenous bone grafting has becomean essential step in treating patients withalveolar cleft, but harvesting autogenous

m adipose-derived stem cell with autogenous

/dx.doi.org/10.1016/j.ijom.2012.10.012

ons. Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ijom.2012.10.012



2 Pourebrahim et al.


bone graft for an alveolar cleft defect maycause short- and long-term complicationsin donor sites.2,3

To reduce these complications, substi-tute biomaterials, such as hydroxyapatites,a- and b-tricalciumphosphates, and demi-neralized bone matrices, are in clinicaluse.4 Combining them with osteoblastsand growth factors (i.e. tissue engineering)provides a new alternative in which bonecells are seeded on 3-dimensional bone-like scaffolds of natural and artificial ori-gin.5 Both synthetic and allograft materi-als allow adhesion and growth ofosteoblastic cells, or osteogenic differen-tiation of precursor cells in vitro.6

Tissue engineering of bone is a rapidlygrowing field and is a promising approach.In tissue engineering, cells, the extra cel-lular matrix, and growth factors are com-bined to design novel graft materialswhich can induce tissue regenerationand repair based on natural healing poten-tial.7 There is much debate about the idealsource of osteoprogenitor cells for use inskeletal tissue engineering.

Embryonic stem cells, the gold standardof multipotency, are derived from theinner cell mass of the preimplantationblastocyte. Embryonic stem cells areknown to have the ability to differentiateinto multiple tissues type from all threeembryonic germ layers.8 Concerns aboutthe possibility of infection, immunogeni-city, and tumourgenicity have limited theapplication of embryonic stem cells.9

Adult stem cells, derived from differenttissues, have the unique ability to self-renew and differentiate into various phe-notypes. These cells have the potential forcell therapy and tissue engineering. Adi-pose tissue is an appropriate source ofmesenchymal stem cells (MSCs) withwide differentiation potential.

Owing to the abundance of stem cells,the ease with which they can be procured,and their rapid expansion in vitro, adiposederived stem cells (ADSCs) are particu-larly desirable candidates for skeletal tis-sue engineering applications.10 Zuk10

originally characterized this populationof cells isolated from adipose tissue andfound that they were able to differentiatetowards osteogenic, adipogenic, myo-genic, and chondrogenic lineages in vitrowhen treated with the appropriate indu-cing factors. Cowan et al.11 demonstratedthe ability of ADSC seeded onto apatite-coated scaffolds to heal critical-sized(4 mm) calvarial defects. This was the firstpublished report of the ability of ADSC toheal critical-sized bony defects. Shi et al.12

compared the biological differences andosteogenic ability between juvenile and

Please cite this article in press as: Pourebrahim N

bone repair in maxillary alveolar cleft model i

adult mice and although differences weredemonstrated, namely greater attachmentand proliferation in juvenile mice, adultADSCs also exhibited robust terminalosteogenic differentiation. The dog offersa valuable experimental model and currentstudies have mainly focused on the osteo-genic potential of canine MSCs in vivo andin vitro.13

MSCs when combined with porous,biphasic calcium phosphate ceramics,namely hydroxyapatite/b-tricalciumphosphate (HA–TCP) ceramics with thecomposition 60% HA/40%TCP (inweight%), have been shown to inducebone formation in large, long bonedefects.14 Yoshikawa et al.15 reported thatHA loaded with MSCs has osteogenicpotential comparable with autogenousparticulate cancellous bone.

This study was designed to compareADSCs based alveolar cleft regenerationwith traditional autogenous bone graftingin a through-and-through canine alveolarcleft model, histologically. ADSCs wereloaded on HA/TCP the bone regenerationof which has been compared histologicallywith traditional autogenous bone grafts.

Materials and methods

Isolation and cultivation of ADSCs

This study was performed in accordancewith the regulation and approval of theInstitutional Animal Care and Use Com-mittee of the Isfahan University of Med-ical Sciences and conformed to itsstandard of animal care. Under generalanaesthesia, 20 mg scapular subcutaneousadipose tissue was isolated from four mon-grel dogs that had undergone maxillaryalveolar cleft creation surgery. The iso-lated adipose tissues were cut into smallpieces and washed with phosphate buf-fered saline (PBS; Gibco, UK). To processthem, 1.5 mg of collagenase type I (Sigma,USA) per gram of fat tissue was added andincubated with continuous shaking for 1 hat 37 8C. To separate stromal cells fromfloating adipocytes multiple centrifuga-tion and washing steps were appliedbefore removing red blood cells by appli-cation of lysis buffer. The separated stro-mal cells were counted using ahaemocytometer and were plated in tissueculture flasks (3000 cells/cm2) containingDulbecco’s modified Eagle’s medium(DMEM; Gibco, UK) supplemented with10% foetal bovine serum (FBS; DainipponPharmaceutical, Osaka, Japan), 1% peni-cillin–streptomycin (Gibco-BRL, LifeTechnologies) and incubated at 37 8Cwith 5% carbon dioxide. After 24 h the

, et al. A comparison of tissue-engineered bone fro

n dogs, Int J Oral Maxillofac Surg (2012), http:/

non-adherent cells were discarded and themedium changed. Culture media werereplaced every 2–3 days and trypsinizationand replating was carried out when thecultures reached about 80% confluency.

MSC characterization

There is no definitive marker to identifyMSCs, so the gold standard procedure toprove their stem cell identity is: theiradherence to cell culture plates after iso-lation; their expression of specific mar-kers; and their differentiation potential toosteoblast, chondrocyte and in vitro.16 Inthis study, the MSC character was provenby flow cytometrical analysis and by theability of the cell to differentiate intovarious lineages. In one tube 1 � 105 cellswere stained simultaneously with phy-coerythrin (PE) conjugated monoclonalantibody to CD44 (ab58754; ABCAMAntibodies, Cambridge Science Park,UK) and fluorecin isothiocyanate conju-gated (FITC) monoclonal antibody toCD90 (ab22541; ABCAM Antibodies,Cambridge Science Park, UK). After incu-bation at room temperature for 15 min thespecimen was analysed by FACS caliber488 (Becton Dickenson, CA, USA). TheFACS analysis showed a distinct popula-tion of CD44 and CD90 positive cells. Theresult of FACS analysis with the result ofthe differentiation assay proves that actualMSCs were transplanted (Fig. 1A and B).

In vitro osteogenic differentiation

For in vitro osteogenic differentiation,confluent passage 3 culture was used.5 � 106 cells were incubated with3 mm � 3 mm � 3 mm HA/TCP (Cera-form, Teknimed, France) (60% HA and40% b-TCP with a mean pore size of 200–800 mm) in specific osteogenic medium at37 8C and 5% carbon dioxide for 21 days.Osteogenic medium consisting of 50 mmlascorbic acid 2-phosphate (Sigma, USA),10 mml b-glycerophosphate (Sigma,USA) and 100 nmg dexamethasone(Sigma, USA).

Cell differentiation was evaluated byreverse transcriptase-polymerase chainreaction (RT-PCR) analysis of osteogenicgen expression. Osteocalcin and collagen Iwere largely produced after 21 days in anosteoinductive medium. The selectedhousekeeping gene was GAPDH(Fig. 2A and B).

Scanning electron microscopy

The morphology of the HA/TCP scaffolds,with and without cells, was observed by


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Bone regeneration from adipose-derived stem cell and autogenous bone 3




Fig. 1. (A) Results of flow cytometry show 99% of isolated cells express CD44 markerpositively. (B) Results of flow cytometry show 91% of isolated cells express CD90 markerpositively.

Fig. 2. (A) Sequences of different primers for reverse transcription polymerase chain reactionanalysis. (B) Reverse transcription polymerase chain reaction analysis. Osteocalcin and collagenI expression after 3 weeks.

scanning electron microscopy (SEM,Vega Tescan, Philadelphia, PA, USA) atan accelerating voltage of 20 kV. Beforethe observation, samples of cell-scaffoldconstructs were fixed with 2% paraformal-dehyde/2.5% glutaraldehyde in 0.1 M Nacacodylate buffer (PH 7.4), then dehy-drated in graded alcohols, and examinedwith SEM (Fig. 3).

Surgical procedure for alveolar cleft

creation

Four adult mongrel dogs (mean age 22month) weighing 20–30 kg were used inthis study. The animals were kept for 2 weekto become acclimatized to the housing anddiet. Throughout the experiments they weremonitored for general appearance andweight. They were starved 24 h before eachsurgery and for 24 h after surgery. Duringthis period they were given serum therapywith lactated Ringer’s solution. After sur-gery, 1 mg ceftriaxon was administeredonce a day. The dogs were given a soft dietup to 3 weeks after surgery. Under generalanaesthesia with ketamine (20 mg/kg) andrampone (2 mg/kg), the animals were pre-pared by removal of two of the three incisorsbilaterally and a 15 mm wide defect wascreated from the alveolar crest to the nasalfloor using a dental handpiece. Nasal boneand membrane were removed to createcomplete alveolar cleft penetration to thenasal cavity. The nasal mucosa was suturedto the oral mucosa (Ethicon, Norderstedt,Germany) and a stent was placed (endotra-cheal tube no. 7, as the stent was placedthrough the cleft on one side, exitingthrough the opposite cleft side, and fixedby wires to the maxillary canine teeth, whichhad been notched on their distal aspect)(Fig. 4). Two months were allowed forhealing and in this way bilateral clefts wereprepared with functional teeth on each sidethat were expected not to heal sponta-neously with new bone.17 To approximatehuman maxillary alveolar cleft more clo-sely, the experimentally created clefts had tofulfil the following five criteria (Fig. 5):bilateral maxillary alveolar cleft had to existin each research animal; each cleft had tohave 15 mm bony width; a demonstrableoronasal communication had to be present;each cleft had to be lined by healthy epithe-lialized mucosa; and there had to be func-tional teeth on each side of every cleft.18

Implantation of scaffold/MSCs

constructs and autograft

After removal of the stent, 2 weeks wereallowed for local inflammation to subside.Following crestal incision at the level of


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Fig. 3. Scanning electron microscope views of the Ceraform1. Cell cultured on the biphasicbone substitute, adipose derived stem cells could be seen everywhere on the surface pore ofCeraform1.

Fig. 6. (A) Repair by HA/TCP-loaded adipose derived stem cells. (B) Repair by autogenoustibial graft.

Fig. 4. Bilateral maxillary alveolar cleft creation and space maintained by the stent (endo-tracheal tube).

Fig. 5. View of bilateral maxillary alveolar cleft defect, 2 months after creation of defectssurgically.

the gingival sulcus, the scar tissues weredissected to reach the bony surface of thecleft walls. The tissue was elevated in thesubperiostal plane. The flaps of the nasalfloor and the oral mucosa formed theceiling and the floor of the cleft, respec-tively. For repair, one side was graftedwith tissue engineered bone from ADSCs(the scaffolds with cells were transfer tothe defect by microforceps) and the otherside was repaired with corticocancelloustibial autograft harvested at the same ses-sion, as the conventional method (Fig. 6Aand B). The wound was closed in a water-tight manner.

Histological examination

Bone regeneration was evaluated by his-tomorphometry 15 and 60 days after graftimplantation. For this purpose, biopsies ofboth grafted sides were taken using a2 mm trephine bur (Messeinger, Dussel-dorf, Germany). Cylindrical specimensacquired from the trephine biopsy(2 mm � 8 mm) were fixed in 10% buf-fered formalin for 5–7 days, and decalci-fied in formic acid and sodium citrate for24 h. The specimens were washed with tapwater, dehydrated with ascending concen-tration of ethyl alcohol, cleared in xylene,infiltrated with paraffin and processed forhistologic evaluation. Decalcified coronal5 mm sections were prepared and stainedusing Masson’s trichrome. On average, 3central sections were used for the histolo-gic evaluation, representing approxi-mately 80 mm of the central aspect ofthe osteotomy defect.

The central sections were chosen forhistomorphometric analysis. Photographswere taken with a light microscope(Olympus, SZX, Tokyo, Japan). Compu-ter-assisted histomorphometric measure-ments of the newly formed bone wereobtained using automated image analysissoftware (IHMMA, Ver.1, Sbmu, Iran).The histomorphometric data obtainedfrom each stained specimen was studiedat magnification of 100� by one calibratedexaminer using polarized light. The per-centage area of newly generated tissue thatconsisted of mineralized bone, marrowspace and collagen synthesis in each his-tological section was assessed by a blindedpathologist and was calculated relative tothe circular area of host bone defect, thislatter volume was take as 100%.

Statistical analysis

All data are presented as means and stan-dard deviation. The data were subjected tostatistical analysis using descriptive and


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Fig. 7. (A) Masson’s trichrome staining showing extensive bone formation in autograftedsample after 60 days. (B) Masson’s trichrome staining showing large amount of bone formationin stem-cell loaded scaffold grafted sample after 60 days. (C) Masson’s trichrome stainingshowing large amount of collagen formation in autografted sample after 15 days. (D) Masson’strichrome staining showing extensive collagen formation in stem-cell loaded scaffold graftedsample after 15 days.

Fig. 8. The integrity of the maxillary alveoli was well established in the autogenous graft andstem-cell graft after 2 months and has provided a suitable bed for future tooth implantation.

paired sample t tests. Differences atp < 0.05 were considered significant. Cal-culations were performed using the SPSSstatistical package (SPSS 11.5, SPSS Inc.,Chicago, USA).

Results

Healing was uneventful in all of the sur-gical sites after 60 days. In both groups,the newly generated tissues varied in sizeand consisted of pieces of mineralizedbone and large marrow spaces with fatcells and some haematopoietic cells.Cuboidal osteoblast-like cells were gener-ally found to be actively laying down boneto varying degrees.

A histological examination of the scaf-fold 60 days after implantation surgeryrevealed that there was an amorphouscalcified matrix lined with many osteo-blasts and abundance of collagen, indicat-ing active bone formation. There was noevidence of foreign body reaction in thehost tissue adjacent to the newly formedbone. In the pores, bone tissue togetherwith cuboidal active osteoblasts wasobserved in contact with the surface ofthe pores in HA/TCP. There was no inter-vening fibrous tissue between the ceramicand new bone. Regenerated bone marrowwas observed in association with the newbone formation inside some porous regionof HA/TCP (Fig. 7A–D). The integrity ofthe maxillary alveoli was established inboth groups (Fig. 8).

Mean bone formation on the autograftsides was higher than on the stem cellsides at 15 and 60 days; 45% and 96%versus 5% and 70%, respectively. Thesides treated with stem cells showed lessbone formation on day 15, but the rateincreased more rapidly after that toapproach an acceptable level of 70% onday 60. The difference between these twogroups on days 15 and 60 was significant(p = 0.004 and p = 0.001, respectively).Mean collagen synthesis in the stem cellgroup was higher than on the autograftedsides on days 15 and 60, 79% and 13%versus 11% and 3%, respectively. Thedifference between these two groups ondays 15 and 60 was also significant(p = 0.006 and p = 0.001, respectively)(Fig. 9 and Tables 1 and 2). Althoughactive osteoblasts were seen only in auto-grafted samples after 15 days, they wereobvious in both stem cell and autograftedsamples after 60 days.

Discussion

The most common presentation of humanmaxillary alveolar cleft is one of a bony



oronasal communication lined by epithe-lialized mucosa. Erupted teeth are usuallyadjacent to the cleft, and partially eruptedor unerupted teeth may be within the cleft.The present study used an experimentalmaxillary alveolar cleft defect to evaluatebone regeneration in dogs. There has beenmuch debate regarding the appropriatemaxillary alveolar cleft defect model forbone regeneration that does not heal spon-taneously. Surgical alveolar cleft modelswith dogs have been developed.17,18 Tomore closely approximate human maxil-lary alveolar cleft despite the creation of athree-walled maxillary defect, the authors’experimentally created clefts had to fulfil



the following five criteria similar to thestudy of Marx et al.18: bilateral maxillaryalveolar cleft had to exist in each researchanimal; each cleft had to have 15 mmbony width; a demonstrable oronasal com-munication had to be present; each clefthad to be lined by healthy epithelializedmucosa; there must be functional teeth oneach side of every cleft. Taken together,the authors think that this experimentalmaxillary alveolar cleft defect model inthe dog is appropriate for evaluating boneregeneration and is similar to the humanmaxillary alveolar cleft.

Although autogenous bone graftingremains the gold standard for reconstructing


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44 44 44 44N =

2.001.00

120

100

80

60

40

20

0

-20

Bone regeneratio n(15th day)

Colla gen regeneration (15th

Bone regeneratio n(60th day)

Colla gen regeneration(60th

Autogr af Stem-cell

Percent

Fig. 9. Histomorphometric data on the percentage of bone regeneration and collagen regen-eration in autografted and stem-cell grafted sides 15 and 60 days after implantation.

Table 1. Mean percentage bone regeneration in the alveolar cleft defect, 15 and 60 days afterrepair by autograft and stem-cell graft surgery.

Mean bone formation � SD15 days after repair

Mean bone formation � SD60 days after repair

HA/TCP + stem cell 5 � 1.75 70 � 16.41Autograft 45 � 14.14 96 � 3.55

Table 2. Mean percentage collagen regeneration in the alveolar cleft defect, 15 and 60 days afterrepair by autograft and stem-cell graft surgery.

Mean collagen formation � SD15 days after repair

Mean collagen formation � SD60 days after repair

HA/TCP + stem cell 78.75 � 4.78 13.50 � 8.65Autograft 11.25 � 4.33 3.25 � 2.95

bone defects, its disadvantages may includethe limited amount of bone and donor sitemorbidity. Tissue engineering approachescan potentially obviate these problems.Bone tissue engineering requires at leastosteoblast-like cells in combination with asuitable scaffold. The use of MSCs for boneregeneration is currently becoming popularpractice.19 Their potential for differentia-tion, their relative availability especiallythose harvested from adipose tissue, andtheir capacity to undergo extensive replica-tion without loss of that multipotency, makethem an attractive source for cell-basedtherapeutic approaches.20 For MSCs direc-ted bone repair to be clinically successful, ascaffold must be identified and optimized tosupport cellular adherence, osteoinduction,and osteoconduction. It has been shown thatMSCs are able to form bone after implanta-tion on porous HA/TCP matrices.21 Theintegrity of the maxillary alveoli was estab-lished in both autogenous and stem-cellgroups but the quantitative measurementindicated more bone formation on the auto-



grafted sides at both assessed times. Morecollagen synthesis was found on the stemcell grafted sides at 15 and 60 days whichindicates a robust effort for bone regenera-tion in this group and which might reach asmuch as 95% like the autografted sides.Behnia et al.22 demonstrated limited boneformation in tissue-engineeredalveolar cleftrepair using human-derived MSCs (derivedfrom bone marrow aspirate) in two patients.The main criticism of their study was thelack of histological or histomorphometricanalysis of bone formation, trabeculationpattern, and direction of bone formation.It was thought that the occurrence of osteo-genesis in bone defects would be the resultof combined action of bone microenviron-ment and MSCs loaded on scaffold materi-als. It is clearly beneficial for any tissue-engineered device to allow the host cells tocontribute to regeneration of the desiredtissue. Comparison of cell free and MSCsloaded matrices in the study of Jafarianet al.23 revealed a clear difference inbone formation, confirming the ostegenic



potency of MSC loaded constructs, and thatcell-free matrices had not formed significantbone formation. The results of this studysuggest that ADSCs have good potency forbone regeneration and the authors encou-rage further research on ADSCs loaded inscaffolds for repair of bony defects espe-cially in human maxillofacial reconstruc-tion.

In conclusion, although autografts arethe gold standard for bone regeneration,tissue engineered bone from ADSCs mayprovide an acceptable alternative espe-cially in the case of limited autograftavailability or donor site morbidity.

Funding

This study was supported by a grant-in-aidof Isfahan University of Medical Sciences,Project No. 388489, 2010.

Competing interests

None declared.

Ethical approval

Not required.

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Address:Shirin ShahnaseriTorabinejad Research CenterDental SchoolIsfahan University of Medical SciencesIsfahanIranTel: +98 9133138074; Fax: +98 3116695189E-mails: [email protected][email protected]


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Documents

Int. J. Oral Maxillofac. Surg. 2012; xxx: xxx–xxxmed.mui.ac.ir/clinical/stemcells/Dr.Shahnaseri.pdftreated with the appropriate indu-the factors. Cowan et al.11 demonstrated coated