Mesenchymal Stem Cells Associated With Porous Chitosan Gelatin Scaffold a Potential Strategy for...

Mesenchymal stem cells associated with porous chitosangelatinscaffold: A potential strategy for alveolar bone regeneration

Suzana C. C. C. Miranda,1* Gerluza A. B. Silva,1* Renato M. Mendes,1

Fernando Antonio M. Abreu,1 Marcelo V. Caliari,2 Jose B. Alves,1,3 Alfredo M. Goes4

1Department of Morphology, Biological Sciences Institute, Federal University of Minas Gerais, Presidente Antonio Carlos

Avenue 6627, 31.270-901, Belo Horizonte, MG, Brazil2Department of Pathology, Biological Sciences Institute, Federal University of Minas Gerais, Presidente Antonio Carlos

Avenue 6627, 31.270-901, Belo Horizonte, MG, Brazil3Laboratory of Biopathology and Molecular Biology, University of Uberaba, Nene Sabino Avenue, 1801, 38.055-500, Uberaba,

MG, Brazil4Department of Biochemistry and Immunology, Biological Sciences Institute, Federal University of Minas Gerais, Presidente

Antonio Carlos Avenue 6627, 31.270-901, Belo Horizonte, MG, Brazil

Received 4 December 2011; revised 15 February 2012; accepted 30 March 2012

Published online 24 May 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34214

Abstract: Tissue engineering has emerged as a novel treat-

ment for replacement of lost bone tissue. This study eval-

uated the effects of a chitosangelatin scaffold seeded with

bone marrow mesenchymal stem cells (BMMSCs) in the

healing process of tooth sockets in rats. BMMSCs isolated

from transgenic rats expressing enhanced green fluorescent

protein (eGFP) were expanded and seeded on a chitosan

gelatin scaffold. These constructs were cultured for three

days and characterized by scanning electronic microscopy

(SEM) and energy dispersion spectroscopy (EDS). Receptor

rats received the implant in the left sockets, after upper

first-molar extraction. Right alveoli served as control. Ani-

mals were sacrificed at days 5, 21, and 35 post-graft for ex-

amination. Morphometry demonstrated increased bone

mineralization after 21 and 35 days in transplanted sockets.

Migration, differentiation, and fate of eGFP-labeled BMMSCs

were monitored by immunohistochemistry. Tartrate-resistant

acid phosphatase staining (TRAP) was carried out at 21

days, to identify the involvement of osteoclastic cells in the

scaffold resorption. The biomaterial was resorbed by TRAP-

negative giant cells in a typical foreign body reaction. Im-

munohistochemical findings showed that BMMSCs contrib-

uted to bone, epithelial, and vascular repair. Together,

results indicate that BMMSCs loaded in the chitosangelatin

scaffold is a strategy for tissue development in bone engi-

neering. VC 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:

100A: 27752786, 2012.

Key Words: chitosangelatin scaffold, mesenchymal stem

cells, tissue engineering, bone healing, tooth socket

How to cite this article: Miranda SCCC, Silva GAB, Mendes RM, Abreu FAM, Caliari MV, Alves JB, Goes AM. 2012. Mesenchymalstem cells associated with porous chitosangelatin scaffold: A potential strategy for alveolar bone regeneration. J Biomed MaterRes Part A 2012:100A:27752786.

INTRODUCTION

The search for strategies to replace or restore bone tissuelost by trauma or pathologies remains a major clinical chal-lenge to biomedical researchers. In this context, bone tissueengineering has emerged as an effective alternative therapyto repair bone defects. Tissue engineering is an interdiscipli-nary eld involving cell biology and materials science thatworks toward the development of biological substitutes torestore, maintain or improve tissue function.1,2 The basicprinciple of tissue engineering is to seed cells onto a biode-gradable scaffold to generate new tissue.35 The choice ofthe biomaterial is critical for the success of such an

approach in bone repair. The ideal scaffold should possesssuitable mechanical properties, biocompatibility, bioperme-ability, three-dimensional (3D) structure, and promote bonetissue growth.6,7

In vitro studies have revealed promising results by usingscaffolds composed of chitosangelatin blends.8,9 This bio-material acts as a stable structural support for mesenchy-mal stem cell colonization and permits their differentiationinto the osteoblastic lineage.10 Chitosan is a nontoxic, bio-compatible, and biodegradable polymer.11 Its associationwith gelatin, a source of collagen type I, enhances cell adhe-sion in culture, due to the afnity of the cells for the

*These authors contributed equally to this work.

Correspondence to: G. A. B. Silva; e-mail: [email protected]

Contract grant sponsors: FAPEMIG (Fundacao de Amparo a Pesquisa de Minas Gerais/Brazil); CNPq (Conselho Nacional de Desenvolvimento

Cientfico e Tecnologico/Brazil)

VC 2012 WILEY PERIODICALS, INC. 2775

adhesion proteins.1214 Adhesion is a fundamental issue ofconcern in bone engineering, as it represents a requisite forthe matrix secretion by osteoblasts, which are anchorage-de-pendent cells.15

The bone marrow mesenchymal stem cells (BMMSCs)remain the most widely used source of osteogenic cells inbone tissue engineering studies.16 In an in vitro study,recently conducted in our laboratory, a porous chitosangel-atin scaffold, cross-linked by glutaraldehyde, was synthe-sized, characterized, and seeded with rat BMMSCs. This bio-material promoted the adhesion, spreading, and in vitroviability of the BMMSCs.17 The transplantation of in vitrocultured tissues, dened as constructs, to experimentalbone defects may provide qualitative and quantitativeimprovements in the newly formed bone. Transplanted stemcells are able to differentiate into osteoblasts in the receptorarea and contribute to bone neoformation.18 However, thenal fate of the transplanted stem cells is difcult to deter-mine. The green uorescent protein (GFP) of the jellysh,Aequorea victoria, has been considered a unique reporterfor monitoring cells in recipient organisms. A mutated GFP,referred to as enhanced GFP (eGFP), has been developed tooptimize GFP labeling. By using this tool, investigators havebeen able to monitor the expression, localization, and differ-entiation of transplanted cells in vivo.19,20

In this study, we proposed a strategy for bone engineer-ing, by the implantation of a chitosangelatin scaffoldseeded with labeled eGFP-BMMSCs in rats with experimen-tal bone defects. We investigated the level of bone neofor-mation by assessing the morphometric data obtained fromcone beam computerized tomography (CBCT). In addition,the expression of eGFP-BMMSCs in the receptor area wasmonitored by immunohistochemistry.

MATERIALS AND METHODS

AnimalsA total of 20 Lewis rats, weighting 230250 g (8-week-old),were used in the study. Four transgenic rats carrying theenhanced green uorescent protein (eGFP) genes (LEW-TgEGFP F455/Rrrc, Rat Resource and Research Center, Univer-sity of Missouri) were used to obtain endogenously labeledbone marrow mesenchymal stem cells (BMMSCs). Sixteenmale, wild-types from the inbred lineage LEWIS/H wereused as the recipient animals. All experimental protocolswere performed in accordance with the guidelines for ex-perimental animals and approved by the Animal Committeeof Federal University of Minas Gerais.

Cell isolation and expansionThe eGFP-rats were anesthetized with a mixture of 10% ke-tamine and 2% xylazine (1:1, 0.1 mL/100 g body weight,i.m.). The epiphysis from both femurs and tibias were dis-sected out and the bone marrow cells were collected byushing the diaphysis using a syringe lled with Dulbeccosmodied Eagles medium (DMEM; Gibco) supplementedwith 10% fetal bovine serum (FBS; Gibco) and antibiotics(100 U/mL penicillin, 100 lg/mL streptomycin and 250 ng/mL amphotericin B; Gibco). A suspension of bone marrow

cells was obtained and resuspended in 10 mL of medium.The cells were plated in culture in 75-cm2 asks. Cell cul-tures were maintained at 37C in an atmosphere containing5% CO2. Nonadherent cells were removed from the culturesby medium change three times a week. The adherent cellswere allowed to grow near to conuence and were thensplit 1:3. The cultures were passage four to six times.

Phenotypic characterization of the BMMSCsby ow cytometryBMMSCs were characterized as previously described,17,21 inaccordance with the minimal criteria to dene a MSC, asproposed by the Mesenchymal and Tissue Stem Cell Com-mittee of the International Society for Cellular Therapy.22 Insummary, cells were released by trypsinization after fourpassages. Approximately 1106 cells/well were incubatedfor 30 minutes (min) at 4C with the following set of mouseantibodies: anti-CD90, anti-CD73, anti-CD54, anti-CD45diluted 1:50 (BD Pharmingen, Franklin Lakes, USA), andrabbit anti-GFP (Green Fluorescent Protein) (Abcam, Cam-bridge, UK). After washing with phosphate-buffered saline(PBS), the cells were incubated with Alexa 488 anti-mouse(Invitrogen, Carlsbad) for 30 min at 4C and then washedwith PBS. The cells were xed with 200 lL of 2% formalde-hyde and acquired, using a uorescence-activated cell sorter(FACS) (FACSCalibur; Becton Dickinson, San Jose). The datawere analyzed with CellQuest Software (BD Biosciences, SanJose).

Preparation and characterization of thecell-scaffold constructThree-dimensional porous chitosangelatin scaffolds wereobtained via a freeze-drying technique23 using glutaralde-hyde cross-linking, as previously described.17 The scaffoldswere produced from natural polymers, chitosan (Sigma, St.Louis) with a degree of de-acetylation of 85% and type Aporcine skin gelatin (Vetec, RJ, Brazil). The chitosangelatinblend at a 3:1 (chitosan:gelatin) ratio was cross-linked with25% glutaraldehyde (Sigma, St. Louis) solution at a 0.1%concentration. One milliliter of the homogenous mixture ofchitosan, gelatin and glutaraldehyde was poured into eachwell of a cell culture plate (24-well) and used as a templateto mold cylindrical standard discs of chitosangelatinsponges. Discs of 8 mm in diameter and 2.5 mm thick wereobtained and cut into four parts, resulting in slices of 4 mmin radius, which were used in all of the in vitro and in vivoprocedures. The samples were distributed in a 24-well cellculture plate, sealed and sterilized at the Nuclear Develop-ment Technology Centre/Nuclear Energy National Commis-sion (CDTN/CNEN) of Minas Gerais Federal University, by cirradiation, 60Co, 20 Grays.

Conuent cultures of the rat bone marrow mesenchymalstem cells labeled with eGFP (eGFP-BMMSCs) were trypsi-nized and the detached cells were suspended in culture me-dium containing 10% fetal bovine serum (FBS). eGFP-BMMSCs were seeded in the scaffold at a density of 5 104/mL in 24-well plates (TPP, Trasadingen, Switzerland)

2776 MIRANDA ET AL. MESENCHYMAL STEM CELLS ASSOCIATED WITH POROUS CHITOSANGELATIN SCAFFOLD

and cultured for three days to obtain a three-dimensionalsystem for in vivo implantation.

The morphological characterization of constructs wasperformed by scanning electronic microscopic (SEM) analy-sis. Three of the cell-scaffold samples were xed in 2.5%glutaraldehyde (0.1 mol/L phosphate buffer - PBS, pH 7.4)for 48 h and post xed with 1% osmium tetroxide for 2 h.After dehydration, the critical-point dried in liquid CO2(CPD-020 Balzers) was performed. The samples weremounted on metallic holders, sputter-coated with gold andobserved in a Zeiss DSM 950 scanning electron microscope,at an accelerating voltage of 15 KV and 750 mA. The chemi-cal composition of both the scaffold and construct wasdetermined by energy dispersion spectroscopy (EDS) after 3days in culture. This analysis was performed to detectchemical differences between the scaffold alone and possi-ble secretions deposited into the construct.

Surgery and construct transplantationThe tooth socket was used as an experimental bone defect,in accordance with Kanyama et al.24 and Mendes et al.25. Allof the recipient male rats were subjected to the extractionof both upper rst molars. Cell-scaffold constructs were im-mediately implanted on the left sockets, while the rightones were used as a control. A micro-amalgam carrier (ABCsurgical instruments) was used to place the constructs into

the tooth sockets. To maintain the constructs in the extrac-tion sites after surgery, the sockets were closed with 6-0sutures (Biosut, BH, Brazil). Animals received a pasty chowfor 2 days after the surgeries and were sacriced after 5,21, and 35 days. The craniums were xed in 10% bufferedneutral formalin solution (pH 7.2 for 48 h) and morphomet-ric analyses were performed. Further, the maxillae were dis-sected and processed for histological and immunohisto-chemical analysis.

Morphometric analysis by computerized tomographyAfter xation, the craniums were scanned by a cone beamcomputerized tomography (CBCT) in an i-CAT (Xoran Tech-nologies, Ann Arbor, MI, and Imaging Sciences International,Hateld, PA) for image acquisition. The position of the coneof the X-ray emitter was standardized, so that the distancefrom the emitter to the maxillae was xed. The CBCTs wereassigned random code numbers to allow the analysis to beperformed without bias. The CBCT was set with the follow-ing parameters: slice thickness of 0.5 mm; pitch, 0.75; tubevoltage, 120 kV; and electrical current-time, 350 mAs. Tobuild the 3D images, Dental Slice software (version 2.1.1)was employed. Figure 1(A) shows a 3D reconstruction ofthe entire cranium. A transversal plane of CBCT exams pass-ing through distal sockets of rst upper molars wasemployed for the study [Fig. 1(B)]. To measure the new-

FIGURE 1. Morphometric analysis using CBCT images. A: Representative 3D reconstruction of a Lewis rat cranium. Note the transversal plane

selected as a reference to morphometry. B: The CBTC showing the study areas of experimental (Exp, left) and control sockets (Co, right). C: The

measurement of total socket area (TSA, delimited by black continuous line). D: The study area (SA, delimited by dashed line) into the socket,

where the weak intensity areas (WIA) were measured. The formula was used to calculate the mean percentage of socket filling. [Color figure can

be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | OCT 2012 VOL 100A, ISSUE 10 2777

formed bone area inside the tooth sockets, three representa-tive serial slices of the experimental and control sides werecaptured from each animal, totaling 15 images per group.The grayscale intensity was counted with the use of theKS300 software coupled to a Carl Zeiss image analyzer (CarlZeiss, Oberkochen, Germany). 8 bits sensor was used to re-cord the data. In 8-bits-per-channel images, the intensityvalues range from 0 (black) to 255 (white). Every pixel of agrayscale image has a brightness value ranging from 0(black) to 255 (white). The gray intensity was measured foreach image and the results were expressed by the averagepercentage of bone formation. Thus, the high intensity areascorresponded to the more mature bone tissue. To quantifythe level of bone lling, the weak intensity areas (WIA) intothe study area [SAFig. 1(D)] were discounted from thetotal socket area, [TSAFig. 1(C)]. The low intensity points

(WIA) corresponded to loose tissues as the brin matrix,blood coat, and inammatory process. The percentage offormed bone was obtained through the following formula:Mean socket lling % [TSA-WIA (mm2)/TSA (mm2)]X100.

Histological and TRAP stainingAfter image acquisition by CBCT, the upper maxillaries weredissected, demineralized in 10% ethylenediamine tetraaceticacid (EDTA; Sigma, St. Louis), pH 7.3, dehydrated throughgraded ethanol solutions and embedded in paraplast. Sagit-tal sections of 6 lm were stained with hematoxylin eosinand tartrate-resistant acid phosphatase staining (TRAP).TRAP histochemistry was carried out only for the experi-mental period of 21 days to identify the involvement ofosteoclastic cells in the process of chitosangelatin scaffold

FIGURE 2. Morphological and chemical characterization of the cell-scaffold construct by SEM. A: The ultrastructural aspect of the scaffold show-

ing the relatively smooth walls and porosity. B: The interconnected pores are indicated by arrows. C: The photomicrography (SEM) of the con-

struct after 3 days in culture. An adherent BMMSC can be seen on the scaffold layer. The images suggestive of extracellular matrices are

indicated by arrows. D: The EDS analysis of the extracellular matrix (white arrow). Note the areas of the calcium peak (black arrow). E: The EDS

analysis on the free-cell scaffold surface (arrow). Only carbon (C) and oxygen (O) peaks were observed; Osmium (Os) and gold (Au) peaks are

usually found due to the processing of the samples for SEM. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


resorption. TRAP activity was detected using a leukocyte-specic acid phosphatase kit (TRAP 387A-1KT Sigma, St.Louis). The sections were stained with TRAP solution for 1h at 37C and counter-stained with Mayers hematoxylin,according to the manufacturers instructions. The TRAP-pos-itive cells appeared dark red or purple.

Cell tracingImmunohistochemistry analysisTo track the fate, distribution and differentiation of thetransplanted BMMSCs after delivery on the scaffold, theeGFP was used as a tag. The slices were deparafnized inxylene, rehydrated through a graded series of ethanol, andwashed in PBS. The sections were then immersed in 3%H2O2 (1 h) to neutralize the endogenous peroxidase activityand incubated with 2% BSA in PBS (1 h) for the blocking ofnonspecic binding sites. Afterward, the sections were incu-bated overnight at 4C with rabbit anti-GFP antibody(1:100, Abcam; Cambridge, UK). The secondary biotinylatedanti-rabbit antibody (Universal LSABTM Kit/HRP, Rb/Mo/Goat K0690-1Dako; Glostrup, Denmark) was applied for 30min at room temperature. After rinses in PBS, the sectionswere incubated with a streptavidinhorseradish peroxidaseconjugate for 30 min at room temperature. The reactionwas revealed with a solution containing 350 lM 3,30- diami-nobenzidine (Sigma Chemical Co.) and 1% H2O2 in PBS. Theslides were counterstained with Mayers hematoxylin. Allsamples were processed at the same time to avoid differen-ces among the assays. Negative controls were obtained bythe omission of the primary antibody.

Statistical analysisData were presented as the percentage means. The morpho-metric results of the CBCT measurements, carried out oncontrol and experimental sides of the same sample unit,were performed using the Wilcoxon test. p-values 21 days > 5 days) cor-respond to larger bone-lling areas, identied by a grayscalein the KS 300 software (Figs. 3 and 4). The treated alveolidemonstrated higher densities for all evaluated time peri-ods. The density observed at 5 days in the implanted side isjustied by the scaffold radiopacity, as shown in detail inFigure 4(A). An occlusal view of the bone repair for eachtime period is illustrated on the 3D reconstruction images(Fig. 4, right panel).

Histological and TRAP-staining analysisAt 5 days post-surgery, histological analysis revealed thepresence of acute inammatory processes in both sockets[Fig. 5(A,B)]. This inammation was more pronounced inthe experimental sockets, where the chitosangelatin meshwas invaded by polymorphonuclear neutrophils and macro-phages cells [Fig. 5(C)]. In addition, neovascularization wereseen in the experimental socket [Fig. 5(B)]. The epithelialhealing and the bone neoformation seemed delayed in thetreated sockets [Fig. 5(B)], when compared with the contra-lateral control sockets [Fig. 5(A)]. Some multinucleated cellswere observed near the chitosan laminae [Fig. 5(D)].

At 21 days, the histological aspects in both the control[Fig. 5(E)] and experimental sides were similar [Fig. 5(F)].Although full mucosal and bone healing had been achieved,the newly formed bone showed an immature aspect withdisorganized lamellae and many marrow spaces [Fig. 5(G)].The rests of the biomaterial, invaded by multinucleatedgiant cells, were seen in the connective tissue of the trans-planted rats, without signs of encapsulation [Fig. 5(F,H,I,asterisks)]; these resorptive giant cells were TRAP-negative[Fig. 5(I)]. Figure 5(J) shows the osteoclasts used as aTRAP-positive control.

FIGURE 3. CBCT morphometric results: The ratio of the new bone fill-

ing by gray-density evaluation. The p values are referred to the signif-

icance of probability of Wilcoxon test. SD, standard deviation.

* Significant difference.

ORIGINAL ARTICLE


The histological ndings after 35 days of implantationwere similar to those described for 21 days. The socketswere totally lled by bone tissue, in both sides, with moredense bone trabeculae and fewer medullary spaces. How-

ever, the bone tissue in the control sockets [Fig. 5(K)] pre-sented some immature areas, with disorganized osteocytessurrounded by newly secreted matrix [Fig. 5(K)]. Most sam-ples of implanted sockets [Fig. 5(L)] showed regular bony

FIGURE 4. Representative CBCT slices of the experimental (left) and control (right) sockets, at 5 (A), 21 (B), and 35 (C) days after surgery. Note

the higher gray density aspect of the experimental side for all of the experimental periods. At day 5, the density was attributed to the scaffold

radiodensity (Fig. A - detail), newly implanted. Respective 3D reconstructions (right) illustrate the occlusal views of alveolar bone repair. The

arrows indicate the experimental side with the better obliteration at only days 21 and 35. [Color figure can be viewed in the online issue, which

is available at wileyonlinelibrary.com.]


FIGURE 5. Representative HE and TRAP staining photomicrographs. Day 5: Distal sockets are delimited with a dotted line. The control socket

(A). The complete epithelial healing (white arrows) and initial bone deposition (black arrows). The experimental sockets (BD). Note the chito-

sangelatin mesh invaded by polymorphonuclear neutrophils (*) in detail (Fig. C). Figure D shows the multinucleated cells (arrows) close to the

biomaterial stained in red. Day 21: The control (E) and experimental (F) sockets. The bone and epithelial healing was complete. The rests of bio-

material are seen in the connective tissue of the experimental group (*). The histological aspect of the immature, newly formed bone observed

in both sides (G). Figure H shows the giant multinucleated cells in the pores of the biomaterial. These cells were TRAP-negative (Fig. I, arrows).

The asterisk (H and I) indicates the chitosan laminae. Osteoclasts (TRAP-positive) were used as a positive control for the TRAP staining (J). Day

35: The histological aspect of the healed bone, immature in the control socket (K) and mature in the experimental socket (L). The biomaterial

can still be observed in the connective tissue, without any signs of inflammation (M). Figure N shows, in detail, the thin laminae of the biomate-

rial. IB, immature bone; MB, mature bone. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


lamella and osteocytes aligned, suggesting a more advancedlevel of bone maturation [Fig. 5(L)]. Residual scaffolds wereseen in perfect integration with loose connective tissue [Fig.5(M)]. The chitosan layers appeared thinner than thoseobserved on day 21 [Fig. 5(N)]. No inammatory granula-tion tissue or brous tissue encapsulation was observed.

Immunohistochemical analysisOn the fth day after implantation [Fig. 6(A)], only a fewisolated eGFP-positive cells were observed between the bio-material layers. This result suggests that the stem cellsmigrated outside of the carrier, possibly in an anterior

phase, before the fth day. The immunostaining was moreevident in the neoformed bone area. Labeled cells wereseen on the surface of newly formed bone trabeculae, aswell as inside the medullary spaces [Fig. 6(B)]. eGFP-posi-tive osteocytes were observed within the bone matrix, per-fectly integrated with the native eGFP-negative osteocytes[Fig. 6(C)]. The periodontal ligament of the adjacent teethpresented several eGFP-positive cells [Fig. 6(E)]. Figure6(D,F) represent images of the negative controls of the boneand periodontal ligament, respectively.

After 21 days, no eGFP-positive cells were seen insidethe scaffold pores. The immunostaining was observed in

FIGURE 6. Immunohistochemical analyses after 5 days of transplantation. The white arrows indicate the eGFP-positive cells observed in the fol-

lowing: (A) on the scaffold (black arrows); (B) inside the medullary spaces; (C) in osteocytes embedded by bone extracellular matrix; and (E) into

the periodontal ligament. Note that the eGFP-positive osteocytes (C - white arrows) are perfectly integrated with the native osteocytes (C black

arrows). (D) and (F) are representative images of the negative control in bone and periodontal ligament, respectively. Scale bar 50 lm. IB, ima-ture bone. PM; periodontal membrane. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


the connective tissue between the biomaterial remnantsand the alveolar healing area [Fig. 7(A)]. The labeled cellswere particularly evident close to the bone matrix andmedullary spaces and in cell aggregates, suggestive ofintramembranous ossication [Fig. 7(B,C)]. eGFP-positivecells were also present in the walls of the blood vesselsin both the connective tissue [Fig. 7(E)] and vascularchannels of the newly formed bone [Fig. 7(F)]. Figure7(D) is a representative image of the negative control for21 days after implantation.

After 35 days, the immunostaining was observed speci-cally in the osteocytes included in the bone matrix of bothimmature [Fig. 8(A)] and histologically organized, maturebone [Fig. 8(B)]. Differentiated eGFP-positive cells were per-fectly integrated with the host osteocytes [Fig. 8(A,B)]. TheeGFP expression was maintained in the osteogenic lineagecells localized at the bone surface [Fig. 8(C)]. Additionally,the eGFP-positive BMMSCs were found into the squamouslayer of the epithelial tissue, suggesting its participation inrepair and epithelial turnover [Fig. 8(E)]. Figure 8(D,F)

FIGURE 7. Immunohistochemical analyses after 21 days of transplantation. The white arrows indicate the eGFP-positive cells observed in the

following: (A) in the connective tissue area between the biomaterial remnants (black arrows) and the alveolar healing area; (B) and (C) close to

the bone surface or inside the medullary spaces in niche suggestive of intramembranous ossification; and (E) and (F) in the walls of the blood

vessels of connective tissue and vascular channels of the newly formed bone, respectively. (D) A representative image of a negative control.

Scale bar 50 lm. IB, immature bone; CT, connective tissue; MS, medullary space; V, vessel. [Color figure can be viewed in the online issue,which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


represent images of the negative control of the bone andepithelial tissue, respectively.

DISCUSSION

In this study, we demonstrated the effects of a BMMSCs-scaffold construct during tooth socket healing. The boneregeneration process after tooth extraction is an interestingexperimental model to study hard tissue repair, whichincludes the well-known phases of inammation, granula-tion tissue formation, angiogenesis, and extracellular matrix

deposition.24,25 By tracing of the eGFP-transplanted cells, itwas possible to visualize their contribution to bone repair,as well as to wound epithelialization and neovascularization.These results can be attributed to factors closely related tothe success of bone engineering. Among them, the composi-tion and the structural characteristics of the biomaterialemployed as the cell carrier. The SEM analysis revealed thehighly porous nature of the chitosangelatin, which favoredits interaction with the seeded cells. In fact, the chitosangelatin scaffold showed a capacity to host the adherence

FIGURE 8. Immunohistochemical analyses after 35 days of transplantation. The white arrows indicate the eGFP-positive cells observed in the

following: (A) some areas of immature bone; (B) the osteocytes typically differentiated in the mature bone areas; (C) the osteoblasts at the bone

surface; and (E) included in the epithelial tissue. The black arrows indicate differentiated osteocytes from the host eGFP-negative cells. (D) and

(F) are representative images of the negative control in the bone surface and epithelial tissue, respectively. Scale bar 50 lm. [Color figure canbe viewed in the online issue, which is available at wileyonlinelibrary.com.]


and differentiation of mesenchymal stem cells, which was inaccordance with previous in vitro and in vivo studies.17,27,28

The BMMSCs were preferably employed in this study; theywere easily isolated from bone marrow, aspirated andexpanded in vitro. Moreover, they present a default pathwayinto the osteogenic lineage.3,27,29 Images suggestive of anextracellular matrix with calcium peaks detected by the EDSwere observed in our construct after 3 days of culture. Thisevidence of osteoblastic differentiation and secretion of amineralized matrix in vitro allows us to infer that the syn-thesized chitosangelatin scaffold mimicked the naturalbone environment and represents an adequate construct forbone tissue engineering. Through our in vivo experiments,the low immunogenicity and biocompatibility of the chito-sangelatin blend, commonly cited in the literature,13,17,30

were conrmed by the histological analyses of the trans-planted alveoli. Neither necrosis of the surrounding tissuesnor scaffold encapsulation was observed, suggesting thatthis construct was nontoxic and biocompatible.

An ideal biomaterial should also be biodegradable andpossess suitable mechanical properties to support thereconstruction of a completely natural tissue without caus-ing an inammatory response. Our histological analysisshowed the presence of resorptive cells in the scaffold poresat all of the evaluated time periods. These multinucleatedgiant cells were more numerous and organized at days 21and 35 than earlier in the experiment. The chitosangelatinwas progressively resorbed, at a slow degradation rate, andthere was, simultaneously, a time-compatible replacementby bone tissue, as has been recommended for an idealscaffold.27

Nakagawa et al.31 has demonstrated the in vitro differen-tiation of osteoclasts on a mineralized construct [MSC-poly(D,L-lactic-co-glycolic acid)]. This nding suggests the possi-ble involvement of osteoclasts in the resorption of biomate-rials that present calcium deposits. The enhancement ofosteoclasts recruitment to the healing site can promote animbalance in the natural process of bone formation/resorp-tion during alveolar healing with prejudices to bone repair.Although calcium deposition was seen on the chitosanlayers in our vitro analysis (EDS), the resorption of the chi-tosangelatin could not be attributed to osteoclastic cells.The multinucleated giant cells observed between the layersof the construct were TRAP-negative. Thus, the resorptionprocess of chitosangelatin seems to be the consequence ofa typical foreign body reaction, which evolved into a favor-able implant-tissue interaction. As also demonstrated byprevious studies,17 the rests of the biomaterial remained inthe connective tissue until day 30, without the formation ofa brous tissue capsule, a negative aspect described in theliterature for others biomaterials.3,32

A more intense inammatory process was observed inthe transplanted alveoli at 5 days postsurgery. This ndingwas interpreted as a normal and expected result responsiblefor the apparent delay of epithelial and bone healing. Theexcessive biomaterial volume overlapping tooth socket wallsrepresented an additional mechanical stress on the connec-tive tissue. According to Brandao et al.,33 the use of materi-

als to improve bone neoformation in alveoli following dentalextraction, can delay bone repair, which was conrmed inour study, during the initial phase of healing. Nevertheless,in a smaller extension than in the control sockets, after 5days, newly formed bone trabeculae could be seen in theapical and medium thirds of the sockets. The presence ofthe scaffold was not able to prevent the healing process. Atdays 21 and 35, the healing was complete and histologicalaspects observed in the control and transplanted socketswere quite similar.

Recent experiments have suggested that cell transplanta-tion has therapeutic value for the treatment of many condi-tions, providing cures to diseases via tissue repair.3437 Intransplantation models, a difcult issue of concern is howto access the fate of the donor cells and maintain theirlabeling in a host environment for the entire experimentalperiod. In our work, we used eGFP-labeled BMMSCs.Although it is considered a marker of choice for many typesof cell transplantation studies,38,39 eGFP may lose its directuorescence during tissue processing. For this reason, im-munostaining was employed, and we achieved an effectivecell tracing. We demonstrated both the ability of theBMMSCs to maintain eGFP labeling until 35 days after theconstruct transplantation in vivo, and the possibility to mon-itor their location by means of immunohistochemical analy-sis. As expected, the transplanted cells migrated and differ-entiated into osteoblasts and osteocytes at the site of theinjury, contributing to the bone regeneration. The immuno-histochemistry showed that the transplanted cells physiolog-ically integrated with the native osteocytes (eGFP-negative)within the bone matrix. These results revealed the potentialof the chitosangelatin scaffold to act as a carrier forBMMSCs and to promote the development of these cellsinto an osteoblastic lineage. Moreover, the transplantedBMMSCs have, for the rst time, been shown to migratethroughout all of the lesioned tissues and also to contributeto epithelial healing and wound neovascularization.

CONCLUSIONS

BMMSCs transplanted in a chitosangelatin carrier into atooth socket environment differentiated into bone, epithelialand vascular cells and contributed signicantly to the matu-ration of newly formed bone. In summary, chitosangelatinseeded with BMMSCs is an efcient construct for bone engi-neering, which can be a promising strategy for craniofacialreconstructions.

ACKNOWLEDGMENTS

The authors would like to thank Helio Chiarini-Garcia, Ph.D.for assisting in the SEM studies andmembers of CONEST (Con-sultoria em Estatstica e PesquisaBelo Horizonte/MG) forstatistical assistance.

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