Mesenchymal stem cells associated with porous chitosangelatin

scaffold: A potential strategy for alveolar bone regeneration

Suzana C. C. C. Miranda,
1
* Gerluza A. B. Silva,
1
* Renato M. Mendes,
1
Fernando Anto nio M. Abreu,
1
Marcelo V. Caliari,
2
Jos e B. Alves,
1,3
Alfredo M. Goes
4
1
Department of Morphology, Biological Sciences Institute, Federal University of Minas Gerais, Presidente Anto nio Carlos
Avenue 6627, 31.270-901, Belo Horizonte, MG, Brazil
2
Department of Pathology, Biological Sciences Institute, Federal University of Minas Gerais, Presidente Anto nio Carlos
Avenue 6627, 31.270-901, Belo Horizonte, MG, Brazil
3
Laboratory of Biopathology and Molecular Biology, University of Uberaba, Nene Sabino Avenue, 1801, 38.055-500, Uberaba,
MG, Brazil
4
Department of Biochemistry and Immunology, Biological Sciences Institute, Federal University of Minas Gerais, Presidente
Anto nio 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 uorescent
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
rst-molar extraction. Right alveoli served as control. Ani-
mals were sacriced 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 ndings 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. Mesenchymal
stem cells associated with porous chitosangelatin scaffold: A potential strategy for alveolar bone regeneration. J Biomed Mater
Res Part A 2012:100A:27752786.
INTRODUCTION
The search for strategies to replace or restore bone tissue
lost by trauma or pathologies remains a major clinical chal-
lenge to biomedical researchers. In this context, bone tissue
engineering has emerged as an effective alternative therapy
to repair bone defects. Tissue engineering is an interdiscipli-
nary eld involving cell biology and materials science that
works toward the development of biological substitutes to
restore, maintain or improve tissue function.
1,2
The basic
principle of tissue engineering is to seed cells onto a biode-
gradable scaffold to generate new tissue.
35
The choice of
the biomaterial is critical for the success of such an
approach in bone repair. The ideal scaffold should possess
suitable mechanical properties, biocompatibility, bioperme-
ability, three-dimensional (3D) structure, and promote bone
tissue growth.
6,7
In vitro studies have revealed promising results by using
scaffolds composed of chitosangelatin blends.
8,9
This bio-
material acts as a stable structural support for mesenchy-
mal stem cell colonization and permits their differentiation
into the osteoblastic lineage.
10
Chitosan is a nontoxic, bio-
compatible, and biodegradable polymer.
11
Its association
with 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: gerluza@terra.com.br
Contract grant sponsors: FAPEMIG (Fundac a o de Amparo a Pesquisa de Minas Gerais/Brazil); CNPq (Conselho Nacional de Desenvolvimento
Cientco e Tecnol ogico/Brazil)
VC 2012 WILEY PERIODICALS, INC. 2775
adhesion proteins.
1214
Adhesion is a fundamental issue of
concern in bone engineering, as it represents a requisite for
the 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 in
bone 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 vitro
viability of the BMMSCs.
17
The transplantation of in vitro
cultured tissues, dened as constructs, to experimental
bone defects may provide qualitative and quantitative
improvements in the newly formed bone. Transplanted stem
cells are able to differentiate into osteoblasts in the receptor
area and contribute to bone neoformation.
18
However, the
nal 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 reporter
for monitoring cells in recipient organisms. A mutated GFP,
referred to as enhanced GFP (eGFP), has been developed to
optimize GFP labeling. By using this tool, investigators have
been 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 scaffold
seeded 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 from
cone beam computerized tomography (CBCT). In addition,
the expression of eGFP-BMMSCs in the receptor area was
monitored by immunohistochemistry.
MATERIALS AND METHODS
Animals
A total of 20 Lewis rats, weighting 230250 g (8-week-old),
were used in the study. Four transgenic rats carrying the
enhanced green uorescent protein (eGFP) genes (LEW-Tg
EGFP F455/Rrrc, Rat Resource and Research Center, Univer-
sity of Missouri) were used to obtain endogenously labeled
bone marrow mesenchymal stem cells (BMMSCs). Sixteen
male, wild-types from the inbred lineage LEWIS/H were
used as the recipient animals. All experimental protocols
were performed in accordance with the guidelines for ex-
perimental animals and approved by the Animal Committee
of Federal University of Minas Gerais.
Cell isolation and expansion
The 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 by
ushing the diaphysis using a syringe lled with Dulbeccos
modied Eagles medium (DMEM; Gibco) supplemented
with 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-cm
2
asks. Cell cul-
tures were maintained at 37

C in an atmosphere containing
5% CO
2
. Nonadherent cells were removed from the cultures
by medium change three times a week. The adherent cells
were allowed to grow near to conuence and were then
split 1:3. The cultures were passage four to six times.
Phenotypic characterization of the BMMSCs
by ow cytometry
BMMSCs were characterized as previously described,
17,21
in
accordance with the minimal criteria to dene a MSC, as
proposed by the Mesenchymal and Tissue Stem Cell Com-
mittee of the International Society for Cellular Therapy.
22
In
summary, cells were released by trypsinization after four
passages. Approximately 110
6
cells/well were incubated
for 30 minutes (min) at 4

C with the following set of mouse

antibodies: anti-CD90, anti-CD73, anti-CD54, anti-CD45
diluted 1:50 (BD Pharmingen, Franklin Lakes, USA), and
rabbit 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 4

C and then washed

with 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 data
were analyzed with CellQuest Software (BD Biosciences, San
Jose).
Preparation and characterization of the
cell-scaffold construct
Three-dimensional porous chitosangelatin scaffolds were
obtained via a freeze-drying technique
23
using glutaralde-
hyde cross-linking, as previously described.
17
The scaffolds
were produced from natural polymers, chitosan (Sigma, St.
Louis) with a degree of de-acetylation of 85% and type A
porcine skin gelatin (Vetec, RJ, Brazil). The chitosangelatin
blend at a 3:1 (chitosan:gelatin) ratio was cross-linked with
25% glutaraldehyde (Sigma, St. Louis) solution at a 0.1%
concentration. One milliliter of the homogenous mixture of
chitosan, gelatin and glutaraldehyde was poured into each
well of a cell culture plate (24-well) and used as a template
to mold cylindrical standard discs of chitosangelatin
sponges. Discs of 8 mm in diameter and 2.5 mm thick were
obtained and cut into four parts, resulting in slices of 4 mm
in radius, which were used in all of the in vitro and in vivo
procedures. The samples were distributed in a 24-well cell
culture plate, sealed and sterilized at the Nuclear Develop-
ment Technology Centre/Nuclear Energy National Commis-
sion (CDTN/CNEN) of Minas Gerais Federal University, by c
irradiation,
60
Co, 20 Grays.
Conuent cultures of the rat bone marrow mesenchymal
stem 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
10
4
/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-dimensional
system for in vivo implantation.
The morphological characterization of constructs was
performed 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 CO
2
(CPD-020 Balzers) was performed. The samples were
mounted on metallic holders, sputter-coated with gold and
observed 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 was
determined by energy dispersion spectroscopy (EDS) after 3
days in culture. This analysis was performed to detect
chemical differences between the scaffold alone and possi-
ble secretions deposited into the construct.
Surgery and construct transplantation
The tooth socket was used as an experimental bone defect,
in accordance with Kanyama et al.
24
and Mendes et al.
25
. All
of the recipient male rats were subjected to the extraction
of both upper rst molars. Cell-scaffold constructs were im-
mediately implanted on the left sockets, while the right
ones were used as a control. A micro-amalgam carrier (ABC
surgical 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 6-0
sutures (Biosut, BH, Brazil). Animals received a pasty chow
for 2 days after the surgeries and were sacriced after 5,
21, and 35 days. The craniums were xed in 10% buffered
neutral 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 tomography
After xation, the craniums were scanned by a cone beam
computerized 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 cone
of the X-ray emitter was standardized, so that the distance
from the emitter to the maxillae was xed. The CBCTs were
assigned random code numbers to allow the analysis to be
performed without bias. The CBCT was set with the follow-
ing parameters: slice thickness of 0.5 mm; pitch, 0.75; tube
voltage, 120 kV; and electrical current-time, 350 mAs. To
build the 3D images, Dental Slice software (version 2.1.1)
was employed. Figure 1(A) shows a 3D reconstruction of
the entire cranium. A transversal plane of CBCT exams pass-
ing through distal sockets of rst upper molars was
employed 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 lling. [Color gure 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 were
captured from each animal, totaling 15 images per group.
The grayscale intensity was counted with the use of the
KS300 software coupled to a Carl Zeiss image analyzer (Carl
Zeiss, Oberkochen, Germany). 8 bits sensor was used to re-
cord the data. In 8-bits-per-channel images, the intensity
values range from 0 (black) to 255 (white). Every pixel of a
grayscale image has a brightness value ranging from 0
(black) to 255 (white). The gray intensity was measured for
each image and the results were expressed by the average
percentage of bone formation. Thus, the high intensity areas
corresponded to the more mature bone tissue. To quantify
the level of bone lling, the weak intensity areas (WIA) into
the study area [SAFig. 1(D)] were discounted from the
total 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 of
formed bone was obtained through the following formula:
Mean socket lling % [TSA-WIA (mm
2
)/TSA (mm
2
)]
X100.
Histological and TRAP staining
After image acquisition by CBCT, the upper maxillaries were
dissected, demineralized in 10% ethylenediamine tetraacetic
acid (EDTA; Sigma, St. Louis), pH 7.3, dehydrated through
graded ethanol solutions and embedded in paraplast. Sagit-
tal sections of 6 lm were stained with hematoxylin eosin
and tartrate-resistant acid phosphatase staining (TRAP).
TRAP histochemistry was carried out only for the experi-
mental period of 21 days to identify the involvement of
osteoclastic 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 gure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
2778 MIRANDA ET AL. MESENCHYMAL STEM CELLS ASSOCIATED WITH POROUS CHITOSANGELATIN SCAFFOLD
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 1
h at 37

C and counter-stained with Mayers hematoxylin,

according to the manufacturers instructions. The TRAP-pos-
itive cells appeared dark red or purple.
Cell tracingImmunohistochemistry analysis
To track the fate, distribution and differentiation of the
transplanted BMMSCs after delivery on the scaffold, the
eGFP was used as a tag. The slices were deparafnized in
xylene, rehydrated through a graded series of ethanol, and
washed in PBS. The sections were then immersed in 3%
H
2
O
2
(1 h) to neutralize the endogenous peroxidase activity
and incubated with 2% BSA in PBS (1 h) for the blocking of
nonspecic binding sites. Afterward, the sections were incu-
bated overnight at 4

C with rabbit anti-GFP antibody

(1:100, Abcam; Cambridge, UK). The secondary biotinylated
anti-rabbit antibody (Universal LSAB
TM
Kit/HRP, Rb/Mo/
Goat K0690-1Dako; Glostrup, Denmark) was applied for 30
min at room temperature. After rinses in PBS, the sections
were incubated with a streptavidinhorseradish peroxidase
conjugate for 30 min at room temperature. The reaction
was revealed with a solution containing 350 lM 3,3
0
- diami-
nobenzidine (Sigma Chemical Co.) and 1% H
2
O
2
in PBS. The
slides were counterstained with Mayers hematoxylin. All
samples were processed at the same time to avoid differen-
ces among the assays. Negative controls were obtained by
the omission of the primary antibody.
Statistical analysis
Data were presented as the percentage means. The morpho-
metric results of the CBCT measurements, carried out on
control and experimental sides of the same sample unit,
were performed using the Wilcoxon test. p-values <0.05
were considered statistically signicant.
RESULTS
Characterization the of eGFP-BMMSCs and the
cell-scaffold construct
In agreement with previous studies performed by our
group,
17,21,26
the high expression of the non-hematopoietic
markers, CD54, CD73, and CD90, conrmed the purity level
of the mesenchymal stem cells. Additionally, CD45, a hema-
topoietic marker, presented a low expression. The ow cyto-
metric analysis showed that the isolated BMMSCs expressed
CD45 (3%), while a signicant percentage were positive for
CD54 (95%), CD73 (94%), and CD90 (87%) (data not
shown).
The synthesized scaffold showed pores interconnected
[Fig. 2(A,B)]. After 3 days (d) in 3D culture, the eGFP-
BMMSCs were seen widely spread on the chitosangelatin
scaffold and had established close contact with the biomate-
rial [Fig. 2(C)]. Images suggesting the deposition of particu-
lates were observed near the cells [Fig. 2(C), arrows]. The
EDS analysis detected calcium peaks in these areas [Fig.
2(D)]. In contrast, the cell-free surface of the chitosangela-
tin scaffold was characterized as an organic composite,
where only carbon and oxygen peaks were detected [Fig.
2(E)]. Residual elements that were used for the SEM proc-
essing (i.e., osmium [Os] and gold [Au]) were also detected
by EDS.
Morphometric analysis
The quantication of the newly formed bone into the
tooth sockets by gray-density evaluation is summarized in
Figure 3. The morphometric analysis expresses the mean
percentages of the areas of new bone formation in relation
to the total alveolus areas. Thus, higher rates observed in
relation to time periods (35 days > 21 days > 5 days) cor-
respond to larger bone-lling areas, identied by a grayscale
in the KS 300 software (Figs. 3 and 4). The treated alveoli
demonstrated higher densities for all evaluated time peri-
ods. The density observed at 5 days in the implanted side is
justied by the scaffold radiopacity, as shown in detail in
Figure 4(A). An occlusal view of the bone repair for each
time period is illustrated on the 3D reconstruction images
(Fig. 4, right panel).
Histological and TRAP-staining analysis
At 5 days post-surgery, histological analysis revealed the
presence of acute inammatory processes in both sockets
[Fig. 5(A,B)]. This inammation was more pronounced in
the experimental sockets, where the chitosangelatin mesh
was invaded by polymorphonuclear neutrophils and macro-
phages cells [Fig. 5(C)]. In addition, neovascularization were
seen in the experimental socket [Fig. 5(B)]. The epithelial
healing and the bone neoformation seemed delayed in the
treated sockets [Fig. 5(B)], when compared with the contra-
lateral control sockets [Fig. 5(A)]. Some multinucleated cells
were 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 with
disorganized lamellae and many marrow spaces [Fig. 5(G)].
The rests of the biomaterial, invaded by multinucleated
giant 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 a
TRAP-positive control.
FIGURE 3. CBCT morphometric results: The ratio of the new bone ll-
ing by gray-density evaluation. The p values are referred to the signif-
icance of probability of Wilcoxon test. SD, standard deviation.
* Signicant difference.
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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | OCT 2012 VOL 100A, ISSUE 10 2779
The histological ndings after 35 days of implantation
were similar to those described for 21 days. The sockets
were totally lled by bone tissue, in both sides, with more
dense 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 osteocytes
surrounded 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 gure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
2780 MIRANDA ET AL. MESENCHYMAL STEM CELLS ASSOCIATED WITH POROUS CHITOSANGELATIN SCAFFOLD
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 inammation (M). Figure N shows, in detail, the thin laminae of the biomate-
rial. IB, immature bone; MB, mature bone. [Color gure 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 2781
lamella and osteocytes aligned, suggesting a more advanced
level of bone maturation [Fig. 5(L)]. Residual scaffolds were
seen in perfect integration with loose connective tissue [Fig.
5(M)]. The chitosan layers appeared thinner than those
observed on day 21 [Fig. 5(N)]. No inammatory granula-
tion tissue or brous tissue encapsulation was observed.
Immunohistochemical analysis
On the fth day after implantation [Fig. 6(A)], only a few
isolated eGFP-positive cells were observed between the bio-
material layers. This result suggests that the stem cells
migrated outside of the carrier, possibly in an anterior
phase, before the fth day. The immunostaining was more
evident in the neoformed bone area. Labeled cells were
seen on the surface of newly formed bone trabeculae, as
well 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 teeth
presented several eGFP-positive cells [Fig. 6(E)]. Figure
6(D,F) represent images of the negative controls of the bone
and periodontal ligament, respectively.
After 21 days, no eGFP-positive cells were seen inside
the 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 gure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
2782 MIRANDA ET AL. MESENCHYMAL STEM CELLS ASSOCIATED WITH POROUS CHITOSANGELATIN SCAFFOLD
the connective tissue between the biomaterial remnants
and the alveolar healing area [Fig. 7(A)]. The labeled cells
were particularly evident close to the bone matrix and
medullary spaces and in cell aggregates, suggestive of
intramembranous ossication [Fig. 7(B,C)]. eGFP-positive
cells were also present in the walls of the blood vessels
in both the connective tissue [Fig. 7(E)] and vascular
channels of the newly formed bone [Fig. 7(F)]. Figure
7(D) is a representative image of the negative control for
21 days after implantation.
After 35 days, the immunostaining was observed speci-
cally in the osteocytes included in the bone matrix of both
immature [Fig. 8(A)] and histologically organized, mature
bone [Fig. 8(B)]. Differentiated eGFP-positive cells were per-
fectly integrated with the host osteocytes [Fig. 8(A,B)]. The
eGFP expression was maintained in the osteogenic lineage
cells localized at the bone surface [Fig. 8(C)]. Additionally,
the eGFP-positive BMMSCs were found into the squamous
layer of the epithelial tissue, suggesting its participation in
repair 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 ossication; 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 gure 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 2783
represent images of the negative control of the bone and
epithelial tissue, respectively.
DISCUSSION
In this study, we demonstrated the effects of a BMMSCs-
scaffold construct during tooth socket healing. The bone
regeneration process after tooth extraction is an interesting
experimental model to study hard tissue repair, which
includes the well-known phases of inammation, granula-
tion tissue formation, angiogenesis, and extracellular matrix
deposition.
24,25
By tracing of the eGFP-transplanted cells, it
was 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 to
the success of bone engineering. Among them, the composi-
tion and the structural characteristics of the biomaterial
employed as the cell carrier. The SEM analysis revealed the
highly porous nature of the chitosangelatin, which favored
its interaction with the seeded cells. In fact, the chitosan
gelatin 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 gure can
be viewed in the online issue, which is available at wileyonlinelibrary.com.]
2784 MIRANDA ET AL. MESENCHYMAL STEM CELLS ASSOCIATED WITH POROUS CHITOSANGELATIN SCAFFOLD
and differentiation of mesenchymal stem cells, which was in
accordance with previous in vitro and in vivo studies.
17,27,28
The BMMSCs were preferably employed in this study; they
were easily isolated from bone marrow, aspirated and
expanded in vitro. Moreover, they present a default pathway
into the osteogenic lineage.
3,27,29
Images suggestive of an
extracellular matrix with calcium peaks detected by the EDS
were observed in our construct after 3 days of culture. This
evidence of osteoblastic differentiation and secretion of a
mineralized matrix in vitro allows us to infer that the syn-
thesized chitosangelatin scaffold mimicked the natural
bone environment and represents an adequate construct for
bone 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 tissues
nor scaffold encapsulation was observed, suggesting that
this construct was nontoxic and biocompatible.
An ideal biomaterial should also be biodegradable and
possess suitable mechanical properties to support the
reconstruction of a completely natural tissue without caus-
ing an inammatory response. Our histological analysis
showed the presence of resorptive cells in the scaffold pores
at all of the evaluated time periods. These multinucleated
giant cells were more numerous and organized at days 21
and 35 than earlier in the experiment. The chitosangelatin
was progressively resorbed, at a slow degradation rate, and
there was, simultaneously, a time-compatible replacement
by bone tissue, as has been recommended for an ideal
scaffold.
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 of
osteoclasts recruitment to the healing site can promote an
imbalance in the natural process of bone formation/resorp-
tion during alveolar healing with prejudices to bone repair.
Although calcium deposition was seen on the chitosan
layers 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 layers
of the construct were TRAP-negative. Thus, the resorption
process of chitosangelatin seems to be the consequence of
a typical foreign body reaction, which evolved into a favor-
able implant-tissue interaction. As also demonstrated by
previous studies,
17
the rests of the biomaterial remained in
the connective tissue until day 30, without the formation of
a brous tissue capsule, a negative aspect described in the
literature for others biomaterials.
3,32
A more intense inammatory process was observed in
the transplanted alveoli at 5 days postsurgery. This nding
was interpreted as a normal and expected result responsible
for the apparent delay of epithelial and bone healing. The
excessive biomaterial volume overlapping tooth socket walls
represented an additional mechanical stress on the connec-
tive tissue. According to Branda o et al.,
33
the use of materi-
als to improve bone neoformation in alveoli following dental
extraction, can delay bone repair, which was conrmed in
our study, during the initial phase of healing. Nevertheless,
in a smaller extension than in the control sockets, after 5
days, newly formed bone trabeculae could be seen in the
apical and medium thirds of the sockets. The presence of
the scaffold was not able to prevent the healing process. At
days 21 and 35, the healing was complete and histological
aspects observed in the control and transplanted sockets
were 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
In
transplantation models, a difcult issue of concern is how
to access the fate of the donor cells and maintain their
labeling in a host environment for the entire experimental
period. In our work, we used eGFP-labeled BMMSCs.
Although it is considered a marker of choice for many types
of cell transplantation studies,
38,39
eGFP may lose its direct
uorescence during tissue processing. For this reason, im-
munostaining was employed, and we achieved an effective
cell tracing. We demonstrated both the ability of the
BMMSCs to maintain eGFP labeling until 35 days after the
construct 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 the
injury, 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 potential
of the chitosangelatin scaffold to act as a carrier for
BMMSCs and to promote the development of these cells
into an osteoblastic lineage. Moreover, the transplanted
BMMSCs have, for the rst time, been shown to migrate
throughout all of the lesioned tissues and also to contribute
to epithelial healing and wound neovascularization.
CONCLUSIONS
BMMSCs transplanted in a chitosangelatin carrier into a
tooth socket environment differentiated into bone, epithelial
and vascular cells and contributed signicantly to the matu-
ration of newly formed bone. In summary, chitosangelatin
seeded with BMMSCs is an efcient construct for bone engi-
neering, which can be a promising strategy for craniofacial
reconstructions.
ACKNOWLEDGMENTS
The authors would like to thank Helio Chiarini-Garcia, Ph.D.
for assisting in the SEMstudies and members of CONEST (Con-
sultoria em Estatstica e PesquisaBelo Horizonte/MG) for
statistical assistance.
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