Bioceramic-collagen scaffolds loaded with human adipose-tissuederived stem cells for bone tissue engineering

Neda Daei-farshbaf • Abdolreza Ardeshirylajimi • Ehsan Seyedjafari •

Abbas Piryaei • Fatemeh Fadaei Fathabady • Mehdi Hedayati • Mohammad Salehi •

Masoud Soleimani • Hamid Nazarian • Sadegh-Lotfalah Moradi •

Mohsen Norouzian

Received: 22 June 2013 / Accepted: 16 December 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The combination of bioceramics and stem cells

has attracted the interest of research community for bone

tissue engineering applications. In the present study, a

combination of Bio-Oss� and type 1 collagen gel as scaffold

were loaded with human adipose-tissue derived mesenchy-

mal stem cells (AT-MSCs) after isolation and characteriza-

tion, and the capacity of them for bone regeneration was

investigated in rat critical size defects using digital mam-

mography, multi-slice spiral computed tomography imaging

and histological analysis. 8 weeks after implantation, no

mortality or sign of inflammation was observed in the site of

defect. According to the results of imaging analysis, a higher

level of bone regeneration was observed in the rats receiving

Bio-Oss�-Gel compared to untreated group. In addition,

MSC-seeded Bio-Oss-Gel induced the highest bone recon-

struction among all groups. Histological staining confirmed

these findings and impressive osseointegration was observed

in MSC-seeded Bio-Oss-Gel compared with Bio-Oss-Gel.

On the whole, it was demonstrated that combination of AT-

MSCs, Bio-Oss and Gel synergistically enhanced bone

regeneration and reconstruction and also could serve as an

appropriate structure to bone regenerative medicine and

tissue engineering application.

Keywords Mesenchymal stem cells � Tissue

engineering � Bone � Bioceramic � Critical-size defect

Introduction

Thyroid hormones have critical functions for bone growth

and turnover process [1] which are induced by acting

directly or indirectly on bone cells including osteoblasts

and osteoclast precursors [2, 3]. In hypothyroidism, bone

resorption is increased because of the unknown mechanism

that causes osteoclast activation and growth, and finally

could lead to the development of osteoporosis. Clinical and

laboratory studies have revealed that in hypothyroid rats,

osteogenesis is decreased in the site of bone defects [4]. In

addition, patients prescribed a high dose of levothyroxine

for the treatment of thyroid hormones lackness, may

experience effects that mimic hyperthyroidism [5]. So

generally, thyroid hormones deficiency is considered to

cause abnormal skeletal regeneration [6].

There are several clinical methods such as bone grafting

and tissue engineering for the treatment of bone defects [7].

Bone graft methods are categorized into three subdivisions

N. Daei-farshbaf � A. Piryaei � F. Fadaei Fathabady �H. Nazarian � M. Norouzian (&)

Departments of Anatomy and Cell Biology, Shahid Beheshti

University of Medical Sciences, Tehran, Iran

e-mail: norouzian@sbmu.ac.ir

A. Ardeshirylajimi � S.-L. Moradi

Departments of Stem Cell Biology, Stem Cell Technology

Research Center, Tehran, Iran

e-mail: r.ardeshiry.62@gmail.com

E. Seyedjafari

Department of Biotechnology, College of Science, University of

Tehran, Tehran, Iran

M. Hedayati

Endocrine Research Center, Shahid Beheshti University of

Medical Sciences, Tehran, Iran

M. Salehi

Department of Biotechnology, Faculty of Medicine, Shahid

Beheshti University of Medical Sciences, Tehran, Iran

M. Soleimani

Department of Hematology, Faculty of Medical Science, Tarbiat

Modares University, Tehran, Iran

123

Mol Biol Rep

DOI 10.1007/s11033-013-2913-8

including autologous, allogenic and xenogenic transplan-

tations. Autografts are in some way a gold standard

because they avoid most problems related to transfection

and rejection. However, they do involve significant donor

site morbidity and chronic donor shortages. Allografts are

made of tissue that derived from other individuals of the

same species. This tissue must be thoroughly sterilized in

order to avoid immunological reactions in the receiver and

infections. Their limitations include donor shortages and

risks of infections as mentioned above. Xenograft advan-

tage of being available in different shapes and sizes, but

they also have a non-negligible risk of immunological

reactions and infection. Therefore, scientists should take

the advantage of new methods referred to as tissue engi-

neering and regenerative medicine for the treatment of

patient’s tissue defects or damages [7, 8].

For tissue engineering, stem cells and scaffolds are the

two essential components [9]. There are many important

requirements for scaffolds such as biocompatibility, bio-

degradability and providing conditions resembling host

tissue in order to prevent the rejection of implanted cells or

tissues [10, 11]. In this study, we used a combination of

Bio-Oss� and type 1 collagen gel as scaffold for bone

regeneration applications. Bio-Oss is a deproteinized

bovine bone material and has unique features such as a

condensed strength of 35 Mpa and its highly porous nature

(75–80 % of the total volume) which increase the surface

area of the scaffolds. Bio-Oss is one of the several bioce-

ramics that are commonly used for treatment of osseous

defects, dental implant therapy, and periodontal defects

[10]. Furthermore, alternative to this sponge-type scaffold,

we used a type 1 collagen gel (from rat tail) which can

enhance the proliferating potential of stem cells and culture

mineralization [12]. Collagen is a fibrous protein that is

mostly found in skin, bone, tendons and other connective

tissues, and is consisted of three alpha-chains which can

combine to form a rope-like triple helix, providing tensile

strength to the extracellular matrix (ECM).

Stem cells have unique features such as intensive

regenerating potential, immunosuppressive features and

strong plasticity required for clinical trial and cell therapy

[13–15]. Mesenchymal stem cells (MSCs), especially with

the origin of bone marrow, are an efficient source for

regenerative medicine and tissue engineering applications

[16, 17]. However, preparation of MSCs from patient’s

bone marrow is an invasive procedure and suffers from

ethical issues. Nowadays, scientists have introduced other

MSCs sources such as human adipose tissue [18–20].

Isolation of MSCs from adipose (AT-MSCs) tissue is a

simple process and fat harvesting is much simpler versus

bone marrow aspiration. In addition, the yield of stem cells

from adipose tissue is higher than those from bone marrow

[21, 22]. Scarce morbidity of AT-MSCs during isolation

and low amount of needed factors for their growth and

expansion are other reasons for the priority of AT-MSCs

over BM.MSCs [19].

The purpose of the present study was to conduct tissue

engineering in hypothyroid models which suffers from

decreased bone regenerating ability using a combination of

Bio-Oss and collagen type I loaded with AT-MSCs.

Materials and methods

Isolation of human Ad-MSCs

The AT-MSCs were isolated from adipose tissue samples

collected at operations or liposuctions from five donors

(mean age 40 ± 5, Erfan Hospital, Tehran, Iran) after

informed consent according to guidelines of the Medical

Ethics Committee, Shahid Beheshti University of Medical

Sciences and Health services (Tehran, Iran). After washing

tissue, it was digested with collagenase type I (Sigma) and

incubated for about 1 h. After centrifugation, the supernatant

was removed and the cell pellet was treated with RBC lysis

buffer (Dako, Glostrup, Denmark) at room temperature (RT)

for 5 min. AT-MSCs were expanded in T-75 polystyrene

flasks in maintenance medium consisting of Dulbecco’s

modified Eagle’s medium (DMEM, Gibco, Grand Island,

NY, USA), 10 % fetal bovine serum (FBS, sigma), and

100 mg/mL streptomycin and 100 U/mL penicillin (1 %

antibiotics, Gibco). They were grown at 95 % air, 37 �C and

5 % CO2 atmosphere. Maintenance medium was replaced by

growth medium with 15 % FBS. Growth medium was

changed every 2 or 3 days. Unattached cells were discarded

by refreshing the medium. After reaching confluence (about

80–85 %) during 10 days, the cells were dissociated with

trypsin (2 min in 37 �C, 5 % CO2) and replated. Cells from

passages two were used for all procedure.

Characterization of isolated human Ad-MSCs

Flow cytometer surface markers

The human AT-MSCs were detached from the tissue cul-

ture flasks after 2 weeks in vitro with trypsin/EDTA and

counted. About 2 9 105 cells were divided into aliquots

and centrifuged at 1,200 rpm for 5 min at RT. The pellet

was resuspended in human serum and incubated for 30 min

on ice. After centrifugation at 1,000 rpm for 5 min, the

pellet was resuspended in 3 % (v/v) human serum albumin

(HSA)/PBS and incubated with appropriate antibodies

including fluorescent isothiocyanate (FITC)-conjugated

mouse anti-human CD45 (leukocyte common antigen),

Phycoerythrin (PE)-conjugated CD105 (Endoglin or SH2)

CD34, CD90and CD10 for 1 h on ice, washed twice in PBS

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and centrifuged for 5 min. The cells were resuspended in

100 ll of PBS and studied by a Coulter Epics-XL flow

cytometer (Beckman Coulter, CA, USA). An isotype con-

trol with FITC- or PE-labeled antibodies was included in

each experiment, and specific staining was measured from

the cross point of the isotype using a specific antibody

graph. The corresponding histograms were created by Win

MDI 2.8 software (Scripps Institute, CA, USA).

Osteogenic and adipogenic differentiation

The potential of the isolated cells to differentiate into

osteogenic and adipogenic lineages was examined. For

osteogenic differentiation, human AT-MSCs were induced

for 3 weeks by DMEM supplemented with 10 % FBS,

0.1 mM dexamethasone, 10 mM b-glycerophosphate, and

50 mM ascorbate 2-phosphate. The medium was replaced

every 2 days up to 3 weeks. The cells were fixed with cold

4 % paraformaldehyde for 20 min at 4 �C and evaluated by

specific histochemical staining for mineralization with

alizarin red staining kit. The staining was examined with a

phasecontrast microscope (Nikon, Tokyo, Japan).

For adipogenesis, the cells were incubated in adipogenic

inductive medium. This medium consisted of DMEM

supplemented with 10 % FBS, 1 mM dexamethasone,

200 mM indomethacin, 500 mM isobutyl-methyl xanthine

and ascorbate 2-phosphate for 18 days. After 18 days, the

cells were evaluated for adipocyte identification, using oil

red O-staining. Briefly, cells were fixed in cold 4 % para-

formaldehyde for 20 min at 4 �C, washed washed with

PBS two times, and stained with oil red O-solution for

5–10 min at 37 �C, the cells were washed again with PBS

three times and depicted by the light microscope. All

control groups without the differentiation inductive med-

ium were maintained in parallel to the differentiation

experiments and stained in the same manner.

Cell labeling

Before the cells seeded on scaffold, in passage two were

labeled. PKH26 red fluorescent cell linker kit (Sigma-

Aldrich) was used according to the manufacturer’s

instructions. In brief, human AT-MSCs were suspended in

a mixture of 1 mL of diluent C (Sigma-Aldrich) and 1 mL

of 4 9 10 - 6 molar PKH26 dye (Sigma-Aldrich) in

polypropylene tubes at room temperature. The samples

were then immediately mixed by pipetting and incubated at

25 �C for 3 min. At the end of this period, the staining

reaction was stopped by adding equal volumes of 1 % fetal

bovine serum for 1 min. Excessive staining solution was

removed by centrifugation of the cells at 400 g for 10 min.

The cells were resuspended in the complete medium and

examined by fluorescence microscopy for labeling.

Cell seeding on Bio-Oss� and type I collagen gel

After labeling stem cells, these cells were seeded on Bio-

Oss� particles (Geistlich Pharma AG, Switzerland) and

were incubated on 37 �C and 5 % CO2 about 8 h for

attaching. Then optimized protein concentration of Colla-

gen type I (GIBCO�) was provided according to the

manufacturer’s instructions. After collagen was provided,

appropriate numbers of AT-MSCs-seeded Bio-Oss� parti-

cles rinsed into it and then implanted in the site of rat

critical size calvarial defect immediately.

Animal model

All animal experiments were performed in accordance with

the Shahid Beheshti University of Medical Sciences and

Health services (Tehran, Iran) guidelines. Nine 6–7 weeks-

old male Wistar rats with a body weight of 190–200 g (five

animals per group, Razi Institute, Karaj, Iran) were used as

transplant recipients. Before surgery, the animals were kept

in clean and standard air conditions at a constant temperature

of 21 �C with a 12-h light/day cycle. They had ad libitum

access to drinking water and a standard laboratory rat food

pellet diet. Rats were anesthetized by intramuscular injec-

tion of 50 mg/kg ketamine hydrochloride with 5 mg/kg

diazepam under sterile conditions. After obtaining blood

samples from corner of their eyes, thyroid hormones (T3,

T4) were measured by radioimmunoassay kit. Digestion of

4 mg powdered methimazole (Tehran, Iran hormone) dis-

solved in 100 cc distilled water for 4 weeks induced hypo-

thyroidism. Thyroid hormones were determined again after

methimazole treatment. After 1 week interval, these rats

were watered with methimazole solution again for 4 weeks

and this schedule was performed all the period of study.

Surgery and transplantation procedure

According to the protocol is mentioned above hypothyroid

rats were anesthetized. Then, skin and periosteum were

raised to expose the calvaria. In all of the rats, an 8 mm

critical-size defect was made in the parietal bone by a

dental bur. Constant saline irrigation was provided and the

dura mater was kept intact. The procedure was performed

under sterile conditions. After transplantation, the skin

incision was closed with nylon sutures. The animals were

kept in sterile condition with enough water and foods. Nine

hypothyroid rats randomly divided into three groups:

control group with empty defect, first experimental group

defect filled with scaffold (Bio-Oss� and type I collagen

gel) only, second experimental group defect filled with

mentioned scaffold loaded with AT-MSCs. The rats were

sacrificed 6 weeks after transplantation and their calvarias

with grafts were harvested.

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Digital mammography and multislice spiral computed

tomography (MSCT) imaging analysis

After 8 weeks, the animals were euthanized, and their

craniums were excised and placed in 10 % formalin. The

cranium samples were then radiographed under direct

digital mammography equipment (KonicaMinolta, Regius

model 110HQ) and were also scanned using a spiral high-

resolution computed tomography (CT) system (Siemens,

SOMATOM Sensation) in multislice mode. The radio-

graph images from digital mammography were scored by

two independent radiologists. To quantify the level of bone

regeneration via MSCT, a 9-mm circular region of interest

was placed in each CT image. The area of newly formed

bone was quantified relative to the original calvarial defect

[23].

Histological analysis

The fixed cranium samples were decalcified in ethylene

di-amine tetra acetic acid/HCl and embedded in paraffin.

For light microscopy studies, histological sections with

3–5 lm thickness were obtained and stained with

hematoxylin and eosin (H&E). The area of newly formed

bone was quantified using a computer-assisted Image-Pro

Plus System (Media Cybernetics, Silver Springs, MD,

USA).

Statistical analysis

Each experiment was performed at least 3 times in vivo.

MSCT and H&E data were reported as mean ± standard

deviation (SD). One-way analysis of variance (ANOVA)

was used to compare the results. All analyses were per-

formed using SPSS 17.0 software. P values of\0.05 were

considered as statistically significant.

Results

Characterization of isolated AT-MSCs

Isolated AT-MSCs were passaged two times and then were

evaluated through its morphology and surface markers.

Flow cytometric analysis demonstrated that the AT-MSCs

expressed CD90, CD10 and CD105, whereas they were

negative for CD34 and CD45 (Fig. 1). Osteogenic differ-

entiation of stem cells was confirmed using alizarin red

staining with the presence of red-colored mineralized area

in the culture (Fig. 2a). Following oil red O-staining, lipid

droplets were obvious after culturing the cells under adi-

pogenic medium after 21 days (Fig. 2b). Undifferentiated

AT-MSCs were negative in both staining (Fig. 2c). These

results showed that the isolated cells had the multipotency-

related properties of genuine AT-MSCs.

Fig. 1 Immunophenotyping of human adipose tissue derived mes-

enchymal stem cells (AT-MSCs) using flow cytometry. Mesenchymal

stem cells (MSCs) were positive for CD10, CD90 and CD105. These

cells were negative for CD34, and CD45. The results are represen-

tative of three independent experiments

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In vivo bone regeneration

Gross examinations

No mortality or sign of complication was observed during

study in any of the animals. As we showed in Fig. 3a, no

sign of wound fester, bleeding, infection, effusion, or scalp

edema was observed at the site of osseous defects after

surgery. After 8 weeks of implantation, all samples were

retrieved for evaluation of new bone reconstruction. No

sign of inflammation or Bio-Oss particle disintegration was

observed at the site of calvarial defects (Fig. 3b). Evalua-

tion of the untreated control group showed negative

spontaneous mineralization and bone healing in the osse-

ous defect after the period of study (Fig. 3b). As we

showed in Fig. 4c, all implanted particles were well inte-

grated into the calvarial osseous defect with no sign of

encapsulation or prominent foreign body reaction. More-

over, the Bio-Oss particles also adhered strongly to the host

bone tissue without any fixation.

Evaluation of bone regeneration

Quantification of newly formed bone on the fixed calvar-

ium specimens after 8 weeks of implantation was per-

formed by digital mammography and MSCT. Results of the

radiological analysis of different groups have been shown

in Fig. 4. Qualitatively, the data revealed reconstruction of

calvarial osseous defects after implantation of particles.

Results of the quantitative analysis of the regenerated bone

areas demonstrated that a higher amount of new mineral-

ized osseous tissue was observed in the groups that

received stem cell-seeded Bio-Oss-Gel compared to Bio-

Oss-Gel and untreated control groups (P \ 0.05). Results

of MSCT have shown that the area of newly formed bone

in stem cell-seeded Bio-Oss-Gel exhibited a complete

regeneration of the calvarial osseous defect compared to

other groups (Fig. 5). In addition, different levels of void

defects were detected in all control groups with no treat-

ment. In both CT and digital mammography, bone regen-

eration was observed to begin from the edges of the

osseous defect toward the center. Since the area of newly

bone formed in untreated groups was not significant, so the

8-mm-diameter osseous defects were demonstrated to be a

critical-size rat calvarial osseous defect in this study.

Finally, histological evaluation was performed to trace

AT-MSCs in the area of newly bone formed in calvarial

osseous defect (Fig. 6). The area of newly formed bone

was revealed as mean ± SD and is shown in Fig. 7. The

highest amount of newly formed bone tissue was perceived

in the rats treated with AT-MSC-seeded Bio-Oss-Gel

(P \ 0.05). Although the healing pattern of calvarial

osseous defects was similar in animals that treated with

Bio-Oss-Gel and AT-MSC-seeded Bio-Oss�-Gel, but in

the latter group, the healing was much more significant

than animals received cell-free Bio-Oss-Gel and control

groups (P \ 0.05).

Discussion

Bone defects are one of the most important and frequent

problems in human health care. Since, bone turnover is a

complicated and long process, biomimetic approach in

tissue engineering and regenerative medicine have been

shown to be an efficient way to design biocompatible and

osteoinductive scaffolds for bone implants applications.

These problems are much increased in patients with

hypothyroidism. Over the four decades, bioceramics have

been known as classic bone graft substitutes. In addition,

there are many polymers that have been used in tissue

engineering and their number are increasing However,

Fig. 2 Morphology of stem cells under two induction medium, after

a 21-day culture under basal medium (a), oil red O-staining of stem

cells after a 21-day culture under adipogenic induction medium

(b) and alizarin red staining of stem cells after a 21-day under

osteogenic induction medium (c), all with low magnification

Mol Biol Rep

123

findings showed that combination of stem cells and bi-

oceramics has been shown more efficient to reconstruct the

bone in osseous defects than when used individually [24–

26].

In the present study, we aimed to evaluate the osteoin-

ductivity of human MSCs seeded on natural bioceramic

implanted in hypothyroid rat calvarial defects. One of the

most important bioceramics that has been studied in bone

tissue engineering and regenerative medicine is calcium-

phosphate based ceramics. Among them, many studies

have reported the highly efficient in vitro and in vivo

performance of hydroxyapatite (HA) as bone implants. HA

could be used in two ways: as scaffolds or as nanoparticles

for coating the surface of other scaffolds. Finding of Bigi

et al. [27] showed the improved adherent of bone to the

surface of HA-coated alloy implant. Dinarvand et al. have

used a combination of bioceramics (HA, bioactive glass

(BG) tricalcium phosphate (TCP) particles) and polymeric

nanofibers (electrospun poly(L-lactic acid) (PLLA) nanof-

ibers) to evaluate their osteogenic potential in vivo. Their

findings showed the highest bone reconstruction in animals

treated with nanofibers coated simultaneously with HA and

BG [28]. In another study, Sollazzo et al. [29] demon-

strated the increased osteogenic differentiation of periph-

eral blood human MSCs seed on Bio-Oss. They evaluated

the in vitro expression of osteoblastic transcriptional

Fig. 3 Critical-size defect created in rat calvaria without (a) or with (b) implanted scaffold and after 8 week treatment (c)

Fig. 4 Digital mammography images of the rat calvarial 8 weeks after treatment: untreated control group (a), Bio-Oss-Gel (b) and MSC-seeded

Bio-Oss-Gel (c)

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factors such as RUNX2 and bone-related genes; SPP1 and

FOSL1. Komlev et al. [30] evaluated bone regeneration

potentials of MSC-seeded Bio-Oss in immunodeficient

mice ectopically and they observed significant bone

regeneration.

In another study, Asti et al. investigated the surface

modification of Bio-Oss with poly-D,L-lactide (PLA) and

then seeded human osteosarcoma cell line SAOS-2 on

these scaffold. Their findings suggested that Bio-Oss-PLA

could be a valuable material for bone tissue engineering

[31]. Kim et al. investigated osteogenic potential of the

apatite-coated PLGA/HA particulates and Bio-Oss in crit-

ical size bone defect model. Bio-Oss showed a little

increased new bone regeneration in comparison to PLGA/

Fig. 5 MSCT images of the rat calvarial after 8 week of treatment: untreated control group (a), Bio-Oss-Gel (b) and MSC-seeded Bio-Oss-Gel

(c)

Fig. 6 Optical micrographs of the defects stained with H&E:

untreated control group (a, d), Bio-Oss-Gel (b, e) and MSC-seeded

Bio-Oss-Gel (c, f) with two magnifications, labeled stem cells by

PKH26 red fluorescent cell linker kit (c), (a, b, c) 10X and (d, e,

f) 40X. Labeled AT-MSCs by PKH26 red fluorescent cell linker kit

after 8 weeks after treatment (g)

Fig. 7 Area of newly formed

bone resulting from the

quantification of MSCT (a) and

H&E (b) data. The significant

difference (P \ 0.05) has been

shown between the groups are

indicated by asterisks. Groups

specified are an untreated

control group (a), Bio-Oss-Gel

(b) AT-MSC-seeded Bio-Oss-

Gel (c)

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HA particulates in the site of defect [32]. From a gross

view, after 8 weeks, no sign of inflammation or bleeding

was observed in the site of implantation for any of the

animals. This observation was confirmed by histological

study and showed the in vivo biocompatibility of our

scaffolds in the animal model. Two independent quantita-

tive methods were used to evaluate the amount of miner-

alization and bone reconstruction during the treatment

period. Interestingly, similar results were found from both

X-ray imaging and MSCT which demonstrated that MSC-

Bio-Oss-Gel scaffold induced the highest level of bone

reconstruction compared to that result from scaffolds

without stem cells. These results demonstrated that not

only human MSCs were not rejected, also bone regenera-

tion was enhanced via these cells in the site of osseous

defect. Presence of human MSCs in the newly formed bone

in the site of defect was confirmed by PKH26 red staining

results.

As the Bio-Oss collagen product has not received suf-

ficient attention in the tissue engineering literatures, in the

present study we used rat critical-size calvarial model for

evaluation of stem cell-seeded Bio-Oss effects on bone

regeneration. By the way, our data demonstrated that

combination of AT-MSCs, Bio-Oss and Gel synergistically

enhanced osseous regeneration and reconstruction higher

than that observed for Bio-Oss and Gel.

Finally, our data from radiology photography and

MSCT were confirmed by pathological analysis. In addi-

tion, penetration of the newly formed bone into the scaf-

folds obviously indicated the capability of AT-MSC-Bio-

Oss-Gel scaffolds to induce an efficient amount of osteo-

integration which is critical for an absolute healing of

osseous lesions and defects.

Conclusion

In this study, we demonstrated that AT-MSC-seeded Bio-

Oss-Gel could be used as an appropriate support to guide

bone reconstruction. In addition, Bio-Oss-Gel could be a

suitable tissue-engineered matrix to support stem cells for

bone regenerative applications.

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