Mol Biol Rep DOI 10.1007/s11033-013-2913-8

Bioceramic-collagen scaffolds loaded with human adipose-tissue derived 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 Ó 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-OssÒ and type 1 collagen gel as scaffold were loaded with human adipose-tissue derived mesenchymal stem cells (AT-MSCs) after isolation and characterization, and the capacity of them for bone regeneration was investigated in rat critical size defects using digital mammography, 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-OssÒ-Gel compared to untreated group. In addition, MSC-seeded Bio-Oss-Gel induced the highest bone reconstruction 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 ATMSCs, 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

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: [email protected] A. Ardeshirylajimi  S.-L. Moradi Departments of Stem Cell Biology, Stem Cell Technology Research Center, Tehran, Iran e-mail: [email protected] 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

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

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including autologous, allogenic and xenogenic transplantations. 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 advantage 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 engineering 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, biodegradability 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-OssÒ 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 bioceramics 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

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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 culture 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 control 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 % paraformaldehyde 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 medium 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 (SigmaAldrich) 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-OssÒ and type I collagen gel After labeling stem cells, these cells were seeded on BioOssÒ particles (Geistlich Pharma AG, Switzerland) and were incubated on 37 °C and 5 % CO2 about 8 h for attaching. Then optimized protein concentration of Collagen type I (GIBCOÒ) was provided according to the manufacturer’s instructions. After collagen was provided, appropriate numbers of AT-MSCs-seeded Bio-OssÒ particles 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 weeksold 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 injection 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) dissolved in 100 cc distilled water for 4 weeks induced hypothyroidism. 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-OssÒ 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 highresolution computed tomography (CT) system (Siemens, SOMATOM Sensation) in multislice mode. The radiograph 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).

Fig. 1 Immunophenotyping of human adipose tissue derived mesenchymal stem cells (AT-MSCs) using flow cytometry. Mesenchymal stem cells (MSCs) were positive for CD10, CD90 and CD105. These

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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 performed 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 differentiation 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 adipogenic 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 multipotencyrelated properties of genuine AT-MSCs.

cells were negative for CD34, and CD45. The results are representative of three independent experiments

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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

In vivo bone regeneration

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 treatment. In both CT and digital mammography, bone regeneration 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-OssÒ-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).

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). Evaluation of the untreated control group showed negative spontaneous mineralization and bone healing in the osseous defect after the period of study (Fig. 3b). As we showed in Fig. 4c, all implanted particles were well integrated into the calvarial osseous defect with no sign of encapsulation or prominent foreign body reaction. Moreover, 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 calvarium specimens after 8 weeks of implantation was performed 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 mineralized osseous tissue was observed in the groups that received stem cell-seeded Bio-Oss-Gel compared to BioOss-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

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,

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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)

findings showed that combination of stem cells and bioceramics 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 osteoinductivity 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 calciumphosphate 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

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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) nanofibers) 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] demonstrated the increased osteogenic differentiation of peripheral blood human MSCs seed on Bio-Oss. They evaluated the in vitro expression of osteoblastic transcriptional

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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-OssGel (c)

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 critical size bone defect model. Bio-Oss showed a little increased new bone regeneration in comparison to PLGA/

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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 quantitative methods were used to evaluate the amount of mineralization and bone reconstruction during the treatment period. Interestingly, similar results were found from both X-ray imaging and MSCT which demonstrated that MSCBio-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 regeneration 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 sufficient 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 addition, penetration of the newly formed bone into the scaffolds obviously indicated the capability of AT-MSC-BioOss-Gel scaffolds to induce an efficient amount of osteointegration which is critical for an absolute healing of osseous lesions and defects.

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Conclusion

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In this study, we demonstrated that AT-MSC-seeded BioOss-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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Bioceramic-collagen scaffolds loaded with human adipose-tissue derived stem cells for bone tissue engineering.

The combination of bioceramics and stem cells has attracted the interest of research community for bone tissue engineering applications. In the presen...
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