Cell Biochem Biophys DOI 10.1007/s12013-013-9726-1
ORIGINAL PAPER
Differentiation of Rabbit Bone Mesenchymal Stem Cells into Endothelial Cells In Vitro and Promotion of Defective Bone Regeneration In Vivo Jinzhong Liu • Chao Liu • Bin Sun • Ce Shi • Chunyan Qiao • Xiaoliang Ke Shutai Liu • Xia Liu • Hongchen Sun
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Ó Springer Science+Business Media New York 2013
Abstract Tissue engineering strategies often fail to regenerate bones because of inadequate vascularization, especially in the reconstruction of large segmental bone defects. Large volumes of vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesis are difficult to obtain. In this study, we simulated bone healing by co-culturing differentiated ECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolic acid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairing rabbit mandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells. After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs. Immunohistochemical staining analysis with CD34 showed that the induced cells had the characteristics of ECs and MSC. The induced ECs were co-cultured in vitro, and the induction of MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR) staining experiments were performed, and the Coomassie brilliant blue total protein and ALP activity were measured. The MSCs proliferated and differentiated
J. Liu B. Sun C. Shi C. Qiao X. Ke X. Liu H. Sun (&) Department of Oral Pathology, School of Stomatology, Jilin University, Changchun 130021, People’s Republic of China e-mail:
[email protected] J. Liu e-mail:
[email protected] C. Liu Najing Stomatological Hospital, Nanjing, Jiangsu, People’s Republic of China S. Liu Yantai Stomatological Hospital, Yantai, Shandong, People’s Republic of China
into osteoblast-like cells through direct contact between the derived ECs and MSCs. The co-cultured cells were seeded on PLGA scaffold to repair 1 cm mandible defects in the rabbit. The effectiveness of the repairs was assessed through soft X-ray and histological analyses. The main findings indicated that MSCs survived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. The results demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis and promoted new bone formation. This study may serve as a basis for the use of in vitro co-culturing techniques as an improvisation to bone tissue engineering for the repair of large bone defects. Keywords Bone tissue engineering Mesenchymal stem cells Endothelial cells Bone repair
Introduction Accidents, tumor resection, or trauma can be the leading causes for large skeletal defects which can inflict tremendous pain to the patient and decrease their quality of life. The development of tissue engineering technology has led to an increasing number of researchers adapting it to repair bone defects [1, 2]. But this technology is also besought with a number of problems owing to the innate complex nature of bone anatomy and physiology. Among the developments that have taken place in this field, use of mesenchymal stem cells (MSCs) have shown a great deal of promise. They offer several advantages such as good histocompatibility reactions and plasticity and have the flexibility to be used as seed cells. MSCs can be conveniently obtained and they can quickly repair tissues. Bone MSCs can proliferate abundantly and have the potential for differentiation into a variety of cells. MSCs possess an
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unexplored plasticity to differentiate into cardiovascular cell types, including endothelial cells (ECs) [3, 4]. Besides this potential one of the most exciting features is their immunomodulatory ability which apparently allows them to regulate and evade the immune system thereby circumventing the need for lifelong immunosuppressive drugs. However, one problem that commonly occurs is the central necroses of the engineered bone tissue because of inadequate blood supply leading to deprivation of nutrition and oxygen [5]. Thus adequate vascularization is a crucial component in repairing large segmental bone defects through tissue engineering and methods to improve local vascularization are not yet popular. In this context ECs have attracted considerable attention owing to their capability to enhance bone vascularization. Previous studies have shown that ECs and growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (BFGF), are important for osteogenesis. These growth factors can also accelerate neovascularization during the regeneration of damaged tissues [6, 7]. ECs can be obtained from blood vessels, kidney or vascular endothelial cell (VEC) line. Mature ECs are terminally differentiated cells with low proliferative potential. They can be used as a replacement for damaged ECs. However, the acquisition of these cells can cause secondary bone lesions, immunologic rejection in case of allografts, and poor long-term limit their clinical application. Hence, deriving sufficient autologous ECs to promote vascularization is crucial [8, 9]. Therefore exploiting the potentials of both MSCs as well as ECs has been the current trend. But the best methodology to obtain the osteogenic stimulation of the MSCs and ECs remains unclear; the choices being a direct contact between the two cells or an interaction between MSCs and ECs in vitro prior to in vivo usage [10]. We hypothesize that MSCs can be induced into ECs in vitro and that the induced ECs can differentiate MSCs into osteoblasts through direct contact or interaction to promote bone formation [11]. Hence to augment the bone’s natural biological response to tissue damage, osteogenic cells, growth factors, and biomaterial scaffolds are incorporated in tissue engineering strategies to achieve repair and restoration of the damaged tissue. An ideal biomaterial scaffold will provide mechanical support to an injured site and also deliver growth factors and cells into a defective site to encourage tissue growth. In addition, this biomaterial should degrade in a controlled manner without causing a significant inflammatory response. A scaffold serves as a matrix for tissue formation and also plays an important role in bone tissue engineering [12, 13]. Polylactide glycolic acid (PLGA) was used as a scaffold in the development of an in vivo substitute for bone [14]. PLGA scaffolds are suitable for tissue engineering because
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of their proper pore size and surface characteristics for cell attachment and proliferation [15]. In this study, we simulated bone healing by co-culturing differentiated ECs and MSCs either on a culture plate or on a PLGA scaffold in vitro. We also evaluated their effectiveness in repairing rabbit mandible defects in vivo and achieving osteogenesis.
Materials and Methods Preparation of PLGA Scaffold We prepared a PLGA scaffold following the method described by Wu et al. [16]. Approximately 1 g of PLGA (85:15) was completely dissolved in dioxane to make a 10 mL solution. The solution was poured into an aluminum dish 5 cm in diameter. The dish was frozen in a refrigerator at -20 °C for 24 h and then freeze dried in a vacuum drying machine for 60 h. The freeze-dried PLGA sheets were cut into small pieces of 10 9 10 9 5 mm3 and then sterilized with gamma radiation (60Co) before use. Pore size was measured using a scanning electron microscope (X650, HITACHI). Isolation and Culture of Rabbit MSCs and Induction of ECs The animal experiments were approved by the Animal Care and Use Committee of Jilin University. Rabbits weighing 2–2.5 kg were anesthetized through intramuscular administration of ketamine (60 mg/kg) and xylazine (8 mg/kg). Bone marrow MSCs were harvested from the femur under sterile conditions through density gradient centrifugation. The cells were suspended in low-glucose Dulbecco’s modified eagle’s medium with 15 % fetal bovine serum (Gibco) and then incubated at 37 °C with 95 % humidity and 5 % CO2. When the cells reached 80 % confluence after day 7 to day 9, they were harvested and then cultured on the surface of the PLGA scaffold. Growth conditions were observed on days 3, 7, 9, and 14. The cells of passage three were used for the induction of ECs. The MSCs of passage three were cultured in M199 medium supplemented with 10–6 M desacortone, 50 ng/ mL VEGF (Invitrogen, Carlsbad, CA, USA), 5 ng/mL BFGF (Invitrogen), 1 % L-glutamine, 10 % fetal bovine serum (Invitrogen), 100 U/mL penicillin G (Invitrogen), and 100 lg/mL streptomycin (Invitrogen). Identification of Induced ECs The MSCs were cultured in M199-conditioned medium for 2 weeks. Anterior ethmoid bone was digested for 2 h with 0.25 % trypsin. CD34 staining was performed using an
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immunohistochemistry staining system (Zhongshan Goldenbridge Biotechnology Co.). The cells were fixed with 4 % paraformaldehyde for CD34 staining. After washing with phosphate-buffered saline (PBS), the cells were permeabilized with 0.1 % Triton-X. The samples were then incubated with primary antibody rabbit anti-human von Willebrand (diluted 1:100) for 30 min, rinsed with PBS, and then incubated with antibody goat anti-rabbit antibody (diluted 1:100) for 30 min in the dark at room temperature. After rewashing thrice with PBS, the sections were incubated in diaminobenzidine reagent and then counterstained. A positive reaction was indicated by the formation of a brown-yellow color in the cytoplasm. All incubations were performed at 37 °C in a humidified 5 % CO2 atmosphere. The cells were observed under a transmission electron microscope (OLYMPUS, Japan). In Vitro Co-culture Models MSCs (104 cells/well) and ECs (104 cells/well) were cocultured in a 24-well plate. Equal quantities of MSCs (2 9 104 cells/well) were cultured in a medium containing osteogenic factors (50 lg/L L-ascorbic acid, 10–8 mol/L VitD3, 100 lg/mL penicillin, 100 lg/mL streptomycin, 0.3 lg/mL amphotericin, 2.2 g/L sodium bicarbonate) and 15 % fetal bovine serum to serve as the control group. After co-culturing cell viability and proliferation was assessed using Coomassie brilliant blue assay and alkaline phosphatase (ALP) activity was measured by an ALP protein assay kit (Nanjing Jiancheng Bioengineering Institute) on days 3, 6, 9, 12, and 15. The cells were collected after centrifugation and then mixed with 4 L of the Coomassie brilliant blue G-250 solution. The absorbance was obtained at 595 nm and cell numbers were linearly correlated with optical density. Similarly the ALP protein assay was also performed but the absorbance was measured at 520 nm. ALP Staining ALP is a vital early stage enzyme marker of osteogenic differentiation. After 2 weeks of co-culturing MSCs and ECs, ALP staining was carried out according to the protocol of the ALP staining kit (Nanjing Jiancheng, Bioengineering Institute, China). The cell-seeded scaffolds were washed twice with PBS and then collected by centrifugation after trypsin digestion. The cell suspension was seeded in a six-well culture plate and then cultured for 6 h for cell adhesion. The cells were washed gently with PBS and then fixed with formalin (4 %, Sigma) for 2 h. After washing twice with PBS, the cells were dyed with a staining reagent for 30 min in the staining kit. The stained cells were photographed using an inverted microscope (Olympus).
Alizarin Red Staining AZR staining was used to identify mineralization nodule formation after 2 weeks in the osteogenic induction culture. In brief, 1 g Tris–HCl was added to 100 mL distilled water and the pH was adjusted to 8.3 with 0.1 M HCl. 0.1 g AZR was added to Tris–HCl solution to obtain 0.1 % AZR staining buffer solution. The cells were digested and collected using the aforementioned method and then fixed with 95 % ethanol for 30 min. After washing twice with PBS buffer, the cells were stained for 30 min and then photographed using an inverted microscope. In Vivo Rabbit Mandible Defect Model The study was authorized by the Animal Ethics Committee of Jinlin University and was carried out in accordance with the Guidelines for the care and use of laboratory animals. Twelve healthy New Zealand white rabbits weighing approximately 2.5–3.0 kg were anesthetized with pentobarbital sodium. A 3 cm incision was made on the inferior border of the mandible, and a critical size defect of 10 9 10 9 5 mm3 was made along the buccolingual direction on both sides of the mandible. Sterilized PLGA pieces of 10 9 10 9 5 mm3 were placed in a 24-well plate. The pieces were divided into two groups depending on the cells that were seeded on them. In Group A, MSCs and VECs co-cultured for 2 weeks were seeded on the surface of pre-wetted PLGA disks. In Group B, the same number of MSCs (2 9 104 cells/well) in osteoblastinduced media was seeded on PLGA disks to serve as the control group. The scaffolds of Groups A and B were implanted into the left and right sides of the mandible defects, respectively. Co-cultured cells of PLGA (10 9 10 9 5 mm3 in size) and PLGA with single MSCs (as the control) were inserted into the mandible defects. Six randomly selected rabbits were sacrificed at 4 and 8 weeks post-implantation. Radiographic Examination After sacrifice, lateral radiographs of the mandibles were obtained using a soft X-ray unit (MR-309M, SHIMAZU), and new bone formation was then measured using an image analysis software (Image pro plus 6.0). Histologic and Histomorphometric Analyses The mandibles were taken out, fixed in 10 % neutral buffered formalin, decalcified in formic acid, dehydrated and then embedded in paraffin. Continuous sections approximately 5 lm thick were prepared and stained with
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hematoxylin and eosin for histological observation and histomorphometric analysis. For the histomorphometric analysis, the newly formed bone volume in the cancellous bone area (%) of the implanted zones was measured. Bone formation was quantified by dividing the newly formed bone volume by the total implanted volume in the measured zone. The mean was calculated for each group and then used for statistical analysis. Statistical Analysis Data were presented as mean ± standard deviation. The differences between groups A and B were evaluated through two-tailed test with Student’s t test using the SPSS 13.0 software. A p value of less than 0.05 was considered to indicate statistical significance.
After day 5 to day 7, the MSCs became multilateral in shape. After the third passage, the cells exhibited homogeneous morphological features and were arranged in a series, like a whirlpool (Fig. 2b). The cells demonstrated high proliferation rate after three passages. Identification of the Induced ECS After 2 weeks of culture in M199-conditioned medium, the appearance of the cells gradually changed from a flat polygonal shape to an oblate or round shape. In addition, the cells were arranged in a typical cobblestone-like morphology (Fig. 2a) when they expressed the typical VEC phenotypic marker CD34 in the cytoplasm (Fig. 2c, d). The MSCs exhibited EC-like characteristics, indicating their differentiation into ECs. In Vitro Effect of Induced VECs on MSC Osteogenic Potential in the Co-culture
Results Characteristics and Biocompatibility of the PLGA Scaffold The scaffold exhibited a highly porous structure (Fig. 1a), with the pore size ranging from 10 to 100 lm. The biocompatibility of the PLGA scaffold was evaluated in vitro by observing the MSCs when cultured on the surface of the PLGA scaffold. After day 14, scanning electron microscopy revealed that the cells attached well to the scaffold (Fig. 1b). This result suggested that the scaffold was biocompatible and noncytotoxic. Evaluation of MSC Cultures On day 1, the MSCs appeared round and showed various sizes. Three days later, the MSCs formed several colonies.
ALP is a specific marker of ostegenic formation in the early stage. After day three in the co-culture system, the morphology of the MSCs changed from a fusiform monolayer to a multilateral shape and multilayer colonies. The secretion of ALP was evident as early as day 7. On day 14, ALP staining (Fig. 2e, f) showed small black granules throughout the cytoplasm. These granules were observed until day 21 seen as positive staining. Significant differences in ALP staining were noted between the co-cultured MSCs and ECs as well as single-MSCs in osteogenic media. The result of AZR staining (Fig. 2g, h) was positive, showing red plaques inside and outside the cells on day 14. By contrast, the ossification induced by MSCs cultured alone slightly showed positive staining. These results indicate that the induced VECs improved the osteogenic differentiation ability of MSCs. As shown in Fig. 3a, the
Fig. 1 SEM of the macro-architecture of PLGA scaffold the pore size of scaffold mainly ranged from 10 to 100 lm (a). Image of MSCs on the PLGA surface as observed under scanning electron microscopy after 14 days. MSCs adhered and extended on the PLGA surface (b)
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Fig. 2 After 2 weeks, differentiating endothelial cells derived from MSCs formed a monolayer with typical cobblestone-like appearance (a). Typical whirlpool-shaped MSCs were observed under phase contrast microscope (b). After 2 weeks, MSCs-derived ECs stained positively for CD34 in cytoplasm (c). Negligible CD34 positive MSC cells seen (d). After MSCs and ECs were co-cultured for 14 days and ALP staining carried out a small black zone diffused distribution in
the cells cytoplasm could be observed (e). When MSCs alone were cultured for 2 weeks, then ALP staining was only weakly positive in the cell (f). After MSCs and ECs co-cultured for 2 weeks, AZR staining was positive as seen in the form of some red plaques (g). After MSCs alone were cultured for 2 weeks, AZR staining was almost negative in the cells (h) (Color figure online)
ALP activity of the co-cultured cells on day 3 was higher than that of the MSCs cultured alone. Subsequently, from day 6 to day 15, the co-cultured cells had significantly
higher ALP activity compared with the cells cultured alone (p \ 0.05). The result of the Coomassie brilliant blue total protein (Fig. 3b) assay showed that the total protein of the
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Cell Biochem Biophys 3 ALP activity assay of co-cultured cells and single cells at various time periods. Values given represent the mean, and the error bars indicate the standard deviation estimated (a). Total protein assay of co-cultured cells and s proliferation of single cells at various time periods (b). Soft X-ray image from Group A (MSCs ? ECs ? PLGA) and Group B (MSCs ? PLGA) after 4 and 8 weeks. The data shown are the mean ± SD from six experiments (c)
b Fig.
the MSCs alone. On day 12, the co-cultured group reached the maximum total protein (0.93 g/L), which is significantly greater compared with that of the control group (0.75 g/L; p \ 0.01). Radiographic Observations At 4 weeks after implantation, new bone formation was observed through soft X-ray at the reconstruction area of the mandibles. The radio density in Group A was significantly higher than that in Group B (Fig. 4a, b). At 8 weeks post-surgery, the border of the bone defects was sclerotic, and the medullary cavities were blocked. Group A demonstrated higher radio-opacity than Group B, indicating enhanced new bone formation in Group A than in Group B. The interface between the implants and the host bone became blurred in Group A as compared to the other group. The previously quadrate-shaped defects in Group A were mostly covered with the projection of new bone and were in sync with the surrounding host bone. However, the intensity was still lower than that of the normal bone (Fig. 4c, d). Densitometry was conducted using image analysis software and the results are as shown in Fig. 3c. By weeks 4 and 8, the radio density in Group A showed acquiescence compared with that in Group B (p \ 0.01). This result suggests that new bone had grown into the scaffold and had well coalesced with the host bone. Histological Analysis
co-cultured cells on day 3 was 0.68 g/L, which was close to that of the control group. From day 6 to day 15, however, the co-cultured cells synthesized higher total protein than
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In all the groups, new bone formation was found in the defective mandible area. At 4 weeks post-surgery, the interface between the implant and the host bone was surrounded by primary matrix fronts, where some lymphocytes and fibroblasts were present around the implant. This phenomenon was regarded as a sign of inflammatory reaction. Group A showed enhanced new trabecular bone formation and new bone matrix deposition as compared to Group B (Fig. 5a, b). At 8 weeks, new bone formation was also observed in Groups A and B. However, Group A still showed much greater and denser newly formed trabecular bone in the central and peripheral regions than Group B. As shown in Fig. 5c, d, the newly formed bone was mature. The results of the histomorphometric analysis for the new bone area are shown in Table 1. By week 4, the area of the newly formed bone was significantly larger in Group A
Cell Biochem Biophys
Fig. 4 Soft X-ray film from rabbit mandibles Group A: MSCs and VECs co-cultured on PLGA disks were implanted into the left side mandible defects. Group B: only MSCs were seeded on PLGA and
were implanted into the right side mandible defects as a control group. At 4 weeks in: a Group A and b Group B. At 8 weeks in: c Group A and d Group B
Fig. 5 Histological photomicrographs of implants (scale bar = 50 lm) Group A: the newly formed bone of MSCs and VECs co-cultured on PLGA in left mandible defects. Group B: the newly
formed bone of MSCs was seeded on PLGA in right mandible defects. At 4 weeks in: a Group A and b Group B. At 8 weeks in: c Group A and d Group B
than in Group B (p \ 0.05). By week 8, the area of the newly formed bone was considerably larger in Group A than in Group B (p \ 0.01).
vessels are well vascularized they provide precursors of osteoblasts, related signaling molecules, nutrient substances, cytokines and growth factors to the local microenvironment while removing the metabolic products of decomposition from the local region [17]. In the present study, we stimulated bone healing by enhancing the vascularization ability of ECs. This was accomplished by exploiting the osteogenic ability of MSCs on a PLGA scaffold and co-culturing them with ECs.
Discussion Bone regeneration is a complex process involving various factors; vascularization being a vital prerequisite. When
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Cell Biochem Biophys Table 1 Ratio of new bone areas at periodic intervals in each group Group (n = 12)
4 weeks
8 weeks
Group A (n = 6) Middle section (%) Interfacical section (%)
8.42 ± 1.28* 12.68 ± 1.67*
12.34 ± 0.87** 32.52 ± 2.33
Group B (n = 6) Middle section (%)
1.48 ± 0.34
2.45 ± 0.34
Interfacical section (%)
7.67 ± 1.45
28.63 ± 1.56
*Compared with Group B p \ 0.05; **compared with Group B p \ 0.01
In tissue engineering, cell adhesion to certain materials is related to the hydrophilic property, structure and chemical composition of the scaffold surface. These factors also affect cellular proliferation, migration, and physiological response [18]. PLGA scaffolds are widely used in bone reconstruction research because of their speedy biodegradation, innocuousness, and tremendous biocompatibility. We took advantage of the benefits of PLGA and adjusted the ratio of PLA:PGA at 85:15. In addition, the PLGA obtained had the ability to degrade in 8–10 weeks, which is the duration close to human bone restoration time. The pore size of the scaffold ranged from 50 to 300 lm, making it conducive for cell attachment and growth into the pores. In the attachment assay, the seeded cell continued to proliferate over 14 days in all the wells in vitro. This phenomenon indicates that the PLGA scaffold can provide a platform suitable for cell adhesion, proliferation, and differentiation. MSCs have received considerable attention because of their ability to differentiate into connective tissue cell types and treat vascular injuries and diseases through tissue engineering [19, 20]. A previous study suggested that MSCs have an unexplored property to differentiate into vascular cell types, including ECs [21]. Previous researchers have reported that indirect interaction occurs between osteoblasts and ECs during osteogenesis. ECs can secrete bone morphogenic protein to promote osteogenesis [21, 22]. However, osteoblasts affect EC activity by releasing VEGF and BFGF, which can enhance the proliferation of VECs. Furthermore, cell–cell interaction mediated by proteins at gap junctions is another communication strategy used between these two cell types [23]. Asahara et al. [24] found that endothelial progenitor cells (EPCs) for angiogenesis are CD34-positive cells. Later studies showed that functional early EPCs are positive for CD133 and CD34 [25]. Hence using these markers we isolated and culture-expanded ECs. After 10 days, the cultured ECs rearranged and organized into microcapillarylike structures. The cells established intercellular connection through cell–cell contact. Immunohistochemistry
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performed on cover glasses after 14 days of incubation demonstrated that the endothelial phenotype marker CD34 was positive. These results indicate that MSCs can differentiate into ECs and that they have the phenotypic characteristics of early ECs. ALP is the marker protein related to the middle stage of osteoblast differentiation. In our previous studies, Hongchen et al. [9] showed that kidney ECs and MSCs cocultured directly on PLGA scaffolds can increase osteogenesis and ALP activity level. After the co-culture of the derived ECs and MSCs, the result of ALP and AZR staining was positive. ALP activity and Coomassie brilliant blue total protein substantially increased from day 6 to day 15, without significant changes in the control group. These results demonstrate that the co-cultured ECs improved the osteogenesis of MSCs, which exhibit the properties of osteoblasts. The present study showed that bone formation is a timedependent process. The key to successful bone regeneration is providing the repaired site with osteogenic cells in a suitable delivery vehicle to ensure osteoblastic differentiation and optimal secretory activity [26, 27]. By week 4, the radio density in Group A was slightly higher than that in Group B and by week 8, Group A showed a significant increase in radio density, which suggests enhanced new bone formation compared with the other group. By week 8, histomorphometric analysis showed that osteointegration in Group A occurred in the implantation area. However, many inflammatory cells and degrading scaffold materials at the center of the implantation remained in Group B. These results are consistent with the report that the most active period in bone formation is the first 2–4 weeks [28]. During the first 4 weeks, the host tissue played the main role in bone healing and remodeling. In a longer period of remodeling, however, the self-repairing capability is limited. Thus, the introduction of co-culture model exhibited greater effect than the control group. By week 8, the area of newly formed bone in Group A was significantly larger than that in Group B (p \ 0.01). Overall, these results suggest that induced MSC-derived ECs co-cultured with MSCs enhanced the osteogenesis of MSCs and accelerated the local mineralization and repair of bone defect regions. In conclusion, we would like to reiterate that MSCs can differentiate into ECs. The induced ECs can promote the osteogenic effect of MSCs, both of which were co-cultured on a PLGA scaffold. This helped in enhancing the ability to repair large bone defects in rabbit in vivo through tissue engineering. Future works should elucidate the interaction between ECs and osteoblasts and determine the mechanism by which vascularization affects osteogenesis. Additional molecular biological studies will add to the existing
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knowledge and help improvise the techniques and thereby vastly help in improved patient satisfaction. Acknowledgments This study was supported by Grants from the National Natural Science Foundation of China (No. 30830108 and No. 81271111), the Science and Technology Development Program of Jilin Province (No. 201101051; No. 200705350), and the Department of Health Program of Jilin Province (No. 2008Z029).
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