| Calvarial bony defects may result from congenital defect, traumatic injuries, neurosurgical procedures or tumor resections. For both of esthetic and functional reasons, successful healing of the defects is quite important for the subjected patients, besides, successful spontaneous calvarial self-repair only occurs in infants younger than 2 years of age. Due to the drawbacks of limited source, immunologic rejection, poor degradation and absorption and disease transmission, the traditional bone grafts and the metal material are limited to be used as routine items in clinic. To avoid these shortcomings, the focus of the present study is to solve the problem of calvarial repair using bone tissue engineering. The purpose of this project is to build a new type of tissue engineered bone for the repair of critical-sized calvarial defect, the seed cells is peripheral blood mesenchymal stem cells, scaffold material is self-assembling peptide nanofiber scaffold and polylactic-co-glycolic acid. This paper is the first to demonstrate the osteogenic differentiation ability of peripheral blood mesenchymal stem cells in vitro and in vivo, and build the tissue engineering material with bionic structure characteristics of skull, the material can support survival of stem cell in vivo for long-term and promote the formation of new bone.At present, the most popular seed cells in bone tissue engineering are mesenchymal stem cells, which have many advantages, such as multipotential differentiation, great proliferation ability, and good repair effect of various tissues. Mesenchymal stem cells are generally obtained from the bone marrow, but the procedure will inevitably lead to patients suffering, tissue damage, the risk of local infection, which limit its application in the basic research and clinical. However, peripheral blood derived mesenchymal stem cells (PBMSCs) permit an autologous low-cost source of stem cells which may be easily harvested from patient’s blood by non-invasive procedures, which are draw increasing attention.Self-assembling peptide nanofiber scaffold (SAPNS) is a new type of nano-materials, which has been developed by Zhang’s group, and proved that it can play a good role in many kinds of tissue repair. So it will be a promising biomaterial for the study of tissue injury repair. Compared with other bone tissue engineering scaffold, SAPNS has many advantages:the diameter of its polypeptide fiber is nano scale, the structure is very close to extracellular matrix (ECM) and provide true 3D nano micro environment for the cells in which to grow. No cytotoxicity and having excellent biocompatibility, biodegradability, and the degradation products are easy to be absorbed. In addition, the material has a good hemostatic effect on the lesion site. No obvious immune rejection and inflammatory reaction. Polylactic-co-glycolic acid (PLGA), as the first batch of biodegradable materials, was approved for the clinical use by the FDA of United States, which is the most widely used biodegradable materials. In this experiment, we made full use of the properties of PLGA that contains the biodegradable, easy for nutrients to penetrate and certain mechanical strength. We use it to make the PLGA membrane, to closed the bone defect sites, which can avoid the harm of brain tissue outside, provide SAPNS hydrogel mechanical support and create good conditions for the survival and differentiation of PBMSCs and promote new bone formation.This main methods of our project are as follows:1. The detection of osteogenic differentiation potential of PBMSCs:P5 PBMSCs were harvested, seeded onto the coverslips in 1×104 cells/coverslip, and cultured within 24-well plates, one slip in one well. The cells were cultured in basic medium for 12 h and then subjected to osteogenic differentiation protocols. The cultures were induced with DMEM medium containing 10%FBS,10" M dexamethasone, l0mM β-glycerophosphate,50mM Ascorbic acid-2-phosphate, and 5×10-8M 1,25-Dihydroxyvitamin D3.21 d later, alkaline phosphatase (ALP), von kossa staining, alizarin red staining and osteocalcin immunofluorescent staining were performed to evaluate osteogenic products.2. Osteogenesis of PBMSCs in a three dimensional (3D) scaffold of self-assembling peptide:1×10 P5 PBMSCs in 5μL DMEM medium were rapidly mixed with 50μL 1% SAPNS solution, the mixture was gently and quickly plated to dish with osteogenic medium (aMEM supplemented with 10-8M dexamethasone, 10mM β-glycerophosphate,50mM Ascorbic acid-2-phosphate, and 5×10-8M 1,25-Dihydroxyvitamin D3).21d later, the culture was fixed with 4% paraformaldehyde for 2 h, cryoprotected in 30% sucrose in PBS for 12 h, embedded in optimum cutting temperature compound (OCT), then cryostat sections (50μm) were cut and mounted onto poly-1-lysine subbed slides. Some slides were used to do ALP staining and remains were immumostained with osteocalcin(OCN) and chondroitin sulfate proteoglycans (CSPG) antibody.3. Scaffold fabrication for calvarial bone tissue engineering:The preparation of PLGA membrane with a thickness of 50 μm. First,0.5 g PLGA (PLA:PGA= 85:15) was dissolved in 10 g dichloromethane with 0.5 g NaCl particles (diameter of 75-95 jμm) added into the solution. The solution was mixed quickly and poured into a Teflon-coated horizontal mold. After evaporation of the solvent in a fume hood for 48 hours, the formed membrane was washed with water thoroughly to remove the NaC1 particles. The membrane was then cut into disc-shapes with a diameter of 8 mm, immersed in 75% alcohol overnight for sterilization, and rinsed three times with saline. By which, the porous PLGA membrane was formed.7 days after osteogenic induction, l0μg/mL BrdU was added to the osteogenic medium for further 48 h culture to label the cells which is conducive to distinguish the grafted cells and host cells after transplantation. Immediately before implantation, a sandwich like PBMSCs/SAPNS/PLGA scaffold was fabricated.4. Animal models and transplantation:Adult male Sprague-Dawley rats (220-250g) were employed. The animals were anesthetized with 1% pentobarbital sodium (40 mg/kg, IP). Under sterile condition, a midline incision in scalp was made to expose parietal bones. A critical size defect (8 mm in diameter) was created in the middle of parietal bones by a sterile drill. Above prepared PMBSCs/SAPNS/PLGA scaffold was implanted into the defect. For control, a scaffold of SAPNS/PLGA which was treated same as PBMSCs/SAPNS/PLGA just replaced the PBMSCs suspension with same value of culture medium, was implanted by same procedures.5. Imaging and histology detection of bone tissue regeneration:12 weeks after surgery, calvarical samples (n=8 for each group) were harvested and fixed in 4% paraformaldehyde for 48 h. Mineral formation within defect area was evaluated using microcomputerized tomography (Micro-CT). After Micro-CT imaging, the tissue was cut in half through the midline of implants and decalcified in 17% EDTA for 4 weeks at room temperature. Then dehydrated in grade ethanol, embedded in paraffin, cut into 5 μm thickness, then sections were deparaffinized and processed for routine hematoxylin/eosin (H&E) staining. Image Pro Plus Software was used to quantify the newly formed bone as a ratio of the new formed bone area to the total defect area.6. In vivo survival and osteogenesis of PBMSCs evaluated with immunohistochemistry:The implant was carefully harvested from the bone defect site and followed by post-fixation at 2 weeks post-surgery. Cryostat sections (20 μm) were cut and mounted onto poly-1-lysine subbed slides. Immunofluorescent staining with OCN antibody was performed to detect the in vivo osteogenic differentiation of transplanted PBMSCs.12 weeks after transplantation, bone defect were acquired, decalcified, paraffin section, we traced the PBMSCs by BrdU before transplantation and immunostained the labeled nuclei with antibody.7. Statistical analysis:All data collected were expressed as mean±standard deviation and statistical comparisons were assessed by Student’s paired t-test using SPSS 20.0 software. A P-value of less than 0.05 was considered statistically significant.Result:1. The experiments of osteogenic differentiation in vitro showed that the harvested peripheral blood mesenchymal stem cells had good osteogenic potential.2. SAPNS could support PMBSCs for growth, survival and differentiation to osteoblast in 3D environment.3. Micro-CT scan showed the effect of bone defect repair on PMBSCs/SAPNS/PLGA transplantation group was significantly better than the SAPNS/PLGA transplantation group and the quantitative detection of BMD and BV found that PMBSCs/SAPNS/PLGA transplantation group was higher than SAPNS/PLGA transplantation group, and the difference is statistically significant.4. HE staining results showed that the amount of bone formation of the bone defect site in the PMBSCs/SAPNS/PLGA transplantation group was significantly higher than that of the SAPNS/PLGA group. To detect quantitative results of the bone area showed that PMBSCs/SAPNS/PLGA transplantation group was significantly higher than SAPNS/PLGA transplantation group, and the difference was statistically significant.5. Through detecting the survival of transplantation cell for 2 weeks and 12 weeks respectively, the results showed that transplanted cells in a skull defect area grew well and most differentiation into osteoblasts after surgery 2 weeks, transplanted cells can survive in skull defect area until 12 weeks and can induce bone formation.Conclusion:The experimental results showed that mesenchymal stem cells harvested from peripheral blood could be used as seed cells for bone tissue engineering, and had good clinical application prospects. PBMSCs/SAPNS/PLGA composite scaffolds is conducive to the repair of calvarial critical defect, becaus e it can simulate diploe like structure in skull. The SAPNS can support survival and osteogenic differentiation of PBMSCs under the three-dimensional culture condition. This study, for the first time, confirmed that PBMSCs can survive in vivo and provided the evidence of direct involvement of bone regeneration by means of GFP and BrdU markers. |
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