| Bone defect is a common but difficult problem in clinics. Traditional treatments are autogenous bone graft and allograft. Autogenous bone graft is restricted by limited source, bleeding and inflammation in the donor site. For the allograft, there is the risk of virus infection and immunoreaction. Recently, tissue engineering has attracted extensive attention for bone regeneration in the defect site. The scaffold, growth factors and seeding cells make a 3D platform to mimic micro-environment in vivo, which is beneficial for cell proliferation, differentiation and new tissue formation. The platform can be further transplanted into the bone defect for regeneration.Poly(lactide-co-glycolide acid) porous microsphere scaffolds (PLGA-pMS) are excellent injectable-scaffolds with the advantages of minimally invasive surgery, better patient adaptability and being suitable for irregular-shaped defects. The PLGA-pMS may be optimized from two aspects. Firstly, the hydrophobicity of PLGA is not favorable for cell attachment on the microspheres. Recently, PLGA-pMS were modified by apatite deposition on the surface of microspheres, which was hard to control and took a long time. Also, it had a negative effect on drug loaded microspheres. Secondly, the drug loaded PLGA-pMS has a rapid in vitro release profile. Non-porous PLGA microspheres are classic drug loading substrates, in which the drugs can be released for over one month, usually. But the porous structure contributes to sever burst release and short releasing period. Rapid release of drugs is disadvantageous towards the new tissue formation. In the fields of tissue engineering, growth factors loaded PLGA-pMS have not been reported.Based on our previous studies on PLGA-pMS, the microspheres were optimized to have better cell affinity and controlled release of growth factors. In this study, PLGA was modified by the GRGDSPC peptide to improve the cell affinity of the PLGA-pMS. Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) loaded chitosan microspheres (CS-MS) were incorporated into the PLGA-pMS for sustained release of BMP-2 and TGF-β1, leading to better new bone formation.To obtain CS-MS with proper size and high encapsulation efficiency (EE), different factors were investigated on morphology, size, EE and in vitro release profile of CS-MS. TPP concentration, chitosan concentration, chitosan molecular weight, cross-linking time and stirring speed were studied, after which an optimized formulation was achieved. BMP-2 and TGF-β1 were encapsulated into the CS-MS based on the optimized formulation.The BMP-2 and TGF-β1 loaded CS-MS had a size of 15.47±0.53μm and 14.78±0.71μm, respectively. The loading efficiency was 1004.27±15.30 ng BMP-2 and 332.46±44.88 ng TGF-βΓ per 1 mg of CS-MS. Both of the growth factors could be released for about 9 days.PLGA-pMS were prepared based on W1/O/W2 double emulsion method and gas foaming method. Stirring speed, PLGA concentration, homogenization speed, NH4HCO3 concentration, PVA concentration and ratio of O/Wl were explored to prepare micro spheres with large surface pore size and proper diameter. The optimized PLGA-pMS had a diameter of 353.32±35.24 μm mand surface pore size of 26.30±3.86μm GRGDSPC was used for PLGA modification. GRGDSPC was first conjugated to PEG, and then reacted with PLGA to obtain PLGA-PEG-GRGDSPC. MG-63 cells were seeded on PLGA-pMS to study the effect of GRGDSPC modification. Results showed that GRGDSPC modified porous microspheres had more spreading cells and higher cell viability. GRGDSPC significantly improved the cell affinity of PLGA-pMS.20% GRGDSPC modified PLGA-pMS were used for further studies.CS-MS were incorporated into the PLGA-pMS during the preparation of the porous microspheres. CLSM and SEM were used to identify the CS-MS-PLGA-pMS. Results showed that CS-MS were partly embedded in the PLGA polymer with a spherical structure. The loading efficiency was 570.27±16.20 ng BMP-2 and 28.58±5.35 ng TGF-β1 per 1 mg of CS-MS-PLGA-pMS. The in vitro release of BMP-2 and TGF-β1 from the CS-MS-PLGA-pMS were studied based on the results of BSA release. The BMP-2 and TGF-β1 loaded CS-MS-PLGA-pMS had a burst release of 12.38±5.84% and 18.18± 0.29% on day 1, respectively. A sustained release of 28 days was achieved. On day 28, the microspheres released 83.71± 5.65% of BMP-2 and 77.47±2.12% of TGF-β1. Compared with CS-MS, CS-MS-PLGA-pMS achieved decreased burst release and prolonged release period.Mesenchymal stem cells (MSCs) were isolated and purified by density gradient centrifugation and adherent cell screening method. The cells were identified based on the multipotent differentiation potential and specific surface antigen. Results showed high expression of alkaline phosphatase (ALP) in the osteogentic medium, and lipid droplets in the adipogenic medium. Results of flow cytometry showed that the cells had surface markers of CD90+CD45-. MSCs were found to be attached on the PLGA-pMS. More cells were distributed on GRGDSPC modified microspheres than unmodified and PEG modified micro spheres, indicating that GRGDSPC remarkably improved the cell affinity of PLGA-pMS.Ectopic bone formation in nude mice and bone regeneration in rabbit calvarial defect model were studied based on the MSCs and the growth factors loaded CS-MS-PLGA-pMS. Ectopic bone formed with, the BMP-2 loaded scaffolds showed larger mass, bone mineral density (BMD) and bone volume/tissue volume (BV/TV) than the control group and blank scaffolds. Results of HE and Masson’s trichrome staining realized more trabecula bone in BMP-2 loaded scaffolds, indicating better bone quality. Without porous microspheres as substrates, the control group resulted in unfavorable new bone. Compared with the control group and blank scaffolds, TGF-β1 baded scaffolds showed improved bone mass. Significant differences were not found between the BMP-2 loaded scaffolds and the BMP-2 and TGF-β1 baded scaffolds in bone mass, BMD and BV/TV. Results of histological evaluation showed plenty of osteoids in the ectopic bone with BMP-2 and TGF-β1 loaded scaffolds. For the rabbit calvarial defect model study, the bone regeneration was not so satisfying because of inflammation caused by surgery which had negative impact on the MSCs. |
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