| Background and objective:The treatment of bone defects is a difficult problem that doctors of orthopedics and research workers often face. Bone defects caused by trauma, infection and bone tumor and other pathogenic factors are difficult to heal on its own. It causes loss of function which will affect the treatment and quality of life of patients. In the clinical treatment of bone defect, it is often encounter structural bone defect, and malunion of fracture, healing difficult as well as not healing, even if bone nonunion. Clinically alternative materials for repairing bone defects including autologous bone graft, allograft and artificial bone materials.Autogenous bone graft is the gold standard for clinical treatment of bone defect, which refers to the body bone tissue transplantation in the same individual from one place to move to another place. Bone tissue in certain parts of the body are taken out that will not left obvious dysfunction, and these sites of bone tissue are often ideal for bone autograft. Cancellous bone that autogenous bone graft required is usually from the clavicle or ilium, and cortical bone is usually from the tibia or fibula. Autologous bone graft with high bone forming ability and very little immune reaction, but there are additional trauma, extremely limited sources, prolong the operation time, increased blood loss and potential surgical infections and persistent chronic pain and other shortcomings, which limit their clinical application.Allogeneic bone graft refers to an individual of the same kind to another individual bone tissue transplantation. Bone tissue transplants have different genetic characteristics, with the more severe immune rejection occurs recipient organization and potential risk of disease transmission. Wherein, by the same binders developed from the two bone tissue transplants between individuals, referred to syngeneic bone tissue transplantation, In this case, the two individual genetic characteristics are close without immune rejection or slight. Although the source of allogeneic bone graft more abundant than autogenous bone graft, but there are still immune rejection, healing slow, leading to the spread of disease, cross-infection and other problems.Artificial bone material refers to a kind of repair, replacement and regeneration for bone tissue which with special function. This kind of artificial material not only provide the possibility for the physiological structure and function in patients with rapid recovery to normal in clinical application, but also to avoid the use of various problems of autologous and allogenic bone grafting brings. According to the structure or properties of the materials, artificial bone material can be classified as inorganic bone material, organic bone material and composite material for bone repair. Artificial bone material can fill the bone defect site, but can not induce the nascent bone tissue formation. Its material properties, biological properties and degradation ability and other issues need to be solved further. Tissue engineering bone in treatment of bone defect is a hotspot in the research of composite bone substitute material. The development of bone tissue engineering has made it an potential to be applied in repairing bone defects, using the biodegradable and controlled-release drug delivery system for treatment of bone defect is a method for more cost-effective method.This research adopts the double emulsion solvent evaporation method for the preparation of alendronate in PLGA microspheres, and with the mixture of calcium phosphate bone cement to prepare the calcium phosphate composite composite tissue-engineering bone containing alendronate-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (Alen-PLGA-CPC composite tissue-engineering bone). Firstly, we evaluate its material characteristics including the drug loading and release characteristics, pore size, porosity and mechanical properties; secondly, we observe cell proliferation and osteogenic ability of BMSCs to evaluate its biocompatibility and osteogenic in vitro study; thirdly, through constructing the model of bone defect in rabbits, we put Alen-PLGA-CPC composite tissue-engineering bone into bone defect site, and evaluate its biocompatibility and osteogenic by radiographic and histological indices. This study was designed to prepare Alen-PLGA-CPC composite tissue-engineering bone and investigate its biocompatibility and osteogenic activities in vitro and vivo. Thus exploring its effects of accelerating the repair of bone defect. This study is expected to provide a new treatment for clinical bone defects repair. This study is divided into the following three parts:(1) The material properties of Alen-PLGA-CPC composite tissue-engineering bone; (2) Biocompatibility and osteogenesis of Alen-PLGA-CPC composite tissue-engineering bone in vitro; (3) Biocompatibility and osteogenesis of Alen-PLGA-CPC composite tissue-engineering bone in vivo. The main content of this study is briefly described as follows.(1) The material properties of Alen-PLGA-CPC composite tissue-engineering boneMethod:Using double emulsion solvent evaporation method (W/O/W) to prepare alendronate-loaded PLGA microspheres. Using JSM-6390 scanning electron microscopic morphology to measure the size and distribution of particle size of the microspheres. Using automated micro-plate reader to evaluate the drug loading and release characteristics of alendronate-loaded PLGA microspheres. Alen-PLGA-CPC composite tissue-engineering bone was prepared by mixing microspheres with calcium phosphate cement. Archimedes method was used to meansure porosity of composite tissue-engineering bone. JSM-6390 scanning electron microscope was used to assess the pore size of composite tissue-engineering bone. RGD-5 universal testing instrument was used to evaluate the mechanical properties (compressive strength) of composite tissue-engineering bone.Result:Alendronate loaded PLGA with unform size 200-300 μm of microspheres were successfully prepared by solid/oil/water (s/o/w) emulsion solvent evaporation method and exhibited good sphericity. Appearance, particle size and distribution of the alendronate-loaded PLGA microspheres and pure PLGA microspheres did not have differently significant. The encapsulation efficiency for PLGA microsphere had a high value of 75.12% for alendronate. The results showed that the Alen-PLGA-CPC composite tissue-engineering bone had a porosity of 67.43±4.2% and pore size of 213±95 μm. The compressive strength for Alen-PLGACPC composite tissue-engineering bone was 5.79±1.21 MPa, which was close to human cancellous bone. There is no obvious difference of Alen-PLGA-CPC composite tissue-engineering bone and PLGA-CPC composite tissue-engineering bone in porosity, pore size and compressive strength.Conclusions:1.PLGA is good alendronate carrier and alendronate can be released after degradation of alendronate-loaded PLGA microspheres.2.Alen-PLGA-CPC composite tissue-engineering bone has good properties, suitable pore structure and mechanical properties.(2) Biocompatibility and osteogenesis of Alen-PLGA-CPC composite tissue-engineering bone in vitroMethod:Percoll density gradient separation method was used to obtain rabbit BMSCs.Then cell cultured, purified and observe the growth state by inverted microscope. The Alen-PLGA-CPC composite tissue-engineering bone and PLGA-CPC composite tissue-engineering bone were placed in the culture plate respectively, and the third generation of BMSCs with good growth status suspensions were seeded on the tissue-engineering bone. Cell morphology was observed by SEM. Cell adhesion and proliferation were determined using CCK-8. The effect of cell cycle was assessed by flow cytometry. The alkaline phosphatase activity and alkaline phosphatase staining was evaluated to determine the osteogenic differentiation activity.Result:The results in vitro also revealed that the Alen-PLGA-CPC composite tissue-engineering bone had osteogenic potential on the BMSCs and exhibited excellent biocompatibility with engineered bone tissue.Alen-PLGA-CPC composite tissue-engineering bone have good cell adhesion of cells with normal morphology.Alen-PLGA-CPC composite tissue-engineering bone promotes BMSCs adhesion and proliferation significantly (P<0.05). After cell seeding, proliferation rate of cells in Alen-PLGA-CPC composite tissue-engineering bone group was significantly higher than that of pure PLGA-CPC composite tissue-engineering bone group and the control group (P＜0.05). The alkaline phosphatase activity and alkaline phosphatase positive staining cells were significantly higher than that of pure PLGA-CPC composite tissue-engineering bone group and the control group (P<0.05).Conclusions:In vitro experiments showed that Alen-PLGA-CPC composite tissue-engineering bone has good biocompatibility and osteogenic activity, thus promoting adhesion, proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cell.(3) Biocompatibility and osteogenesis of Alen-PLGA-CPC composite tissue-engineering bone in vivoMethod:Experimental Animal Center of Shandong University offered 45 healthy adult New Zealand white rabbits (23 female,22 male; weight 2.0-3.0 kg). Femoral condyle bone defects model was established by surgery and randomly divided into group A, B and C,15 rabbits in each group. One group for sham-operation; one group for the implantation of pure PLGA-CPC composite tissue-engineering bone; and the other one for Alen-PLGA-CPC composite tissue-engineering bone. Only one side of each animal for the surgical operation. Gross anatomy observation, X-ray examination, micro-CT measurement (bone coverage and bone mineral density(BMD)) and histomorphology of rabbit femoral condyle were performed at 6 and 12 weeks post-implantation, respectively.Result:The Alen-PLGA-CPC composite tissue-engineering bone group demonstrated more active in vivo biocompatibility and osteogenesis in the defect area compared with other groups during various implantation periods. The sham-operation group showed no obvious healing of bone defects; X-ray exhibited significant bone defects translucent. The sham-operation group demonstrated a bone coverage of 3.40±2.25% and 6.10±4.48% at 6 weeks and 12 weeks; a BMD of 31.04±5.8mg/cm3 and 50.80±8.8mg/cm3 at 6 weeks and 12 weeks. HE staining showed a large number of fibrous encapsulation were found inside bone defects, but only with a few newly formed bone tissue in the sham-operation group. The PLGA-CPC composite tissue-engineering bone group showed partly healing of bone defects; X-ray exhibited week bone defects translucent.The PLGA-CPC composite tissue-engineering bone group demonstrated a bone coverage of 12.89±5.74% and 20.24±9.25% at 6 weeks and 12 weeks; a BMD of 57.08±8.4mg/cm3 and 81.80±10.8mg/cm3 at 6 weeks and 12 weeks. HE staining showed in the PLGA-CPC scaffold group, besides a small amount of fibrous tissue, partly neogenetic osteoid was found with weak new bone trabecular. The Alen-PLGA-CPC composite tissue-engineering bone group showed the bone defect was almost repaired; X-ray exhibited the most week bone defects translucent. The Alen-PLGA-CPC composite tissue-engineering bone group demonstrated a bone coverage of 25.78±6.89% and 45.06±11.62% at 6 weeks and 12 weeks; a BMD of 88.92±9.9mg/cm3 and 115.98±12.2mg/cm3 at 6 weeks and 12 weeks. HE staining showed in the PLGA-CPC scaffold group, besides little fibrous tissue, new bone structure with more and thicker trabeculae regenerated.Conclusions:Animal experiments confirmed that Alen-PLGA-CPC composite tissue-engineering bone can promote new bone formation in bone defect of rabbit femoral condyle and accelerate the repair and healing of bone defects. |
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