| Background and objective:Bone defect caused by trauma, infection, tumor and other diseases is a common disease in department of orthopedics, and it is also a difficult problem for doctors and scientific research workers. Large section of bone defects often heal slowly or difficult to heal, it may cause serious nonunion, pseudarthrosis, secondary fractures and even endanger the lives of patients. Traditional methods for treating bone defects are bone autograft, allograft and artificial bone graft.Autologous bone graft is currently the gold standard for the treatment of bone defects. Since the transplanted bone tissue was taken from the patient’s own, which has the desired therapeutic effect. However, limited sources, increased risk of infection, trauma and surgery and postoperative chronic pain and other issues, limiting its widespread application. The bone tissue transplantation of allogeneic bone graft comes from different individual of the same kind. Because of transplantation of bone tissue from different individuals, it is possible to transplant the immune rejection and the potential of the disease, resulting in the failure of transplantation, thus affecting the effect of treatment.Artificial bone transplantation is a synthetic bone tissue replacement material for the repair of bone defect site, avoiding the shortcomings of autologous bone graft and allograft bone transplantation. In 1995, Crane et al proposed the concept of bone tissue engineering, using cells, drugs and biological active factors mixed into composite materials to prepare tissue engineering bone for repair and treatment of bone defect. In recent years, the research of materials science and medical researchers have made rapid progress in bone tissue engineering.Tissue engineering bone includes four factors, which are the matrix material of the scaffold, osteoblasts, activation factors and mechanical properties. Ideal tissue engineering bone should have the following characteristics:(Ⅰ) Good biocompatibility and plasticity, which could fill the defect site immediately without toxicity, inflammatory response; (Ⅱ) Having a three-dimensional structure, suitable pore structure and porosity, thus contributing to tissue adhesion and proliferation, as well as infiltration of nutrients and discharge of metabolites; (Ⅲ) Has a certain mechanical properties and good biodegradability. It can be provide immediate support at defect site after implantation, new organization gradually repaired and replaced after the gradual degradation of the matrix material, then replace the tissue engineering bone completely; (IV) Osteoconduction and osteoinduction.After implantation the scaffold provides support micro environment, thus guiding and supporting surrounding active stem cells, bone cells, vascular ingrowth, induce undifferentiated mesenchymal stem cells into osteoblasts, and promoting new bone formation and precipitation. The traditional single matrix material such as calcium alginate, hydroxyapatite, and so on only have single phase biological and material properties, it is difficult to meet the complex requirements. However, if we combine the drugs of osteogenic effectiveness with the carrier material to make a sustained-release drug performance,then mixed it with the matrix material to prepare the composite bone tissue engineering in line with the requirements of biology and materials science.Poly lactic acid polymer (PLGA) is composed of two monomers, lactic acid and hydroxyl groups, and can be adjusted component ratio according to demand. It has excellent biocompatibility and drug loading performance, which is now widely used in the pharmaceutical, medical materials and tissue engineering.Calcium phosphate cement (CPC) is a bone repair materials which is widely used in clinic with good biological compatibility. Its solidification process heat production is extremely small,that can be equipped to ensure the efficacy and activity of the drug is not affected [3].Simvastatin is a kind of statin drug. It is a hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor which can block the conversion of HMG-CoA to mevalonate. At room temperature, simvastatin is a white powdery substance, which insoluble in water and easily soluble in organic solvents. Simvastatin is widely used clinically to lower blood cholesterol and low density lipoprotein. However, recent studies have found that statins also promote osteoblast proliferation and differentiation, inhibition of osteoclast activity, promote new bone formation. The mechanism includes:(I) It can promote expression of bone morphogenetic protein-2 (BMP-2), thus facilitate bone marrow mesenchymal stem cells induced to differentiate into osteoblasts and stimulate bone formation and mineralization. Mundy et al found that statins can activate BMP-2 gene promoter to promote its expression, it would statins skull subcutaneous injection in mice, found that local injection site obvious new bone formation. They inject statins in the skin of mouse skulls and found that local injection site of new bone formation significantly. (II) Inhibition matrix metalloproteinase-9 (MMP-9) activity. MMP-9 is a member of the matrix metalloproteinase family, which plays an important role in the pathological process of bone diseases, such as bone arthritis, etc. Thunyakitoisal et al found that simvastatin can reduce expression of MMP-9 of osteoblast and human HT1080 fibrosarcoma cells and related to dose and duration of action, that demonstrated simvastatin can inhibit MMP-9 to promote osteoblast response and inhibit osteoclast response. (Ⅲ) Upregulation of the expression of calcium binding protein (Calcyclin). Hwang et al analysed the expressed protein of stimulated by simvastatin and found that Calcyclin was significantly increased, thus significantly stimulate the proliferation and expression of markers of bone alkaline phosphatase mRNA. In addition, simvastatin can be used to promote bone repair by inhibiting osteoclast activity,and enhancing the function of macrophages and other immune cells. Part of the mechanism of simvastatin was dose related. However, the utilization of oral simvastatin is low,and there were liver toxicity and muscle toxicity. The side effect is more obvious with long-term medication. The tissue engineering bone with sustained release property and high bioavailability was prepared by using the carrier material with a sustained release drug delivery performance can reduce the toxic side effects and play the maximum drug efficacy in the bone defect site.Solid/oil/water (s/o/w) emulsion solvent evaporation method was used to prepared simvastatin loaded microspheres. The particle size and distribution, encapsulation efficiencyg and drug release rule of the simvastatin loaded microspheres was observe. Then, PLGA microspheres was added to CPC powder to obtain a calcium phosphate composite scaffolds containing simvastatin-loaded PLGA microspheres (SIM-PLGA-CPC scaffolds). Firstly, the properties of the materials, such as the size and distribution of the aperture, the porosity and the compressive properties, are meansured. Secondly, rabbit bone marrow mesenchymal stem cells (BMSCs) were extracted, purificated and cultured in vitro. And the proliferation and osteogenic differentiation of BMSCs seeded on tissue engineering bone were observed to assess its biocompatibility and osteogenic activity. Finally, to build rabbit femoral condyle bone defect model after anesthesia, then laced SIM-PLGA-CPC scaffolds into the lateral femoral condyle defect. Its biocompatibility and osteogenic activity in vivo were meansurd by radiography (Micro-computed tomographic, Micro-CT) and histology (Hematoxylin-Eosin, HE staining).The purpose of this study is to prepare the simvastatin-loaded PLGA microspheres, and then prepare tissue engineering bone with calcium phosphate bone cement. Subsequently, to determinate its material properties, evaluate the biocompatibility and osteogenic activity both in vitro and in vivo, explore the repair effect of rabbit femoral condyle bone defect. The innovation of this study is use PLGA microspheres as a sustained release carrier firstly, and prepared a sustained release of simvastatin tissue engineering bone successfully, and expected to provide a new approach for the treatment of bone defect.This study is divided into three parts:(I) Preparation and material properties of SIM-PLGA-CPC composite tissue engineering bone; (II) Biocompatibility and osteogenesis of SIM-PLGA-CPC composite tissue engineering bone in vitro; (III) Biocompatibility and osteogenesis of SIM-PLGA-CPC composite tissue engineering bone in vivo. The main contents of this study are as follows. Part I Preparation and material properties of SIM-PLGA-CPC composite tissue engineering boneMethod:Solid/oil/water (s/o/w) emulsion solvent evaporation method was used to prepared simvastatin unloaded and loaded microspheres. The morphology, particle size and distribution of the simvastatin unloaded and loaded microspheres was observed by scanning electronic microscopy (SEM). The encapsulation efficiency and drug release rule of simvastatin-loaded PLGA microspheres was evaluated by suspending microspheres into buffer solution. Pure PLGA microspheres and simvastatin loaded PLGA microspheres were mixed with calcium phosphate, then cured to prepare calcium phosphate composite scaffolds containing simvastatin-loaded or unloaded PLGA microspheres (SIM-PLGA-CPC scaffolds or PLGA-CPC scaffolds). Scaffolds containing simvastatin-loaded and no drug loaded PLGA microspheres were scanned and the data were analyzed by Micro-CT analyzer to assess pore size and distribution as well as porosity. The test instruments was used to meansure the compressive strength of two kinds of scaffolds.Result:The pure PLGA microspheres (221.89±70.20μm) and simvastatin-loaded PLGA microspheres (217.95±63.37μn) were fabricated utilizing solid/oil/water (s/o/w) emulsion solvent evaporation technique, respectively. There were no significant differences in appearance, particle size and distribution of the two kinds of microspheres. The encapsulation efficiencies of simvastatin-loaded PLGA microspheres is about 85.30±4.3%. Simvastatin-loaded PLGA microspheres shows a significant initial burst of simvastatin with>60% released within the first 7 days. The release of simvastatinis almost complete in 21 days. The PLGA-CPC scaffold exhibited 64.50±4.27% total porosity, pore diameter of 234.65±65.36 μm and compressive strength of 6.34±1.41Mp. The SIM-PLGA-CPC scaffold exhibited 65.95±3.30% total porosity, pore diameter of 225.74±57.02μm and compressive strength of 6.52±1.73Mp. There were no significant differences in the porosity, pore size and compressive strength of the two kinds of tissue engineering bone.Conclusion:4. The simvastatin-loaded PLGA microspheres with the requirements of material science could be fabricated utilizing solid/oil/water (s/o/w) emulsion solvent evaporation technique.5. PLGA microspheres can be used as controlled release carrier of simvastatin. With the drug-loaded microspheres degrade, can produce simvastatin burst effect and slow release of up to 21 days.With the degradation of drug loaded microspheres, simvastatin could produce burst effect and sustained release up to 21 days.6. The calcium phosphate composite scaffolds containing simvastatin-loaded or unloaded PLGA microspheres has excellent drug release properties, good three-dimensional connectivity, suitable pore size and compressive properties.Part II Biocompatibility and osteogenesis of SIM-PLGA-CPC composite tissue engineering bone in vitroMethod:One-month-old New Zealand rabbits were anesthetized and sacrificed, then tibia and femur condyle were separated, for extracting BMSCs. Ficoll density gradient centrifugation method was used to extract BMSCs. Cells were cultured and passaged in vitro, and was observed by inverted microscope. Well growing BMSCs of the fourth generation were seeded on the PLGA-CPC scaffolds and SIM-PLGA-CPC scaffolds. After cultured for 7 days, morphology of BMSCs on the PLGA-CPC and simvastatin-loaded PLGA-CPC scaffolds were observed by SEM; cell adhesion and proliferation was assessed using the CCK-8 assay; flow cytometry analysis permits the detection of the effects of scaffolds on cell cycle; osteogenesis of scaffolds was investigated by measuring the alkaline phosphatase (ALP) activity and alizarin red S staining.Result:Both PLGA-CPC and simvastatin-loaded PLGA-CPC scaffold have a good biocompatibility on BMSCs. However, simvastatin-loaded PLGA-CPC scaffold exhibited more osteogenic differentiation activity. This two kinds of tissue engineering bone are all good in cell adhesion. Cells seeded on the simvastatin-loaded PLGA-CPC scaffold expressed more angular morphology and with greater number. Compared with PLGA-CPC scaffold group and blank control group, simvastatin-loaded PLGA-CPC scaffold group demonstrated more cell proliferation, more cells in cell division stage, higher levels of alkaline phosphatase activity and more and larger mineralized nodule with positive Alizarin Red S staining (P<0.05).Conclusion:The calcium phosphate composite scaffolds containing simvastatin-loaded or unloaded PLGA microspheres has excellent biocompatibility and osteogenic differentiation activity in vitro. Its sustained release of simvastatin could promote the proliferation of rabbit BMSCs and induce osteogenic differentiation.Part III Biocompatibility and osteogenesis of SIM-PLGA-CPC composite tissue engineering bone in vivoMethod:Thirty rabbits (6-week-old; 15 males,15 females; mean weighting 1.0 kg; provided by the Experimental Animal Center of Shandong University, license number; SYXK 2013-0001) were randomly divided into three groups, one group for sham-operation, one group for the implantation of pure PLGA-CPC scaffolds, the other one for SIM-PLGA-CPC scaffolds. Group A was the blank control group (sham operation group), defect on lateral femoral condyle was made and implanted nothing; group B for pure PLGA-CPC scaffolds group, and pure PLGA-CPC scaffold was implanted into the bone defect site; group C for SIM-PLGA-CPC scaffolds group, SIM-PLGA-CPC scaffold was implanted into the bone defect site. The general observation, Micro-CT measurement (2D scan analysis and 3D reconstruction) and histomorphology of rabbit femoral condyle were performed at 6 and 12 weeks post-implantation, respectively, to evaluated the healing of bone defect, and analyze whether there were significant differences in the degree of healing of bone defects in each group.Result:At 6 and 12 weeks post-implantation, the speed and quality of bone defect repair in SIM-PLGA-CPC scaffolds group were significantly better than that in blank control group and pure PLGA-CPC scaffolds group. The general observation:At 6 weeks and 12 weeks after operation, the healing of bone defect in C group was better than that in A group and B group. Micro-CT scan analysis:An average bone coverage of 25.78±6.89% and 68.06±11.62% of each bone defect had been repaired in group C at 6 and 12 weeks, respectively, which was significantly more than group A (3.40± 2.25% at 6 weeks and 6.10±4.48% at 12 weeks) and group B (12.89±5.75%at 6 weeks and 29.24±9.25% at 12 weeks). Bone mineral density (BMD) of these groups quantitatively demonstrated that group C (90.92±9.85 mg/cm3 at 6 weeks and 201.98 ±12.23 mg/cm3 at 12 weeks) resulted in more new bone tissue formed within the bone defect compared with group A (31.04±5.75 mg/cm3 at 6 weeks and 52.80± 8.84 mg/cm3 at 12 weeks) and group B (59.28±5.75 mg/cm3 at 6 weeks and 52.08± 10.78 mg/cm3 at 12 weeks). Micro-CT 3D reconstruction:The speed and quality of the healing of the femoral condyle bone defect in C group were significantly higher than those in the A group and the B group. At 12 weeks, the bone defect has completely healed, and there was no obvious boundary between the bone tissue and scaffold. In group A, there was no obvious healing of bone defect, and still had a large defect at 12 weeks. In group B, the bone defect was partially healed, and left part defects at 12 weeks. Histological examination (HE staining):The speed and quality of bone trabecula and bone matrix in C group were superior to those in A group and B group, and a large number of new bone was observed. The thickness of the bone trabecula was samilar with normal bone tissue. Rarely new bone trabecular was seen in group A in bone defect site, filling with a lot of fibroblasts. Weak new bone trabecular was found in group B with a small amount of fibroblasts.Conclusion:The calcium phosphate composite scaffolds containing simvastatin-loaded PLGA microspheres has excellent biocompatibility and osteogenic activity in vivo, which could improve the speed and quality of bone defect repair. |
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