Research On The Tissue Engineering Treatment For Osteonecrosis Of Femoral Head

Background:Osteonecrosis of femoral head, caused by numorous factors, is a common disease in orthopedics, of which pathogenesis is poorly understood. Depite the artroplasty of the hip, there is no common and effective way to treat the early and medle stage osteonecrosis. According to the pathological characteristic of early and middle femoral head osteonecrosis, it is very important to prevent the collapse of femoral head in the early necrosis of femoral head, and rebuild a functional osteochondral composite in the medle stage of osteonecrosis of femoral head, which is characterized by osteochondral defect.Objective:To determine the effects of local delivery of specific dose of zoledronate (ZOL) on reducing the femoral head deformity following ischemic osteonecrosis. use the laser punching and decellularization technology to fabricate the scaffold derived from osteochondral composite matrix. And investigate the physical properties, biocompatibility and biocompatibility of the scaffold. And test the applicability of the scaffold by implanting the scaffold into the osteochondral defect of a rabbit to evaluate the repair effectiveness of osteochondral defect. This may provide a good scaffold for the tissue engineering treatment of the medle age of osteonecrosis.Methods:Both the osteoclasts and osteoblasts were separated into two groups: control group and ZOL group. TRAP staining and quantitive analysis were carried on to detect the proliferation and activity of osteoclasts. ALP staining and MTT were used to detect the morphological characteristics and proliferation of osteoblasts. PLGA (poly lactic-co-glycolic acid. PLGA) was chosen for the carrier for the zoledronate. Every PLGA cylinder contains 30 ug ZOL.30 SD rats were divided into Drilling group, PLGA group and PLGA-ZOL group randomly after the model of the osteonecrosis of the femoral head being created by placing a ligature tightly around the femoral neck and transecting the round ligament. A tunnel was drilled on the top of the femoral head and received an implantation of PLGA cylinder with 30μg ZOL in PLGA-ZOL group and without ZOL in PLGA group compared with that of without PLGA cylinder as drilling group. The rats were sacrificed six weeks after operation and femoral heads were used to analysis of the deformity and calculate the bone mineral density (bone mineral density, BMD), bone volume fraction (bone volume fraction, BVF), trabecular thickness (trabecular plate thickness, Tb.Th) and trabecular bone (trabecular spacing, Tb.Sp) with X-ray and micro-CT scans in each group. HE staining and Masson trichrome staining were carried out to measure histological changes. Fabricate the scaffold derived from natural pig osteochondral composite by using laser punching and chemical decellularizition method. And then test its mechanical, physical and chemical properties; Use CCK-8 assay to evaluate the cell compatibility and the effects on the proliferation of the cells after seeding the rabbit articular chondrocytes into the scaffold; Seed the rabbit chondrocytes into the scaffold and implant the scaffold-cell composite into the osteochondral defect in non-weight bearing area of rabbit knee joint. Specimens were harvested at 12 and 24 weeks, and then performed gross observation, histological staining, microscopic CT scan, and biomechanical evaluation.Results:After a week, osteoclasts had their typical morphological characteristics. And bone resorption lacunas were found in vitro. ZOL group had significantly decreased number of osteoclasts (p<0.05), number of lacunae (p<0.05), and area of lacunae (p<0.05), compared with control group. However, the optical density of osteoblasts was dramatically higher in ZOL group (3.37±0.11) than control group (2.87±0.12). BMD (535.95±15.08, p<0.05), BVF (0.77±0.04, p<0.05) and Tb.Th (0.64±0.05, p<0.05) increased significantly in zoledronic acid group compared with the model group, drilling group and PLGA group, while Tb.sp (0.37±0.06, p<0.05) significantly reduced. However, no significant difference was found between drilling group and PLGA group. Mover, PLGA-ZOL group have a better shape of femoral head and more new bone formation. A new scaffold was successfully fabricated, which retained the matrix of subchondral bone and cartilage. The scaffold is characterized by a parallel vertical pore structure, which is tapered and with a hole spacing of 300μm. Mechanical tests showed the scaffold had a good resistance to compressive strength, which is similar to nature cartilage. Results showed that the osteochondral composite has a good compression modulus of 1.56 ± 0.288MPa, which is similar to the compression modulus of nature articular cartilage 1.983 ± 0.354MPa, no statistical difference (p< 0.05,n=7). The porosity of the cartilage is 59.75±3.73%.2) The chondrocytes were proliferated well in the scaffold, which was examined by the CCK 8 assay at day 1, day 3, day 5, and day 7. No obvious inflammatory reaction and cell toxicity were found after implanting cells-scaffolds subcutaneously 4 weeks later, by which the scaffold showed a good biocompatibility. 3) Histologic results showed that the repair of scaffold-chondrocytes group is significantly better than pure scaffold and control group (p< 0.05). At 24 weeks postoperative, the repaired cartilage tissue thickness was similar to nature cartilage and the specific staining is positive, and the interface of the subchondral bone and cartilage was regenerated and bone remodeling was completely integrated with surrounding normal tissues; Micro-CT and bone metrology analysis showed a high quality subchondral bone formation; Indentation test shows that the repair area has good mechanical properties, which is similar to the normal cartilage (p> 0.05).Conclusions:The low concentration (10-6mol/L) ZOL can inhibit proliferation and activity of osteoclasts and promote the proliferation of osteoblasts. Therefore, it is important to choose right administration and dose of ZOL to enhance the osteogenesis and inhibit the bone resorption. Local use of zoledronic acid can inhibit bone resorption and promote local bone formation, by which zoledronate can prevent collapse of the femoral head. The bionic scaffold was made by natural articular osteochondral composite matrix by laser punching and decellularization technology, which retained the natural bone and cartilage matrix and complete osteochondral interface. The porous scaffolds have good cell compatibility, which promotes cell adhesion and proliferation. The osteochondral defects in rats are successfully repaired by this porous osteochondral composite derived scaffold. This scaffold can be a good method to treat medle-age osteonecrosis of femoral head, which is characterized by osteochondral lesions or osteochondral defect.