Preparation And Biological Evaluation Of Bone-like Collagen Fibril Based Porous Tricalcium Phosphate/collagen Composites

The repair of bone defect and nonunion has always been the challenging problem in orthopedics.Many methods and materials have been employed to repair bone defect,such as autograft,allograft,and xenograft bone transplantations,metal and ceramic grafts as well as grafts of synthetic polymers.However,each of these methods and materials has its own limitations,for example,the limited donor supply, immune rejection responses,non-degradability of materials,toxicity and other side-effects.In recent years,biomimetically designed bone matrix materials have provided a brand new way for the repair of bone defect and nonunion,which has permeated into almost every area of orthopedics and has become one of the hottest subjects of research.Biomimetic matrix material design is to construct matrix material that mimics the natural matrix both in structure(structure mimetics or bionic structure)and in functional mimetics or functional bionics).Such a biomimetic matrix material should not only have good degradability and bioactivity but also promote and accelerate bone repair.In this thesis,based on the compositional and microstructural characteristics of natural bones,bone-like collagen fibrils(collagen fibrils integrated with nano-tricalcium phosphate(TCP)particles)are proposed as units to construct porous TCP/collagen composites.Such microstructure is believed to make best use of both biological advantages of TCP and collagen in bioactivity,biodegradability, morphology control,carrier ability and cytocompatibility.The resulting composite scaffold was evaluated,through a series of in vitro and in vivo experiments,for its physico-chemical properties,biocompatibility,osteoinductivity and osteoconductivity, and is expected to become a novel bone repair/regeneration biomaterial.In this thesis,the researches include the preparation,characterization and physico-chemical properties of the bone-like collagen fibril based porous TCP/collagen composite and cytocompatibility and bioactivity evaluation of the resulting composites.Eventually,in vivo studies of biomimetic composite for the repair of rabbit femoral condyle defects were done to preliminarily evaluate its ability for bone repair.These researches are summarized as:(1)Collagen fibers could be disassembled into fibrils in acid solution.When nano-β-TCP were added into the collagen fibril suspension,nano-β-TCP particles were integrated with fibrils to form bone-like collagen fibrils.After freeze-drying, bone-like collagen fibrils based porous TCP/collagen composites were obtained.The morphology,porous size and porosity of the composite scaffolds were examined by scanning electron microscopy(SEM),morphological change ofβ-TCP/collagen composites was observed after immersing in PBS buffer solution.Results revealed that the pore walls in the porous composite showed to have mircopores,the optimized acidity of pH 2,the pore size of of 100μm,and porosity of 90%;After soaked in phosphate buffer solution,cluster-shaped HA crystals were formed on the surface of the composite in situ which could accelerate osteoblast proliferation and differentiation.(2)Using in vitro cell culture technology,bone-like collagen fibril based porous TCP/collagen composites was co-cultured with osteoblast-like cells,TCP and MG63 used as positive and negative control respectively.Cell attachment and proliferation were investigated by MTT assay,influence of composites on cell differentiation was investigated by detecting alkaline phosphotatse(ALP)activity,morphological change of cells grew on the material surface was observed by scanning electron microscopy and Laser Scanning Confocal Microscope analysis,bone-specific mRNA expression in cells on different substrate surface was analyzed by RT-PCR and semi-quantification assay,effect of the composites on cell cycle was investigated by Flow cytometry.Results showed that①the number of cells attached on the TCP/Col materials increased higher than TCP control(p＜0.05)and had no significant difference compared with blank control(p＞0.05).②on the fifth day,the number of cell proliferation on the TCP/Col materials was higher than TCP control(p＜0.05)and had no significant difference compared with blank control(p＞0.05).③from the fifth day on,ALP activity in ceils on the TCP/Col materials began to rise and increase significantly than TCP control and have no significant difference compared with blank control.④SEM showed cells on the composites were shuttle-shaped and distributed uniformly and extended well;much matrix secreted accumulated on the surface with time going;LSCM also confirmed the results.⑤RT-PCR and semi-quantification assay showed osteocalcin(OC)and collagen I-mRNA expression in MG63 on TCP/Col composite surface was significantly higher than control.⑥cell cycle analysis demonstrated percentage of cells in S period was improved largely from 35.618%to 59.480%in TCP/Col and DNA content synthesized was also increased.(3)Twenty-four healthy New Zealand rabbits were used to create 48 sides of femoral condyle defects.The defects were implanted with TCP/Col composites and no implant as the control group.X-Ray radiophotography was taken at weeks 2,4,8,12 after operation,and tissue samples were taken for histomorphological and SEM examination.Bone density in defect area was also determined to evaluate the ability of bone repair primarily.Results revealed:①X-ray showed density of bone defect began to rise while no obvious degradation for materials occurred at 2 weeks;the outline of the implant area turned blurred at 4 weeks and its brightness began to decline and the density of material reduced slowly;at 8 weeks,the border of bone defect was unclear and its density continued to decrease,new bone bridge was seen between implant and host bone and new bone grew into the scaffold,most of materials degraded;at 12 weeks,shape of materials disappeared,density of the defect area was comparative to natural bone tissue and bone defect was repaired,while at 12 weeks only little callus occurred for control.②examination of tissue slices and SEM found large quantities of osteoblasts,fibroblasts and new blood vessels growing into the pores of the composite at 2 weeks;at 4 weeks,much osteoid appeared in the defect area;at 8 weeks,most of scaffold were absorbed and substituted for quantities of bone islands;large quantities of new bone grew and connected with each other and Haversian System appeared at 12 weeks while no obvious bone tissue was observed for control.③the rate of bone mineralization for experiment group was higher than control after 4 weeks and by 12 weeks,the density of new bone had been close to natural bone(p＜0.05).Based on above researches,bone-like collagen fibril based porous TCP/collagen composites can be constructed and has similar three-dimensional,porous microstructure with natural bone.Also,the composites show good cytocompatibility and bioactivity,and have positive effects on bone regeneration and accelerate bone repair and have excellent osteoconductivity.Hence,the porous TCP/Col composites could become a new bone repair material.