Experimental Study On Repaired Bone Defects By BMP-7derived-peptide/chitosan/nanometerhydroxya Patite/collagen Biomimetic Composite

Charpter I Effects of neotype bone morphogenetic protein-7derived-peptide on biological behavior of bone marrow stromal stem cellsObjectives:To explore the effect of BMP-7derived-peptide on proliferation, osteogenic differentiation and mineralization of bone marrow stromal stem cells (BMSCs).Methods:Through FMOC/tBu solid-phase peptide synthesis method of BMP7derived-peptide synthesis.BMSCs were harvested from rats and identified with flow cytometry instrument CD90, CD34. The3rd generation of BMSCs was divided into experimental and control groups. The medium of experimental group was supplemented with100mg/L BMP-7derived-peptide. The medium of control group was not supplemented with BMP-7derived-peptide. After2,4,6,8, and10days, the proliferation of BMSCs was analyzed by MTT assay. After7,14, and21days, the calcium contents and alkaline phosphatase activities were measured to assess the differentiation of BMSCs towards osteoblasts. The mineralization of BMSCs was determined by calcein staining.Results:The purity of the BMP-7derived-peptide (KQLNAISVLYFDD, GL Biochem, Shanghai, China) was97%determined by high performance liquid chromatography (HPLC). BMSCs grew well in primary culture and the purity of BMSCs was91.5%. There was no significant difference in proliferation between experimental and control groups (P>0.05). On days7,14, and21, calcium contents and alkaline phosphatase activities in the experimental group were significantly higher than the control group (P<0.05). The mineralization level of BMSCs in the experimental group was significant higher than in the control group.Conclusions:BMP-7derived-peptide has no influence on the proliferation of BMSCs, but it can promote osteogenic differentiation and mineralization of BMSCs significantly. Charpter II Preparation of BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic composite and its biocompatibility in vitroObjectives:To prepare BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic composite and explore the effect of the adhesion, proliferation and osteogenic differentiation of bone marrow stromal cells(BMSCs) on BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen composite.Methods:The chitosan/nanometer hydroxyapatite/collagen composite was prepared and the microcosmic appearance of the composite was observed by scanning electron microscope. The BMP-7derived-peptide was introduced into the composite by vacuum adsorption. The released peptide content from the scaffold was detected using high performance liquid chromatography (HPLC)at different set times. The third generation of BMSCs was seeded onto the composites loaded BMP-7derived-peptide and the unloaded composites as control. The proliferation, adhesion and osteogenic differentiation of BMSCs were analyzed.Results:On scanning electron microscopy, the chitosan/nanometer hydroxyapatite/collagen scaffold had a uniformly distributed and interconnected pore structure, with pore size ranging from several10μm to about100μm. The releasing character of BMP-7derived-peptide belonged to slow release. the release curve exhibited a sharp initial burst at the first day with about (32.5±1.22)%of BMP-7derived-peptide released from the scaffold. After an initial burst, the protein was released in a sustained manner, and the rate decreased with time. At14days, (64.02±4.21)%of total BMP-7derived-peptide was released. There is no significant difference on proliferation of the BMSCs between the experimental group and the control group(P>0.05), but the abilities of adhesion and osteogenic differentiation of the BMSCs in the experimental group were significantly greater than those in the control group (P<0.05)Conclusions:BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic composite is a kind of ideal scaffold material for bone tissue engineering and has good biocompatibility. Charpter III Experimental study on advanced bone regeneration by BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic compositeObjectives:To investigate the effect of BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic composite on repairing rat critical-sized cranial defects.Methods:The chitosan/nanometer hydroxyapatite/collagen composite was prepared and the microcosmic appearance of the composite was observed by scanning electron microscope. The BMP-7derived-peptide was introduced into the composite by vacuum adsorption. The released peptide content from the scaffold was detected using high performance liquid chromatography at different set times. Critical-sized cranial defects were created on both sides of the parietal bone in24adult Sprague-Dawley rats. The BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic composites were implanted on the right side as experimental group and the left side was implanted with chitosan/nanometer hydroxyapatite/collagen biomimetic composites alone as control group. The rats of both groups were killed in batch respectively after the6and12weeks,. Macroscopic observation, three-dimensional reconstruction of computed tomography and histological observation were performed on these samples. The sections from the specimens at the12th week were stained with Masson trichrome methods to evaluate the synthesis of collagen fibrils. Biomechanics detection was performed on the12weeks.Results:The results of scanning electron microscope showed that the surface of the scaffold was porous. The releasing character of BMP-7derived-peptide belonged to slow release. The result of animal experiment showed that the BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic composite can more effectively promote the repair of cranial bone defects comparing with the chitosan/nanometer hydroxyapatite/collagen biomimetic composite alone. Representative Masson trichome staining photographs of regenerated bone at the12th week were shown. Massive calcified bones stained red color were observed in the experimental group. However, in the control group there was only slight amounts of fresh-formed bone stained blue color. Biomechanics detection indicated the maximum compression loading and loading/strain ratio on repairing tissue of defects in the experimental group was obviously superior to those in the control group. The difference was statistically significant (P<0.05)Conclusions:The BMP-7derived-peptide/chitosan/nanometer hydroxyapatite/collagen biomimetic composite can effectively promote bone regeneration of bone defects. The composite is a kind of ideal scaffold material for bone tissue engineering.