| Background: Recently,the incidence of congenital hip dysplasia,osteopenia,femoral head necrosis,osteoarthritis,and infected osteonecrosis caused by genetics,diseases,tumors,and trauma has been high.Such diseases can lead to incomplete bones and bone regeneration difficulties,and affecting the body’s normal activities.For skeletal problems caused by this type of etiology,artificial bone tissue materials are usually used clinically to fill or support bone defects,the osteogenic properties of the materials are emplaned to promote bone self-healing and restore normal activities of the organism.Bone is mainly composed of organic collagen and the inorganic component hydroxyapatite(HA).HA is an essential inorganic component that makes up bone tissue,and can be synthesized artificially by chemical methods.Synthesized HA possess excellent biocompatibility and osteoinductive ability,and is widely used in bone tissue regeneration.To optimize the bone repair ability of synthetic HA,HA can be doped and modified by different ions for overcoming the deficiencies as a bone tissue replacement material.The formation of new bone tissue requires adequate blood supply,because highly vascularized tissue is necessary for artificial bone grafting.Cobalt(Co)is an inhibitor of hypoxia-inducible factor prolyl hydroxylase that activate the hypoxiainduced factor-1(HIF-1)signaling pathway,which in turn activates vascular endothelial growth factor(VEGF)to promote angiogenesis.Polymers(amino acids)(PAA)are composed of natural and synthetic amino acid monomers,and show similar chemical structure to collagen,with strong flexibility,degradability,and high activity.PAA can be processed into various shapes according to different needs.In this study,organic PAA was synthesized by melt polymerization,and inorganic cobalt hydroxyapatite(Co-HA)was added by in situ method to obtain a new polymer(amino acid)/cobalt hydroxyapatite(PAA/Co-HA)composite with excellent biocompatibility and the ability to promote angiogenesis and bone growth.Methods:1.In this experiment,Co-HA and PAA/Co-HA composites were synthesized by hydrothermal and in situ melt polymerization methods,respectively.Moreover,the two materials were characterized materialistically by X-ray Electron Diffraction,Scanning Electron Microscopy,Fourier Transform Infrared Spectroscopy,X-ray Photoelectron Spectroscopy,Differential Scanning Calorimetric analysis,Thermogravimetric analysis and Mechanical performance tests.2.The effects of Co-HA and PAA/Co-HA composites on the proliferation and morphology of BMSCs were investigated by CCK-8 assay and cell staining.The qualitative and quantitative Alkaline phosphatase(ALP),qualitative and quantitative calcium nodules,Western Blotting and Real-time Fluorescence Quantitative Polymerase Chain Reaction(RT-PCR)were researched of evaluating the osteogenesis and angiogenesis of PAA and PAA/Co-HA composites at proteins and m RNA levels.3.Femoral condyle and muscle defect model of New Zealand rabbit was used for in vivo implantation of PAA and PAA/Co-HA composites,and the biocompatibility and osteogenic properties of PAA and PAA/Co-HA composites were investigated by histological observation and fluorescent staining observation.Result:1.The results showed that the Co-HA synthesized by hydrothermal method is an irregular granular with good crystallinity and HA characteristics.the 0.1% Co2+ doped Co-HA possess the smallest crystallinity and the largest microcrystal size among all Co2+doped HA samples.The results of cell experiments confirmed that Co-HA could promote the proliferation of BMSCs,and the optimal proliferation ratio of cobalt/calcium was 0.1 % Co-HA.ALP results showed that 0.1% Co-HA exhibits higher ALP activity.Calcium nodules experiments showed that 0.1% Co-HA can promote mineralization deposition and increase the number of calcium nodules during osteogenic differentiation of BMSCs.Western Blotting and RT-PCR results showed that0.1% Co-HA increased the protein and m RNA expression of HIF-1α,VEGF,heme oxygenase,and osteopontin(OPN),which could effectively promote osteogenic differentiation of BMSCs.2.The materialistic characterization of the PAA/Co-HA composite showed that the Co-HA was uniformly distributed in the PAA matrix and chemical bond was formed between the interface of PAA and Co-HA.The addition of Co-HA led to an increase in the melting point and crystallization temperature of PAA.The PAA/Co-HA composites containing 30% of Co-HA(PAA/Co-HA30)shows good stability with a final decomposition temperature of 441.88 ℃.The compressive strength of PAA/Co-HA30 is 125.57 Mpa,which is higher than that of the natural bone.And the bending and yield strengths of PAA/Co-HA30 are 85.125 Mpa and 110.28 Mpa,respectively,which can be well-matched with natural bone.The results of cell experiments showed that the PAA/Co-HA composites could promote the proliferation of BMSCs,and the PAA/CoHA30 possessed the best pro-proliferative ability.In comparison with other Co-HA doped PAA,the calcium nodules experiments showed that the PAA/Co-HA30 complex increased the mineralization deposition of calcium nodules during the osteogenic differentiation of BMSCs.Western Blotting and RT-PCR results showed that the PAA/Co-HA30 composites increased the protein and m RNA expression of Runt-related transcription factors,osterix,integrin binding sialoprotein,and OPN,which effectively promoted osteogenic differentiation of BMSCs.3.PAA and PAA/Co-HA composites showed no adverse reactions and good biocompatibility when implanted into muscle of rabbits.PAA and PAA/Co-HA composites showed no adverse reactions when implanted into femoral condyle defects.PAA/Co-HA30 composites could bond directly with the new bone tissue and promote new bone formation.Fluorescent staining results showed that PAA and PAA/Co-HA composites guided new bone growth in the femoral condyle defects.Conclusion:1.Co-HA showed typical characteristics of HA with excellent cytocompatibility and pro-angiogenic ability,and it can promote osteogenic differentiation of BMSCs.2.PAA/Co-HA composites possessed good in mechanical properties,vitro cytocompatibility and ability of osteogenic induction.3.PAA/Co-HA composites display excellent in vivo biocompatibility,osseointegration,and pro-new bone regeneration. |
