Study Of Biomimetic Mineralized Collagen Bone Materials Modified With Hyaluronic Acid Oligosaccharides

Bone defect caused by trauma,fracture,congenital disease and tumor is always the troublesome problem in the orthopaedics field.Bone transplantation has become the most demanding graft besides blood transfusion and has a rising trend.At present,there are some deficiencies in the clinical application of bone repair materials.Based on the existing problems of bone repair materials and the physiological characteristic of low molecular weight hyaluronic acid(HA)to promote wound healing and angiogenesis,this thesis first proposed the utilization of angiogenic hyaluronan oligosaccharides(oHAs)to modify collagen(Col)to gain glycosylated collagen(Col/oHAs,Col/HA)as the template for modulating calcium phosphate mineralization by mimicking the main components and microstructure of natural bone matrix.The glycosylated mineralized collagen was prepared in vitro by simulating the biomineralization process and fabricated by electrospinning to mimick natural bone with similar components,microstructure and function.Besides,we evaluated the biocompatibility of the mineralized fibers by culturing entothelial cells(PIEC)and pre-osteoblasts(MC3T3-E1)on these materials.The main results of this study are as follows.(1)Both the EDC/NHS crosslinking and the reductive amination reaction were conducted to achieve the collagen modification with hyaluronan oligosaccharides.According to the different molecular weight of HA,the amount of immobilized HA is about 1.18-4%by EDC/NHS crosslinking method,and for the reductive amination the HA content is about 0.91-5.27%.Considering the convenience of subsequent mineralization and molding,the glycosylated collagen prepared by reductive amination was utilized as the template of mineralization in following experiments.(2)According to the principle of biomineralization and self-assembly,the oHAs modified mineralized collagen were prepared by providing Ca2+,PO43-and suitable condition with glycosylated collagen as template.FTIR analysis showed Col/oHAs,Col/HA and Col could provide the nucleation sites by the coordination effect between carboxyl or carbonyl groups and Ca2+,triggering the process of mineralization and deposition of calcium phosphate salt.XRD diffraction and TME analysis showed the formation of nano hydroxyapatite,the morphology analysis of SEM and TEM also indicated the glycosylated collagen achieved mineralization in such biomimetic system,and formed mineralized fiber bundle with oriented arrangement,which is similar to the microstructure of natural bone.(3)With collagen or polylactic acid(PLA)as forming agent,the glycosylated mineralized collagen was generated into porous nanofiber by electrospinning.The processed material has high porosity and nanofiber morphology and thus mimic natural extracellular matrix to a certain extent.(4)PIECs were seeded on the fibrous membrane to test the bioactivity of the glycosylated mineralized collagen.The SEM morphology of the cells showed that PIECs could adhere and spread well on the mineralized fibers,and maintained the normal growth.Meanwhile the cells connected closely and even some cells could be observed to migrate into the fibrous membrane,this phenomenon indicates that the scaffold molded by electrospinning is favorable for the cell migration.MTT results showed that the proliferation activity of PIEC on Col/HA/HAP,Col/oHAs/HAP was relatively better than that on Col/HAP.Moreover,SEM observation showed that PIECs were highly proliferative on some parts of oHAs or HA(5K)modified fibrous materials and they aggregated into pieces on these areas,while such phenomenon had not been seen on material without glycosylation.The result further indicats that the mineralized material modified by oHAs or HA in low molecular has potential for proliferation of PIEC,and the later improvement in increasing the oHAs content in scaffold is expected to evidently promote the proliferation of PIEC.(5)MC3T3-E1 was seeded on the fibrous material to study the effect of glycosylated mineralized collagen on cell growth and preliminarily evaluated the osteogenic properties of the materials.The SEM morphology showed that MC3T3-E1 adhered and stretched well with good growth form on Col/HAP,Col/oHAs/HAP,Col/HA/HAP.In addition to form pseudopods to tightly stick on material to maintain good interaction with material,the cells were also connected with each other,which was conducive to proliferation,differentiation and signal transmission.Compared with the non-glycosylation material,the scaffolds modified with oHAs or HA could obviously promote the stereo migration growth of MC3T3-E1,especially in oligosaccharide group.MTT results indicated that the oligosaccharide modified material was more conducive to the proliferation of MC3T3-E1.Analysis of Col I,OCN related genes and detection of ALP expression revealed that the glycosylated mineralized fibers could meet the physiological function of osteoblasts,especially the oligosaccharide modified mineralized fiber is beneficial to the differentiation of MC3T3-E1 cells into osteoblasts and the secretion of bone matrix.In general,the study of the oHAs modified bionic bone material and its biocompatibility is an important pre-work to construct ideal tissue engineering bone scaffold.It is expected to achive the effective repair and reconstruction of bone defect by promoting angiogenesis and the good osteogenesis performance after the later improvement,and such approach may provide a new strategy for developping vascularized tissue engineering scaffold.