Effect Of Heparan Sulfate On Bone Defect Repair

Heparan sulfate(HS)is a highly sulfated linear anionic polysaccharide,which can be covalently linked to the core protein to form heparan sulfate proteoglycans(HSPGs)on the surface of cell membrane and basement membrane.Although previous studies have shown that HSPGs can combine with growth factors related to fracture healing,thus directly involved in the regulation of growth factor signal transduction and bone cell function.However,the structure-activity relationship and mechanism of HS osteogenesis are not clear,and the application of HS in 3D printing porous composite scaffolds has not been reported.Therefore,heparan sulfate was prepared by separation and purification,and a series of derivatives were prepared by selective desulfation.The osteogenic activity,structure-activity relationship and related pathways of heparan sulfate were studied in vitro.New scaffold materials were prepared by 3D printing technology for the treatment of bone defect repair.The main results are as follows:(1)Heparan sulfate was isolated and purified from crude heparin sodium by relatively mature ion exchange chromatography.The molecular weight of HS was 16.52 k Da and the dispersity was 1.56,which showed good homogeneity.The molar ratio of sulfate to carboxylate of HS was calculated to be 1.35 by Conductometric titration.Then,heparin sodium was selectively desulfated by chemical modification to obtain three selectively desulfated heparin derivatives:2-O-desulfated heparin(2-O-DS),6-O-desulfated heparin(6-O-DS)and N-desulfated heparin(N-DS).The molecular weights of 2-O-DS,6-O-DS and N-DS measured by GPC-MALS were 87.68 k Da,33.93 k Da and 9.61k Da,respectively.The chemical shifts of H atoms corresponding to each peak in the ~1H-NMR spectra were consistent with the previous studies in the laboratory.(2)In terms of osteoblast proliferation,HS/DS can inhibit osteoblast proliferation to a certain extent.The inhibitory effect of 6-O-DS and N-Ds on osteoblast proliferation was slightly stronger than that of 2-O-DS and HS.After 72 hours of HS/DS intervention,the activity of alkaline phosphatase(ALP)in osteoblasts was significantly increased compared with the control group,and the ALP activity in HS group was higher than that in other heparin sulfate free groups.The increase of ALP activity indicates that HS/DS has a positive effect on promoting bone differentiation,and HS can promote the differentiation of osteoblasts more obviously.The suitable concentration of HS is 50?g/ml.(3)The virtual and real binding forces were analyzed by computer simulation docking method and surface plasmon resonance(SPR)technology,and it was found that HS could be tightly combined with ER-?.Then,the ER-beta protein expression level of MC3T3-E1 cells was intentionally downregulated by short interfering RNAs(si RNAs),and the transfected and untransfected osteoblasts were treated with specific concentrations of HS.It was found that ER-beta is a key biomarker to regulate the expression of RANKL and has a certain effect on the generation of osteoclasts.ER-beta mediated the inhibition of osteoclastogenesis by HS through the RANKL/RANK/OPG pathway.HS thus regulated bone tissue metabolism,which might be produced by the direct binding of HS to ER-?.(4)The PCL-HA scaffold prepared by 3D printing technology had good porosity and high biocompatibility,which ensured stable bone ingrowth.Moreover,the scaffolds had high compressive capacity and ductility,which could meet the requirements of different levels of repair of bone defects.PCL-HA scaffolds with low concentration of HS had the best effect in promoting osteoblast maturation and bone defect repair.PCL-HA scaffolds with high concentration of HS(500?g/m L)inhibited osteoblast proliferation in vitro,but this effect was not observed in animal experiments.Therefore,compared with PCL-HA scaffolds alone,HS-loaded PCL-HA scaffolds have higher osteoinductive activity,significantly promote the repair process of bone defects in vivo,and have broad clinical application prospects.Therefore,compared with PCL-HA scaffolds alone,HS-loaded PCL-HA scaffolds have higher osteogenic induction activity,can significantly promote the repair of bone defects in vivo,and have excellent clinical application prospects.