The Osseointegration Property Of N-HA/PA66 And Its Immunomodulatory Effect On Mediating Macrophage Polarization

PART1 COMPARISON OF THE BONE-IMPLANT INTERFACE AND OSSEOINTEGRATION PROPERTY BETWEEN N-HA/PA66 AND PEKKObjectiveTo compare the bone-implant interface between n-HA/PA66 and PEEK implant,and evaluate their osseointegration property.MethodsPEEK and n-HA/PA66 implants were prepared and implanted into the femoral condyles of adult New Zealand white rabbits to create a line-to-line bone-implant interface.The interfaces were systematically investigated using X-ray radiography,histological analysis,scanning electron microscopy(SEM),elemental mapping analysis(EDX),micro-computed tomography,and push-out tests at 4,8,12,24,and 52 weeks.ResultsAnalysis of X-ray films and histological sections indicated a radiolucent gap around the margin of n-HA/PA66 in the early weeks after implantation(4 to 8 weeks).The gap narrowed and decreased gradually from 24 to 52 weeks.Histological analysis and SEM results suggested that the bone could integrate and adhere in the implant surface.In addition,a better bone-like apatite layer was formed between the bone and the n-HA/PA66 interface.Push-out tests conducted at 24 and 52 weeks showed that the n-HA/PA66 implants have better bonding strength compared with PEEK.ConclusionTherefore,the n-HA/PA66 composite exhibits good osseointegration properties and an improved integrated bone-implant interface.PART2 THE EFFECT OF N-HA/PA66 ON THE POLARIZATION OF MACROPHAGES AND ITS IMMUNOMODULATORY EFFECT OF OSTEOGENIC EFFECT IN VITROObjectiveIn order to further evaluate the difference of osseointegration property between n-HA/PA66 and PEEK.In this study,the immunomodulatory capability of n-HA/PA66 and PEEK on macrophage polarization and its effects on osteogenic differentiation are investigated.MethodsThe n-HA/PA66 and PEEK samples were co-cultured with macrophage was conducted,we tested the immunomodulatory effect of two materials through the cell proliferation experiment and scanning electron microscope and flow cytometer.Flow cytometry was used to identify the M1/M2 phenotype of macrophages;real-time PCR was employed to detect the inflammation-related genes expression of macrophages.Then the macrophage-conditioned medium of n-HA/PA66 and PEEK was prepared,after co-cultured with r BMSCs for 7 and 14 days to explore their osteogenic differentiation ability through alkaline phosphatase(ALP)and alizarin red staining,and RT-PCR tests.ResultsOur results indicated that both n-HA/PA66 and PEEK showed good biocompatibility with macrophages.But n-HA/PA66 can induce macrophages polarization to M2 phenotype,secreting osteogenic cytokines to generate a microenvironment favoring the osteogenesis of r BMSCs in vitro.ConclusionIn summary,n-HA /PA66 can induce the macrophages polarization to M2 phenotype to some extent,and the immune microenvironment induced by this is conducive to the osteogenic differentiation of mesenchymal stem cells.PART3 THE IMMUNOMODULATORY EFFECT OF N-HA/PA66 IN VIVOObjectiveTo better assess the immunomodulatory performance of n-HA/PA66 and PEEK in vivo,Methodswe using air pouch model in SD rats,then the skin tissue around the two materials was harvested,and by HE staining to observe the inflammatory reaction and by immunofluorescence of the surrounding skin tissue to validate the immunomodulatory ability of n-HA/PA66 and PEEK.ResultsThe results of HE staining showed that in the n-HA /PA66 group,the infiltration of inflammatory cells was less than PEEK group.The immunofluorescence results showed that n-HA /PA66 potential enhanced the fraction of CD206+ M2 macrophages,while the PEEK increased the fraction of CCR7+ M1 macrophages.ConclusionThis result suggested that n-HA /PA66 could ameliorate the inflammation and increased the fraction of CD206+ M2 macrophages to build a pro-osteogenic microenvironment compared with PEEK implant.