Study Of A Coating Of Silicon Nitride On Porous Polyetheretherketone On Behaviors Of Bone Repair

As a synthetic non-oxide bioceramic,silicon nitride(Si3N4,SN)exhibits good biocompatibility and excellent osteogenic activity.SN possesses favorable surface bioactivity that could significantly promote cellular responses(such as proliferation and differentiation),which has been confirmed by in vitro experiments using both osteosarcoma cells and human osteoblast-like MG63 cells.The chemical and spectroscopy analyses of the SN implants revealed that the silicon and nitrogen elements were deposited into the crystal lattice of native hydroxyapatite by action of the osteoblasts.Previous studies have shown that SN was not only biocompatible but also osteoconductive,which exhibited excellent osteogenesis in vivo.The presence of Si and N could stimulate progenitor cells’differentiation and promote osteoblastic activity in vivo,which ultimately accelerates new bone formation.Porous silicon,silicon/silica nanoparticles,and silicon-substituted hydroxyapatites have been developed for bone regeneration;all of which exhibited the common mechanism of Si-enhanced osteogenic activity.Nitrogen(N)is a complementary element,which is essential for protein synthesis and bone growth as well as tissue regeneration.Polyetheretherketone(PK),a semicrystalline thermoplastic polymer with good biocompatibility,mechanical strength,and an elastic modulus similar to that of human bone,has been developed as bone implants in orthopedic and dental applications.However,the bioinert nature of PK displays poor osteogenic activity,which cannot integrate with the host bone and so decreases the primary fixation as well as long-term stability of implants.Porous biomaterials have been identified as a potential alternative given their ability to allow bone tissue ingrowth,indicating good primary stability during early osteogenesis.Therefore,a strategy to improve osteogenesis and osseointegration with the host bone is to incorporate porosity into PK,which can promote the ingrowth of new bone tissues into the porous structures,thereby causing more biological anchorage with the host bone to improve the stability of the implants.During the bone healing process,osteogenesis is always coupled with angiogenesis,and osteogenesis is always accompanied by vascular invasion.Good blood supply and angiogenic capacity are essential for bone regeneration.Therefore,finding a method for locally promoting angiogenesis for porous implants is also a challenge for designing novel biomaterials for bone repair.In this study,polyetheretherketone(PK)was used as the matrix material,and the macroporous structure PK(PPK)was prepared by cold pressing sintering and salt-leaching method and then a bioactive SN coating on a PPK(CSNPPK)surface was fabricated by using suspension coating and melt binding.The objective of this study is to promote the vascularization and osteogenesis of CSNPPK by taking advantage of the macroporous structures and bioactive SN coating on PPK.Therefore,the effects of the SN coating of CSNPPK on the behaviors of MC3T3-E1 cells in vitro and on the vascularization,osteogenesis,and osseointegration of CSNPPK in vivo were evaluated.The relationship between the CSNPPK surface characteristics and structural composition and the bone repair were analysed.In this study,we explore the mechanism of CSNPPK to promote bone ingrowth and osseointegration,and provide new ideas and methods for the development of new special functions bone implant materials.Our study consists of three parts:Parts 1,The preparation and characterization of CSNPPK biomaterials;Parts 2,The effect of CSNPPK on the behavior of MC3T3-E1 cells in vitro;Parts 3,The vascularization,osteogenesis,and osseointegration of CSNPPK in vivo.Parts 1,The preparation and characterization of CSNPPK biomaterialsObjectives:The surface morphology、chemical composition、the three-dimensional(3D)surface roughness、pore size、porosity、compressive strength、water absorption、ion release and PH values properties of CSNPPK were studied.Methods:Firstly,macroporous PK(PPK)was prepared by pressing sintering and salt leaching by using NaCl particles as the porogen and then SN coating was prepared on the surface of PPK(CSNPPK)by suspension coating and melt binding.DPK and PPK were selected as the control group and CSNPPK as the experimental group.The surface morphology of the samples was observed by scanning electron microscopy(SEM;S-4800,Hitachi,Tokyo,Japan)with an acceleration voltage of 10 kV.The chemical compositions of the specimens were determined by X-ray diffraction(XRD,D/MAX 2550 VB/PC,Rigaku Co.,Japan)and Fourier transform infrared spectrometry(FTIR,Nicolet 6700,Nicolet,U.S.A.).The element distribution of the samples was measured using energy dispersive spectroscopy(EDS,S-4800,Hitachi,Japan).The three-dimensional(3D)surface topography and roughness of the specimens were characterized by a laser microscope(VK-X 110,Keyence Co.,Japan).The compressive strength of the samples was measured using a mechanical testing machine(Instron-3345 U.S.A.).Average pore size was measured by using the CTAn program(Skyscan Company,Belgium).The water absorption and porosity of samples were measured using the liquid displacement method.The concentration of Si ions was measured by an inductively coupled plasma emission spectrometer(ICP-OES,U.S.A.)at different times(1,3,7,10,14,and 28 days)after the specimens were immersed in a simulated body fluid(SBF)solution.At each time point(6,12,24,72,and 168 h),the pH values were determined by using a flat-membrane microelectrode(PB-10,Germany).Results:From the SEM images,the SN microparticles(agglomeration of nanoparticles)were found on the macroporous surface of CSNPPK.From the XRD spectra of the specimens,the characteristic peaks at 20=13.4,23.3,27.0,33.6,and 36.00 originated from SN on the surface of CSNPPK.From the FTIR spectra of the samples,the characteristic stretching vibration bands(Si-N)of SN for CSNPPK were observed at 884 cm-1.Furthermore,carbon,oxygen,silicon,and nitrogen elements were observed on the macroporous surface of CSNPPK from the EDS analysis of the samples.3D laser microscopy analysis showed that the roughness of CSNPPK was 187.08±0.21um,while the roughness of PPK was 155±0.18um.The compressive strength of CSNPPK(7.7±1.4MPa)was higher than that of PPK(6.8±1.9MPa).The average pore sizes of PPK and CSNPPK were 347.0±27.6 μm and 339.0±31.2 μm,The water absorption of CSNPPK(476.8±23.1%)increased compared to PPK(411.7±10.5%)and the porosity of CSNPPK(68.2±2.6%)exhibited no obvious decrease compared with PPK(71,4±3.1%).The Si-ion concentration for CSNPPK rapidly increased from 1 to 14 days followed by stabilization of values from 14 to 28 days,and the Si-ion concentration was stable at approximately 3.4 mg/L at 28 days.The pH value for CSNPPK slightly increased from 6 to 168 h,and the pH value was approximately 7.5 at 168 h.Conclusions:By the method of suspension coating and melt binding,the SN coating was successfully prepared on the surface of CSNPPK.The SN components were observed on the macroporous surface of CSNPPK from the XRD、FTIR and EDS analysis of the samples.With the addition of silicon nitride,the surface roughness、water absorption、compressive strength of CSNPPK was improved compared to PPK,while the pore size and porosity of CSNPPK exhibited no obvious changes.The ion release experiment showed that CSNPPK could release a small amount of silicon ions continuously from 1 to 28 d and PH value for CSNPPK slightly increased from 6 to 168 h.Parts 2,The cytocompatibility of CSNPPK in vitroObjectives:The effect of CSNPPK on the behavior of MC3T3-E1 cells in vitroMethods:Mouse osteoblast-like MC3T3-E1 cells were selected for cytocompatibility evaluation in vitro.DPK and PPK were selected as the control group and CSNPPK as the experimental group.Cell counting kit-8(CCK-8 Dojindo,Kumamoto,Japan)was utilized to determine cell adhesion at 6 and 12 h and to determine cell proliferation at 1,4,and 7 days.The cells morphology on samples at 24 h were observed by SEM and CLSM images.The osteogenic differentiation ability of MC3T3-E1 cells on materials was analyzed by ALP staining and quantitative analysis of ALP activity at 7,14,and 21 days.Mineralized nodules of the cells on samples were analyzed by alizarin-red staining at 28 days.The mRNA expression of MC3T3-E1 osteogenic differentiation-related genes,including alkaline phosphatase(ALP),collagen type 1(COL1),osteopontin(OPN),and osteocalcin(OCN),was quantitatively analysed by a real-time polymerase chain reaction(PCR).Results:The number of cells adhered on CSNPPK was obviously higher than on PPK and DPK at 12 h.The relative proliferation rates of cells on CSNPPK were obviously higher than on DPK and PPK at 4 and 7 days.SEM shows that the cells on the DPK surface exhibited a sphericity morphology,while the cells on PPK and CSNPPK displayed a flattened morphology.Moreover,more cells with more lamellipodia adhered and spread well on CSNPPK compared with PPK and DPK at 24h.CLSM images shows that more cells adhered and spread well on CSNPPK compared with PPK and DPK at 24h.The areas of ALP staining as well as ALP activity of the cells on CSNPPK were higher than on PPK and DPK at each time point.The values of the alizarin-red staining area and mineralized nodules of the cells on CSNPPK were higher than on PPK and DPK at 28 days.7 days,the expressions of ALP and COL1 of the cells on CSNPPK were higher than on DPK and PPK.At 14 days,the expressions of ALP,COL1,and OPN for CSNPPK were higher than for DPK and PPK.At 21 days,the expressions of the four genes for CSNPPK were higher than for DPK and PPK.Conclusions:CSNPPK with the bioactive SN coating remarkably stimulated the cellular responses(adhesion,proliferation,and differentiation as well as osteogenically related gene expression)in vitro.Parts 3,The vascularization,osteogenesis,and osseointegration of CSNPPK in vivoObjectives:To study the vascularization,osteogenesis,and osseointegration of CSNPPK in vivo.Methods:A rabbit model of femoral condyle defect was used as an animal experiment in vivo.The samples containing newly formed bone tissue and implants were imaged with X-ray and micro computed tomography(micro-CT,Scanco Medical,uCT-80,Switzerland)at 4 and 8 weeks after operation.The decalcified samples were imaged with micro-CT to evaluate the vascularization.Meanwhile,the immunohistochemistry of vascular endothelial growth factor(VEGF)staining was conducted to assess vascularization(vessel ingrowth).The specimens stained with H&E and Masson’s trichrome were observed by a microscopic system for descriptive histology of new bone formation.The fluorescence labeling was used to study the formation rate of new bone during bone healing.The sections were stained by using Van Gieson’s picrofuchsin to observe newly formed bone.A push-out test was performed to assess the osseointegration of the samples by using a mechanical testing machine(Instron-3345 U.S.A.)at 8 weeks after implantation.Results:Compared with PPK,more newly formed bone tissues(NB)were found to have grown into CSNPPK at 4 and 8 weeks after implantation.No vessel formation was found for DPK.However,the formation of vessels for CSNPPK was more than for PPK.Some loose fibrous connective tissues around DPK were found.However,some NB grew into macropores of PPK and CSNPPK in which more NB was found for CSNPPK than for PPK at 4 and 8 weeks.It was found that the area of VEGF staining for CSNPPK was higher than for PPK at 4 and 8 weeks after implantation.Histological images labeled by fluorescence revealed that the NB area for CSNPPK was higher than for PPK and DPK.Histological images of Van Gieson staining indicated that the NB area for CSNPPK was higher than for PPK and DPK at 4 and 8 weeks after implantation.At 8 weeks after implantation,the push-out load for CSNPPK was higher than for PPK and DPK.Conclusions:CSNPPK with the SN coating enhanced obviously vascularization and osteogenesis and promoted osseointegration in vivo.