Design Of Blood-Derived Hybrid Hydrogels Based On Bone Microenvironment For Bone Regeneration

Background:Bone defects caused by aging,trauma and various desease have always been common lesions in clinical settings.The current gold standrad for bone defect repair is autologous bone grafting because of its high immunocompatibility and favorable osteoconductive activities.However,the limited availability and donor site morbility of the autologous bone graft used for bone repair have become the major obstacles to the clinical applications.With the development of bone tissue engineering,by modulating the microenvironment of defect area for bone regeneration has become popular.The bone microenvironment plays an essential part of bone tissue engineering,which is also accordance with the reasonal design of biomaterials for augment bone regeneration.The study of the mechanism of bone microenvironment for bone regeneration will make a graet progress in tissue engineering technology.In order to achieve better therapeutic effect,the ideal biomaterials are suggested to modulate multi-microenvironment for bone regeneration.In recent years,autologous blood-derived hybrid hydrogels inclouding platelet rich plasma(PRP),platelet rich fibrin(PRF)and concentrated growth factors(CGF)have been widely applied in Oral Implantology and have already achieved excellent therapeutic effect.Injectable platelet rich fibrin(i-PRF)has emerged as a novel platelet concentrate with no additives,which is injectable before the gel formation and has shown positive effects on bone regeneration with unique advantages.However,due to its inferior mechanical strength,burst release of growth factors and undesirable immunomodulatory,the osteogenesis of i-PRF is still unsatisfied.Thus,it is necessary to develop ideal blood-derived biomaterials for bone regeneration.Objective:By grinding the Polydopamine(PDA)coated Polycaprolactone(PCL)/Nano-hydroxyapatite(n HA)nanofibers and mixed with ungelled i-PRF to prepare PnP-iPRF hybrid hydrogel.Evaluating therapeutic effect of the prepared PnP-iPRF hybrid hydrogel in modulating the bone micro-environment for osteogenesis in vivo and vitro.Methods:1.By electrospinning and PDA coating to prepare PCL/n HA/PDA nanofibers.2.Collecting 5ml blood from human veins and centifugaling for gathering i-PRF.3.Characterizing PnP NFs and PnP-iPRF hybrid hydrogel by using Scanning Electron Microscope(SEM),Universal mechanical testing machine,rheological test,X-ray diffraction pattern(XRD),X-ray photoelectron spectroscopy(XPS),contact angle test and Fourier transform infrared spectroscopy(FTIR)etc.4.The cytotoxicity and biocompatibility of PnP-iPRF hybrid hydrogel was tested by CCK-8(in vitro)and H&E staining of rat main organs.5.The condition of growth factors release and capability of osteogenesis was detected by Enzyme-linked immunosorbent assay(ELISA)and fluorescent real-time quantitative PCR technology(qRT-PCR)to evaluate whether PnP-iPRF hybrid hydrogel was better.6.The stiffness effected osteogenesis of BMSCs was examined by seeding BMSCs on i-PRF and PnP-iPRF hybrid hydrogel with different stiffness.7.Immunofluorescence staining and qRT-PCR was used to verify in vitro mimicking of degradation of PnP-iPRF hybrid hydrogel in vivo while modulating bone immunomicroenvironment.8.Evaluating the therapeutic effect of PnP-iPRF hybrid hydrogel in vivo.Results:1.PCL/n HA/PDA(PnP NFs)evenly dispersed in i-PRF,and we had also proved that PnP-iPRF hybrid hydrogel has higher mechanical strength.2.CCK-8 and the H&E staining of rat main organs were indicated that PnP-iPRF hybrid hydrogel has good biocompability and low cytotoxicity.3.ELISA and qRT-PCR had proved the sustained release of growth factors and better osteogenesis owed by PnP-iPRF hybrid hydrogel.4.The osteogenesis-related gene was much more higher in PnP-iPRF hybrid hydrogel than that of i-PRF was verifyed by Yap-associated mechanical stimulation pathways.5.During degradation,PnP-iPRF hybrid hydrogel could induce macrophages M2-polarization persistently,and made BMSCs osteogenic differentiation indirectly.6.The osteogenesis was much more higher in PnP-iPRF hybrid hydrogel than that of i-PRF in vivo,and PnP-iPRF hybrid hydrogel could also modulate bone immunomicroenvironment in vivo.Conclusions:PnP-iPRF hybrid hydrogel was prepared by uniformly dispersing ground PnP NFs in i-PRF,and PnP-iPRF had longer-lasting growth factor release,higher mechanical strength and better immunomodulatory ability.By developing this hydrogel with multiple regulatory effects on bone microenvironment provides new insights for the development of blood-derived hybrid hydrogels in bone regeneration.