Biomimetic Intrafibrillarly Silicified Collagen Scaffold Promotes In Situ Bone Regeneration By Monocyte Modulation

In recent years,researches focus on bone regenerative materials have gained great development for clinical use.However,the different physicochemical properties,mechanical properties and biological application of these materials usually cause large diversity in the restorative efficacies.Host immune response is one of the most critical factors affecting the fate of biomaterials in vivo.Previously,tissue regeneration strategies focused on minimizing host immune responses to promote engraftment efficacy.However,it is now generally accepted that the immune system exerts positive immunomodulatory effects on tissue regeneration,leading to the progress of defect repair and tissue regeneration.Monocytes are crucially involved in the coupling process of osteoblast bone formation and osteoclast bone resorption,which are essential for bone regeneration.The interaction between monocytes and the implanted biomaterials directly affect the fate of the materials and engraftment efficacy.Biomimetic intrafibrillar silicified collagen is a kind of biomaterials with favorable physicochemical properties and mechanical properties.3-D silicified collagen scaffold(SCS)based on this material is infiltrated with highly-ordered,intrafibrillar amorphous silica nanoparticles that are derived from the condensation of poly(amine)-stabilized orthosilicic acid.Our previous in vitro study showed that the scaffold has great potential in promoting bone regeneration.In our present study,we evaluate the biological application potential of the intrafibrillar silicified collagen scaffold in bone regeneration.Animal models were employed to validate the bone repair efficiency of this scaffold.The interaction of the scaffold and host immune response were also investigated,including the effect of SCS on monocytes,as well as promoted angiogenesis,cell homing and bone regeneration by this monocyte modulation.1.Scheme 1)With the help of transmission electron microscopy(TEM)and micro computed tomography(Micro-CT),we characterized the morphology festures of the intrafibrillar silicified collagen scaffold.Cumulative release profile of Si derived from immersion of silicified collagen scaffolds in phosphate-buffered saline over a 30-day period was also calculated.The in vivo subcutaneous implant model was employed to evaluate the biocompatibility of the scaffold.In vitro proliferation of lymphocytes was evaluated to determine the immunogenicity of the scaffold.We also used flow cytometry to evaluate the effect of scaffold on the level and the activity of circulatory lymphocytes.ELISA was used to evaluate the level of circulatory inflammatory factors.HE staining was used to observe the infiltration of inflammatory cells in situ.2)In the second part of this study,we built a mice calvarial defect model and the intrafibrillar silicified collagen scaffold was used for defect repair.Micro-CT scanning was used to detect the level of bone regeneration,volume of new bone formation and bone mineral density immediately,1 month and 3 months after scaffold implantation,respectively.Fluorescent double-labeling of calcein and alizarin red were used to evaluate the activity of bone regeneration at 3 months after implantation.And the undecalcified sections were made for Van Gieson staining and von Kossa silver staining to evaluate the efficiency of bone formation.Micro-CT angiography was used to evaluate angiogenesis of the defect site at 3 month after implantation.For further analysis,immunofluorescence and immunohistochemistry were used to investigate sections 1 month after implantation,and evaluate monocytes & macrophages,cytokines involved in bone regeneration.TRAP staining was used to observe the osteogenesis and the activity of osteoclasts.3)In the third part of this study,we further examinated the effect of the intrafibrillar silicified collagen scaffold on monocyte.MTT cell proliferation test,cell apoptosis test and intracellular ROS level test were used to examine the effect of SCSs on the cell fate of monocyte.TRAP cell staining was used to examine the effect of SCSs on the differentiation of monocyte.Expression of certain cytokines secreted by monocytes were evaluated by cell immunofluorescence,q RT-PCR and western blot at both m RNA and protein level.Transwell cell migration test was employed to determine the effect of interaction between SCSs and monocyte on the migration of BMSCs and EPCs.Matrigel tube formation test was used to evaluate the angiogenesis potential of EPCs affected by SCSs or monocyte-conditioned SCSs.Neutralizing antibodies were used to determine the level of monocyte cytokine expressions which might be involved in cell migration and angiogenesis exposed to SCSs.4)In the last part of the study,we built a rat femur partial defect model and the SCSs were used for defect repair.This part was aimed to further investigate signaling pathways changes involved in monocyte exposed to SCSs.Micro-CT,immunohistochemistry and Van Geison staining were used to evaluate the efficiency of angiogenesis and bone regeneration in bone defect site at 1 month after implantation.Immunofluorescence was used to investigate the distribution patterns and cytokines expression of monocyte in situ.In further in vitro study,western blot was used to examine the changes of signaling pathways.Monocytes inhibited by certain pathway were further examined by western blot,TRAP cell staining to investigate cell differentiation and secretion.Pathway inhibitors were also used in vivo to investigate signaling pathways involved in SCSs bone regeneration.2.Results Part I Physicochemical properties and biocompatibility of biomimetic intrafibrillar silicified collagen scaffold1)Collagen fibrils of SCSs contained an ordered deposition of intrafibrillar amorphous silica that creates a banded appearance.SCSs could release silicic acid sustainingly.Silicic acid release from the homoegeneously silicified SCSs was characterized by an initial burst release,followed by a slower continuous release.30 days after expose to PBS(10m L),concerntration of silicic acid could reach 1.2mmol/L per 100 mg SCS.2)Intrafibrillar silicified collagen scaffold is biocompatible.We have demonstrated that SCS has less immunogenicity,and induce minimum proliferation of lymphocytes after twice stimulation(p>0.05 when comparing with control group).Lymphocytes numbers and activity,as well as inflammatory cytokines were kept in general level after SCSs implantation(p>0.05 when comparing with control group).HE staining of in situ sections showed that collagen fibers were absorbed obviously 7 and 14 days after inplantation,and there are minimum inflammatory cells infiltration around the scaffolds,which proves SCSs is biocompatible and could be further used in in vivo bone repair.Part II Application of SCS in mice calvarial defect model 3)We found that intrafibrillar silicified collagen scaffold could promote bone regeneration in the mice calvarial defect.3 months after surgery,more new bone formation and more active bone regeneration could be observed in SCSs group when comparing with the control groups(p<0.05).4)3 months after implantation,SCSs group generate more new vessels in defect site(p<0.05).Vessel length,thickness and connectivity of SCSs groups is significantly more than the negative control group(p<0.05).5)One month after surgery,there could be observed more CD31+Endomucin+ subtype vessels in SCSs group(p<0.05),as well as more PDGF-BB expression(p<0.05).TRAP positive mononuclear cells were significantly more(p<0.05)and these cells express more SDF-1 and TGF-β1(p<0.05).In SCSs group,expression of BMSC marker Nestin and VEGF were increased in defect site(p<0.05),which indicated that there is an enhaced seed cell homing effect.Part III The effect of SCSs on monocyte modulation6)The SCSs did not affect monocyte proliferation,apoptosis and intracellular ROS level(for each experiment,p>0.05 when comparing with control group).Conditional medium of monocytes exposed to SCSs did not affect the migration of BMSCs and EPCs(p>0.05 when comparing with control group).7)We demonstrated that monocytes could differentiate into TRAP positive mononuclear cells after exposed to SCSs.And this kind of monocytes could express more SDF-1,TGF-β,VEGF and PDGF-BB in both m RNA and protein level(p<0.05 when comparing with control group).8)Conditional medium of monocytes exposed to SCSs could significantly promote BMSCs and EPCs migration(p<0.05),as well as EPCs tube formation(p<0.05).Neutralizing antibodies verification demonstrated that SDF-1,TGF-β and PDGF-BB were the key factors that affect cell migration,and TGF-β,VEGF,PDGF-BB were the key factors that promote EPCs tube formation.Part IV Cell signaling pathways of monocyte involved in SCSs modulation9)We demonstrated that intrafibrillar silicified collagen scaffold is favorable for different bone defect models.In the rat femur partial defect model,SCSs could significantly promote vessels formation and trabecular bone formation(p<0.05),which indicated that SCSs promote wound healing and bone repair.Immunofluorescence double labeling showed that numbers of CD31+Emcn+ subtype vessels(p<0.05)as well as TRAP positive monocytes and PDGF-BB expression were increased(p<0.05)in the SCS group.10)In vitro study showed that the P38 and ERK1/2 of monocyte were activated by SCSs.Monocyte differentiation towards TRAP positive mononuclear cell and secretion of SDF-1,TGF-β,VEGF,PDGF-BB were decreased(p<0.05)when P38 and ERK1/2 were inhibited,respectively.Conditional medium of SCS-exposed monocytes using P38 or ERK1/2 inhibitor could not promote BMSCs and EPCs migration(p>0.05),nor EPCs tube formation(p>0.05).Further in vivo study demonstrated that when P38 inhibitor was used,CD31+Emcn+ subtype vessels formation was significantly declined(p<0.05 when comparing with SCS group).TRAP positive monocytes in defect site and expression of PDGF-BB were declined either(p<0.05 p<0.05 when comparing with SCS group).This indicated that P38 signaling pathway were the key factor in the modulation of monocyte by SCSs,which directly affects bone regeneration.3.ConclusionFirstly,our findings demonstrated that biomimetic intrafibrillar silicified collagen scaffold has minimum immunogenicity.After implantation,host inflammation response could be controlled in low degree,and cause no inflammatory cells infiltration,nor increased inflammatory cells/factors in circulation.These findings indicate that intrafibrillar silicified collagen scaffold is biocompatible,and is favorable for biological application.Secondly,intrafibrillar silicified collagen scaffold could be used for different bone defect type(both mice calvarial defect model and rat femur partial defect model).SCSs promote local angiogenesis and bone regeneration significantly,which leads to advanced outcome.Thirdly,in the early stage of defect repair,intrafibrillar silicified collagen scaffold could modulate monocytes to differentiate into TRAP positive mononuclear cells by activating P38 signaling pathway,which leads to an enhanced secretion of certain cytokines(SDF-1,TGF-β,VEGF,PDGF-BB).As a result,this could promote local angiogenesis,especially CD31+Emcn+ subtype vessels formation,which is critical in the coupling of angiogenesis and osteogenesis.Moreover,this could also enhance host BMSCs and EPCs homing towards the defect site,and promote local vessel and bone formation.In conclusion,the biomimetic intrafibrillar silicified collagen scaffold is biocompatible,and has great potential in biological application.SCSs show favorable efficency in bone defect model repair,and could promote local angiogenesis and osteogenesis significantly.By modulating monocytes,SCSs could promote local neovessels formation and host seed cells migration.Comparing with traditional bone grafts which need extra loading of cells or cytokines,SCSs show much advantage in convenience and potential clinical application.