| Early inflammation plays an irreplaceable role in bone repair facilitating vascularization and bone remodeling.Persistent inflammation will obstruct endogenous bone marrow mesenchymal stem cells(MSCs)homing and aggravate bone tissue destruction.However,the role of macrophage(Mφ)lipid metabolism pathway in anti-inflammatory/pro-resolving effects and accelerating bone healing has gradually attracted the attention of scholars.Although biomechanical stimulations have been comfirmed to regulate the lipid metabolic axis of inflammatory cells to produce specialized pro-resolving mediators(SPMs),how matrix stiffness affects early inflammatory response regulated by Mφ lipid metabolism in bone repair remains unclear.Strategies to accelerate the resolution of inflammation in early bone repair include not only improving the biomechanical microenvironment of bone regeneration,also preventing acute inflammation from developing into chronic inflammation.Therefore,enhancing the "cross-talk" between endogenous MSCs and host Mφs by promoting inflammation resolution can improve bone regenerative microenvironment.In this study,by investigating the mechanical response of Mφs to demineralized bone matrix(DBM)scaffolds with different matrix stiffness,the regulation effect of matrix stiffness on Mφ lipid metabolism pathway was expounded.Then,the low stiffness difunctional DBM scaffold that could not only endogenously recruit MSCs but also promote inflammation resolution was designed,so as to explore the pro-resolving mechanism of MSCs and Mφs in bone repair.Finally,the immunoregulatory effects of maresin 1(MaR1)on M2-subtype Mφ lipid metabolism pathway were investigated.At the same time,the effects of Mφ nuclear receptor peroxisome proliferator-activated receptor-γ(PPAR-γ)on the biological behaviors of MSCs in bone repair microenvironment were further explored.The main contents and results are as follows:(1)Immunological regulatory role and mechanism of matrix stiffness on Mφ lipid metabolism remodelingThe MSCs were successfully isolated from mouse bone marrow of femur and tibia.The effect of DBM scaffolds with different matrix stiffness on Mφ subtypes polarization,expression of inflammatory cytokines,expression of key enzyme 12-lipoxygenase(12-LOX)in Mφ lipid metabolism,and biosynthesis of lipid mediators(LMs)were analyzed.The obtained results showed that the low matrix stiffness could polarize Mφs into an anti-inflammatory phenotype,and promote the expression of anti-inflammatory cytokines,SPMs,and 12-LOX protein,which finally confirmed that matrix stiffness regulated bone repair by modulating 12-LOX-mediated early inflammation.(2)Preparation and characterization of the difunctional DBM scaffoldThe low stiffness DBM scaffold with no obvious immunological rejection was selected to immerse in 1.25%(w/v)sodium alginate(SA)solution,then the amine-modified aptamer 19S(Apt19S)was combined with DBM scaffold by chemical amide bond.The MaR1 was loaded onto the DBM scaffold by physisorption method to fabricate the low stiffness difunctional DBM scaffold successfully.Finally,the internal pore structure,cytotoxicity,cumulative release curves of Apt19S and MaR1,amide bond formation of difunctional DBM scaffold were detected.The results indicated that the difunctional DBM scaffold was successfully prepared and characterized.It was verified that the difunctional DBM scaffold could promote MSCs proliferation,recruitment,osteogenic differentiation,and the polarization of Mφs towards M2 subtype in vitro.(3)Effects of the difunctional DBM scaffold on biological behaviors of Mφs and MSCsThe effects of MaR1 on the expression of 12-LOX and PPAR-γ,and lipid droplets(LDs)biosynthesis in Mφs were investigated.The effects of nuclear receptor PPAR-γ on Mφ subtypes polarization,expression of inflammatory cytokines,and lipid metabolites in Mφs cultured on the difunctional DBM scaffold were detected.Meanwhile,the proliferation,migration,and osteogenic differentiation of MSCs in the co-culture system were further investigated.The results showed that MaR1 up-regulated the expression of PPAR-γ protein,12-LOX and its metabolite 12S-hydroxy-eicosatetraenoic acid(12S-HETE),and LDs biogenesis in Mφs.Furthermore,the difunctional DBM scaffold enhanced the anti-inflammatory function of Mφs by modulating nuclear receptor PPAR-γ-mediated early inflammation and further promoted the proliferation,migration,and osteogenic differentiation of MSCs in the co-culture system.(4)Immunomodulatory effect of the difunctional DBM scaffold on mouse skull defect repairThe mouse skull defect model(Φ=4 mm)was established,and the difunctional DBM scaffold was implanted to detect its effects on the biological behaviors of MSCs and Mφs in early bone repair.Subsequently,the pro-resolving mechanism of difunctional DBM scaffold was investigated in early inflammation of bone repair,and its effects on bone morphological repair and functional reconstruction were evaluated systematically.The results indicated that the difunctional DBM scaffold promoted the expression of anti-inflammatory cytokines,inhibited the expression of pro-inflammatory cytokines,enhanced the expression of chemokines,induced the polarization of Mφs towards M2 subtype,and up-regulated the expression of PPAR-γprotein,12-LOX protein and its metabolite 12S-HETE in vivo.Finally,the difunctional DBM scaffold promoted MSCs recruitment,collagen deposition,osteogenic proteins expression,and angiogenic marker CD34 protein expression,indicating a good osseointegration and bone repair ability in vivo.In conclusion,this study elaborated the immunoregulatory mechanism of matrix stiffness on Mφ lipid metabolism by investigating the biomechanical response of Mφs to the DBM scaffolds with different matrix stiffness.The difunctional DBM scaffold was successfully prepared and characterized.It was confirmed to regulate the expression of nuclear receptor PPAR-γ in Mφs and promote the proliferation,migration,and osteogenic differentiation of MSCs in bone repair,thereby providing theoretical reference for clinical treatment in bone repair. |
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