Induction Of M2-type Macrophage Differentiation For Bone Defect Repair By GO-CMC/PEGDA Nanocomposite Hydrogel With An IPN Structure

Objective:In recent years,bone immune-induced biomaterials have become a hotspot in bone tissue engineering.The purpose is to reduce the body’s inflammatory response after biomaterials are implanted and improve the repair effects.Traditional hydrogel scaffolds are unable to provide good mechanical support for tissue growth due to their poor mechanical properties.Therefore,the purpose of this research is to construct an interpenetration network(IPN)composite hydrogel with using graphene oxide(GO)-carboxymethyl chitosan(CMC)/polyethylene glycol diacrylate(PEGDA).Compared with traditional gel scaffold,the interpenetration network hydrogel have excellent mechanical properties.At the same time,the GO release system loaded with IL-4 and BMP-2 in the gel scaffold can not only induce the differentiation of macrophages into M2 type and reduce inflammation,but also enhance BMSCs osteogenic growth and new bone formation.Materials and Methods:First,we used whole bone marrow adherence method and peritoneal lavage to separate,extract and culture rat bone marrow mesenchymal stem cells(BMSCs)and peritoneal macrophages,and identify them;secondly,we used graphene oxide to load interleukin 4(IL-4)and bone morphogenetic protein 2(BMP-2)and then add it into the mixed solution of carboxymethyl chitosan(CMC)and polyethylene glycol diacrylate(PEGDA),and then add a cross-linking agent(EDC/NHS)and light to gel(added photoinitiator 2959)to prepare a hydrogel with interpenetration network structure(IPN),and performed electron microscopy(SEM),Fourier transform infrared(FTIR),X-ray energy spectroscopy(XPS),therm ogravimetry(TG),rheological test and Young’s modulus test,while tested the swelling and slow-release performance of the gel scaffold,as well as cell adhesion,proliferation and live-death staining.Then,the composite gel scaffold loaded with IL-4 and BMP-2 was co-cultured with macrophages through a Transwell plate to induce macrophages differentiated into M2 type,IL-10 and IL-12 protein expression was detected by ELLISA in the supernatant,gene expression(iNOS,TNF-α,Arg-1,CD206)were detected by Rt-qPCR,iNOS and CD206 immunofluorescence detection of cell surface expression after induced;And then,the composite gel scaffold and BMSCs were co-cultured to induce their osteogenic differentiation,and alkaline phosphatase staining(ALP)and alizarin red staining(ARS)were conducted after induction for several days,and Rt-qPCR was used to detect osteogenic genes(ALP,RUNX-2,COL 1,OCN);The composite gel scaffold was again co-cultured with macrophages through the Transwell plate,and the induction supernatant was collected and combined with osteogenic induction medium to form conditioned medium,and then BMSCs were cultured for osteogenic induction using the conditioned medium.ALP and ARS staining were performed after 7 and 21 days of induction,and Rt-qPCR was used to detect osteogenic related genes(ALP,RUNX-2,COL1,OCN)expression;Finally,we embedded the factor-loaded gel scaffold into the rat subcutaneously,and took samples for histological staining(HE,Masson,immunohistochemical staining),observe its osteogenesis(ALP,RUNX-2),angiogenesis(CD31)and immune inflammation(CD68,CD206,iNOS),and at the same time,implanted the scaffold into the rat skull defect model,samples were taken at 4 and 8 weeks after implantation,and micro-CT detection and histological staining were performed to observe the formation of new bone(ALP,COL 1,OCN),angiogenesis(CD31),and inflammation(CD68,CD206,iNOS)).Results:1.We successfully extracted,cultured and identified rat bone marrow mesenchymal stem cells and peritoneal macrophages;2.The GO-CMC/PEGDA composite hydrogel was successfully prepared,and Fourier transform infrared(FTIR)and X-ray spectroscopy(XPS)confirmed that the composite hydrogel has an interpenetration network(IPN)structure.Compared with pure hydrogel,it has better mechanical properties and stability performance;At the same time,the GO sustained-release system not only showed the function of sustained and slowly release of factors,but also can minimize the dosage of factors;3.The hydrogel loaded with IL-4(pure IL-4,IL-4 and BMP-2)had the function of inducing macrophages differented into M2 type;4.Hydrogels loaded with factors(IL-4 and BMP-2)can enhance the osteogenic differentiation of BMSCs,and the hydrogel loaded with IL-4 and BMP-2 has the strongest osteogenic ability;IL-4 has the ability to enhance the osteogenic differentiation of BMSCs,but the specific mechanism is not clear;5.By culturing the conditioned medium with BMSCs,it is found that it can enhance the osteogenic differentiation of BMSCs,and the hydrogel loaded with IL-4 and BMP-2 factors has the strongest osteogenic ability;and IL-4 can also promote BMSCs osteogenic differentiation;6.The results of the subcutaneous implantation experiment in rats found that the gel scaffold loaded with IL-4 has the function of reducing local inflammation.The expression of M1 type inflammatory macrophages around the materials was decreased,and the expression of M2 type anti-inflammatory macrophages was increased;while the skull defect model also indicated that in the gel scaffold group loaded with IL-4 factor,the expression of M2 macrophages in the local microenvironment was increased,and immunohistochemical staining showed that the staining of bone formation and angiogenesis was significantly better than other groups.Micro-CT results showed that in the gel scaffold group loaded with IL-4 and BMP-2,the bone defect area was completely repaired at 8 weeks.Conclusion:The hydrogel scaffold loaded with IL-4 and BMP-2 has a synergistic effect on bone regeneration.This kind of biological material with induction and immune regulation provides a new research direction for the future development of bone immune regulation and tissue engineering applications.