Nanofish Bone For Bone Tissue Engineering Via Osteoimmunomodulatory And Antioxidant Activities

In the past,the major principle for developing bone biomaterials is to manipulate the in vitro osteogenic differentiation of the osteoblastic lineage and then investigating the potential osteogenic biomaterials in an in vivo model.With this principle,many strategies have been developed to fabricate an ideal bone biomaterial that can gain desired osseointegration and osteogenesis.However,this principle often does not lead to clinically useful bone implant materials,with many candidates failing to make it beyond the confines of the laboratory.Osteoimmunology has revealed the vital role of immune cells in regulating bone dynamics.Neglecting the importance of the immune response is a major shortcoming of the traditional strategy,and may explain inconsistencies between in vitro and in vivo conditions.Here,we proposed osteoimmunomodulation(OIM)in recognition of the importance of the immune response during biomaterial-mediated osteogenesis.By selecting gelatin with good biological properties as the matrix and introducing animal-derived nano-fish bone to prepare the hybrid hydrogel,the osteoimmunomodulation ability and bone regeneration effect were studied.In addition,oxidative stress can also affect immune reaction and bone regeneration,the bone graft materials supplemented by antioxidant properties were used in this paper to regulate immune response to promote better bone regeneration.By combining arginine,which has antioxidant properties,with nano fish bone to form nanocomplex,the ability of osteoimmunomodulation and osteogenic effect were studied.This article starts with fish bone,which is the main by-product of fish processing industry,has enriched mineral elements such as magnesium(Mg),calcium(Ca)and phosphorus(P)and contains rich in proteins,lipids and other nutrients which could be potential biomaterials for bone regeneration.In addition,nanofish bone is widely available and inexpensive.Based on the results from previous studies,nanofish bone was obtained by High-energy wet media milling and bone grinding machine and,its application in bone tissue engineering was explored.The SEM results showed that the NFB particles were nearly spherical shape.The DLS results demonstrated that the NFB with a negative charge.These results indicated that NFB can be applied for bone tissue engineering.Based on the preliminary exploration of NFB,the feasibility of the NFB in bone repair was further explored in this paper.According to the characteristics of the NFB,gelatinmethacryloyl/NFB(Gel-MA/NFB)hybrid hydrogels were prepared successfully by combining Gel-MA and NFB particles,and used as a scaffold for bone repair.The physicochemical characterization displayed that the Gel-MA/NFB hydrogels revealed an obvious color change when enhancing the NFB proportion in hybrid hydrogels.The SEM results exhibited that the pore size of Gel-MA hydrogels was around 10-50 ?m and the incorporation of NFB did not change its morphology and pore size.The storage modulus of Gel-MA/NFB hybrid hydrogels was increased when the concentration of NFB increased.In vitro cytocompatibility experiments results indicated that the Gel-MA/NFB hybrid hydrogels showed not toxic and could promote cell proliferation.The results of cell staining showed that the Gel-MA/NFB hydrogels could promote cell proliferation and adhesion of the cells when the concentration of the NFB was increased.Before the investage of osteogenesis in vitro,the immune response had a great influence on the osteogenesis.First,we characterized the morphology of macrophages on the materials.The Gel-MA/NFB hybrid hydrogels could stimulate the macrophages polarization and show different morphology.Subsequently,the immune cytokines and proteins released by macrophages on the material were tested.The results showed that the concentration of NFB increased,the expression of pro-inflammatory factors was downregulated and the expression of anti-inflammatory factors was upregulated.Furthermore,the effects of Gel-MA/NFB hybrid hydrogels stimulated macrophages produced conditioned medium on the regulation of osteogenesis showed that the proportion of NFB increased,the calcium nodules,alkaline phosphatase activity,and osteogenic cytokines and proteins expression were increased.These studies show that the new bone formation can be promoted by regulating the immune microenvironment.To further validate these in vitro results,we performed in vivo experiments.The results revealed that the inflammation and osteogenesis expression were consistent with in vitro results.The Gel-MA/NFB hybrid hydrogels could regulate the immune microenvironment and promote new bone formation.All these results indicate that GelMA/NFB hybrid hydrogels had been successfully constructed and could regulate the immune microenvironment,and improved bone repaired effect had been achieved by the help of Gel-MA/NFB hybrid hydrogels.In addition,a mass of free radicals would be produced through complex reactions when the bone is drilling.Some free radicals would react with oxygen to produce peroxyl radical metabolites.With the inadequate anti-oxidant capacity of the cells,it would lead to the production of excessive ROS,which would result in the damage on cell adhesion,proliferation and even cell apoptosis,and further cause oxidative stress during bone healing process.Hence,L-arginine/nanofish bone(Arg/NFB)nanocomplex has been developed to attenuate oxidative stress in early stage,and then regulate bone immune microenvironment for bone regeneration.The L-arginine(Arg)has exhibited good biocompatibility property as antioxidant in previous studies.In addition,arginine has negative electrical properties.Hence,Arg/NFB nanocomplex was fabricated via electrostatic force between the Arg and NFB to research its application in bone tissue engineering.The physicochemical characterization displayed that the morphology of Arg/NFB nanocomplex revealed cuboidal shapes by SEM.The average diameter of Arg/NFB nanocomplex analysis by DLS demonstrated that the particle size distribution of the NFB combined with arginine significantly decreased.XPS results analyze suggested that the arginine was successfully combined with NFB.The results of in vitro antioxidant performance showed that the Arg/NFB nanocomplex have a good DPPH and hydroxyl radical scavenging activity.Then,the immune microenvironment produced by the materials stimulated macrophages was researched.Morphological characterization of macrophages displayed that the Arg/NFB nanocomplexs could promote the polarization of macrophages and the macrophages grown on Arg/NFB nanocomplex surfaces were slender spindle shape and pseudopodia.Immunomodulated effects of macrophages on materials were further studied by ELISA and RTqPCR.These results demonstrated that the Arg/NFB nanocomplex could significantly downregulated the expression of proinflammatory cytokines,and the anti-inflammatory factors expression was upregulated especially the expression of interleukin 10(IL-10).After research the immune microenvironment produced by the interaction of macrophages and Arg/NFB nanocomplex,the influence of microenvironment for osteogenic and angiogenesis effect was the next step test.The results indicated that the Arg/NFB nanocomplex groups could increase the calcium nodule,alkaline phosphatase activity,osteogenic and angiogenesis factors and proteins expression.In vivo animal experiments showed that the inflammation and osteogenesis expression were consistent with in vitro cell experiments,and Arg/NFB nanocomplexs could promote bone regeneration.The results above indicated that the Arg/NFB nanocomplexs had been successfully constructed and have good antioxidant activity and immunomodulatory osteogenesis effect.Besides,a good therapeutic effect has been achieved in the application of skull repair in rats.In conclusion,NFB materials were used in bone tissue engineering in this paper.We investigated the Gel-MA /NFB hybrid hydrogels stimulated macrophages to produce immune microenvironment influence on osteogenesis.Besides,oxidative stress will cause the failure of bone repair.Hence,the NFB combined with arginine to enhance oxidation capacity to reduce oxidative stress and regulate the immune microenvironment to investigate osteogenesis effect of it.All these results showed that it had good antioxidant and immunomodulatory osteogenesis ability.It is of great significance to the development of bone biomaterials and the better prediction of bone repair effect.