Construction Of Silk Fibroin Hydrogel Incorporated With Bioengineered Black Phosphorus Nanosheets For Efficient Bone Regeneration

BackgroundBone trauma,surgical tumor resection,and infectious diseases could cause sever bone defects.Large bone defects could lead to persistent pain,high incidence of complications,and low quality of life.Treatment of critical bone defects is a major clinical challenge for orthopedic surgeons.Currently,autogenous bone graft is the golden standard for critical-sized bone defects treatment in clinical practice.However,its clinical adoption has been stifled by insufficient donor tissue supply,donor site morbidity,and size mismatch.Although allogeneic bone grafting will not cause donor site morbidity,it also has some drawbacks,such as limited osteoinductive potential,immunological reactions,and disease transmission.Therefore,developing new osteoinductive biomaterials that can treat critical-sized bone defects is urgent.Osteoimmunology and immune microenvironments have significant role in bone tissue regeneration.Immune cells（e.g.,macrophages and T cells）can significantly impact bone healing via cytokine secretion.In different microenvironments,macrophages can be activated and switched into different polarization status,which is known as the classically activated macrophages（M1 phenotype）and alternatively activated macrophages（M2 phenotype）.The pro-inflammatory macrophages（M1）can activate inflammation and promote fibrosis while the pro-regenerative macrophages（M2）can promote tissue regeneration.Therefore,efficient and timely activation of pro-regenerative macrophages（M2）at the early stage of bone injury is significant for bone regeneration.Black phosphorus（BP）is an emerging two-dimensional（2D）nano material with a unique folded and layered structure.BP has excellent biocompatibility and biodegradability,because the degradation products of BP in the physiological environment are commonly non-toxic phosphate ions.However,BP is susceptible to oxidation in aqueous environments and has limited immunomodulation ability.Tannic acid（TA）is a naturally plant polyphenol and possesses many free gallic acid groups.TA has superior antioxidation and anti-inflammation ability.TA can modify material surfaces through multiple interactions,such as hydrogen-bond andπ-πinteractions.Magnesium ion(Mg²⁺)is important for bone metabolism and homeostasis and has excellent osteoimmunomodulatory properties.TA can chelate with paramagnetic Mg²⁺property.Therefore,coated TA-Mg²⁺chelate on BP can prevent the fast oxidation of BP nanosheets in aqueous environments as well as improve the osteogenesis and osteoimmunomodulation ability of BP.Furthermore,the coating of TA also enhances the interfacial bonding between nanosheets and hydrogel matrix.Therefore,modified 2D BP nanosheets were integrated into 3D silk fibroin hydrogel for immunomodulation and promoting bone defect regeneration.ObjectiveIn this study,we developed a bioengineered black phosphorus nanosheets-incorporated silk fibroin hydrogel.We demonstrated the effects of our new designed hydrogel on the immunomodulation,angiogenesis and osteogenesis.In addition,we transplanted this new engineering hydrogel into rats to examine its ability of immunoregulation and bone regeneration in critical-sized calvarial defect model in vivo.Our results could provide theoretical foundation for further clinical translation of this new designed hydrogel.MethodsPart Ⅰ:A layer of TA-Mg²⁺chelate networks were coated on BP nanosheets via a simple one-step method.Then we performed transmission electron microscopy（TEM）,energy dispersive spectrometer（EDS）and X-ray photoelectron spectroscopy spectra to show the morphology and elemental composition.Atomic force microscope（AFM）,zeta potential,Fourier transform infrared spectroscopy（FTIR）and UV–vis spectroscopy was tested to show the successful synthesis of BP@TA-Mg.Part Ⅱ:To confirm the best concentration of nanosheets among hydrogel and the characteristic of methacrylate silk fibroin（SFMA）with BP@TA-Mg.We conducted Live/dead staining and CCK-8 assays to show the cytotoxicity of different concentration of nanosheets among hydrogel.Then we performed quantitative real-time polymerase chain reaction（qRT-PCR）and immunofluorescence staining to show the immunomodulation ability of different concentration of nanosheets among hydrogel.According to the above results,we built the best concentration of nanosheets among hydrogel and then conducted scanning electron microscope,FTIR,Raman spectra,gravimetric method and compression test to show the characteristic of different hydrogels.Part Ⅲ:In this part,we tried to show the biocompatibility,immunomodulation,angiogenesis and osteogenesis ability of different hydrogels.The experiments were divided into five group:control group,SFMA group,SFMA–BP hydrogel（SFBP）,SFMA–BP@TA hydrogel（SFBT）and SFMA–BP@TA-Mg hydrogel（SFBTM）.We conducted Live/dead staining,CCK-8 assays and lactate dehydrogenase（LDH）release experiment to show the cytotoxicity of different hydrogels.Then we conducted enzyme-linked immunosorbent assay（ELISA）,qRT-PCR and immunofluorescence staining to figure out the immunomodulation ability of different hydrogels.The supernatants of RAW264.7 cells co-cultured with different hydrogels were collected to obtain different conditioned media（CM）.Different conditioned media was added into HUVECs for further culture,tube formation assessment and immunofluorescence staining were conducted to show the angiogenesis ability.Different conditioned media was added into BMSCs for further culture,qRT-PCR immunofluorescence staining,alkaline phosphatase（ALP）staining and alizarin red staining were conducted to show the osteogenesis ability.Part Ⅳ:In this part,we further verified the biocompatibility,immunomodulation,angiogenesis and osteogenesis ability in vivo.After different hydrogels implanted in the rats for 7 days,immunohistochemistry and immunofluorescence staining were conducted to figure out the immunomodulation in vivo.After different hydrogels implanted in the rats for4 and 8 weeks,Micro-CT test,HE staining,Masson staining,immunohistochemistry and immunofluorescence conducted to figure out the angiogenesis and osteogenesis in vivo.After different hydrogels implanted in the rats for 8 weeks,the major organs（heart,liver,spleen,lung,and kidney）of the rats were collected to conduct HE staining to show the biocompatibility.ResultsPart Ⅰ:The result of TEM confirms that BP@TA-Mg still maintain the sheet-like morphology after coating.TEM-associated EDS also found that phosphorus（P）,carbon（C）,oxygen（O）,and magnesium（Mg）elements are well distributed over the entire nanosheets.The AFM images showed the thickness of BP@TA-Mg is much higher than BP nanosheets.We further investigated the zeta potential of BP nanosheets,BP@TA and BP@TA-Mg.The zeta potential of BP nanosheets,BP@TA,and BP@TA-Mg is approximately 17.4 m V,21.7m V,and 12.8 m V,respectively.UV–vis spectroscopy results show a strong optical absorption on near 277 nm,which is resulted from TA,emerged in the optical absorbance of BP@TA-Mg aqueous dispersions.Above results showed the successful synthesis of BP@TA-Mg.Part Ⅱ:The live/dead cell staining and CCK-8 results also shown that the dead cell was slight increased after incubation with SFBTM（900μg/m L）for 72 h.Therefore,we chose the concentration of 0,100,300,500,700μg/m L for further biological effects experiments.The immunofluorescence and qRT-PCR results indicated that the concentration of 500μg/m L has the best effect of immunomodulation ability.Different hydrogels contained interconnected porous structures with a large pore diameter.After incorporating BP@TA-Mg,there existed a big pore size（approximately 100μm）and a small pore size（approximately 40μm）.The results of FTIR and Raman spectrum indicates that BP@TA-Mg was successfully incorporated into the SFMA matrix.We also found that compressive strength evidently improved after BP@TA-Mg was incorporated.Part Ⅲ:The live and dead staining,LDH release experiment and CCK-8 assays showed that different hydrogels maintained good cell compatibility.We found that the secretion of pro-inflammatory cytokine（IL-1βand TNF-α）was significantly higher in the control and SFMA groups.By comparison,the secretion of anti-inflammatory cytokine（TGF-βand IL-10）was significantly higher in SFBP,SFBT,and SFBTM groups.As immunofluorescence results showed our hydrogels could down regulate the proportion of M1 macrophages（marked by CD86 and INOS）and upregulate the proportion M2macrophages（marked by CD206 and ARG-1）.Analysis of qRT-PCR indicated similar results that SFBTM could downregulate the gene expression of pro-inflammatory（INOS）and upregulate the gene expression of M2 macrophages marker（CD206）.Our results found that the introduction of BP@TA-Mg could significantly improve the tubes formation and the expression of VEGF and HIF-1α.We also found that the relative expression of OPN was significantly increased after being stimulated by SFBTM CM.In addition,the qRT-PCR results showed that SFBTM CM could significantly increase the expression levels of osteogenesis-related genes.ALP staining and alizarin red staining showed that SFBTM CM could significantly increase the expression of ALP and forming of mineralization nodules.Part Ⅳ:The hydrogels were injected into SD rats with a critical-sized calvarial bone defect followed by the gelation induced by the irradiation at the defect area.In our in vitro study,we showed that SFBTM hydrogel has excellent immunoregulation ability.We found IL-10 positive cells were significantly increased after being treated with SFBTM hydrogel.The expression of M1 macrophages marker（INOS）and M2 macrophages marker（CD206）in the bone defect site was further investigated.The SFBTM hydrogel has a great potential in changing M1 macrophages into M2 macrophages.After implantation for 4 and 8 weeks,Micro-CT were conducted to investigate new bone growth in the calvarial bone defects.There was more infiltrated and newly regenerated bone around the edge of the defects in SFBTM group,as compared with control or pure SFMA groups.After implantation for 4and 8 weeks,more collagen fibers and new bone were observed in the rats treated with SFBTM than other groups in both HE staining and Masson staining.We also found that SFBTM group had higher expression of Col1,OCN and OPN as shown in immunohistochemical and immunofluorescence staining.The expression of CD31 were investigated since it is deemed as typical marker of angiogenesis during bone regeneration.We found the expression of CD31 significantly increased in week 4 after implantation of SFBTM hydrogel in vivo.Finally,histological analysis of five major organs（heart,spleen,liver,kidney,and lung）of different hydrogels indicated that different hydrogels all had no systemic toxicity in vivo after 8 weeks of implantation.ConclusionTA-Mg²⁺chelate networks coated on BP improves the stability of BP and enhances the osteogenesis and osteoimmunomodulation ability of BP.Introducing BP@TA-Mg into SFMA hydrogel can significantly enhance the mechanical capacity and the biological activity.The SFBTM hydrogel exhibits the potential to polarize macrophages towards M2phenotype,enhance angiogenesis in HUVECs,and promote osteogenic differentiation in BMSCs.In vivo results also demonstrate that the SFBTM hydrogel significantly enhances bone regeneration by modulating the immune microenvironment and macrophage phenotype,thereby promoting the vascularization and bone regeneration at the site of bone defects.Besides,the SFBTM hydrogel had no systemic toxicity in vivo.To sum up,this new designed hydrogel possesses excellent osteoimmunomodulation ability and could significantly enhance bone regeneration.Our results could provide theoretical foundation for further clinical translation of this new designed hydrogel.