| Hydrogels for intradermal/subcutaneous soft tissue repair and reconstruction have broad clinical needs.Sodium hyaluronate(SH)hydrogel has the advantages of strong hydrophilicity,water retention and shaping ability,however,in vivo degradation rate is extremely fast and the cell affinity is weak,the ability to guide regenerative and repair of defective soft tissue is limited.Silk fibroin(SF),as a kind of fibrous protein,has a slow biodegradation rate and ability to adhere to and guide the migration of cells.However,the water retention and compressive resistance of regenerated SF hydrogels still need to be improved.The respective deficiencies of SH or SF hydrogels can be improved by blending SH and SF.Whereas,the properties of SH/SF blend hydrogels directly depend on the blending method,the cross-linking method and the internal network structure of the hydrogel.The key issues to be solved in the field of soft tissue repair are to seek new blending and crosslinking methods for SH and SF natural polymers,to construct SH/SF blend hydrogels that can give full play to the respective performance advantages derived from SH and SF,to improve their mechanical properties and reduce their biodegradation rate.In this study,a novel scheme for the construction of SH/SF blend hydrogels with double network structure was proposed.SH was first mercaptosylated to obtain mercaptosodium hyaluronate(MSH).Then,the MSH/SF hydrogel with covalently crosslinked double network structure(referred to as double network MSH/SF hydrogel)was prepared by forming MSH-MSH network between MSH macromolecules and SF-SF network between SF macromolecules,respectively,under the dual catalysis of horseradish peroxidase(HRP)on sulfhydryl groups on the side chain of MSH and phenolic hydroxyl groups on the side chain of SF.The influence of MSH/SF weight ratio on the internal network structure,pore structure,aggregated structure,viscoelasticity,water absorption,swelling,compression performance,in vitro biodegradability,cell adhesion and proliferation of the hydrogel was systematically investigated.The structure,physicochemical properties and biological properties were further compared with that single mixed network hydrogel formed by carbodiimide-mediated crosslinking(named as single network MSH/SF(C)hydrogel).Finally,an injectable microgel with particle size of 300-500 μm was obtained by the"liquid nitrogen freezing-pulverization method",and the single network and double network MSH/SF microgels were injected subcutaneously into rats,respectively,to investigate the influence of internal single and double network structure of hydrogels on biodegradation rate,cell infiltration,tissue regeneration and inflammatory response in vivo.Firstly,double network MSH/SF blend hydrogels were prepared by a "one-step method" under the dual catalysis of HRP on the sulfhydryl groups grafted on the side chain of MSH and the phenolic hydroxyl groups of tyrosine residues on the side chain of SF.When the weight ratio of MSH/SF reached 50/50,MSH and SF could obviously inhibit the self-aggregation of each other,and achieved uniform dispersion between two components in the system.The observation results after fluorescence labeling showed that MSH-MSH network and SF-SF network penetrated and interspersed into each other at this time,forming the most obvious double network structure.The results of infrared spectroscopy showed that MSH macromolecules were connected by disulfide bonds,and SF macromolecules were connected by di-tyrosine bonds.The micro-commercial thermogravimetric analysis also confirmed that MSH and SF can inhibit aggregation from each other and form a double network structure,from the side.With the decreasing of MSH/SF weight ratio,the hydrophilicity and swelling ratio of the blend hydrogels gradually decreased,the pore size declined,and the porosity increased.The formation of MSH-MSH and SF-SF double network structures can significantly improve the compression strength and compression modulus of single component MSH hydrogel as well as SF hydrogel.Secondly,the influence of MSH/SF weight ratio of the blend hydrogel on the in vitro degradation rate and cytocompatibility of hydrogels was investigated.The results showed that the double network structure formed between MSH-MSH network and SF-SF network of the blend hydrogels has a certain protective effect on the two components of MSH and SF.When the weight ratio of MSH/SF was 50/50,the in vitro degradation rate of double network hydrogels in PBS buffer,hyaluronidase solution,and type I collagenase solution could be significantly slowed down.At this time,the inhibitory effect of MSH on the molecular conformation transition of SF from random coil to β-sheet was the most significant.With the decreasing of the weight ratio of MSH/SF,namely,the increasing of SF proportion,the adhesion properties of the blend hydrogels to mouse fibroblast(L929)cells and human umbilical vein endothelial cells(HUVECs)were significantly improved,the cells spreading was more fully,and the cell proliferation viability was significantly improved.Then,the structure and properties between the double network hydrogel and single network hydrogel with the MSH/SF weight ratio of 50/50 were compared.The results showed that compared with single network hydrogel MSH/SF(C),the internal porosity the of double network hydrogel MSH/SF increased,compressive strength,compressive modulus and storage modulus G’ of double network hydrogel MSH/SF were significantly improved and in vitro biodegradation rate was significantly slowed down,the ability to promote L929 proliferation was enhanced.Subsequently,single and double network microgels with particle sizes mainly distributed in the range of 300-500 μm were prepared by "liquid nitrogen freezingpulverization method".The effects of microgels on adhesion,proliferation and growth of stem cells were investigated by co-culturing the microgels with human adipose mesenchymal stem cells.The results showed that compared with the single network microgel MSH/SF(C),the double network microgel MSH/SF was more favorable for the adhesion,spreading of stem cells and to guide the uniform distribution of stem cells within the hydrogel.It was found that double network microgel MSH/SF could provide physical protection for cells and significantly maintain the proliferative viability of stem cells,compared with direct injection for naked cells.Finally,the single network and double network MSH/SF microgels were injected subcutaneously into rats to investigate the effects of single and double network structures on the degradation performance,cell infiltration,tissue regeneration and inflammatory response of the hydrogel in vivo.The results showed that,unlike the single network microgels MSH/SF(C),which had already collapsed due to degradation at 2 weeks of implantation,and were completely biodegraded at 4 weeks,double network microgels MSH/SF could be basically sustained the original particle morphology,the phenomenon of microgels collapse arisen form degradation did not occur during the whole experimental period of 8 weeks when microgels were implanted into the body,the in vivo biodegradation rate was significantly slower than that of single network microgels.Within 4 weeks of double network microgels MSH/SF implantation,cells and tissues were not only able to grow along the gaps between microparticles,but also infiltrated into the microparticles and deposited collagen.The inflammatory reactions of microgels in each group implanted into the body were mild,and the positive expression rates of inflammatory related factors TNFa and IL-6 gradually decreased after 1 week,and reached a low level after 4 weeks,and the fibrous capsule around the microgels disappeared completely.The above study results demonstrated that the double network microgel MSH/SF can not only resist biodegradation continuously in vivo,but also has good histocompatibility,indicating potential for soft tissue repair.In this paper,MSH/SF blend hydrogels with double network structure was constructed by utilizing the dual catalytic activity of HRP on the sulfhydryl group on the side chain of MSH and the phenolic hydroxyl group of tyrosine residue on the side chain of SF.The results showed that MSH-MSH network and SF-SF network inside the hydrogel have a mutual protection effect after sufficient penetration,which can significantly enhance the biostability of the hydrogel.Compared with single network hydrogel,the mechanical properties were significantly improved,and the degradation rates in vitro and in vivo are significantly slowed down.SF component and high porosity of the double network hydrogel can significantly enhance the adhesion,proliferation,and tissue infiltration of host cells within the hydrogels,and accelerate collagen deposition.MSH/SF microgels have good biocompatibility in vitro and in vivo.The above results provide a systematic experimental basis for the development and clinical application of novel microgels for soft tissue repair. |
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