| It is difficult to repair once the damage occurred due to the poor self-healing ability of articular cartilage.Stem cells assisted with hydrogel exhibited obvious superiority in regeneration cartilage defects in situ with the development of cartilage tissue engineering and regenerative medicine.Among them,the selection of stem cells and the biomimetic gelation carrier were the pivotal factors in cartilage regeneration.However,a large number of hydrogels used in cartilage repair were unable to reconstruct the different layers of cartilage.Previous studies had confirmed that cells migrated from osteochondral defects with bioactive type I collagen(Col ?)could promote cartilage repair.Therefore,the injectable self-crosslinking Col I/HA-SH hydrogel with biological lubrication and inflammatory regulation functions was designed,combining with human umbilical cord mesenchymal stem cells(HUC-MSCs)to explore the repair effect of cartilage defects in situ.The effects of Col I/HA-SH composition and properties on cartilage repair and related biological mechanisms were further explored.Col I/HA-SH injectable hydrogels could match different shapes of cartilage defects.They could greatly enhance biological lubrication and inflammatory regulation based on highly biomimetic extracellular matrix of chondrocytes.By regulating HUC-MSCs,newly tissues were regenerated,similar to natural cartilage in cell arrangement and collagen fiber structure.The current problem of hydrogel in complicated structure and ambiguous biological function were solved.This was favorable of clinical application and industry transformation in a simple and efficient treatment strategies.The main research contents and conclusions were as follows:1)Collagen regulates endogenous stem cells to repair cartilage defects: On behalf of researching the properties and influence of endogenous stem cells with the different bioactive material on cartilage repair,osteochondral defects model was constructed.Regulation ability of bioactive Col I and biologically inert agarose for endogenous stem cells to repair cartilage were evaluated.These results indicated that:1.these cells were defined as MSC-like cells because they revealed similar features with BMSCs,as demonstrated by the cell morphological observation,H&E staining of the regenerated tissue,typical MSC surface markers,and the differentiation potential assays in osteogenesis,adipogenesis,and chondrogenesis;2.Bioactive Col ? could significantly promote the migration and aggregation of MSC-like cells in the defect site,and induce cartilage repair;3.The quality of regenerated cartilage induced by Col? implantation still needed to be improved,which might be related to the deficiency of endogenous stem cells enriching to the material and the insufficient optimization of the the material.2)Injectable Col ?/HA-SH hydrogel regulates chondrogenic differentiation of HUC-MSCs: In order to further enhance the chondrogenic effect of Col ? and improve the repair effect of complex cartilage defects,a series of injectable selfcrosslinking Col ?/HA-SH hydrogels were established by using collagen and thiolated hyaluronic acid.The microstructure,mechanical properties,crosslinking degree and degradation performance were optimized by modifying the composition ratio.The effects of hydrogels on the proliferation,cell phenotype maintenance and differentiation of HUC-MSCs were investigated by cell experiments in vitro.The experimental results showed as follows: 1.The shortest gelation time was 10 s-30 s and the injectable thrust was 1.6-4.6 N of Col ?/HA-SH,which was controllable,suitable,convenient and efficient for injection in situ in clinical application;2.C5HS5 had good biocompatibility and could promote the proliferation and differentiation of HUC-MSCs,maintaining the phenotype of chondrocyte and facilitating the high expression of chondrogenic related genes.3)Injectable Col ?/HA-SH hydrogels motivate HUC-MSCs to reconstruct structure and recover the biological function of articular cartilage: For the purpose of further exploring the performance of injectable Col ?/HA-SH hydrogel loaded with HUC-MSC to repair cartilage defects in vivo.A rabbit model of articular cartilage defects was constructed.Herein,bioinspired HUC-MSCs-loaden C5HS5 injection was developed to rebuild cartilage defects at only 4.25×104 cells/cm2 in situ.The cartilage defect was suffused with implant seamlessly.The effects of injectable Col I/HA-SH hydrogels loaded with HUC-MSCs on cartilage structural and functional recovery were evaluated by MRI,morphological observation,histological evaluation,IHC/IF,biomechanics,PCR,WB and transcriptome analysis.The results showed that: 1.The cartilage defect can be highly matched by the self-crosslinking Col I/HA-SH hydrogel in situ;2.HUC-MSCs without induction in vitro loaded with C5HS5 hydrogel could regenerate cartilage tissue in situ,which was highly practical and convenient in clinical application;3.The C5HS5 hydrogel as biomimetic matrix of chondrocytes could promote the differentiation of HUC-MSCs in vivo and generate hyaline cartilage similar to nature cartilage at 6 months after surgery;4.The introduction of highly selfcrosslinking HA-SH could recover the surface biolubrication function of superficial cartilage;5.Analysis of transcriptome data showed significant changes of the expression of inflammatory factors during the repair process,so it was speculated that the microenvironment of injectable C5HS5 hydrogels might promote cartilage repair by regulating the inflammatory response.4)Immune regulation capacity of injectable Col ?/HA-SH hydrogels and their effect on cartilage repair: For the sake of deeply analyzing the reasons of the differences between regenerated cartilage tissue and nature cartilage,based on the above results,the interaction between the material microenvironment,stem cel l differentiation and inflammatory response were further explored to influence the mechanism of cartilage repair.Firstly,the expression of inflammatory related genes in HUC-MSCs encapsulated within Col ?/HA-SH hydrogel was investigated in vitro.Secondly,the effects of C5HS5 hydrogel cultured in the supernatant of M0/M1 macrophages on chondrocytes and stem cells were investigated by research the interaction between Col ?/HA-SH hydrogel and macrophages in vitro.Finally,cartilage defect model was constructed and repaired by C5HS5 hydrogel loaded with HUC-MSCs to evaluate the expression of inflammatory factors in repaired tissues and verified their repair ability of cartilage defects at 5 months after surgery in vivo.The results showed that: 1.C5HS5 hydrogel showed relatively high anti-inflammatory properties;2.C5HS5 could inhibit the chemotactic behavior and M1 polarization of macrophages;3.Culture in the supernatant of M1 type macrophages was not conducive to the secretion matrix of chondrocytes;4.Culturing in the supernatant of M0 type macrophages was conducive to chondrogenic differentiation of stem cells and promoted the secretion of extracellular matrix;5.The C5HS5 hydrogel loaded with HUC-MSCs was beneficial to cartilage repair and secreted low proinflammatory factors.These results suggested that C5HS5 hydrogel could prevent the proinflammatory development and promote cartilage repair.The findings highlighted the potential of injectable Col ?/HA-SH hydrogels loaded with HUC-MSC as a novel therapeutic strategy for cartilage tissue engineering.In conclusion,the applicability of injectable Col ?/HA-SH hydrogels combining with HUC-MSCs in cartilage regeneration process was confirmed.The important influence of the inherent biological properties of carrier materials for stem cell(such as the biological lubrication and anti-inflammatory regulation involved in this paper)on cartilage repair process was proposed and verified.Therefore,so as to solve the biological problems in the process of cartilage defects repair,optimizing design of functional implants based on the inherent biological characteristics of the materials was conducive to recover structure and biological function of cartilage in the long term,and was ulteriorly expected to achieve the clinical application transformation. |
