Fabrication Of Gelatin/Hyaluronic Acid Microgel Assembly And Its Application In Articular Cartilage Repair

Articular cartilage injury is a common clinical disease,caused by trauma,tumor,infection and osteoarthritis.Since there are no blood vessels,nerves and lymph,it is difficult to self-heal once damaged.Cartilage tissue engineering has become a research hotspot in the field of cartilage regeneration and repair,but this method still has many problems.For example,most of the scaffold materials for cartilage tissue engineering are sponges,fibers or bulky hydrogels.The bulky size of implants results in a large surgical wound and hence is inconducive for postoperative recovery.Besides,failure to fill irregular defects after implantation and lack of effective adhesion with the host cartilage/bone makes the implants easy for malposition.The limited source of autologous chondrocytes and the risk of losing chondrocyte phenotype during the long-term in vitro expansion culture further restrict their clinical application.Therefore,the functional design of scaffold materials and the selection of appropriate physicochemical signals to regulate the chondrocyte phenotype of cells are essential to improve the effect of cartilage regeneration.This subject starts from the three elements of cartilage tissue engineering,makes unique selection and design around scaffolds,cells and factors(physical signals or chemical signals),eventually solves the current problems of cartilage tissue engineering products.We fabricate a series of microgel assemblies with injectable,self-healing,tissue adhesion,controlled drug release and interconnected microporous structure by using droplet microfluidic technology,selecting gelatin and hyaluronic acid as the main materials,applying suitable chemical reaction.Meanwhile,appropriate mechanical signal and small molecule drugs are utilized to promote the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and achieve good effect of articular cartilage repair.Firstly,the gelatin/hyaluronic acid(Gel-HA)microgels are successfully prepared in the microfluidic chip,which are used as a research model to explore the effect of the 3D mechanical microenvironment on the behavior of BMSCs,especially the chondrogenic differentiation behavior.Through the thiol-Michael addition reaction between thiolated gelatin(Gel-SH)and ethylsulfated hyaluronic acid(HA-VS)with different degrees of substitution,three types of Gel-HA microgels are prepared with low,medium and high crosslinking densities,which are named Gel-HA(L),Gel-HA(M)and Gel-HA(H),respectively.Experimental results show that the proliferation,distribution and chondrogenic differentiation behavior of BMSCs are closely dependent on the mechanical microenvironment in the microgel.It is worth noting that BMSCs in Gel-HA(L)show obvious differentiation into hyaline cartilage,while BMSCs in Gel-HA(M)and Gel-HA(H)show an obvious trend of fibrocartilage differentiation.Transcriptome RNA sequencing shows that the mechanical microenvironment of Gel-HA microgel affects the differentiation behavior of BMSCs through the TGF-?/Smad signaling pathway,the Hippo signaling pathway and the integrin/YAP/TAZ signaling pathway.On the basis of the above materials,we construct Gel-HA microgel self-assembly and propose a new cartilage repair strategy,which means BMSCs-laden microgels are injected into the defect site and spontaneously form a cartilage-like microgel self-assembly by cell-cell interaction.In vitro tests show that BMSCs-laden microgels can be injected without sacrificing the viability of BMSCs.More importantly,they can self-assemble into cartilage-like scaffolds through cell-cell interaction.In vivo experiments further confirm that the microgel can form microgel self-assembly in situ.Besides,there is a large amount of cartilage matrix deposition in the microgel self-assembly without obvious vascularization and hypertrophy.After successfully constructing the microgel self-assembly,we explore the construction of other forms of microgel assembly to design new cartilage repair strategies and provide a variety of options for cartilage regeneration and repair.By using methacrylic anhydride and phenylboronic acid-modified hyaluronic acid(HAMA-PBA)and methacrylic anhydridemodified gelatin(Gel MA)as the main materials,microgels are prepared in the droplet microfluidic chip.Then,with the help of dynamic phenylboronic acid ester reaction between the phenylboronic acid on the microgel and the dopamine-modified hyaluronic acid(HA-DA),the microgels connect together to construct a dynamic covalently bonded microgel assembly(DC-MA).DC-MA has good shear thinning and mechanical strength,hence can be used as a bio-ink.DC-MA can be stably extruded microfilaments up to 70 mm and can be stably printed into a 20-layer ring structure without collapse.In addition,DC-MA with tissue adhesion,microporosity and self-healing properties,which is conducive to cell adhesion,migration and extracellular matrix deposition.We integrate the kartogenin-laden cyclodextrin nanoparticles(KGN@CD NPs)with the above microgel assembly to construct a nanocomposite microgel assembly.The nanocomposite microgel assembly has injectable,tissue adhesion,sustained drug release and connected microporous structure.Since KGN@CD NPs are anchored in the gel network through the free radical reaction of double bond,the cytotoxic effect caused by endocytosis can be avoided.Meanwhile,the release of KGN can be maintained up to 28 days.The nanocomposite microgel assembly can promote the expression of cartilage marker genes(such as runx1,sox9,col2a1 and aggrecan)and the secretion of cartilage matrix(such as Col I,Col II and aggrecan).In vivo,the nanocomposite microgel assembly can significantly promote the deposition of articular cartilage matrix and achieve a good effect of articular cartilage regeneration.