Multiple Hydrogen Bonds Generate Supramolecular Hydrogels Toward Cartilage Tissue Regeneration

Articular cartilage has limited self-repair capability.Once the cartilage damage is not treated in time,the degeneration of articular cartilage could lead to osteoarthritis and even disability.Tissue engineering combining cells,scaffold and environmental factors has opened up new avenues for cartilage regeneration repair.Hydrogels are a new class of ideal seed cells scaffold materials,as they can effectively mimic the functions of native extracellular matrix(ECM).However,traditional covalently cross-linked hydrogels still face several shortcomings,such as limited durability and stability,poor biocompatibility,and the elastic stresses from hydrogel matrix could resist the expansion and confine cell volume.Spatial confinement of cell volume could inhibit proliferation of the cell and formation of a greater volume of interconnected cartilage matrix.Supramolecular hydrogels based on reversible non-covalent bonding could not only recapitulate the dynamic ECM,but also have the capabilities of syringeability,self-healing and shear-thinning,which is in favor of providing mechanical protection for delivering cells.Supramolecular hydrogels can quickly recover to the original elastic modulus via the reformation of reversible hydrogen bonds,which is able to improve in situ cell retention as well as long-term cell survival.Additionally,the formed dynamic threedimensional microenvironment of supramolecular hydrogels is crucial for chondrocyte phenotype maintaining and cartilage-specific matrix deposition.Based on aforementioned advantages,we have conducted comprehensive studies and the contents are outlined below:Firstly,hyaluronic acid(HA)supramolecular polymer was designed to develop the thermosensitive and self-healing hydrogel through the combination of quadruple hydrogen bonds with thermo-triggered hydrophobic self-assembly.This branched supramolecular polymer,consist of HA backbone,poly(di(ethylene glycol)methyl ether methacrylate)(PDEGMA)segment and ureido-pyrimidinone(UPy)moiety,was synthesized through one-pot free radical polymerization at low monomer concentration.The hydrophobic microphases formed by short PDEGMA side chains thermal assembly above LCST significantly enhance the stability of quadruple hydrogen bonds under polar water environment condition,endowing the supramolecular hydrogel with unique thermo-responsive and self-healing properties.Dynamic rheological properties measurements indicate that the formed HDU supramolecular hydrogels display completely reversible sol-gel transition and excellent self-healing.Additionally,it is suggested that the prepared HDU hydrogel displays good cytocompatibility and in vivo tissue biocompatibility.Additionally,gelatin was introduced into HDU solution for the preparation of macro-porous and water-triggered shape-recoverable HDU/Gelatin hybrid supramolecular hydrogels via ice templating.The unique macro-porous and water-triggered shape-recoverable property are in favor of cell homogeneous distribution throughout the gel construct.It is worth noting that the formed HDU/Gelatin hybrid supramolecular hydrogels could promote entrapped chondrocytes to acquire a spherical morphology through the regulation of microstructure,which is important for maintaining chondrocyte function.Secondly,we further evaluated the effectiveness of cartilage tissue formation based on the injectable chondrocyte-laden HDU supramolecular hydrogels in vitro and in vivo,respectively.Experimental results of PCR show that the formed three-dimensional culture systems are in favor of maintaining chondrocytes phenotypes.The cell immunofluorescence and histological staining were next performed to investigate cartilaginous matrix deposition.That results demonstrate that the cartilaginous specific matrix deposition was clear along with the extension of the culture time,and the formed cartilaginous matrix is mainly type II collagen and glycoprotein.Additionally,chondrocyte-laden HDU hydrogel for cartilage defect repair of SD rat and New Zealand white rabbit in vivo were subsequently explored.The results showed that chondrocyte-laden HDU hydrogel administered via injection through a needle effectively facilitate the regeneration of hyaline cartilage.Finally,this study further explored the potential application of HDU supramolecular hydrogels for efficient repair of osteochondral defect.Laponite nano-clays are able to enhance cellular functions including adhesion,proliferation and differentiation,most notably for osteogenesis.We utilized Laponite to develop a type of shear thinning HDU/Laponite nanocomposite hydrogel based on the physical interactions.The resulting HDU/Laponite nanocomposite hydrogel has stronger mechanical properties than the HDU supramolecular hydrogels alone.The rheological properties measurements suggest that the formed HDU/Laponite nanocomposite hydrogel display excellent shear thinning property that was in favor of three-dimensional BMSCs culture.Additionally,self-integrating HDU/HDU-Laponite composite hydrogels are particularly advantageous as separate hydrogel components that allowed for the relatively simple spatial localization of BMSCs and chondrocytes.The tissue biocompatibility of bi-phase hydrogels and osteochondral regeneration was determined in vivo.