Bioactive Glass/Gellan Gum Thermosensitive Injectable Gradient Hydrogel With Potential For Integrated Osteochondral Repair

Osteochondral tissue with a gradient structure plays a crucial role in the movement of articular cartilage and adjacent bone.Osteochondral defects caused by disease and trauma have a serious impact on the living quality of patients.Hydrogel is the preferred material for cartilage tissue engineering because its structure and moisture content are similar to cartilage.Thermosensitive gradient hydrogels can be injected into the body and gel in response to temperature changes in situ.Without the need for crosslinkers and organic solvents,they are expected to overcome the limitations of traditional hydrogels that cannot seamlessly fill the defect in a less invasive manner and are hard to mimic the gradient structure of osteochondral tissue.However,there were few reports on the thermosensitive gradient hydrogel as a less invasive integrated osteochondral repair material.In view of the above problems,first of all,this thesis proposed the idea of multi-component gradient-mixing injection.A temperature-controlled gradient-mixing injection device consisting of a controller,micro-injection pumps,and temperature-controlled silicone plates was assembled for the first time.It achieved dual-channel gradient-mixing injection and could be personalized to prepare multicomponent thermosensitive injectable gradient hydrogels.The parameters of gradient mixed injection were designed and optimized.It was found that the sol flowed smoothly and the injection volume was accurate when choosing 1 mL syringes,pipe with 0.8 mm inner diameter and the maximum injection speed of each channel was not exceeding 0.04 mL/s.Then,thermosensitive injectable gradient hydrogels(B_0.5G,B₁G)were firstly prepared using natural thermosensitive polymer gellan gum（GG）as gel matrix and bioactive glass（BG）with bone repair ability as additive components based on the temperature-controlled gradient-mixing injection device.Their structure and a series of properties were studied.By optimizing the concentration and ratio of GG and BG,the upper critical solution temperature（UCST）of the hydrogels based on 1.5 wt.%GG could be controlled in the range of37.7-40.2℃,which was slightly higher than the human body temperature.These hydrogels had excellent thermosensitivity and injectability.B_0.5G and B₁G gradient hydrogels had gradient changes in composition,structure,and morphology using scanning electron microscopy（SEM）,energy dispersive spectroscopy（EDS）,micro-computed tomography（micro-CT）,and other analyses,qualitative and quantitative results.The top and bottom parts of B_0.5G and B₁G gradient hydrogels simulated the cartilage and subchondral bone,respectively.BG content gradually increased from the top to the bottom,simulating the gradient structure of osteochondral tissues.The top surface of hydrogels had good lubrication and met the requirements of surface lubrication of articular cartilage.The mechanical properties of gradient hydrogels were significantly higher than pure GG hydrogels.The compressive strength and elastic modulus of B_0.5G gradient hydrogels reached（103.6±1.9）kPa and（416.4±36.9）kPa respectively.Finally,the effects of gradient hydrogels on cell proliferation and differentiation were studied.Hydrogels in this thesis had excellent cytocompatibility,especially the B_0.5G gradient hydrogel significantly promoted the proliferation of bone marrow mesenchymal stem cells（BMSCs）.During the process of injection,the sol had an excellent protective effect on BMSCs and chondrocytes,and the cell viability was significantly higher than the PBS group.The B_0.5G gradient hydrogel was suitable for the three-dimensional culture of BMSCs.The qRT-PCR showed that GG up-regulated the expression of chondrogenesis-related genes（Sox9,COL Ⅱ,Aggrecan）in BMSCs,and BG（1%）/GG significantly up-regulated the expression of osteogenesis-related genes（OCN,OPN,RUNX2,COL Ⅰ）.These results indicated that B_0.5G gradient hydrogel obtained by gradient-mixing GG and BG（1%）/GG induced osteogenic and chondrogenic differentiation of BMSCs simultaneously.Moreover,the injection experiment of porcine articular osteochondral defects also proved that B_0.5G gradient hydrogel could seamlessly fill irregular osteochondral defects through less invasive injection.In summary,thermosensitive injectable BG/GG gradient hydrogels prepared in this thesis had excellent thermosensitive injectability,gradient structure,high mechanical properties,and the potential for the integrated repair of osteochondral defects through less invasive surgery.