Study Of Injectable Thermo-sensitive Hydrogel Designed Based On Extracellular Matrix

Articular cartilage injury is a common disease which often induced by osteoarthritis.The regeneration ability of articular cartilage is limited.The clinical treatment of osteoarthritis cannot meet the demands of repairing the damaged cartilage.BMP-2 serves as the basis for tissue engineering due to induction of osteogenesis to repair bone defects.In this work,we have prepared a series of injectable thermo-sensitive composite hydrogels,composed of Pluronic F127,glycosaminoglycan(GAG,hyaluronic acid or chondroitin sulfate)and bone morphogenetic protein(BMP-2)which was designed to mimic extracellular matrix(ECM).This composite thermo-sensitive gel system(PF/GAG@BMP-2)can achieve the effect of long-term release for BMP-2 and prevent it from degradation in tissues.The composite thermo-sensitive gel is obtained by mixing BMP-2,PF127 with three kinds of molecular weight hyaluronic acid(10K,90 K,800K)or chondroitin sulfate by cold method.The rheological properties and dissolution rate of composite hydrogels containing chondroitin sulfate or with different hyaluronic acid molecular mass(10k,90 k,800k)were investigated.Meanwhile,bovine serum albumin(BSA)or FITC-BSA was chosen as model drug loaded into PF/GAG hydrogels to study their sustained release behavior in vitro.The toxicity of composite thermo-sensitive hydrogels to rat osteoblasts MC3T3-E1 was investigated by MTT assay.In order to further investigate cytotoxicity,MC3T3-E1 cells were seeded on composite thermo-sensitive hydrogels for 4 days,and the proliferation of cells in the hydrogels was investigated.Finally,the effect of composite thermo-sensitive gel on the repair of cartilage injury in rats with osteoarthritis was investigated.The rat model of osteoarthritis was established.After 1 week,different composite thermo-sensitive hydrogels PF/GAG@BMP-2 were injected into the joint respectively.3 weeks after the treatment,the joint tissue was sliced and stained with safran O-solid green.The histological analysis and the Mankin score was carried out.The results showed that the gelation temperature of the composite thermo-sensitive hydrogel is 25 o C.Less than 90 s was needed for the composite thermo-sensitive hydrogel to undergo phase transition from solution state to gel state.Hydrogels could maintain shapes for more than 16 days.The in vitro release of BSA in the composite thermo-sensitive gel showed that,compared with PF127@BSA,the PF/GAG@BSA with glycosaminoglycan had a significant effect on inhibiting the detonation of protein drugs which due to the affinity between BSA and GAG.However,after 9 days,the dissolution of the composite thermo-sensitive gel dominated the release process,and the hydrophilicity of glycosaminoglycan accelerated the water molecules intruded in the gelstructure.Finally,the release rate of the protein drugs was accelerated resulting in the faster dissolution rate of the composite thermo-sensitive gels.Furthermore,the hydrophilic property of hyaluronic acid is related to its molecular weight,increasing the molecular weight of hyaluronic acid correspondingly increased hydrogel dissolution rate and BSA release in the hydrogels.Subsequently,MTT experiments were performed to investigate the toxicity of the hydrogels on mouse pre-osteoblast cell MC3T3-E1.The results demonstrated that introduction of glycosaminoglycan eventually reduced the toxicity of the composite gels and the better the compatibility of the hydrogel with cells was caused by higher the molecular weight and increased the concentration of hyaluronic acid..The cell proliferation test showed that the PF/GAG composite thermo-sensitive gel could promote the proliferation of MC3T3-E1 cells,while more dead cells were found in the PF127 thermos-ensitive gel,which proved that the glycosaminoglycan molecules had excellent cellular compatibility.In vivo anti-inflammation results showed that PF/GAG@BMP-2 composite hydrogels had the most efficient efficacy on recovery of injured cartilage which induced by osteoarthritis,compared to the control groups(PF127@BMP-2 or BMP-2 saline solution).Therefore,the PF/GAG injectable thermo-sensitive hydrogels may be act as an appropriate biological vector for BMP-2 in tissue reparation due to their long-term effect,good biocompatibility and excellent phase transition ability in the formation of protein reservoirs locally.