| Objective:To creat an injectable temperature-sensitive hydrogel loaded with growth factors,that is implanted in the damaged area of the meniscus to promote the repair of the damaged meniscus.Method:100ng/ml TGF-β1 is added to the HA/HPCH hydrogel with mass ratio of m(HPCH):m(HA)=12:1,the mass fraction of the hydrogel is 2%(m/v).The sol-gel transition temperature of each group of hydrogels was established by bottle inversion method.The degradation of each group of hydrogels was established by the residual mass weighing method in vitro.The morphological characterization of each group of hydrogels is photographed with a SEM.We cultured meniscus fibrochondrocytes.The CCK-8 method was used to determine the biocompatibility of each group of hydrogels.The Live-Dead method was used to identify the cell adhesion of each group of hydrogels.Meniscus fibrochondrocytes cells were incubated under conditions of a three-dimensional,using CCK-8 method to detect the cell proliferation rate in each group,and the morphological characterization of each group was photographed by SEM.Using ELISA kit to detect GAG secreted by meniscal fibrochondrocytes treated with HA and TGF-β1.Thirty six New Zealand white rabbits were divided into four groups and made meniscus full-thickness injury mode.In group A,normal saline was injected into the meniscus defect,in group B,pure HPCH hydrogel was injected into the meniscus defect,in group C,HA/HPCH hydrogel was injected into the meniscus defect,in group D,TGF-β1+HA/HPCH hydrogel was injected into the meniscus defect.Histological analysis was conducted after staining with HE,Safranin O-fast green,and toluidine blue after 4 weeks,8 weeks,and 12 weeks.Results:The prepared hydrogels of each group have good temperature sensitivity,the sol-gel transition temperature of the hydrogel is about 21.3℃,the sol-gel transition temperature of the HPCH hydrogel rises slightly after adding HA.The degradability of the hydrogel in vitro is good,the degradation rate reaches more than 90%after 4 weeks,and there are basically no residual hydrogel components.The SEM image indicates that the composite hydrogel material is loose and porous,and the pores are related to each other,which is conducive to the attachment and growth of cells.It can be observed that there are spherical cell structures on the wall of the hydrogel,and silk foot-like structures can be seen around the cells after three-dimensional culture.Each group of hydrogels has small cytotoxicity and good biosafety to meniscus fibrochondrocytes.Pure HPCH hydrogel are basically non-adhesive for meniscus Fibrochondrocytes,the addition of HA and TGF-β1 improves the adhesion of the material.TGF-β1+HA/HPCH hydrogel has the best adhesion to cells.After cell-hydrogel three-dimensional culture,the cell proliferation rate was about 43%after 24 hours,and cell proliferation rate exceeded 115%after 48 hours,and the cell proliferation rate in the HA/HPCH group and TGF-β1+HA/HPCH group was greater than that in the HPCH group.The results of GAG suggest that low concentrations of HA have no significant effect on the secretion of GAG from MFCs.High concentrations of HA significantly promote the secretion of GAG,low concentrations of TGF-β1 promote MFCs to secrete GAG,and high concentrations of TGF-β1 promote MFCs to secrete more GAG.And its effect is more obvious after working with HA After modelling,Samples were taken at 4 weeks,8 weeks,and 12 weeks.Gross observation,HE staining,toluidine blue staining,and safranin O-fast green staining indicated that the TGF-β1+HA/HPCH group had the best repair effect on meniscus injuries,followed by the HA/HPCH group,the pure HPCH group has no obvious repair effect on meniscus injury.Conclusion:The injectable TGF-β1+HA/HPCH hydrogel has well degradability,heat sensitivity,biological safety,cell agglutination,and cell proliferation is normal after three-dimensional culture,which has a good meniscus damage repair effect in animals and favorable prospects. |
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