基于丝素蛋白的可注射水凝胶在肌肉再生工程的应用

Application of injectable hydrogel based on silk fibroin in muscle regeneration engineering

针对丝素蛋白水凝胶可注射性较差、机械性能不稳定、无法植入细胞等问题,文章制作了一种新型的可注射丝素蛋白水凝胶。通过弱碱法脱去蚕丝的丝胶蛋白,经过溶解、透析、离心、过滤后获得丝素蛋白溶液,利用磷脂诱导丝素蛋白溶液形成水凝胶。将水凝胶和细胞通过注射的方式植入湖羊耳部,分别在1、6、11 d取出水凝胶—肌肉细胞生长样品,通过Masson染色研究了水凝胶的降解规律和肌肉再生的过程。结果表明:这种基于丝素蛋白的新型水凝胶凝胶时间可控,制备简单,具有可注射性。水凝胶可以植入细胞,并且细胞在水凝胶中形成新的肌肉组织,说明这种基于丝素蛋白的新型水凝胶可以用作肌肉再生工程。进一步梳理丝素蛋白水凝胶的制备过程和切片结果发现:水凝胶制作简单,细胞在水凝胶中可以增殖分化,只需要注射就可以进入动物体内,在动物体内还可以降解。最终结果表明:基于丝素蛋白的水凝胶可以用肌肉组织再生工程,磷脂诱导的方式使水凝胶具备可注射性。

Silk fibroin,as one of the most potential materials for tissue engineering,is widely used in the field of regenerative medicine.With the gradual aggravation of China’s aging population,the number of muscle degenerative diseases has gradually increased.For muscle degenerative diseases,implanting renewable stem cells at the lesion site is an effective treatment.After the traditional artificial synthetic scaffold is implanted into the human body,it often produces acidic products,which will increase the immune level at the implantation site and cause the risk of related complications.Therefore,people need a new scaffold with certain mechanical strength,green degradation and zero immunogenicity that can support the attachment,proliferation and migration of corresponding stem cells.It is better to implant it into the human body without surgery,so as to minimize secondary injury.We hope to remove sericin protein on the surface of silk fibroin by treating silk with weak alkali method of sodium bicarbonate,so as to minimize immunogenicity.For the problems of poor injectability,unstable mechanical properties and inability to implant cells in silk fibroin hydrogels,we made a novel injectable silkin hydrogel.Then,we prepared the silk fibroin hydrogel by inducing the silk fibroin aqueous solution to accelerate the gel reaction with myristoyl phosphatidylglyceride(DMPG)and controlling the gel time.We co-cultured the cells with silk fibroin hydrogel extract,and tested the cytotoxicity with CCK-8.From the results of CCK-8,it can be seen that this DMPG-induced silk fibroin hydrogel has no cytotoxicity.Finally,we mixed the stem cells with silk fibroin hydrogel and injected the mixture into the subcutaneous skin of Hu sheep ears.We took out the hydrogels on different days for HE staining and Msaaon trichrome staining,and calculated the degradation rate of silk fibroin hydrogel and muscle regeneration rate by ImageJ software.It can be seen from the results that this silk fibroin hydrogel is simple to prepare,can be degraded in vivo,and has no immunogenicity.Through the above experiments,a highly efficient,simple,immunogenic,degradable silk protein hydrogel cell scaffold is obtained.It is the first attempt to use phospholipid to induce the preparation of hydrogels.The production method is simple.Cells can proliferate and differentiate in the hydrogels.They can enter animals only by injection and can be degraded in animals.Silk fibroin,as a very representative material in tissue engineering,will shine brightly in muscle tissue engineering and bone tissue engineering in the future.Therefore,it is necessary to explore the degumming method of silk fibroin and a means suitable for factory production.Silk fibroin hydrogels prepared by the above method provide certain reference and practical guidance for injectable silk fibroin hydrogels in the future.