Abstract: BACKGROUND: Acellular amniotic membrane is a natural biomaterial scaffold, which has been widely used in related fields of tissue engineering. Scleraxis gene plays an important regulatory role in the formation of connective tissues such as ligaments.
OBJECTIVE: To verify the biocompatibility of Scleraxis modified human amniotic mesenchymal stem cells and human acellular amniotic membrane scaffold complex in rats.
METHODS:  The amniotic membrane tissues of the placenta at term were taken, and the fresh human amniotic membrane was decellularized by chemical-enzymatic digestion method. Two-step enzyme digestion method was used to separate human amniotic mesenchymal stem cells from human fresh amniotic membrane. The third-generation human amniotic mesenchymal stem cells were transfected with Scleraxis lentivirus, which were then cultured with human decellularized amniotic membrane for 5, 10, and 15 days so as to detect mRNA expression of related genes. The 18 Sprague-Dawley rats were randomly divided into three groups. In the experimental group, the prepared cell scaffold complex was implanted into the subcutaneous fascia of the back of the rat. In the negative control group, an incision was made on the back of the rat, without implanting materials. In the blank control group, no treatment was performed. Hematoxylin-eosin staining was performed on the operation area tissues 1 and 4 weeks after surgery. Immunohistochemical staining of CK protein was performed on the operation area tissues 4 weeks after surgery. 
RESULTS AND CONCLUSION: (1) Scleraxis gene transfection of human amniotic mesenchymal stem cells and human acellular amniotic membrane compound culture could significantly up-regulate the mRNA expression levels of ligament differentiation-related genes type I collagen, type III collagen, fibronectin and cytocontin C. (2) The neonatal granulation tissue was seen in the local tissue of the experimental group at 1 week after surgery, and the inflammatory response was heavier than that of the negative control group and the blank control group. At 4 weeks after surgery, the local tissue arrangement of the experimental group tended to be neat; the granulation tissue was reduced; and the inflammation subsided obviously. (3) The CK protein expression was positive in the experimental group at 4 weeks after the operation; the local tissues were neatly arranged; and a large number of cells were attached around the amniotic membrane tissue. (4) Results suggested that co-culture of Scleraxis gene transfected human amniotic mesenchymal stem cells with human acellular amniotic membrane scaffold can promote the differentiation of human amniotic mesenchymal stem cells into ligaments in vitro, and the cell scaffold complex shows good biocompatibility in animals.

Key words: stem cell, mesenchymal stem cell, amniotic membrane, acellularization, ligament, scaffold, gene, rat