Abstract: BACKGROUND: In recent years, using tissue engineering technology to repair intervertebral disc degeneration has become a research hotspot in the field of spinal surgery.  
OBJECTIVE: To compare the effect of silk fibroin/nano hydroxyapatite composite material combined with icariin and silk fibroin/nano hydroxyapatite composite in the repair of intervertebral disc degeneration.
METHODS:  Silk fibroin/nano hydroxyapatite hydrogel was prepared by physical blending and porogen. The hydrogel was directly physically mixed with icariin to prepare a hydrogel containing icariin (10 mmol/L). Animal models of intervertebral disc degeneration in New Zealand white rabbits were established by nucleus pulposus aspiration method. Thirty-six rabbits were randomly divided into 3 groups, with 12 in each group. The model group was injected with PBS using microinjector into intervertebral disc. The control group was injected with silk fibroin/hydroxyapatite hydrogel and bone marrow mesenchymal stem cell suspension. The experimental group was injected with fibroin/hydroxyapatite hydrogel containing icariin and bone marrow mesenchymal stem cell suspension. The normal group (n=12) was not modeled and did not receive any treatment. X-ray and MRI examinations were performed 4, 8, and 12 weeks after surgery. Histology (hematoxylin-eosin staining and toluidine blue staining), RT-PCR detection (type II collagen, proteoglycan, Sox9 mRNA), and western blot assay (type II collagen, proteoglycan, Sox9 protein expression) were performed 12 weeks after surgery.  
RESULTS AND CONCLUSION: (1) X-ray films showed that the height of the intervertebral disc in the model group gradually decreased at 4, 8, and 12 weeks after the operation, while the height of the intervertebral disc in the control group and the experimental group increased gradually. Among them, the rabbit disc height in the experimental group recovered better than the control group. (2) MRI demonstrated that at 4, 8, and 12 weeks after surgery, the T2 weighted image signal value of the rabbit intervertebral disc in the model group gradually decreased, and gradually increased inthe control group and the experimental group, of which the experimental group increased more significantly. (3) Hematoxylin-eosin staining showed that the nucleus pulposus tissue of the model group was obviously damaged, and the number of nucleus pulposus cells was remarkably reduced; the control group was less complete with a small amount of nucleus pulposus-like cell proliferation in the central nucleus pulposus. In the experimental group, nucleus pulposus tissue was intact, and a large number of nucleus pulposus-like cell proliferation could be seen in the central nucleus pulposus. (4) Toluidine blue staining showed that the nucleus pulposus of the model group was not stained, moderately stained in the control group, and deeply stained in the experimental group. (5) RT-PCR and western blot assay showed that the expression levels of type II collagen, proteoglycan and Sox9 in the model group were lower than those in the normal group (P < 0.05). The expression levels of type II collagen, proteoglycan and Sox9 in the control and experimental groups were higher than those of the model group (P < 0.05). The expression levels of type II collagen, proteoglycan and Sox9 in the experimental group were higher than those in the control group (P < 0.05). (6) The results have shown that the silk fibroin/hydroxyapatite hydrogel combined with icariin can promote the proliferation and differentiation of bone marrow mesenchymal stem cells into nucleus pulposus like cells, increase the synthesis of cell matrix and promote the repair of degenerative intervertebral disc.

Key words: material, silk fibroin, nano-hydroxyapatite, hydrogel, intervertebral disc degeneration, icariin, experimental animal