Active bone material containing modified recombinant human bone morphogenetic protein-2 induces bone regeneration in rabbit spina bifida

Abstract

This study focuses on spina bifida, which is a high incidence among the current clinical manifestations of human birth defects. Because in the treatment of bone defects, the source of autologous bone is limited and it is easy to cause secondary injury to the patient. At the same time, since the bone tissue in animals needs to perform a variety of biological functions, its complex structure cannot be replaced by a single material. Therefore, in this study, we used Japanese white rabbits to establish an animal model similar to human congenital spina bifida. The established animal model is used to screen the best regenerative repair products for the treatment of congenital spondylolisthesis defects, and to evaluate the safety of regenerative repair products. The results show that bone morphogenetic protein (BMP)-2 combined with collagen material has a better regeneration effect than collagen material alone, and it did not negatively affect the health of animals. This study is not only suitable for the screening of large-scale biomaterials, accelerating the research progress of regenerative repair products, but also conducive to the research on the mechanism of regeneration and repair of various materials.

Keywords: Japanese white rabbits; Spina bifida; active bone; bone morphogenetic protein-2; collagen material; damage repair.

Figure 1.

Surgical process. (a) Bone material and collagen membrane, (b to f) skin and muscle tissue at the surgical site are cut and removed, (g to j) expose the spinal cord, (k) add bone material, (l) add collagen membrane, and (m and n) suture the skin. (A color version of this figure is available in the online journal.)

Figure 2.

Gross observation of vertebrae morphology and structure. (A) The general appearance of the vertebrae three months after surgery. The red box and red plus sign assist in displaying the recovery of the transplanted area. (B) The statistics of bone defect area ratio, each group was compared with the control group, and the difference was denoted by * (*P < 0.05). (C) X-ray analysis of vertebral bodies was performed three months after surgery. (D) HE staining results – (a to d) and (a1 to d1) HE staining results three months after surgery. (a) and (a1) Normal group; (b) and (b1) control group; (c) and (c1) material group; (d) and (d1) BMP group. (a) to (d) Tissue scan results after HE staining. (a1) to (d1) The result of HE staining after 100× magnification. Scale bar is 100 μm. (A color version of this figure is available in the online journal.) CS: cavitation structure; FT: fibrous tissue; NB: new bone.

Figure 3.

Sirius red staining results (100×). (A) (a) to (d) Sirius red staining results of each group three months after surgery. (B) The percentage of type 1 collagen three months after surgery. All groups were compared with the normal group, and the statistical difference was denoted by *. Scale bar is 100 μm. (A color version of this figure is available in the online journal.) **P < 0.01.

Figure 4.

Expression of glycogen and alkaline phosphatase in vertebral tissue. (A) PAS staining results (100×). (a) to (d) PAS staining results three months of each group after surgery. (B) ALP staining results (100×). (a) to (d) ALP staining results three months of each group after surgery. Mark the area of the positive signal with a red arrow. Scale bar is 100 μm. (A color version of this figure is available in the online journal.)

Figure 5.

TUNEL results (200×). (A) (a) to (d) TUNEL staining results three months after surgery. The areas with positive signals are marked with red arrows. (B) The percentage of positive signal in each group at three months. Scale bar is 50 μm. *Represents the statistical difference between each group and the normal group (*P < 0.05).

Figure 6.

IHC test results in vertebral tissue three months after surgery. (A and B) BMP-2; (C and D) Runx2; (E and F) Vegf; (G and H) Hif-1α. (A, C, E and G) The picture of IHC test result (200×), the areas with positive signals are marked with red arrows. (B, D, F and H) The percentage of positive signal in each group at three months. Scale bar is 50 μm. (A color version of this figure is available in the online journal.) *Represents the statistical difference between each group and the normal group (*P < 0.05). #Represents the statistical difference between each group and the control group (#P < 0.05).

Figure 7.

Serum BGP concentration three months after surgery.