Through histological analysis, immunofluorescence staining, electrophysiological detection and Sensory and motor function evaluation to investigate the effects of 3D printed hydrogel scaffold combined with bone marrow mesenchymal stem cells (BMSCs) in promoting functional recovery of spinal cord injury.

10% GelMA hydrogel and 10⁶ U stem cell suspension were prepared into bioink of appropriate concentration to construct the biomimetic spinal cord scaffold through 3D printing platform. The scaffold was placed in the medium and cultured in an environment of 37 ℃ CO₂ incubator. The microstructure of the scaffolds and the distribution of BMSC in the scaffolds was observed by scanning electron microscope. CAM/PI staining and confocal microscopy were used to observe the survival of stem cells in the scaffolds and determine the biocompatibility of the scaffolds. The scaffolds were implanted into the subcutaneous tissues of the back of rats, and the subcutaneous tissues were determined by HE staining to detect the immunogenicity of the scaffolds. After the rat model of hemicytoma defect was made, stents were transplanted for treatment, and confocal microscopy was used to evaluate the regeneration of neurons and axons in local area of spinal cord injury. At the same time, BBB score was used to evaluate motor function, mechanical pain score was used to evaluate sensory function, and surface electrode detection method was used to evaluate electrophysiological recovery weekly.

The long spindle shaped BSMC were uniformly distributed in the scaffold with a loose reticular structure. The scaffolds had good biocompatibility, and the cell survival rate of the prepared scaffolds reached 96% after 24 hours of printing. After 28 days of subcutaneous transplantation, the immune rejection was mild and immunogenicity was low. It was shown that the regenerated spinal cord tissue in the treatment group was significantly increased compared with the control group, which was widely distributed with cells after 28 days by HE staining. It was confirmed that part of the regenerated spinal cord tissue was neurons by immunohistochemical staining.Compared with the injured group, the regeneration of neurons and axons in the treatment group were significantly increased by immunofluorescence staining and confocal microscopy. In the treatment group, the BBB score recovered to 10 points, while the control group only recovered to about 1 point in the first week, which was statistically significant. And it recovered to 17 in the fourth week, while the control group only recovered to about 4 point in the four week, which was statistically significant. The Angle of inclined plate support of the treatment group was restored to 40 degrees, while it was only restored to 22 degrees in the control group. The pain threshold of the treatment group decreased to 18.5 points, which was not statistically different from that of the control group. The latent recovery effect of electrophysiology in the treatment group was the same as that in the sham operation group and better than that in the control group.

3D printing hydrogel scaffold with loose network structure is suitable for cell proliferation. It has well biological survival, low cytotoxicity and low immunogenicity, which promoted neurons and axons to recovery and extend so as to effectively promote the recovery of motor function, sensory function and neural signal transmission rate after spinal cord injury.

Tissue Engineering; Stem cell transplantation; Spinal cord injuries; Mesenchymal stem cells; Histological techniques