ABSTRACT
Objective: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.Methods:10% GelMA hydrogel and 10 6 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 ℃ CO 2 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. Results: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.Conclusion: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.
ABSTRACT
BACKGROUND:Lymph-targeted tracing and therapy based on carbon nanotubes have been one of the hottest researches on targeting tumor diagnosis and treatment. To evaluate the accumulation of carbon nanotubes in axil ary lymph node can provide experimental evidences for developing nano-tracers and drug carriers which are more lymph-specific and more biocompatible. OBJECTIVE:To study the accumulation of the intravenously injected carboxylated single-wal ed carbon nanotubes in axil ary lymph nodes of Sprague-Dawley rats, and to evaluate their effect on blood cel s. METHODS:Sixty-four Sprague-Dawley rats were randomly divided into two groups. Rats in testing group were injected with carboxylated single-wal ed carbon nanotubes suspension (2 mg/kg), while those in control group were injected with 5%glucose solution (1 mL/kg), both through the tail vein, three times per week. Four periods of 7, 60, 90 and 120 days were set (the 120-day period referred to 90 days of administration fol owed by 30 days of drug withdrawal). At the end of each period, eight rats from each group were randomly picked out, to col ect blood samples via the abdominal aorta for blood routine test. Final y the axil ary lymph nodes were observed, and the lymph node samples of rats in the testing group were col ected and analyzed at 120 days by transmission electron microscope. RESULTS AND CONCLUSION:Compared with the control group, black staining of axil ary lymph nodes of rats in testing group was not obvious at the end of the 7-day period. However, with the increase of the dosing periods, the lymph nodes of the rats in the testing group became enlarged, firm and black stained, coupled with a significant rising in the percentage of blood neutrophils. After 30 days of drug withdrawal, the size of the rat axil ary lymph nodes was reduced and black staining partly faded, with the decreasing of blood neutrophil percentage. Under the transmission electron microscope, abundant carboxylated single-wal ed carbon nanotubes were uptaken by lymphocytes to form a large number of phagocytic vacuoles after drug withdrawal for 30 days. It indicates that the short-term tracing of rat axil ary lymph nodes by carboxylated single-wal ed carbon nanotubes injected through the tail vein is relatively weak, while the long-term intravenous injection can cause their accumulation in rat axil ary lymph nodes, coupled with the increase of neutrophils;after drug withdrawal, the carboxylated single-wal ed carbon nanotubes can be slowly cleared by the lymph nodes.