Objective: To prepare bionic spinal cord scaffold of collagen-heparin sulfate by three-dimensional (3-D) printing, and provide a cell carrier for tissue engineering in the treatment of spinal cord injury.
Methods: Collagen- heparin sulfate hydrogel was prepared firstly, and 3-D printer was used to make bionic spinal cord scaffold. The structure was observed to measure its porosity. The scaffold was immersed in simulated body fluid to observe the quality change. The neural stem cells (NSCs) were isolated from fetal rat brain cortex of 14 days pregnant Sprague-Dawley rats and cultured. The experiment was divided into 2 groups: in group A, the scaffold was co-cultured with rat NSCs for 7 days to observe cell adhesion and morphological changes; in group B, the NSCs were cultured in 24 wells culture plate precoating with poly lysine. MTT assay was used to detect the cell viability, and immunofluorescence staining was used to identify the differentiation of NSCs.
Results: Bionic spinal cord scaffold was fabricated by 3-D printer successfully. Scanning electron microscope (SEM) observation revealed the micro porous structure with parallel and longitudinal arrangements and with the porosity of 90.25% ± 2.15%. In vitro, the value of pH was not changed obviously. After 8 weeks, the scaffold was completely degraded, and it met the requirements of tissue engineering scaffolds. MTT results showed that there was no significant difference in absorbence (A) value between 2 groups at 1, 3, and 7 days after culture (P > 0.05). There were a lot of NSCs with reticular nerve fiber under light microscope in 2 groups; the cells adhered to the scaffold, and axons growth and neurosphere formation were observed in group A under SEM at 7 days after culture. The immunofluorescence staining observation showed that NSCs could differentiated into neurons and glial cells in 2 groups; the differentiation rate was 29.60% ± 2.68% in group A and was 10.90% ± 2.13% in group B, showing significant difference (t = 17.30, P = 0.01).
Conclusion: The collagen-heparin sulfate scaffold by 3-D-printed has good biocompatibility and biological properties. It can promote the proliferation and differentiation of NSCs, and can used as a neural tissue engineered scaffold with great value of research and application.