Research Article

Construction of a tissue engineered intervertebral disc with high biological activity using an allogeneic intervertebral disc supplemented with transfected nucleus pulposus cells expressing exogenous dopamine beta-hydroxylase

Published: September 09, 2015
Genet. Mol. Res. 14 (3) : 10658-10671 DOI: 10.4238/2015.September.9.6

Abstract

This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 105 and 1 x 106 groups vs the 1 x 104 group were significantly increased (P < 0.05); no significant difference was observed between the 1 x 105 and 1 x 106 groups (P > 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 104 and the control groups at various times, while the protein content was significantly higher in the 1 x 105 vs the control and the 1 x 104 groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 105 transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 105 and 1 x 106 groups vs the 1 x 104 group were significantly increased (P < 0.05); no significant difference was observed between the 1 x 105 and 1 x 106 groups (P > 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 104 and the control groups at various times, while the protein content was significantly higher in the 1 x 105 vs the control and the 1 x 104 groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 105 transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

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