Objective: To evaluate the cell biological features and the effect of transplantation of transforming growth factor beta3 (TGF-beta3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro.
Methods: NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-beta3 (Ad-TGF-beta3) gene (Ad-TGF-beta3 group), and then the cell biological features were observed by cell vitality assay, the expression of the TGF-beta3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was established by needling L(3, 4), L(4, 5), and L(5, 6) in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-beta3-transfected rabbit degenerative nucleus pulposus cells (100 microL, 1 x 10(5)/mL, group A, n=12), no gene-modified nucleus pulposus cells (100 microL, 1 x 10(5)/mL, group B, n=12), and phosphate-buffered saline (PBS, 100 microL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L(3, 4), L(4, 5), and L(5, 6) disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR.
Results: The viability of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-beta3 gene. At 3, 7, and 14 days after transfected, TGF-beta3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-beta3 group, and the positive rate was significantly higher than that of Adv group and blank group (P < 0.05). The disc degeneration in group A was slighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P < 0.05).
Conclusion: TGF-beta3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.