[Increased synthesis of extracellular matrix in passaged nucleus pulposus cells by transfection with adenoviral vectors containing human transforming growth factor beta1].
OBJECTIVE
To determine whether the transforming growth factor beta1 (TGF-beta1) is a key regulatory molecule required for an increase or a balance of extracellular matrix (ECM) and DNA synthesis in the goat passaged nucleus pulposus (NP) cells.


METHODS
The NP cells isolated from the goat intervertebral discs were cultured in vitro for a serial of passages and transfected with the replication-incompetent adenoviral vectors carrying the human TGF-beta1 (hTGF-beta1) or lacZ genes. Then, they were cultured in monolayer or alginate bead 3-dimensional (3-D) systems for 10 days. The changes in the production and the molecular components of ECM that occurred in the NP cells transfected with Ad/hTGF-beta1 or the controls were evaluated by Western-blot and absorbance of glycosaminoglycan (GAG)-Alcian Blue complexes. Differences of DNA synthesis in the variant cells and culture systems were assessed by fluorometric analysis of the DNA content.


RESULTS
DNA quantitation in the variant culture systems indicated that in monolayers the NP cells at Passage 3 transfected with Ad/hTGF-beta1 had a much higher cell viability and more DNA synthesis (P < 0.05); however, in the alginate 3-D culture system, the NP cells transfected with Ad/hTGF-bea1 did not have any significant difference from the controls (P > 0.05). The Western blotting analysis of the protein sample isolated from the variant cells for TGF-beta1, type II collagen, and Aggrecan expression indicated that in the monolayers and alginate 3-D culture systems the NP cells at Passage 3 transfected with Ad/hTGF-beta1 revealed much higher protein levels than the controls (P < 0.05); whereas the type I collagen content was much lower than the controls (P < 0.05), but a significatly increased ratio of type II /type I collagen was found in both of the cell culture systems (P < 0.05). The GAG quantification also showed a positive result in both the cell culture systems and the NP cells at Passage 3 transfected with Ad/hTGF-beta1 had a much higher GAG content than the controls (P < 0.05).


CONCLUSIONS
To a greater extent, hTGF-beta1 can play a key role in maintaining the phenotype of the NP cells and can still have an effect of the phenotypic modulation after a serial of the cell passages. The NP cells that are genetically manipulated to express hTGF-beta1 have a promising effect on the restoration of the intervertebral disc defects. The NP cells transfected with Ad/ hTGF-beta1 cultured in the 3-D alginate bead systems can show a nearly native phenotype.
Li
Xu
X
Department of Orthopedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong, 510515, PR China. drlxyinger@yahoo.com.cn
Hou
Xiaokui
X
Tang
Tingting
T
D000256:Adenoviridae / Q000235:genetics; D053674:Aggrecans / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D005109:Extracellular Matrix / Q000378:metabolism*; D005822:Genetic Vectors; D006041:Goats; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics* / Q000378:metabolism
D053674:Aggrecans; D053773:Transforming Growth Factor beta1; D003094:Collagen; D004247:DNA