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Abstract
Study Design
In- vitro experiments using human mesenchymal stem cells (MSCs), intervertebral disc (IVD) cells and type 5 adenovirus/transforming growth factor- β1 construct (A d/TGF- β1).
Summary of Literature Review
MSCs are known to be multipotent in tissue regeneration. In degeneration of IVD, cellular replacement with genetic modification other than that of IVD cells may prove an enhanced mechanism for the regeneration of IVD cells.
Materials and Methods
MSCs and IVD cells were cultured and an adenovirus construct containing TGF- β1 cDNA (A d/TGF- β 1) was also produced. In the first step, the MSCs were transduced with A d/TGF- β1, then mixed with IVD cells in various proportions and three dimensionally cultured. [methyl-3 H]Thymidine and [35 S]Sulfur incorporation for DNA and proteoglycan synthesis, respectively, were measured. RT-PCR was performed to assess the aggrecan and collagen types I and II mRNA expressions
Results
Mixed cultures of MSC and IVD cells showed relatively similar amounts of newly synthesized proteoglycan compared with cultures of IVD cells only. In mixed cultures transduced with A d/TGF- β1, there were significant decreases in newly synthesized proteoglycan with increasing the proportions of MSCs, which was also found with the aggrecan and collagen type II mRNA expressions. However, the collagen type I mRNA expression increased with increased proportions of MSCs transduced with A d/TGF- β1.
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Fig. 1.
(A) total DNA content in three dimensional culture of IVD cells and MSCs, (B)Newly synthesized proteoglycan in three dimensional culture of IVD cells and MSCs presented as percent control. Mixed cultures with IVD cells and MSCs equalled or quite decreased in newly synthesized proteoglycan comparing only IVD cell cultures (p 〈0.05).
Fig. 2.
(A) total DNA content in three dimensional culture of IVD cells and Ad/TGF-β1-transduced MSCs, (B) Newly synthesized proteoglycan in three dimensional culture of IVD cells and Ad/TGF-β1-transduced MSCs presented as percent control (p 〈0. 05).
Fig. 3.
RT-PCR for aggrecan, collagen type I, and collagen type II in three dimensional culture of IVD cells and MSCs. β-actin was used for normalization. Mixed ratio of IVD cells and MSCs are a) 1:0, b) 0:1, c)4:1, d) 1:1, e) 1:4.
Fig. 4.
RT-PCR for aggrecan, collagen type I, and collagen type II in three dimensional culture of IVD cells and Ad-transduced MSCs. β-actin was used for normalization. a) only IVD cells, b) only MSC, c) only Ad/luc-transduced MSC [Mock], d) only Ad/TGF-β1-transduced MSC [Ad/TGF-β1-MSC], e) [IVD cells : Ad/TGF-β1-MSC] 1:1, f) [IVD cells : Ad/TGF-β1-MSC] 1:4.
Table 1.
Primer sequence and reaction temperature
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