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Abstract
Study Design
This is an in-vitro experiment using rabbit intervertebral disc (IVD) cells and growth factors.
Objectives
We wanted to determine the effect of types I,and II atelocollagen and growth factor gene therapy for matrix regeneration of rabbit IVD cells.
Summary of the Literature Review
Adenovirus-medicated growth factor gene therapy is efficient for matrix regeneration of the IVD. Atellocollagen has provided a favorable environment for matrix synthesis. However, a combined approach using gene and cell therapy in an atelocollagen scaffold has not yet been attempted.
Materials and Methods
Rabbit IVD cells were transduced with Ad/TGF-β1 and Ad/BMP-2. The cells were then implanted to the atelocollagen scaffold. The [methyl-3 H]thymidine incorporation for DNA synthesis and the [35 S]sulfur incorporation for proteoglycan synthesis were measured. RT-PCR was performed for assessing the aggrecan, collagen type I, collagen type II and osteocalcin mRNA expressions.
Results
The rabbit IVD cells with Ad/TGF-β1 and that were cultured in type I atelocollagen showed a 130% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/TGF-β1 and that were cultured in type II atelocollagen showed a 180% increase in new proteoglycan synthesis (p<0.05). The rabbit IVD cells with Ad/BMP-2 and that were cultured in type I atelocollagen showed a 70% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/BMP-2 and that were cultured in type II atelocollagen showed a 95% increase (p<0.05). Rabbit IVD cells with Ad/TGF-β1 and Ad/BMP-2 and that were cultured in type I and II atelocollagen demonstrated increased collagen type I and II mRNA expressions without an osteocalcin mRNA expression (p<0.05).
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Fig. 1.
Proteoglycan synthesis([S35] incorporation) normalized by DNA synthesis([H3] incorporation) in rabbit nucleus pulposus cells seeded on atelocollagen type I and II scaffolds for culture period of 1 week. Ad/TGF-b1, Ad/BMP-2 (75MOI) were administered. Control is only cell seeded scaffold without virus infection. (∗ P<0.05)
Fig. 2.
Densitometry of RT-PCR products by beta-actin expression. (A) mRNA expression of aggrecan, collagen I, II, osteocalcin for 1 week culture in atelocollagen type I matrix and (B) atelocollagen type II.
Fig. 3. (A)
RT-PCR of beta-actin, aggrecan, collagen type I, collagen type II, and osteocalcin on atelocollagen type I matrix for culture period of 1 week, (B) atelocollagen type II.- : Control (Only cell), 1 : Ad/TGF-β1, 2 : Ad/BMP-2t
Fig. 4.
Scanning electron microscopy of the surfaces of atelocollagen type I matrix for 1 week culture (A) and atelocollagen type II (B). Note that the cells had newly synthesized extracellular matrix (single arrow). Magnification is ×500 (A-1, B-1), ×1,000 (A-2, B-2), ×3,000 (A-3, B-3).
Table 1.
Sequences of primers used for PCR amplification of cDNA.
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