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Abstract
Objectives
To examine the cellular proliferation, synthetic activity and phenotypical expression of intervertebral disc (IVD) cells seeded on types I and II atelocollagen scaffolds, with the stimulation of TGF- β1 and BMP- 2.
Summary of Literature Review
Recently, tissue engineering is regarded as a new experimental technique for the biological treatment of degenerative IVD diseases, and has been highlighted as a promising technique for the regeneration of tissues and organs in the human body. Research on cell transplantation in artificial scaffolds has provided that the conditions for tissue engineering have to be equilibrated, including the cell viability and proliferation, maintenance of characteristic phenotype, suitable scaffolds in organisms and biologically stimulated growth factor.
Material and Method
Lumbar IVD cells were harvested from 10 New Zealand white rabbits, with the nucleus pulposus cells isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96- well plate (diameter 5 mm), frozen at - 70°C, and then lyophilized at - 50°C. Fabricated porous collagen matrices were made using the cross- linking method. Cell suspensions were imbibed by surface tension into a scaffold consisting of atelocollagen. The cell cultured scaffolds were then treated with TGF- β1(10 ng/ ml) or BMP- 2 (100 ng/ ml) or both. A fter 1 and 2 week culture periods, the DNA synthesis was measured by [3 H] thymidine incorporation, and newly synthesized proteoglycan by incorporation of [35 S] sulphate. Reverse transcription- polymerase chain reactions for the mRNA expressions of type I and II collagen, aggrecan and osteocalcin were performed. The inner morphology of the scaffolds was determined by scanning electron microscopy (SEM).
Results
The IVD cultures in collagen type II with TGF- β1 demonstrated an increase in proteoglycan synthesis and up regulation of aggrecan and types I and II collagen mRNA expressions compared to the control. IVD cultures in the type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of the type II atelocollagen mRNA expression. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no up regulation of the osteocalcin mRNA expression. Furthermore, there was no synergistic effect of TGF- β1and BMP- 2 in the matrix synthesis or for the mRNA expression of the matrix components.
Conclusions
Nucleus pulposus cells from rabbit were viable in atelocollagen types I and II atelocollagen scaffolds. The type I atelocollagen scaffold was suitable for cell proliferation, but the type II atelocollagen scaffold was more suitable for extracellular matrix synthesis. The IVD cells in both scaffolds were biologically responsive to growth factors. Taken together, nucleus pulposus cells in atelocollagen scaffolds, with anabolic growth factors, provide a mechanism for tissue engineering of IVD cells.
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Fig. 1.
Rabbit nucleus pulposus cells seeded on atelocollagen type I scaffold. (A) DNA synthesis, (B) Proteoglycan synthesis (∗p>0.05)
Fig. 2.
Rabbit nucleus pulposus cells seeded on atelocollagen type II scaffold. (A) DNA synthesis, (B) Proteoglycan synthesis (∗p>0.05)
Fig. 3.
RT-PCR of beta-actin, aggrecan, collagen type I and type II, osteocalcin. (A) Atelocollagen type I scaffold. (B) Atelocollagen type II scaffold.
Fig. 4.
Densitometry of mRNA expression in atelocollagen type I scaffold. (A) Aggrecan, (B) Collagen type I, (C) Collagen type II.
Fig. 5.
Densitometry of mRNA expression in atelocollagen type I scaffold. (A) Aggrecan, (B) Collagen type I, (C) Collagen type II.
Fig. 6.
Morphology of porous atelocollagen scaffolds on scanning electron microscopy (SEM). (A, B) Atelocollagen type I scaffold (× 100, × 3,000), (C, D) Atelocollagen type II scaffold (× 100, × 3,000).
Table 1.
Sequences of primers used for PCR amplification of cDNA
Table 2.
PCR conditions
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