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Abstract
Objectives
To determine the proteoglycan synthesis of the rabbit nucleus pulposus cells in various concentration of extracellular collagen type I and II under the stimulation of TGF- β1.
Summary of Literature Review
Therapeutic effect of growth factor and gene therapy can be altered by composition of extracellular matrix. However, the effect of extracellular collagen types I and II on synthetic activity of intervertebral disc cells is not thoroughly studied before.
Materials and Methods
The nucleus pulposus cells were isolated and cultured from 10 skeletally mature rabbits. Cultures were trypsinized and incorporated into alginate beads with different concentration of extracellular collagen type I and II (0.5%, 1.0% and 1.5%). Those cultures with TGF- β1 (10 ng/ml) served stimulated condition of matrix synthesis. Newly synthesized proteoglycans were assessed by 35 S- sulfate incorporation using chromatography on Sephadex G- 25 in PD- 10 columns. Scintillation count was normalized with DNA content by Hoechst dye method.
Results
In basal condition, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II were statistically insignificant. In stimulated condition with TGF- β1, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II was also statistically insignificant. However, cultures in stimulated condition with TGF- β1 showed increased amount of newly synthesized proteoglycans compared to those of basal condition regardless of the concentration of extracellular collagen type I and II (p <0.05).
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Fig. 1.
The effect of type I collagen on the synthesis of proteoglycan by rabbit disc cells encapsulated in alginate beads. Incorporation of 35S-sulfate into disc cells cultured in basal medium or in medium supplemented with transforming growth factor-β1 (TGF-β1) was measured. Data represent mean ± SD of three experiments performed in triplicate. The values of Y-axis were content of newly synthesized proteoglycan, which was divided by the amount of deoxyribonucleic acid. Rabbit intervertebral disc cells cultured in alginate beads, treated by TGF-β1 (10 ng/ml) showed 3 fold increase in newly synthesized proteoglycan compared to basal condition (p<0.05), while increased concentration of extracellular type I collagen failed to affect proteoglycan synthesis (p>0.05). *: p<0.05, NS: statistically non-significant.
Fig. 2.
The effect of type II collagen on the synthesis of proteoglycan by rabbit disc cells encapsulated in alginate beads. Incorporation of 35S-sulfate into disc cells cultured in basal medium or in medium supplemented with transforming growth factor-β1 (TGF-β1) was measured. Data represent mean ± SD of three experiments performed in triplicate. The values of Y-axis were content of newly synthesized proteoglycan, which was divided by the amount of deoxyribonucleic acid. Rabbit intervertebral disc cells cultured in alginate beads, treated by T GF-β1 (10 ng/ml) showed 3 fold increase in newly synthesized proteoglycan compared to basal condition (p<0.05), while increased concentration of extracellular type II collagen failed to affect proteoglycan synthesis (p>0.05). *: p<0.05, NS: statistically non-significant.
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