IADR Abstract Archives
Characterization and Cytotoxicity of the Acid Soluble, Porcine Skin Extracted Collagen on Peridontal Ligament Cells
Objectives: To characterize acid soluble collagen extracted from porcine skin and investigate its cytotoxicity in primary periodontal ligament cells
Methods: The native collagen was extracted using an enzymatic-acid technique. The porcine skins were washed thoroughly in ice-cold normal saline solution. After removing hair, the skins were stripped of remaining fat, noncollagenous proteins and polysaccharides using sodium hydroxide, and the mixture of acetic acid and 0.1% (w/w) enzyme pepsin, respectively. The acid soluble collagen extracts were further precipitated with 3 M sodium chloride, and collected by centrifugation. The white precipipant was dialyzed in 20 volumes of ice-cold dH2O, frozen, and lyophilized. The lyophilized extracted collagen was further characterized using the amino acid content analysis and SDS-PAGE. Primary periodontal ligament cells were treated with the conditioned media of 5, 10, 25, 50, and 100 mg extracted collagen for 24 h. The cytotoxicity was evaluated using MTT based colorimetric assay. Three independent experiments were performed.Data were collected and analyzed using SPSS program. p value less than 0.5 was considered significant.
Results: The major composition of the extracted collagen were glycine, proline, and hydroxyproline. The approximately 100, 200, and 300 KDa polypeptide bands were observed. The MTT assay demonstrated that the 100 mg collagen conditioned media significant reduced cell vaibility at 24 h (p<0.05). The condition medium of 5 , 10, and 25 mg collagen slightly increased cell viability, while that of 50 mg collagen slightly decreased cell viability.
Conclusions: The extracted collagen at 5, 10, 25, and 50 mg were biocompactibilty to primary periodontal ligament cells.
Methods: The native collagen was extracted using an enzymatic-acid technique. The porcine skins were washed thoroughly in ice-cold normal saline solution. After removing hair, the skins were stripped of remaining fat, noncollagenous proteins and polysaccharides using sodium hydroxide, and the mixture of acetic acid and 0.1% (w/w) enzyme pepsin, respectively. The acid soluble collagen extracts were further precipitated with 3 M sodium chloride, and collected by centrifugation. The white precipipant was dialyzed in 20 volumes of ice-cold dH2O, frozen, and lyophilized. The lyophilized extracted collagen was further characterized using the amino acid content analysis and SDS-PAGE. Primary periodontal ligament cells were treated with the conditioned media of 5, 10, 25, 50, and 100 mg extracted collagen for 24 h. The cytotoxicity was evaluated using MTT based colorimetric assay. Three independent experiments were performed.Data were collected and analyzed using SPSS program. p value less than 0.5 was considered significant.
Results: The major composition of the extracted collagen were glycine, proline, and hydroxyproline. The approximately 100, 200, and 300 KDa polypeptide bands were observed. The MTT assay demonstrated that the 100 mg collagen conditioned media significant reduced cell vaibility at 24 h (p<0.05). The condition medium of 5 , 10, and 25 mg collagen slightly increased cell viability, while that of 50 mg collagen slightly decreased cell viability.
Conclusions: The extracted collagen at 5, 10, 25, and 50 mg were biocompactibilty to primary periodontal ligament cells.