The sequence of the first fifteen N-terminal amino acids was determined and was found to be MDPVVLQRRDWENPG. Amino acid %Asp 0.39; Thr 0.16; Ser 5.70; Pro 18.87; Gly 29.17; Ala 13.38; Val .71; Met .18; Ile 0.16; Leu 8.04; Tyr 0.90; Phe 0.15; Lys 0.27; His 0.15; Arg 0.03; Gln 17.40; Hydro Pro 4.34.
A. Bio-Absorption
B. Cell-Adhesion
C. Mini-immunogenicity and neovascularization
D. Promoting the formation of epithelial cell
E. Biocompatibility
1. Cultivation of BHK-21 and 2BS cell line
BHK-21 or 2BS cells were plated onto the bottom of the 96-well cell culture plates at the density of 1*103cells/well, respectively, with 0.1ml RPMI1640 contained 10% fetal bovine serum in the well. BHK-21 or 2BS cells were incubated in standard conditions (at 37oC with 5% CO2 in a humidified incubator) for 24 hours. 2. Biochemical activity analysis of Human-like Collagen Complete culture media was removed after 24 hours, Collagen was added at various concentrations 0.01%,0.03%,0.05%,0.07%,0.09%,0.10%,0.11%,0.13% and a complete medium was used as blank control. Cells were incubated in standard conditions (at 37oC with 5% CO2 in a humidified incubator). The morphology of cell was observed by reverse microscope after 24hr, 48hr, 72hr, 96hr, and 120hr.
Thereafter, the viable cell count was determined by a colorimetric method using MTT (3-4,5dimethylthiazol-2yl-2, 5 diphenyl tetrazolium bromide) after 24hr, 48hr, 72hr, 96hr and 120hr. A490 was evaluated by ELISA reader. 3. Results
The results demonstrate that Collagen has no significant effect on cell morphology. MTT analysis shows that Collagen has remarkable effect on cell proliferation when its concentration is higher than 0.03%. Cell proliferation was improved by 40% to 60% compared to control.
The protein was lyophilized after from a sterile solution containing no additives.
Sterile Filtered White lyophilized (freeze-dried) powder.
Greater than 95.0% as determined by:
(a) Analysis by RP-HPLC.
(b) Analysis by SDS-PAGE.
It is recommended to reconstitute the lyophilized Collagen in sterile 18MΩ-cm H2O not less than 100ug/ml, which can then be further diluted to other aqueous solutions.
Lyophilized Collagen although stable at room temperature for 3 weeks, should be stored desiccated below -18oC. Upon reconstitution Collagen should be stored at 4oC between 2-7 days and for future use below -18oC.
For long term storage it is recommended to add a carrier protein (0.1% HSA or BSA).
Please prevent freeze-thaw cycles.
