Modification Of Fish Scale Collagen Matrix And Its Preliminary Application In Wound Repair

Aims:Recently, faced by wound infection, slow wound repair, repeated cracking,severe scarring and other problems, external trauma treatments are still a great chanllenge. Collagen(Coll) materials have became the general method to solve those problems because of its good structure and biological properties. However, because of the high price and body immune response of Coll materials, it is of great importance to find better Coll materials. In this study, fish scale Coll was chosen as the matrix,because of its cheaper price, more extensive sources, lower antigenicity and anaphylaxis. On the basis of the integrity of its classical triple-helical structure, the fish scale Coll was modified by composing with other Colls and adding natural materials, in order to improve the Coll’s thermal stability and mechanical properties.Methods:Coll was extracted with pepsin at p H 2-3 for 3-5 days at 4 °C. The structure and purity of Coll was analyzed by UV-absorption spectra, Fourier transform infrared spectroscopy(FTIR) and SDS-PAGE gel electrophoresis. Coll sponges were prepared by dissolving Coll gel in dilute acetic acid, followed by lyophilization to form sponges. Then, the modification of Coll sponges were carried out: 1. composing the fish scale typeⅠColl with bovine and porcine typeⅠColl to study the properties of recombinant Coll sponges. 2. Using chitosan(CTS) and alginate dialdehyde(ADA) as dual-crosslinker to modifythe recombinant Coll gel. These modified Coll specimens were analyzed with the variations of the structure and properties. The FTIR and in vitro fiber form analysis demonstrated the retention of triple-helical structure of Coll sponges; the differential scanning calorimetry(DSC) and thermogravimetry(TG)measurements indicated the improvement of thermal stability and crosslinking degree;the scanning electron microscopy(SEM) and water-absorbing quality evaluated the micro-fibrils structure interactions between Coll structure. The antibacterial activity and the biocompatibility of Coll/CTS mixture were analyzed by the antibacterial test and cell proliferation assay, and the wound repair ability was studied by the wound healing test. Meanwhile, the influence of two common reaction temperatures(4℃ and room temperature) was also taken into consideration.Results:The maximum absorption wavelength in UV, absorption peaks in FTIR and the stripes in SDS-PAGE gel electrophoresis indicated that the Coll we extracted was the typical type ⅠColl, which has high purity and wholly triple-helical structure. To the recombinant Coll sponges, the FTIR results showed that the recombinant Coll kept the intact triple-helical structure. Compared with pure Coll, the water absorption of recombinant Coll sponges was improved by ten-fold, the micro-fibril structure became more compact and the pore structure turned more uniform. Thermostability and enzymatic hydrolysis stability indicated that composing with bovine typeⅠColl could improve the fish scales Coll’s properties and the best ratio of fish scale Coll to bovine type I Coll was 2:1, with enzymatic hydrolysis over 24 h was reduced from90% to 50%, thermal denaturation temperature(Td) improved from 48.7℃ to 63℃ in DSC analysis, and weight loss of modified Coll from 84% reduced to 59% in TG analysis. On the other hand, based on the results of the recombinant Coll study, 2:1was chosen as the ratio of fish scale Coll to bovine Coll for matrix in further modification with CTS and ADA. The results showed that the modified Coll mixtures had kept the intact triple-helical structure of Coll and had a higher in vitro fiber formation capability. At the same time, the water absorption, thermostability of Coll sponges also got significant improvement, Td improved from 63℃ to 86℃,weight loss from 67.5% reduced to 51%. The two common reaction temperatures turned out to have little effect on Coll’s structure and the modified Coll sponges has better wound healing ability. The antibacterial measurement results indicated the Coll/CTS and their mixtures sponges modified by ADA have good antibacterial activity(over 70%). Simultaneously, the biocompatibility test showed that modified Coll sponges had good biocompatibility and could function as the medical hemostatic dressing.Conclusions:Recombinant Coll mixtures could keep the intact triple-helical structure of Coll and composing with bovine Coll, can highly improve the enzymatic hydrolysis stability and thermostability of the fish scale Coll. Compared with pure Coll, the micro-fibril structure become more compact and the crosslinking degree of Coll become higher. Meanwhile, the recombinant Coll owned more uniform structure,which benefited the water absorption and maintenance.The modification of recombinant Coll matrix by CTS and ADA had no destruction on the triple-helical structure of Coll, and in vitro fiber formation capability and thermostability of Coll sponges also got significant improvement. At the same time, the content of ADA in the Coll/CTS mixtures had no significant influence on the antibacterial activity of CTS, and the best concentration of ADA was10%. The two reaction temperatures did not effect the structural and thermal properties of Coll sponges. In conclusion, biological evaluation of ADA-modified Coll/CTS mixture sponges suggested that ADA can be a safe choice to produce biocompatibility Coll dressing with excellent physicochemical properties. The fish scale Coll is worth of further study.