Chondrosia reniformis marine-sponge collagen membranes for skin re-epithelialization

Introduction

Chondrosia reniformis collagen has been identified as mainly of type IV. Being collagen IV the main component of the epidermal basal layer [1], C. reniformis represents a valuable source to be explored in the skin regeneration field. This work envisaged the production of C. reniformis collagen membranes for the selection of rapidly adherent epidermal cells, like the commercial collagen coatings, and for their subsequent culture. This approach would permit a single system for culturing and carrying basal epidermal cells aimed at re-epithelialize skin wounds.

Materials and Methods

The collagen of C. reniformis marine-sponge was extracted with 100mM Tris-HCl, 10mM EDTA, 8M urea and 100mM 2-mercaptoethanol. To define the best re-solubilization conditions, the obtained precipitate was dissolved in five different solutions: Solution A: 100mM Tris-HCl+8M Urea+10mM EDTA (pH 9.5); Solution B: 50mM Tris-HCl+1M NaCl (pH 7.4); Solution C: 100mM Tris-HCl (pH 7.4); Solution D: 0.5% H2O2 (v/v) (pH 11) and Solution E: 100mM Tris-HCl (pH 9.5). Solutions of 1% collagen were prepared and cross-linking was performed with HMDI, genipin and EDC/NHS at different concentrations. The membranes were obtained by solvent-casting and/or freeze-drying, and their stability was tested both in PBS and culture medium, for at least 7 days. Morphological characterization of the membranes was carried out by scanning electron microscopy (SEM). Cytotoxicity, based on metabolic activity (MTS assay) and cell proliferation (DNA quantification) analysis of the 100mM Tris-HCl (pH 9.5) and 8mM EDC/NHS cross-linked collagen membranes, was assessed with L929 cells.

Results were analyzed by IBM SPSS Statistics Version 20 using one-way ANOVA and Kruskall-Wallis test. Significance was set for p<0.05.

Results

The re-solubilization of the collagen was achieved with solutions A, D and E. Nonetheless, although it was possible to obtain the 1% collagen membranes, those prepared from A and D solutions were not stable and collapsed during the tests. Stable membranes were obtained after re-solubilization of the collagen in solution E and upon cross-linking by immersion in 8mM and 25mM EDC/NHS. Additionally, the freeze-dried membranes were analyzed by SEM (Fig. 1), revealing no porosity but some roughness on their surface.

The in vitro tests showed a reduced cytotoxicity, confirming the capability of the membranes to support cell adhesion and proliferation.

Fig. 1. Freeze-dried 8mM (left) and 25mM (right) EDC/NHS cross-linked membranes.

Discussion and Conclusions

The obtained results suggests that the100mM Tris-HCl (pH 9.5) and 8mM EDC/NHS cross-linked collagen membranes are good candidates for further proceeding with the envisaged rational for skin re-epithelialization

References

1. Kaur P, Li A., J Invest Dermatol, 114 (2000) 413

Acknowledgments

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. KBBE-2010-266033 (project SPECIAL) andfrom FEDER through POCTEP Project0687_NOVOMAR_1_P.