Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at -20 degrees C and lyophilized to obtain a sponge, which could be stored at -80 degrees C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2-3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.

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Collagen is a widely used material in biomedical applications. Although processes that prepare collagen and collagen-based materials that show suitable properties after extraction exist, a ready-to-use, easily stockable, with tailored collagen concentration has not yet been developed. Using rat tail tendons, acid soluble collagen solutions were prepared by two different methods. To improve cell viability of pure collagen films, solutions with physiological pH were also prepared by mixing with NaOH solution. Specimens in the form of thin sheets were then fabricated by solvent evaporation. Next, IR spectroscopy, tensile testing techniques as well as human fibroblast cell morphology and cytotoxicity were used to validate the significant variations in the processes. The results demonstrated that, during the synthesis of collagen stock solution, lyophilization and mechanical blending had little effect on the final properties and therefore offers a method for obtaining solutions with a more homogeneous and modifiable collagen concentration and longer storage time. Neutralizing the stock solution with aqueous NaOH prior to solvent evaporation provided films that had lower mechanical properties but significantly improved biological performance.

In the past 25 years, several efforts have been focused on developing vascular substitutes showing long-term patency when implanted in humans. In this study, we present our last findings in an ongoing project aiming to develop a tissue-regenerated blood vessel from collagen-based scaffolds. A collagen-based scaffold with adequate biological properties was developed by directly assembling collagen and cells in a cylindrical geometry. The technique mainly involves the use of a ventilated rotating tube allowing smooth muscle cells to grow in a cylindrical collagen matrix. Scanning electron microscopy (SEM), histology, and cell cycle analyses were carried out in order to assess the biological potential of the cell-based scaffold for further maturation. Compliance results showed that this technique allows cells to assemble in the collagen matrix, thus providing enough rigidity to the structure to be handled and mounted in a perfusion bioreactor for further growth and maturation.

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