A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size‐exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel‐filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S‐100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. © 1996 John Wiley & Sons, Inc.

Porcine dermal collagen permanently crosslinked with hexamethylene diisocyanate was investigated for its suitability as a dermal tissue engineering matrix. It was found that the chemically crosslinked collagen had far fewer free lysine groups per collagen molecule than did the uncrosslinked matrix. The ability of the matrix to support human primary fibroblast outgrowth from explants was compared for matrices that had been presoaked in various solutions, including fibroblast media, cysteine and phosphate buffered saline (PBS). It was found that superior cell outgrowth was obtained after soaking with fibroblast media and PBS. The fibroblast attachment properties of the matrix were compared against tissue culture plastic and PET. The collagen matrix showed the least amount of cell retention compared to the other to matrices, however, the general trends were similar for all three scaffolds. Longer term cultures on the collagen showed fibroblasts covering the matrix stacking up on each other and bridging natural hair follicles. However, it was also observed that the fibroblasts were not able to penetrate into the matrix structure. This was believed to result from the chemical crosslinking, as shown by the resistance of the matrix to degradation by collagenases.

Biomaterials derived from tissue continue to offer viable alternatives to synthetic materials when autologous materials are unavailable for transplantation due to their unique chemical and mechanical properties. Tissue processing aims to stabilize the material against host degradation and render it immunologically inert by removing cellular material and crosslinking the structural proteins. It is clear that different approaches taken to achieve these goals have very different chemical and mechanical effects on the material. We describe herein the development of a tissue processing methodology to generate acellular scaffolds for tissue engineering small-diameter vascular grafts. Carotid arteries were isolated from Great White pigs and exposed to various solvent treatments, xylene, butanol, and ethanol to determine optimal parameters for the extraction of host lipids. The tissue was then exposed to a limited proteolysis with trypsin to disrupt cellular protein. This resulted in a controlled digestion that disrupted porcine nuclear DNA and cleared bulk cellular protein, leaving the more resistant structural proteins largely intact and retaining the bulk mechanical properties of the matrix. Histological analysis and scanning electron microscopy illustrated the complete removal of intact cells and nuclear material. The decellularized graft was stabilized by crosslinking with the photooxidative dye methylene green in the presence of 30,000 LUX of broad-band light energy. High-performance liquid chromatography analysis showed that the crosslinked tissue yielded 78.6% less hydroxyproline, compared with control tissue, after 20 h incubation with pepsin. Analysis of the crosslinked vessels' burst-pressure and stress-strain characteristics have shown comparable mechanical properties to those of control vessels. Assessment of in vitro cell adhesion and compatibility was conducted by seeding primary human umbilical vein endothelial cells and adult human vascular smooth muscle cells onto the lumenal and ablumenal surfaces, respectively; these cells were shown to adhere and proliferate under traditional static culture conditions.

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