Preparation of collagen scaffolds from different skin sources for fibroblast seeding

Abstract

The repair of damaged tissues in the body can be accelerated by replacing or regenerating such tissues. Regeneration can be achieved by using biological structures such as 3D porous scaffolds which are seeded with cells and maintained under the necessary environmental conditions including growth factors. Tissue scaffolds can be generated from a variety of materials, one being type I collagen which is the most prominent structural protein found in the majority of hard and soft tissues in animals. The aim of the project was to investigate the potential of different animal skin to yield free Type I collagen and a decellularized collagen matrix. Specific objectives were to find the optimal source of animal tissue from which Type I collagen can be extracted, and to optimize the experimental conditions to maximize the quality and quantity of collagen that can be extracted from the selected animal skin source. The extraction was performed using acid hydrolysis, enzyme hydrolysis, and a combination of both Type I collagen was extracted from the skin and tendons of porcine and chicken feet using varying concentrations of 0.3 to 0.7mol/L acetic acid, hydrolysis times (24-72 hours), and concentrations of 0.1% or 0.2% pepsin to find out which combination maximized yield. Overall, the porcine trotters were found to provide a better collagen yield than chicken feet. With acetic acid only, porcine tissues yielded best when using 0.5mol/L acetic acid for 72 hours. Chicken feet showed optimal extraction with 0.7mol/L acetic acid for 24 hours. Incorporating pepsin, the best yield from both animal tissues was achieved using 0.7mol/L acetic acid and 0.2% pepsin for 48 hours of hydrolysis. Additionally, porcine skin was decellularized using Triton X-100 and trypsin, monitoring the process at various reaction periods. This was done to further understand the efficiency at which a collagen scaffold can be produced by decellularization, compared to extracting collagen. The use of 200µl Triton X-100 with 5ml 2.5M trisodium citrate and 1ml 0.25% trypsin on porcine dermis indicated the possibility of successful denaturation, but this could not be quantified. Both extraction of type I collagen and dermal decellularization showed promise for further applications in scaffold engineering.