Developing a Scaffold from Porcine Adipose Tissue
Abstract
Tissue engineering aims to address the critical lack of immunocompatible tissues and organs available for grafting and transplantation. Scaffolds are three dimensional, porous structures that provide shape and attachment sites for cells during tissue growth and are a critical component of tissue engineering. Naturally derived scaffolds have seen significantly greater clinical usage than synthetic scaffolds and have marked advantages, including naturally present growth factors and ideal morphology. In addition, scaffolds derived from xenogeneic tissues are advantageous because human tissues are difficult and costly to obtain. However, careful decellularization is required to prevent immune rejection. Porcine adipose tissue (PAT) is inexpensive and readily obtained. This study's objective was to develop a general tissue scaffold from PAT while maintaining the structure of its extracellular matrix (ECM). Maintaining PAT's ECM intact is expected to improve nutrient distribution and cell ingrowth. Two decellularization methods were attempted: methanol-chloroform submersion, and freeze-thawing. Methanol-chloroform submersion destroyed the tissue and was discontinued; freeze-thawing was successful and pursued: the number of freeze-thaw steps (1 - 7), the tissue surface area and thickness, and the trypsin incubation time (1 - 3 hours) were evaluated and optimized. Moreover, following an initial cell seeding study during which cell attachment and ingrowth did not occur, a lipid removal strategy using sonication (20 - 60 minutes with water, trypsin, or SDS) and immersion in xylene (20 seconds to 20 minutes) was also devised to remove all lipids and thereby create a hydrophilic environment conducive to cell seeding. Processed scaffold mechanical strength and morphology were examined using histology slides and SEM digital micrographs. An additional cell seeding study using CFDA-SE stained cells was conducted. An average ultimate tensile strength of 87.4 kPa, an average break strain of 53.9 kPa, an average elastic modulus of 324 kPa, 30% relaxation per ramp, and intact morphology, including tubular vascular channels were found. Cells examined in micrographs of seeded tissue demonstrated successful cell ingrowth and uniform distribution at 8 days. Overall, an optimized decellularization process and a lipid removal processes were developed that retained natural tissue morphology. Moreover, obtained scaffolds compared favorably with small intestine submucosa (SIS), a clinically available scaffold.
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- OSU Theses [15752]