Development and Characterization of an Acellular Porcine Medial Meniscus for Use in Tissue Engineering

Abstract

The objectives of this study were to characterize fresh porcine menisci and develop a decellularization protocol with a view to the generation of a biocompatible and biomechanically functional scaffold for use in tissue engineering/regeneration of the meniscus. Menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic acid and final washing in phosphate-buffered saline. Histological, immunohistochemical, and biochemical analyses of the decellularized tissue confirmed the retention of the major structural proteins. There was, however, a 59.4% loss of glycosaminoglycans. The histoarchitecture was unchanged, and there was no evidence of the expression of the major xenogeneic epitope, galactose-alpha-1,3-galactose. Biocompatibility of the acellular scaffold was determined by using contact cytotoxicity and extract cytotoxicity tests. Decellularized tissue and extracts were not cytotoxic to cells. Biomechanical properties were determined by indentation and tensile tests, which confirmed the retention of biomechanical properties following decellularization. In conclusion, this study has generated data on the production of a biocompatible, biomechanically functional scaffold for use in meniscal repair.

Development and Characterization of Acellular Extracellular Matrix Scaffolds from Porcine Menisci for Use in Cartilage Tissue Engineering

“…The acellular menisci demonstrated in vitro biocompatibility for murine NIH 3T3 embryonic fibroblast. Stapleton et al 49,50 decellularized menisci by exposing them to several freeze-thaw cycles and incubating them in hypotonic Tris buffer and SDS in hypotonic buffer plus protease inhibitors, nucleases, and hypertonic buffer followed by disinfection with peracetic acid. The menisci scaffold showed immunobiocompatibility, no evidence of the expression of the major xenogeneic epitope (galactose-a-1,3-galactose), and was capable of supporting the attachment and infiltration of human fibroblasts and porcine meniscal cells.…”

Whole-organ engineering with natural extracellular materials

“…While verification of cell removal is typically quantified by DNA content post-decellularization by means of spectrophotometry [29][30][31][32], further characterization of the remaining major histocompatibility complexes (MHCs) is essential for clinical human allograft studies. For the decellularized human whole-trachea graft, MHC class I and II removal was monitored.…”

“…Other imaging techniques such as scanning-electron microscopy have been used to visualize and compare nanostructures (e.g., the weave, coil and strut fibers in the decellularized heart study) in decellularized and non-decellularized cadaveric tissue [32]. Together, these studies show that with optimal treatment, decellularized tissues can maintain ECM antibody epitope expression in such molecules as collagens, laminin and fibronectin in immunohistochemistry and immunofluorescence studies [13,15,18,21,30,36]. Table 2 summarizes the various ECM molecules characterized in select decellularized organ and tissue studies.…”

Meniscus repair and regeneration: review on current methods and research potential

“…The collagen meniscus implant (CMI) (ReGen Biologics, Franklin Lakes, NJ) is the first regenerative technique applied to meniscal tissue in clinical practice (Stone et al, 1992;Stone et al, 1997;Steadman and Rodkey, 2005;Zaffagnini et al, 2007). Since an outer rim of meniscal tissue is needed for CMI implantation, it is indicated only for partial and not total meniscus regeneration.…”