Effect of trypsin on biochemical and functional properties of decellularized porcine articular cartilage

Abstract

Objective: to study the effect of trypsin pretreatment in the porcine articular cartilage decellularization protocol on the ability to restore the biochemical composition and functional properties of the resulting finely dispersed tissue-specific scaffold when co-cultured with human adipose-derived stem cells (hADSCs).Materials and methods. Porcine articular cartilage was micronized to a maximum size of 250 μm. The resulting porcine articular cartilage microparticles (CMps) were treated with trypsin (0.05, 0.25, 0.50%) / EDTA solution at +37 °C for 24 hours. Then, the CMps were successively incubated for 24 hours in three surfactant solutions containing 0.1% sodium dodecyl sulfate and increasing concentration of Triton X-100 (1, 2, 3%) at room temperature and in DNase I solution at +37 °C for 48 hours. The degree of change in the biochemical composition and the ability of decellularized CMps (DCMps) scaffolds within cell-engineered constructs (CECs) to support hADSC adhesion and proliferation, as well as their potential ability to exert a stimulatory regenerative effect, were then assessed. DNA, glycosaminoglycans (GAGs) and collagen content in the DCMps and CECs were examined. The morphology of the samples was examined using histological and immunohistochemistry staining.Results. Histological analysis showed that there were no cells and detritus in the DCMp samples. Pretreatment of CMps samples гыштп a solution with the lowest content of trypsin (0.05%) / EDTA in the samples retained 5.14 ± 0.87 ng/mg DNA in the samples, while GAG content decreased to 5.34 ± 0.9 μg/mg and collagen to 154 ± 34 μg/mg. By day 28 of CEC cultivation, adherent cells had produced their own extracellular matrix (ECM) containing GAGs and collagen. The amount of DNA in it was 6.30 ± 0.11 μg/CEC and that of GAGs was 19.36 ± 0.73 μg/CEC.Conclusion. Pretreatment with trypsin allows achieving uniformly complete decellularized CMps. At the same time, onset of changes in the ECM composition indicates a decrease in the ability of hADSCs to synthesize GAGs and type II collagen during co-culturing with DCMps. The increased proliferative activity of adherent hADSCs, as well as the tissue specificity of the DCMp scaffold will allow further research towards a hydrogel matrix capable of enhancing the specific and stimulating regenerative potential when co-cultured with cells of the same phenotype.

Keywords

• cartilage tissue
• decellularization
• trypsin
• mesenchymal stromal cells
• tissue engineering