The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds

Abstract

Biologic materials from various species and tissues are commonly used as surgical meshes or scaffolds for tissue reconstruction. Extracellular matrix (ECM) represents the secreted product of the cells comprising each tissue and organ, and therefore provides a unique biologic material for selected regenerative medicine applications. Minimal disruption of ECM ultrastructure and content during tissue processing is typically desirable. The objective of this study was to systematically evaluate effects of commonly used tissue processing steps upon porcine dermal ECM scaffold composition, mechanical properties, and cytocompatibility. Processing steps evaluated included liming and hot water sanitation, trypsin/SDS/TritonX-100 decellularization, and trypsin/TritonX-100 decellularization. Liming decreased the growth factor and glycosaminoglycan content, the mechanical strength, and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for all). Hot water sanitation treatment decreased only the growth factor content of the ECM (p ≤ 0.05). Trypsin/SDS/TritonX-100 decellularization decreased the growth factor content and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). Trypsin/Triton X-100 decellularization also decreased the growth factor content of the ECM but increased the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). We conclude that processing steps evaluated in the present study affect content, mechanical strength, and/or cytocompatibility of the resultant porcine dermal ECM, and therefore care must be taken in choosing appropriate processing steps to maintain the beneficial effects of ECM in biologic scaffolds.

Fig. 1

Dermis was processed at the time of harvest with either no liming or hot water sanitation (sample d), liming but no hot water sanitation (sample d-L), hot water sanitation but no liming (sample d-H) or both liming and hot water sanitation (sample d-LH). Dermis sample d was treated with either the 0.25% trypsin/0.1% SDS/1% Triton X-100 decellularization protocol (d-TST) or the 0.25% trypsin/1% TritonX-100 decellularization protocol (d-TT).

Fig. 2

Decellularization of porcine dermis was assessed by imaging and analysis of DAPI and Hematoxylin and Eosin (H&E) stained sections (A), agarose gel analysis (B), and dsDNA per mg dry weight as measured with the Quant-iT PicoGreen dsDNA Assay Kit (C).

Fig. 3

Liming, hot water sanitation, and decellularization treatments affected the growth factor and glycosaminoglycan contents of the porcine dermis samples. The bFGF (A), VEGF (B), TGF-β1 (C) and glycosaminoglycan (D) contents of the dermis were measured. Results are plotted as mean ± standard error. * signifies a p value of ≤.05 as compared to d; ** signifies a p value of ≤.05.

Fig. 4

The ball burst test was used to assess the mechanical strength of porcine dermis samples. Results are plotted as mean ± standard deviation. * signifies a p value of ≤.05 as compared to d; ** signifies a p value of ≤.05.

Fig. 5

Processing treatments affected the ability of the porcine dermis samples to support the growth of NIH-3T3 mouse fibroblasts in vitro. Results are shown as average scores ± standard error. * signifies a p value of ≤.05 as compared to d; ** signifies a p value of ≤.05.