oa Gelatin and Decorin are Suitable Candidates for Application in Tissue-Engineered Matrices - an Immunological In Vitro Study

Inflammatory and immune-mediated responses to extracellular matrix proteins (ECMp) or biomaterials mainly determine the acceptance of tissue-engineered scaffolds like heart valve grafts. Therefore, we focused on detailed analysis of those responses induced either by the single ECMp gelatin and the proteoglycan decorin or by electrospun scaffolds composed of both proteins. Proliferation assays were performed to analyze response of T cell subsets by flow cytometry. Therefore, human peripheral blood mononuclear cells (PBMCs) were co-cultured with bovine decorin, porcine gelatin or with electrospun scaffolds generated of poly-ε-caprolacton (PCL) with either gelatin alone or in combination with decorin under low-dose anti-CD3 treatment for 5 days. Moreover, monocyte-derived immature dendritic cells (DC) were co-cultured with decorin and gelatin to determine maturation effects by specific surface markers and flow cytometric analysis compared to a Lipopolysaccharride (LPS) stimulated control. Supernatants of both T cell and DC cultures were analyzed for the pro-inflammatory cytokines IFNγ, TNFα and IL-6 by cytometric bead arrays or ELISA. Additionally, complement activation by decorin and gelatin was screened by incubation with pooled human serum and measuring induced C5a release by standard ELISA. In general, a slight reduction of the T cell subset proliferation compared to the anti-CD3 stimulated positive control in CFSE-based assays could be demonstrated for bovine decorin and porcine gelatin. Both electrospun scaffold types did not alter the proliferation response. Additionally, neither decorin nor gelatin induced a distinct proinflammatory cytokine secretion of IFNγ and TNFα relative to the control. The overall high secretion level of IL-6 was not affected by both proteins and electrospun scaffolds. Remarkably, a trend for reduced IFNγ and TNFα release could be observed for both scaffold types. Moreover, the surface marker expression pattern of DC revealed that decorin as well as gelatin were not able to induce DC maturation due to low expression levels for CD83 and fairly low TNFα secretion when compared to the LPS control. Analyzing complement activating capacities of both ECMp as part of the first line innate immune response, decorin as well as gelatin did not induce enhanced C5a release compared to controls. The results presented here illustrate the important role of evaluating complex immune responses to single ECMp as well as to electrospun scaffolds to facilitate an efficient selection of immunological inert tissue-engineered matrices for their future application as heart valve substitutes. Thus, gelatin as well as decorin are promising candidates for tissue engineering scaffolds as seen by their low immunogeneic properties as single ECMp or as part of electrospun blends.