Tissue Engineering of Ureteral Grafts: Preparation of Biocompatible Crosslinked Ureteral Scaffolds of Porcine Origin

Abstract

The surgical reconstruction of ureteric defects is often associated with post-operative complications and requires additional medical care. Decellularized ureters originating from porcine donors could represent an alternative therapy. Our aim was to investigate the possibility of manufacturing decellularized ureters, the characteristics of the extracellular matrix (ECM) and the biocompatibility of these grafts in vitro/in vivo after treatment with different crosslinking agents. To achieve these goals, native ureters were obtained from pigs and were decellularized. The success of decellularization and the ECM composition were characterized by (immuno)histological staining methods and a DNA-assay. In vitro: scaffolds were crosslinked either with carbodiimide (CDI), genipin (GP), glutaraldehyde, left chemically untreated or were lyophilized. Scaffolds in each group were reseeded with Caco2, LS48, 3T3 cells, or native rat smooth muscle cells (SMC). After 2 weeks, the number of ingrown cells was quantified. In vivo: crosslinked scaffolds were implanted subcutaneously into rats and the type of infiltrating cells were determined after 1, 9, and 30 days. After decellularization, scaffold morphology and composition of ECM were maintained, all cellular components were removed, DNA destroyed and strongly reduced. In vitro: GP and CDI scaffolds revealed a higher number of ingrown 3T3 and SMC cells as compared to untreated scaffolds. In vivo: at day 30, implants were predominantly infiltrated by fibroblasts and M2 anti-inflammatory macrophages. A maximum of MMP3 was observed in the CDI group at day 30. TIMP1 was below the detection limit. In this study, we demonstrated the potential of decellularization to create biocompatible porcine ureteric grafts, whereas a CDI-crosslink may facilitate the remodeling process. The use of decellularized ureteric grafts may represent a novel therapeutic method in reconstruction of ureteric defects.

A review of decellurization methods caused by an urgent need for quality control of cell‐free extracellular matrix' scaffolds and their role in regenerative medicine

“…Additionally, it should be observed that the presence of active metalloproteinases may lead to the degradation of the ECM. 4 A number of chemicals that do not lead to cell removal, but are used to crosslink the collagen-rich material, are being considered, 125 the dominant ones being: glutaraldehyde, formaldehyde, polyepoxy compounds and polyurethane, asylum azide, carbodiimidecompounds, hexamethylene diisocyanate, tannic acid, tartaric acid, vitamin B2-induced UVA crosslinking, and genipin 63,91,[125][126][127][128][129][130][131][132] (Table IV). As it is often described, the procedure for processing the dermis can consist of a two-step method based on enzymatic digestion of cellular elements and then cross-linked structure of the extracellular matrix with glutaraldehyde in order to mask residual cellular structures.…”

Naturally derived biomaterials for addressing inflammation in tissue regeneration

“…Scaffolds derived from decellularized matrix (from both allogeneic and xenogeneic sources) have been investigated as materials for regeneration in a range of tissues: heart valve, 24,25,51,52 nasal cartilage, 53 skeletal muscle, 26 gastrointestinal tract, 27,54 ureters, 28 liver, 55 and flexor tendons. 56 Both segmented and whole tissues can be decellularized.…”

“…27 Conversely, carbodiimide cross-linked porcine ureteral scaffolds implanted in a subcutaneous rat model showed a macrophage phenotypic switch to the anti-inflammatory M2 phenotype. 28 Cross-linking with glutaraldehyde or genipin stimulated a pro-inflammatory M1 macrophage switch in vivo. 28 These data are consistent with glutaraldehyde cross-linking of decellularized bovine pericardium which resulted in limited attachment and survival of macrophage-like cells (U937) and increased release of pro-inflammatory cytokines (MMP-1).…”

“…28 Cross-linking with glutaraldehyde or genipin stimulated a pro-inflammatory M1 macrophage switch in vivo. 28 These data are consistent with glutaraldehyde cross-linking of decellularized bovine pericardium which resulted in limited attachment and survival of macrophage-like cells (U937) and increased release of pro-inflammatory cytokines (MMP-1). 52 Future work should carefully consider the balance between scaffold degradation and the inflammatory wound healing response in the use of decellularized matrix for tissue regeneration applications.…”

“…17,26–28,56 The overwhelming observation, detailed in the following paragraphs, is that decellularized matrix can modulate the immune response by exhibiting anti-inflammatory effects and promoting a shift in macrophage phenotype from M1 to M2.…”