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

Koch H, Hammer N, Ossmann S, Schierle K, Sack U, Hofmann J, Wecks M, Boldt A - Front Bioeng Biotechnol (2015)

Bottom Line: 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.TIMP1 was below the detection limit.

View Article: PubMed Central - PubMed

Affiliation: Translational Centre for Regenerative Medicine (TRM), University of Leipzig , Leipzig , Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Reseeding of crosslinked or chemical untreated ureteral scaffolds with LS48 (A), 3T3 (B), aco2 (C), and smooth muscle cells (D). Cells are able to grow on chemically untreated and crosslinked scaffolds after incubation for 2 weeks. Effects of crosslinking on relative amount of cells could not be detected under seeding with LS48 [(A), n = 9] and Caco2 [(C), n = 9] cells. However, 3T3 cells show an increased infiltrating rate into CDI and GP crosslinked scaffolds, compared to the untreated group [(B), n = 9]. Furthermore, crosslinking with GP caused an increase of smooth muscle cells, grown on surface and infiltrate scaffolds [(D), n = 7]. Data were calculated based on the scoring by two blinded observers: 1 = no cells; 2 = in total <20 cells; 3 = partially 1 cell layer, in total <50 cells, 4 = 1 cell layer around the scaffold; 5 = cells are arranged in multilayer, with matrix-infiltration. Untreated, untreated decellular ureteral tissue; GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01, ***P < 0.001 vs. decellular ureteral scaffolds.
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Figure 4: Reseeding of crosslinked or chemical untreated ureteral scaffolds with LS48 (A), 3T3 (B), aco2 (C), and smooth muscle cells (D). Cells are able to grow on chemically untreated and crosslinked scaffolds after incubation for 2 weeks. Effects of crosslinking on relative amount of cells could not be detected under seeding with LS48 [(A), n = 9] and Caco2 [(C), n = 9] cells. However, 3T3 cells show an increased infiltrating rate into CDI and GP crosslinked scaffolds, compared to the untreated group [(B), n = 9]. Furthermore, crosslinking with GP caused an increase of smooth muscle cells, grown on surface and infiltrate scaffolds [(D), n = 7]. Data were calculated based on the scoring by two blinded observers: 1 = no cells; 2 = in total <20 cells; 3 = partially 1 cell layer, in total <50 cells, 4 = 1 cell layer around the scaffold; 5 = cells are arranged in multilayer, with matrix-infiltration. Untreated, untreated decellular ureteral tissue; GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01, ***P < 0.001 vs. decellular ureteral scaffolds.

Mentions: After incubation for 2 weeks, the ureteral scaffolds were increasingly infiltrated by tested cell lines (Figures 4 and 5). LS48 cells were uniformly distributed at the scaffold surface (Figures 5A–D). No significant effects of crosslinking were observed on cell growth (Figure 4A). 3T3 cells showed a higher infiltration after crosslinking with CDI or GP ([F(3,24) = 6.84, P = 0.002]; CDI: P < 0.05, GP: P = 0.001; Figure 4B). Furthermore, multilayer formation (GA, CDI, GP) and moderate scaffold infiltration (CDI, GP) were detected in crosslinked scaffolds, whereas only sporadic cells could be detected in the untreated scaffold group (Figures 5E–H). After incubation with Caco2 cells, the relative amount of cells did not differ between any of the groups (Figure 4C). However, scaffold infiltration and multilayer formation were predominant after crosslinking with GP or CDI (Figures 6A–D). Furthermore, SMC showed a preference for crosslinked scaffolds (Figures 6E–H), whereas the highest infiltration was observed after crosslinking with GP ([F(3,18) = 5.32, P < 0.008]; P < 0.01; Figure 4D). However, reseeding of lyophilized scaffolds failed; neither cell lines nor native cells were grown on dried scaffolds (data not shown). Interestingly, lyophilized implants were degraded by cell lines within 2 weeks.


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

Koch H, Hammer N, Ossmann S, Schierle K, Sack U, Hofmann J, Wecks M, Boldt A - Front Bioeng Biotechnol (2015)

Reseeding of crosslinked or chemical untreated ureteral scaffolds with LS48 (A), 3T3 (B), aco2 (C), and smooth muscle cells (D). Cells are able to grow on chemically untreated and crosslinked scaffolds after incubation for 2 weeks. Effects of crosslinking on relative amount of cells could not be detected under seeding with LS48 [(A), n = 9] and Caco2 [(C), n = 9] cells. However, 3T3 cells show an increased infiltrating rate into CDI and GP crosslinked scaffolds, compared to the untreated group [(B), n = 9]. Furthermore, crosslinking with GP caused an increase of smooth muscle cells, grown on surface and infiltrate scaffolds [(D), n = 7]. Data were calculated based on the scoring by two blinded observers: 1 = no cells; 2 = in total <20 cells; 3 = partially 1 cell layer, in total <50 cells, 4 = 1 cell layer around the scaffold; 5 = cells are arranged in multilayer, with matrix-infiltration. Untreated, untreated decellular ureteral tissue; GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01, ***P < 0.001 vs. decellular ureteral scaffolds.
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Related In: Results  -  Collection

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Figure 4: Reseeding of crosslinked or chemical untreated ureteral scaffolds with LS48 (A), 3T3 (B), aco2 (C), and smooth muscle cells (D). Cells are able to grow on chemically untreated and crosslinked scaffolds after incubation for 2 weeks. Effects of crosslinking on relative amount of cells could not be detected under seeding with LS48 [(A), n = 9] and Caco2 [(C), n = 9] cells. However, 3T3 cells show an increased infiltrating rate into CDI and GP crosslinked scaffolds, compared to the untreated group [(B), n = 9]. Furthermore, crosslinking with GP caused an increase of smooth muscle cells, grown on surface and infiltrate scaffolds [(D), n = 7]. Data were calculated based on the scoring by two blinded observers: 1 = no cells; 2 = in total <20 cells; 3 = partially 1 cell layer, in total <50 cells, 4 = 1 cell layer around the scaffold; 5 = cells are arranged in multilayer, with matrix-infiltration. Untreated, untreated decellular ureteral tissue; GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01, ***P < 0.001 vs. decellular ureteral scaffolds.
Mentions: After incubation for 2 weeks, the ureteral scaffolds were increasingly infiltrated by tested cell lines (Figures 4 and 5). LS48 cells were uniformly distributed at the scaffold surface (Figures 5A–D). No significant effects of crosslinking were observed on cell growth (Figure 4A). 3T3 cells showed a higher infiltration after crosslinking with CDI or GP ([F(3,24) = 6.84, P = 0.002]; CDI: P < 0.05, GP: P = 0.001; Figure 4B). Furthermore, multilayer formation (GA, CDI, GP) and moderate scaffold infiltration (CDI, GP) were detected in crosslinked scaffolds, whereas only sporadic cells could be detected in the untreated scaffold group (Figures 5E–H). After incubation with Caco2 cells, the relative amount of cells did not differ between any of the groups (Figure 4C). However, scaffold infiltration and multilayer formation were predominant after crosslinking with GP or CDI (Figures 6A–D). Furthermore, SMC showed a preference for crosslinked scaffolds (Figures 6E–H), whereas the highest infiltration was observed after crosslinking with GP ([F(3,18) = 5.32, P < 0.008]; P < 0.01; Figure 4D). However, reseeding of lyophilized scaffolds failed; neither cell lines nor native cells were grown on dried scaffolds (data not shown). Interestingly, lyophilized implants were degraded by cell lines within 2 weeks.

Bottom Line: 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.TIMP1 was below the detection limit.

View Article: PubMed Central - PubMed

Affiliation: Translational Centre for Regenerative Medicine (TRM), University of Leipzig , Leipzig , Germany.

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.

No MeSH data available.


Related in: MedlinePlus