Limits...
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

(A) Shows the comparison of remnant DNA in decellular ureteral tissue with and without DNA digestion (both n = 9). Significant differences between both groups were not found. Native, untreated ureteral tissue served as control (n = 9). In (B), gel electrophoresis of native ureter sample (2) and corresponding decellular scaffold without (3) and with DNase (4) is shown. Lane numbers indicate the respective sample. (C) shows the analysis of residual SDS before and after washing in distilled water (n = 12). Decellular ureteral scaffolds showed high SDS-concentrations (8.45 ± 0.43 mg/g dry tissue) before washing compared to non-toxic SDS-concentration after washing with distilled water (0.01 ± 0.01 mg/g dry tissue). Washed scaffold pieces show residual SDS-concentrations of 0.07 ± 0.05% compared to scaffolds before washing (P < 0.001; not shown). ***P < 0.001 vs. native group. +++P < 0.001 vs. decellular ureteral scaffolds before washing.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4477215&req=5

Figure 3: (A) Shows the comparison of remnant DNA in decellular ureteral tissue with and without DNA digestion (both n = 9). Significant differences between both groups were not found. Native, untreated ureteral tissue served as control (n = 9). In (B), gel electrophoresis of native ureter sample (2) and corresponding decellular scaffold without (3) and with DNase (4) is shown. Lane numbers indicate the respective sample. (C) shows the analysis of residual SDS before and after washing in distilled water (n = 12). Decellular ureteral scaffolds showed high SDS-concentrations (8.45 ± 0.43 mg/g dry tissue) before washing compared to non-toxic SDS-concentration after washing with distilled water (0.01 ± 0.01 mg/g dry tissue). Washed scaffold pieces show residual SDS-concentrations of 0.07 ± 0.05% compared to scaffolds before washing (P < 0.001; not shown). ***P < 0.001 vs. native group. +++P < 0.001 vs. decellular ureteral scaffolds before washing.

Mentions: The analysis of the DNA content revealed significant differences among native ureters, decellular ureters, and decellular ureters + DNA digestion [P < 0.001; each n = 9]. The DNA content in decellular scaffolds was significantly lower (85.01 ± 3.1% = 966.1 ± 188.2 ng/mg tissue; P < 0.001) compared to that in native ureter samples (100% = 6,468.11 ± 646.9 ng/mg tissue). An additional DNA digestion further reduced the amount of the DNA to 97.32 ± 0.7% (173.28 ± 36.6 ng/mg tissue) compared to native ureter samples (P < 0.001; Figure 3A. Differences between decellular scaffolds and decellular scaffolds+ DNA digestion did not reach a level of significance (P = 0.31). The percentage of remaining DNA after both procedures was decreased by about 91.2% compared with native ureteral tissue (P < 0.001). Qualitative analysis by gel electrophoresis showed intact DNA bands in the native samples with a size larger than 3,000 base pairs. Decellularization caused a gross but incomplete removal of this band, accompanied by a visible DNA-smear. When treating the decellular scaffolds with DNase, the smear was grossly removed (Figure 3B).


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)

(A) Shows the comparison of remnant DNA in decellular ureteral tissue with and without DNA digestion (both n = 9). Significant differences between both groups were not found. Native, untreated ureteral tissue served as control (n = 9). In (B), gel electrophoresis of native ureter sample (2) and corresponding decellular scaffold without (3) and with DNase (4) is shown. Lane numbers indicate the respective sample. (C) shows the analysis of residual SDS before and after washing in distilled water (n = 12). Decellular ureteral scaffolds showed high SDS-concentrations (8.45 ± 0.43 mg/g dry tissue) before washing compared to non-toxic SDS-concentration after washing with distilled water (0.01 ± 0.01 mg/g dry tissue). Washed scaffold pieces show residual SDS-concentrations of 0.07 ± 0.05% compared to scaffolds before washing (P < 0.001; not shown). ***P < 0.001 vs. native group. +++P < 0.001 vs. decellular ureteral scaffolds before washing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4477215&req=5

Figure 3: (A) Shows the comparison of remnant DNA in decellular ureteral tissue with and without DNA digestion (both n = 9). Significant differences between both groups were not found. Native, untreated ureteral tissue served as control (n = 9). In (B), gel electrophoresis of native ureter sample (2) and corresponding decellular scaffold without (3) and with DNase (4) is shown. Lane numbers indicate the respective sample. (C) shows the analysis of residual SDS before and after washing in distilled water (n = 12). Decellular ureteral scaffolds showed high SDS-concentrations (8.45 ± 0.43 mg/g dry tissue) before washing compared to non-toxic SDS-concentration after washing with distilled water (0.01 ± 0.01 mg/g dry tissue). Washed scaffold pieces show residual SDS-concentrations of 0.07 ± 0.05% compared to scaffolds before washing (P < 0.001; not shown). ***P < 0.001 vs. native group. +++P < 0.001 vs. decellular ureteral scaffolds before washing.
Mentions: The analysis of the DNA content revealed significant differences among native ureters, decellular ureters, and decellular ureters + DNA digestion [P < 0.001; each n = 9]. The DNA content in decellular scaffolds was significantly lower (85.01 ± 3.1% = 966.1 ± 188.2 ng/mg tissue; P < 0.001) compared to that in native ureter samples (100% = 6,468.11 ± 646.9 ng/mg tissue). An additional DNA digestion further reduced the amount of the DNA to 97.32 ± 0.7% (173.28 ± 36.6 ng/mg tissue) compared to native ureter samples (P < 0.001; Figure 3A. Differences between decellular scaffolds and decellular scaffolds+ DNA digestion did not reach a level of significance (P = 0.31). The percentage of remaining DNA after both procedures was decreased by about 91.2% compared with native ureteral tissue (P < 0.001). Qualitative analysis by gel electrophoresis showed intact DNA bands in the native samples with a size larger than 3,000 base pairs. Decellularization caused a gross but incomplete removal of this band, accompanied by a visible DNA-smear. When treating the decellular scaffolds with DNase, the smear was grossly removed (Figure 3B).

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