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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

Immunohistochemical analysis of the degree of scaffold infiltration by CD68 + (A) and CD163 + (B) macrophages after 1, 9, and 30 days, as well as the change of the CD163/CD68 ratio from day 9 to day 30 (C). An increase of CD68 + and CD163 + macrophages could be observed in all groups from day 1 to day 9, whereas the amount remains constant to day 30 post implantation. In addition to the standard group, untreated and CDI-crosslinked scaffolds showed a macrophage M2 phenotype switch, indicated by a positive CD163/CD68 ratio at day 30 compared to ratio at day 9. All data were represented as ratios of specific cells/total cells ± SEM. Five microscopic fields (magnification × 000) of one slide per rat were analyzed (n = 6/group and day). An average of 54 ± 1.59 total cells per microscopic field was counted to generate the ratio of cells/total cells. Untreated, untreated scaffold; BP, bovine pericardium (St. Jude, USA); GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01 vs. untreated decellular ureteral tissue.
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Figure 12: Immunohistochemical analysis of the degree of scaffold infiltration by CD68 + (A) and CD163 + (B) macrophages after 1, 9, and 30 days, as well as the change of the CD163/CD68 ratio from day 9 to day 30 (C). An increase of CD68 + and CD163 + macrophages could be observed in all groups from day 1 to day 9, whereas the amount remains constant to day 30 post implantation. In addition to the standard group, untreated and CDI-crosslinked scaffolds showed a macrophage M2 phenotype switch, indicated by a positive CD163/CD68 ratio at day 30 compared to ratio at day 9. All data were represented as ratios of specific cells/total cells ± SEM. Five microscopic fields (magnification × 000) of one slide per rat were analyzed (n = 6/group and day). An average of 54 ± 1.59 total cells per microscopic field was counted to generate the ratio of cells/total cells. Untreated, untreated scaffold; BP, bovine pericardium (St. Jude, USA); GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01 vs. untreated decellular ureteral tissue.

Mentions: A detailed macrophage analysis showed only a few number of CD163 positive cells at the periphery of BP and CDI crosslinked samples at day 1 after implantation. Furthermore, the number of CD68 positive cells was higher in the BP group compared to that in untreated scaffolds ([F(4,25) = 9.80, P < 0.001]; P < 0.001, Figure 12A). Scaffold analysis at day 9 after implantation revealed a significant increase of CD68 positive macrophages after crosslinking with GA compared to the untreated group ([F(4,25) = 4.40, P = 0.008]; P < 0.05). At day 30 post-operative, there were no significant effects of crosslinking on the number of CD68 and CD163 positive cells (Figure 12B). Additionally, in most groups, the amount of the anti-inflammatory, pro-remodeling macrophage M2 phenotype increased from day 9 to day 30, indicated by a positive CD163/CD68 ratio (untreated: +10.05%; BP: +65.37%; CDI + 114.03%), whereas it decreased in the GP (−28.31%) and GA (−58.48%) groups (Figure 12C).


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)

Immunohistochemical analysis of the degree of scaffold infiltration by CD68 + (A) and CD163 + (B) macrophages after 1, 9, and 30 days, as well as the change of the CD163/CD68 ratio from day 9 to day 30 (C). An increase of CD68 + and CD163 + macrophages could be observed in all groups from day 1 to day 9, whereas the amount remains constant to day 30 post implantation. In addition to the standard group, untreated and CDI-crosslinked scaffolds showed a macrophage M2 phenotype switch, indicated by a positive CD163/CD68 ratio at day 30 compared to ratio at day 9. All data were represented as ratios of specific cells/total cells ± SEM. Five microscopic fields (magnification × 000) of one slide per rat were analyzed (n = 6/group and day). An average of 54 ± 1.59 total cells per microscopic field was counted to generate the ratio of cells/total cells. Untreated, untreated scaffold; BP, bovine pericardium (St. Jude, USA); GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01 vs. untreated decellular ureteral tissue.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Immunohistochemical analysis of the degree of scaffold infiltration by CD68 + (A) and CD163 + (B) macrophages after 1, 9, and 30 days, as well as the change of the CD163/CD68 ratio from day 9 to day 30 (C). An increase of CD68 + and CD163 + macrophages could be observed in all groups from day 1 to day 9, whereas the amount remains constant to day 30 post implantation. In addition to the standard group, untreated and CDI-crosslinked scaffolds showed a macrophage M2 phenotype switch, indicated by a positive CD163/CD68 ratio at day 30 compared to ratio at day 9. All data were represented as ratios of specific cells/total cells ± SEM. Five microscopic fields (magnification × 000) of one slide per rat were analyzed (n = 6/group and day). An average of 54 ± 1.59 total cells per microscopic field was counted to generate the ratio of cells/total cells. Untreated, untreated scaffold; BP, bovine pericardium (St. Jude, USA); GP, genipin; GA, glutaraldehyde; CDI, carbodiimide. *P < 0.05, **P < 0.01 vs. untreated decellular ureteral tissue.
Mentions: A detailed macrophage analysis showed only a few number of CD163 positive cells at the periphery of BP and CDI crosslinked samples at day 1 after implantation. Furthermore, the number of CD68 positive cells was higher in the BP group compared to that in untreated scaffolds ([F(4,25) = 9.80, P < 0.001]; P < 0.001, Figure 12A). Scaffold analysis at day 9 after implantation revealed a significant increase of CD68 positive macrophages after crosslinking with GA compared to the untreated group ([F(4,25) = 4.40, P = 0.008]; P < 0.05). At day 30 post-operative, there were no significant effects of crosslinking on the number of CD68 and CD163 positive cells (Figure 12B). Additionally, in most groups, the amount of the anti-inflammatory, pro-remodeling macrophage M2 phenotype increased from day 9 to day 30, indicated by a positive CD163/CD68 ratio (untreated: +10.05%; BP: +65.37%; CDI + 114.03%), whereas it decreased in the GP (−28.31%) and GA (−58.48%) groups (Figure 12C).

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