Successful Islet Transplantation Into a Subcutaneous Polycaprolactone Scaffold in Mice and Pigs

Abstract

Islet transplantation is a promising treatment for type 1 diabetes. It has the potential to improve glycemic control, particularly in patients suffering from hypoglycemic unawareness and glycemic instability. As most islet grafts do not function permanently, efforts are needed to create an accessible and replaceable site, for islet grafts or for insulin-producing cells obtained from replenishable sources. To this end, we designed and tested an artificial, polymeric subcutaneous transplantation site that allows repeated transplantation of islets.

Methods: In this study, we developed and compared scaffolds made of poly(D,L,-lactide-co-ε-caprolactone) (PDLLCL) and polycaprolactone (PCL). Efficacy was first tested in mice' and then, as a proof of principle for application in a large animal model, the scaffolds were tested in pigs, as their skin structure is similar to that of humans.

Results: In mice, islet transplantation in a PCL scaffold expedited return to normoglycemia in comparison to PDLLCL (7.7 ± 3.7 versus 16.8 ± 6.5 d), but it took longer than the kidney capsule control group. PCL also supported porcine functional islet survival in vitro. Subcutaneous implantation of PDLLCL and PCL scaffolds in pigs revealed that PCL scaffolds were more stable and was associated with less infiltration by immune cells than PDLLCL scaffolds. Prevascularized PCL scaffolds were therefore used to demonstrate the functional survival of allogenic islets under the skin of pigs.

Conclusions: To conclude, a novel PCL scaffold shows efficacy as a readily accessible and replaceable, subcutaneous transplantation site for islets in mice and demonstrated islet survival after a month in pigs.

Graphical abstract

FIGURE 1.

Transplantation outcome of mouse study. Kaplan–Meier graph for rates of diabetes reversal (<150 mg/dL) after transplantation of 800 rat islets into prevascularized subcutaneous PDLLCL (n = 4) and PCL (n = 3) scaffolds in diabetic nude mice (A). Transplantation of the same islet dosage under the kidney capsule was used as control (n = 6). Statistical analysis was carried out using a log-rank (Mantel–Cox) test with multiple comparison correction (*<0.05 compared with the kidney capsule control). In addition, the long-term nonfasting blood glucose levels of these mice are depicted (B). After 65 d, the islet grafts were removed to prove that the reduction in blood glucose levels was graft dependent and not caused by pancreas regeneration. The mean and standard error of the mean are plotted. PCL, polycaprolactone; PDLLCL, poly(D,L,-lactide-co-ε-caprolactone).

FIGURE 2.

In vitro viability of porcine islets after culturing on PDLLCL and PCL. The viability of porcine islets was measured by calcein AM/propidium iodide staining after 1 (A), 3 (B), and 7 (C) d of culture. Mean and standard error of mean are plotted (n = 4), statistical analysis was carried out using a 1-way ANOVA with a Tukey posthoc test (*P < 0.05). ANOVA, analysis of variance; PCL, polycaprolactone; PDLLCL, poly(D,L,-lactide-co-ε-caprolactone).

FIGURE 3.

In vitro functionality of porcine islets after culturing on PDLLCL and PCL. Glucose-stimulated insulin secretion of porcine islets after 1 (A), 3 (B), and 7 (C) d of culture. The white bars indicate the amount of insulin secreted during the first low-glucose (2.8 mM) incubation, the black bars represent the amount of insulin produced during high-glucose (28 mM) incubation, and the grey bars show the return to basal insulin secretion during the second low-glucose (2.8 mM) incubation. The mean and standard error of the mean are plotted (n = 4), statistical analysis was carried out using a 2-way ANOVA (P < 0.05). ANOVA, analysis of variance; PCL, polycaprolactone; PDLLCL, poly(D,L,-lactide-co-ε-caprolactone).

FIGURE 4.

Histology of PDLLCL and PCL scaffolds after 12 wk implantation in pigs. Sections were stained (pink) for CD3-positive cells, macrophages, MPO, and CD31. MPO, myeloperoxidase; PCL, polycaprolactone; PDLLCL, poly(D,L,-lactide-co-ε-caprolactone).

FIGURE 5.

Relative gene expression of several vascular and tissue response genes. The mean and standard error of the mean are plotted (n = 4), statistical analysis was carried out using the Kruskal–Wallis test with Dunn’s post hoc test (*P < 0.05). aSMA = alpha-smooth muscle actin; Col1a1 = collagen 1 alpha 1; CSF1 = colony-stimulating factor 1; TGFb = transforming growth factor beta; VEGFa = vascular endothelial growth factor A.

FIGURE 6.

The transplantation procedures. A large (polycaprolactone) PCL scaffold with 6 islet channels was developed using the salt leaching technique (A), which was implanted subcutaneously. After 12 wk of prevascularization, an incision was made next to the implanted scaffold to remove the tubing from the islet channels, and islets were injected into the channels (B). One month after the islet transplantation, an in vivo glucose tolerance test was performed by injecting glucose into a subcutaneous pocket around the scaffold and at a sham site (C) while the animal was anesthetized. The mean insulin concentration (± standard error of the mean) of the in vivo and in vitro glucose tolerance tests are plotted (D; n = 2). Histology shows insulin-positive cells (pink) within the scaffold after 1 mo (E).