Extracellular matrix protein-coated scaffolds promote the reversal of diabetes after extrahepatic islet transplantation

Abstract

Background: The survival and function of transplanted pancreatic islets is limited, owing in part to disruption of islet-matrix attachments during the isolation procedure. Using polymer scaffolds as a platform for islet transplantation, we investigated the hypothesis that replacement of key extracellular matrix components known to surround islets in vivo would improve graft function at an extrahepatic implantation site.

Methods: Microporous polymer scaffolds fabricated from copolymers of lactide and glycolide were adsorbed with collagen IV, fibronectin, laminin-332 or serum proteins before seeding with 125 mouse islets. Islet-seeded scaffolds were then implanted onto the epididymal fat pad of syngeneic mice with streptozotocin-induced diabetes. Nonfasting glucose levels, weight gain, response to glucose challenges, and histology were used to assess graft function for 10 months after transplantation.

Results: Mice transplanted with islets seeded onto scaffolds adsorbed with collagen IV achieved euglycemia fastest and their response to glucose challenge was similar to normal mice. Fibronectin and laminin similarly promoted euglycemia, yet required more time than collagen IV and less time than serum. Histopathological assessment of retrieved grafts demonstrated that coating scaffolds with specific extracellular matrix proteins increased total islet area in the sections and vessel density within the transplanted islets, relative to controls.

Conclusions: Extracellular matrix proteins adsorbed to microporous scaffolds can enhance the function of transplanted islets, with collagen IV maximizing graft function relative to the other proteins tested. These scaffolds enable the creation of well-defined microenvironments that promote graft efficacy at extrahepatic sites.

FIGURE 1

Protein adsorption to scaffolds. Photomicrographs of scaffolds stained with picrosirius red after 1 mg/mL collagen IV was adsorbed. The scaffolds were treated by base hydrolysis (A) or were untreated (B). Negative control for base-hydrolyzed scaffold by incubation with PBS (C). Indicator marks at bottom of images are 1 mm apart.

FIGURE 2

Glucose regulation after islet transplantation. (A) Nonfasting blood glucose levels from day 0 (day of transplant) through day 300 posttransplant. ●, collagen IV group (n=7); ■, fibronectin group (n=8); ○, laminin group (n=8); ▲, serum group (n=8); □, no islet group (n=8). Data are presented as mean glucose level±SEM (one-sided error bars used for clarity). (B) The fraction of diabetic animals that converted to euglycemia over time for scaffolds coated with collagen IV (solid line), fibronectin (dashed line), laminin (dash-dot line), and serum proteins (dot-dot line). ***P<0.001, collagen IV vs. all other conditions.

FIGURE 3

Change in body weight after islet transplantation. Percent change in body weight from day 0 through day 300 posttransplant. ●, collagen IV group (n=7); ■, fibronectin group (n=8); ○, laminin group (n=8); ▲, serum group (n=8); □, no islet group (n=8). Data are presented as mean percent change in weight±SEM (one-sided error bars used for clarity).

FIGURE 4

Intraperitoneal glucose tolerance tests. An IPGTT was performed at 4 (A, B) and 40 weeks (C, D) after islet transplantation on animals that were euglycemic at that time. (A, C) Blood glucose levels as a function of time after glucose injection. ●, collagen IV group (n=7 at 4 weeks, n=7 at 40 weeks); ■, fibronectin group (n=5 at 4 weeks, n=8 at 40 weeks); ○, laminin group (n=6 at 4 weeks, n=8 at 40 weeks); ▲, serum group (n=4 at 4 weeks, n=6 at 40 weeks); □, normal control group (n=3 at 4 weeks, n=3 at 40 weeks). Data are presented as mean glucose level±SEM. ||, P<0.05, collagen IV vs. fibronectin; §, P<0.05, collagen IV vs. laminin; ¶, P<0.05, collagen IV vs. serum. (B, D) Areas under the glucose challenge curves (AUC) were calculated. Data are presented as mean AUC±SEM. *, P<0.05, collagen IV vs. fibronectin (at 40 weeks); **, P<0.01, collagen IV vs. serum (at 40 weeks); ***, P<0.001, collagen IV vs. fibronectin, laminin and serum groups (at 4 weeks). For clarity, statistically significant differences between the normal group and all other groups are not explicitly displayed.

FIGURE 5

Histopathological assessment of explanted islet grafts. Immunohistochemical staining (brown) for insulin on a cryosection (cut perpendicular to the islet-seeded surface) taken from a collagen IV-coated scaffold explanted 7 days after implantation (A), or paraffin sections of collagen IV- (B), fibronectin- (C), laminin- (D) or serum-coated (E) scaffolds explanted 297 days after implantation. All scale bars indicate 200 μm. In panel A, two example islets are indicated by “Is,” black arrows indicate the edge of the scaffold, and red arrows indicate the scaffold surface in direct contact with the islets.

FIGURE 6

Quantification of islet area and vascular density. (A) An example H&E stained section indicating how islet boundaries were established (dashed line) to calculate area and intraislet vessels (yellow arrows). Examples of large vessels seen adjacent to islets and periislet vessels are marked with an asterisk. Total islet area per tissue section (B), area per islet (C), vessels per islet (D), and vessel density (E) for the four conditions tested. Data are presented as mean±SEM. *P<0.05; ***P<0.001. Scale bar indicates 100 μm.