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. 2013 Nov;19(21-22):2361-72.
doi: 10.1089/ten.TEA.2013.0033. Epub 2013 Jun 27.

Collagen IV-modified scaffolds improve islet survival and function and reduce time to euglycemia

Woon Teck Yap  1 , David M Salvay, Michael A Silliman, Xiaomin Zhang, Zachary G Bannon, Dixon B Kaufman, William L Lowe Jr, Lonnie D Shea
Affiliations

Collagen IV-modified scaffolds improve islet survival and function and reduce time to euglycemia

Woon Teck Yap et al. Tissue Eng Part A. 2013 Nov.

Abstract

Islet transplantation on extracellular matrix (ECM) protein-modified biodegradable microporous poly(lactide-co-glycolide) scaffolds is a potential curative treatment for type 1 diabetes mellitus (T1DM). Collagen IV-modified scaffolds, relative to control scaffolds, significantly decreased the time required to restore euglycemia from 17 to 3 days. We investigated the processes by which collagen IV-modified scaffolds enhanced islet function and mediated early restoration of euglycemia post-transplantation. We characterized the effect of collagen IV-modified scaffolds on islet survival, metabolism, and insulin secretion in vitro and early- and intermediate-term islet mass and vascular density post-transplantation and correlated these with early restoration of euglycemia in a syngeneic mouse model. Control scaffolds maintained native islet morphologies and architectures as well as collagen IV-modified scaffolds in vivo. The islet size and vascular density increased, while β-cell proliferation decreased from day 16 to 113 post-transplantation. Collagen IV-modified scaffolds promoted islet cell viability and decreased early-stage apoptosis in islet cells in vitro-phenomena that coincided with enhanced islet metabolic function and glucose-stimulated insulin secretion. These findings suggest that collagen IV-modified scaffolds promote the early restoration of euglycemia post-transplantation by enhancing islet metabolism and glucose-stimulated insulin secretion. These studies of ECM proteins, in particular collagen IV, and islet function provide key insights for the engineering of a microenvironment that would serve as a platform for enhancing islet transplantation as a viable clinical therapy for T1DM.

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Figures

FIG. 1.
FIG. 1.
Localization of islets seeded on microporous poly(lactide-co-glycolide) (PLG) scaffolds. (A) View of the interior of an islet-seeded scaffold (top-to-bottom cut made through scaffold midline) showing the extent of islet infiltration into the scaffold through the interconnected pore structure of the scaffold. (B) View of the top surface of an islet-seeded scaffold. Scale bar=1 mm, n=3. Color images available online at www.liebertpub.com/tea
FIG. 2.
FIG. 2.
(A) Metabolic activity of islets cultured on extracellular matrix (ECM)-modified microporous PLG scaffolds. Fifty islets were cultured for 24, 48, and 72 h on collagen IV-, fibronectin-, or laminin-modified scaffolds. A fluorescence assay, based on the metabolic reduction of resazurin to resorufin, was performed to quantify islet cell metabolic activity relative to that of islets seeded onto control scaffolds. Fluorescence was measured using excitation and emission wavelengths of 560 nm and 590 nm, respectively. *p<0.05, n=5 for each condition. (B) Insulin secretion, in response to a glucose challenge, by islets cultured on ECM protein-modified microporous PLG scaffolds. Fifty islets were cultured for 24, 48, and 72 h on collagen IV-, fibronectin-, or laminin-modified scaffolds. A radioimmunoassay for insulin was performed to quantify islet insulin secretion in response to a glucose challenge relative to that of islets seeded onto control scaffolds. The glucose challenge was performed by incubating islets that had been cultured on scaffolds, in low glucose concentration media (2.8 mM) for 1 h, followed by incubation in high glucose concentration media (28 mM) for 1 h. The stimulation index is defined as the amount of insulin secreted under the high glucose condition divided by the amount of insulin secreted under the low glucose condition. *p<0.05, n=3 for each condition.
FIG. 3.
FIG. 3.
(A) Viability/cytotoxicity of islets cultured on ECM protein-modified microporous PLG scaffolds. Fifty islets were cultured for 24, 48, and 72 h on collagen IV-, fibronectin-, or laminin-modified scaffolds. A live/dead fluorescence assay, based on the presence of specific proteases in intact (viable) or lysed (dead) cells in culture, was performed to quantify islet cell viability and cytotoxicity relative to that of islets seeded onto control scaffolds. For the live cell marker, fluorescence was measured using excitation and emission wavelengths of 400 and 505 nm, respectively. For the dead cell marker, fluorescence was measured using excitation and emission wavelengths of 485 and 520 nm, respectively. *p<0.05, n=5 for each condition. (B) Apoptosis of islets cultured on ECM protein-modified microporous PLG scaffolds. Fifty islets were cultured for 24, 48, and 72 h on collagen IV-, fibronectin-, or laminin-modified scaffolds. A luminescence assay, based on caspase-3/7 activities, was performed to quantify islet cell apoptosis relative to that of islets seeded onto control scaffolds. Luminescence signal was integrated over 1 s. *p<0.05, n=6 for each condition.
FIG. 4.
FIG. 4.
(A) Nonfasting blood glucose regulation after islet transplantation. Nonfasting blood glucose levels from day 0 (day of transplantation) through day 113 post-transplantation. ■, islets transplanted on collagen IV-modified scaffolds (n=6); □, islets transplants on control scaffolds (n=6). Data are presented as mean glucose level±standard error of the mean (SEM). Euglycemia is defined as a sustained nonfasting blood glucose value of≤200 mg/dL as indicated by the dashed line. (B) Mean time required for restoring euglycemia after islet transplantation. ■, islets transplanted on collagen IV-modified scaffolds (n=6); □, islets transplants on control scaffolds (n=6). Data are presented as mean time to euglycemia±SEM.
FIG. 5.
FIG. 5.
Islets transplanted on PLG scaffolds are morphologically and architecturally similar to islets in native pancreata. (A–E) Representative images of islets stained using immunofluorescence (IF) demonstrate the maintenance of islet morphologies and architectures 16 and 113 days post-transplantation, n=4 per group. Green=insulin, β-cell marker, red=somatostatin, δ-cell marker, blue=glucagon, α-cell marker. (A) Islets in native stroma of pancreas, scale bar=133 μm. (B, C) Islets transplanted on collagen IV-modified scaffolds or control scaffolds, respectively, and explanted on day 16 post-transplantation, scale bar=100 μm. (D, E) Islets transplanted on collagen IV-modified scaffolds or control scaffolds, respectively, and explanted on day 113 post-transplantation, scale bar=200 μm. (F) Total islet area per section was quantified and presented as mean±SEM for islets transplanted on collagen IV-modified scaffolds or control scaffolds and explanted on days 16 and 113 post-transplantation, n=5 for each condition on day 16, n=4 for each condition on day 113. Color images available online at www.liebertpub.com/tea
FIG. 6.
FIG. 6.
Proliferating β-cells in islets transplanted on PLG scaffolds. (A–D) Representative images of islets stained using IF demonstrate the presence of proliferating β-cells in islets 16 and 113 days post-transplantation, n=4 per group. Green=insulin, β-cell marker, red=Ki-67 antigen, proliferating cell nuclear marker. Nuclei counterstaining with Hoechst 33258 was omitted from the overlay to enhance clarity of insulin and Ki-67 staining. (A, C) Islets transplanted on collagen IV-modified scaffolds and explanted on days 16 or 113 post-transplantation, respectively, scale bar=100 μm. (B, D) Islets transplanted on control scaffolds and explanted on days 16 or 113 post-transplantation, respectively, scale bar=133 μm. (E) Percentage of β-cells that were proliferating was quantified and presented as mean±SEM for islets transplanted on collagen IV-modified scaffolds or control scaffolds and explanted on days 16 and 113 post-transplantation, n=5 for each condition on day 16, n=4 for each condition on day 113. Color images available online at www.liebertpub.com/tea
FIG. 7.
FIG. 7.
Functional revascularization of islets and maintenance of VBMs in islets transplanted on PLG scaffolds. (A) Representative image of islets stained using IF demonstrate functional vasculature in islets post-transplantation, scale bar=200 μm. Green=perfused tomato lectin, functional vasculature marker, red=insulin. Nuclei counterstaining with Hoechst 33258 was omitted from the overlay to enhance clarity of insulin and perfused tomato lectin staining. (B–F) Representative images of islets stained using IF demonstrate the maintenance of VBMs in islets 16 and 113 days post-transplantation, n=4 per group, scale bar=133 μm. Green=insulin, β-cell marker, red=laminin α4, VBM marker. Nuclei counterstaining with Hoechst 33258 was omitted from the overlay to enhance clarity of insulin and laminin α4 staining. (B) Islets in native stroma of pancreas. (C, D) Islets transplanted on collagen IV-modified scaffolds or control scaffolds, respectively, and explanted on day 16 post-transplantation. (E, F) Islets transplanted on collagen IV-modified scaffolds or control scaffolds, respectively, and explanted on day 113 post-transplantation. (G) Functional vascular density (vascular area per unit islet area) per section was quantified and presented as mean±SEM for islets transplanted on collagen IV-modified scaffolds or control scaffolds and explanted on days 16 and 113 post-transplantation, n=5 for each condition on day 16, n=4 for each condition on day 113. Color images available online at www.liebertpub.com/tea
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