The rational design of immunoprotective hydrogel barriers for transplanting insulin-producing cells requires an understanding of protein diffusion within the hydrogel network and how alterations to the network structure affect protein diffusion. Hydrogels of varying crosslinking density were formed via the chain polymerization of dimethacrylated PEG macromers of varying molecular weight, and the diffusion of six model proteins with molecular weights ranging from 5,700 to 67,000 g/mol was observed in these hydrogel networks. Protein release profiles were used to estimate diffusion coefficients for each protein/gel system that exhibited Fickian diffusion. Diffusion coefficients were on the order of 10−6 to 10−7 cm2/s, such that protein diffusion time scales (td = L2/D) from 0.5 mm thick gels vary from 5 minutes to 24 hours. Adult murine islets were encapsulated in PEG hydrogels of varying crosslinking density, and islet survival and insulin release was maintained after two weeks of culture in each gel condition. While the total insulin released during a one hour glucose stimulation period was the same from islets in each sample, increasing hydrogel crosslinking density contributed to delays in insulin release from hydrogel samples within the one hour stimulation period.
Controlled matrix interactions were presented to pancreatic b-cells in three-dimensional culture within poly (ethylene glycol) hydrogels. Dispersed MIN6 b-cells were encapsulated in gel environments containing the following entrapped extracellular matrix (ECM) proteins: collagen type I, collagen type IV, fibrinogen, fibronectin, laminin, and vitronectin. In ECM-containing gels, b-cell survival was significantly better than in gels without ECM over 10 days. Correspondingly, apoptosis in encapsulated b-cells was less in the presence of each matrix protein, suggesting the ability of individual matrix interactions to prevent matrix signaling-related apoptosis (anoikis). MIN6 b-cells cultured in gels containing collagen type IV or laminin secreted more insulin in response to glucose stimulation than b-cells in all other experimental conditions. Variations in collagen type IV or laminin concentration between 10 mg=mL and 250 mg=mL did not affect insulin secretion. Finally, b-cell function in hydrogels presenting both collagen type IV and laminin revealed synergistic interactions. With a total protein concentration of 100 mg=mL, three gel compositions of varying ratios of collagen type IV to laminin (25:75, 50:50, and 75:25) were tested. In the presence of 25 mg=mL of collagen type IV and 75 mg=mL of laminin, bcell insulin secretion was greater than with laminin or collagen type IV individually. These results demonstrate that specific, rationally designed extracellular environments promote isolated b-cell survival and function.
The individual and synergistic effects of extracellular matrix interactions on isolated islet function in culture were investigated within a three-dimensional poly(ethylene glycol) (PEG) hydrogel encapsulation environment. First, we observed similar glucose-stimulated insulin secretion from unencapsulated murine islets and islets photoencapsulated in PEG gels. Then islets were encapsulated in gels containing the basement membrane proteins collagen type IV and laminin, individually and in combination, at a total protein concentration of 100 μg/ml, and islet insulin secretion in response to high glucose was measured over time. Specific laminin interactions were investigated via islet encapsulation with adhesive peptide sequences found in laminin as well as via functional blocking of cell surface receptors known to bind laminin. Over 32 days, islet interactions with collagen type IV and laminin localized within the three-dimensional extracellular environment contributed to twofold and four-fold increases in insulin secretion, respectively, relative to islets encapsulated without matrix proteins. Hydrogel compositions containing both matrix proteins and > 75% laminin further increased islet insulin secretion to approximately six-fold that of islets encapsulated in the absence of matrix proteins. Encapsulation with the peptide sequence IKVAV resulted in increased islet insulin secretion, but not to the extent observed in the presence of whole laminin. Increased insulin secretion in the presence of laminin was eliminated when islets were exposed to functionally blocking anti-α 6 integrin antibody prior to islet encapsulation with laminin. Our results demonstrate the potential of specific matrix interactions within an islet encapsulation microenvironment to promote encapsulated islet function.
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