Effects of PEG hydrogel crosslinking density on protein diffusion and encapsulated islet survival and function.

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 5700 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)-10(-7) cm(2)/s, such that protein diffusion time scales (t(d) = L(2)/D) from 0.5-mm thick gels vary from 5 min to 24 h. 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 1 h 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 1 h stimulation period.

Hydrogel encapsulation environments functionalized with extracellular matrix interactions increase islet insulin secretion.

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 microg/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 two-fold 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-alpha6 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.

Cell-matrix interactions improve beta-cell survival and insulin secretion in three-dimensional culture.

Controlled matrix interactions were presented to pancreatic beta-cells in three-dimensional culture within poly(ethylene glycol) hydrogels. Dispersed MIN6 beta-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, beta-cell survival was significantly better than in gels without ECM over 10 days. Correspondingly, apoptosis in encapsulated beta-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 beta-cells cultured in gels containing collagen type IV or laminin secreted more insulin in response to glucose stimulation than beta-cells in all other experimental conditions. Variations in collagen type IV or laminin concentration between 10 microg/mL and 250 microg/mL did not affect insulin secretion. Finally, beta-cell function in hydrogels presenting both collagen type IV and laminin revealed synergistic interactions. With a total protein concentration of 100 microg/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 microg/mL of collagen type IV and 75 microg/mL of laminin, beta-cell insulin secretion was greater than with laminin or collagen type IV individually. These results demonstrate that specific, rationally designed extracellular environments promote isolated beta-cell survival and function.

Multifunctional pancreatic islet encapsulation barriers achieved via multilayer PEG hydrogels.

The diverse requirements for a successful islet encapsulation barrier suggest the benefit of a barrier system that presents differing functionalities to encapsulated cells and host cells. Initially, multifunctional hydrogels were synthesized via the sequential photopolymerization of PEG hydrogel layers, each with different isolated functionalities. The ability to achieve localized biological functionalities was confirmed by immunostaining of different entrapped antibodies within each hydrogel layer. Survival of murine islets macroencapsulated within the interior gel of two-layer hydrogel constructs was then assessed. Maintenance of encapsulated islet survival and function was observed within multilayer hydrogels over 28 days in culture. Additionally, the functionalization of the islet-containing interior PEG gel layer with cell-matrix moieties, with either 100 microg/ml laminin or 5 mM of the adhesive peptide IKVAV found in laminin, resulted in increased insulin secretion from encapsulated islets similar to that in gels without an exterior hydrogel layer. Finally, through cell seeding experiments, the ability of an unmodified, exterior PEG layer to prevent interactions, and thus attachment, between nonencapsulated fibroblasts and entrapped ECM components within the interior PEG layer was demonstrated. Together the presented results support the potential of multilayer hydrogels for use as multifunctional islet encapsulation barriers that provide a localized biologically active islet microenvironment, while presenting an inert, immunoprotective exterior surface to the host environment, to minimize graft-host interactions.

The effects of cell-matrix interactions on encapsulated beta-cell function within hydrogels functionalized with matrix-derived adhesive peptides.

The influence of matrix-derived adhesive peptide sequences on encapsulated beta-cell survival and glucose-stimulated insulin release was explored by covalently incorporating synthetic peptide sequences within a model encapsulation environment. Photopolymerized poly(ethylene glycol) (PEG) hydrogels were functionalized via the addition of acrylate-PEG-peptide conjugates to the polymer precursor solution prior to beta-cell photoencapsulation. Individual MIN6 beta-cells were encapsulated in the presence of the laminin-derived recognition sequences, IKLLI, IKVAV, LRE, PDSGR, RGD, and YIGSR, and the collagen type I sequence, DGEA. In the absence of cell-cell and cell-matrix contacts, encapsulated MIN6 beta-cell survival diminishes within one week; however, in PEG hydrogel derivatives including the laminin sequences IKLLI and IKVAV, encapsulated beta-cells exhibit preserved viability, reduced apoptosis, and increased insulin secretion. Interactions with the laminin sequences LRE, PDSGR, RGD, and YIGSR contribute to improved viability, but insulin release from these samples was not statistically greater than that from controls. MIN6 beta-cells were also encapsulated with various concentrations of IKLLI and IKVAV (0.05-5.0mm), individually, and the peptide combinations IKLLI-IKVAV, IKVAV-YIGSR, and PDSGR-YIGSR to explore synergistic effects. The presented results give evidence that synthetic peptide epitopes may be useful in the design of an islet encapsulation environment that promotes cell survival and function via targeted cell-matrix interactions.

Multifunctional Pancreatic Islet Encapsulation Barriers Achieved via Multilayer PEG Hydrogels.

The diverse requirements for a successful islet encapsulation barrier suggest the benefit of a barrier system that presents differing functionalities to encapsulated cells and host cells. Initially, multifunctional hydrogels were synthesized via the sequential photopolymerization of PEG hydrogel layers, each with different isolated functionalities. The ability to achieve localized biological functionalities was confirmed by immunostaining of different entrapped antibodies within each hydrogel layer. Survival of murine islets macroencapsulated within the interior gel of two-layer hydrogel constructs was then assessed. Maintenance of encapsulated islet survival and function was observed within multilayer hydrogels over 28 days in culture. Additionally, the functionalization of the islet-containing interior PEG gel layer with cell-matrix moieties, with either 100 µg/ml laminin or 5 mM of the adhesive peptide IKVAV found in laminin, resulted in increased insulin secretion from encapsulated islets similar to that in gels without an exterior hydrogel layer. Finally, through cell seeding experiments, the ability of an unmodified, exterior PEG layer to prevent interactions, and thus attachment, between nonencapsulated fibroblasts and entrapped ECM components within the interior PEG layer was demonstrated. Together the presented results support the potential of multilayer hydrogels for use as multifunctional islet encapsulation barriers that provide a localized biologically active islet microenvironment, while presenting an inert, immunoprotective exterior surface to the host environment, to minimize graft-host interactions.

PEG-based hydrogels as an in vitro encapsulation platform for testing controlled beta-cell microenvironments.

An in vitro encapsulation platform for systematically testing the effects of microenvironmental parameters on encapsulated islets was developed. The base encapsulation matrix was a biocompatible hydrogel formed via the photoinitiated polymerization of dimethacrylated poly(ethylene glycol) (PEGDM). The resulting inert encapsulation matrix affords control over the biochemical and biophysical cellular microenvironment and the introduction of systematic changes to this environment. The compatibility of the PEG-based encapsulation platform with pancreatic beta-cells was first established using a murine beta-cell line, MIN6. When cell-cell contacts were introduced via aggregation of MIN6 beta-cells prior to encapsulation, MIN6 beta-cells remained viable within the PEG hydrogel platform throughout 3weeks of in vitro culture. Proliferating cells were observed within encapsulated MIN6 aggregates qualitatively with bromodeoxyuridine staining and quantitatively by measuring the DNA content of encapsulation samples with time. MIN6 beta-cells were encapsulated in hydrogels formed from three PEGDM macromers of varying molecular weights (M (n)=4,000, 8,000, 10,000g/mol), and the resulting differences in hydrogel crosslinking density, which influences transport properties, did not affect encapsulated beta-cell survival. Encapsulated MIN6 beta-cells transplanted into diabetic mice returned blood glucose levels to normal levels, indicating in vivo function. Finally, the compatibility of the PEG encapsulation system with freshly isolated islets was confirmed.