Enhanced function of pancreatic islets co-encapsulated with ECM proteins and mesenchymal stromal cells in a silk hydrogel
Introduction
Type 1 diabetes (T1D) is characterized as an autoimmune-mediated destruction of insulin-producing β-cells within pancreatic islets, leading to insufficient regulation of blood glucose levels. Islet cell transplantation offers therapeutic potential for patients with T1D to normalize glucose metabolism and prevent the complications of the disease [1]. Despite many advances, transplantation success rates have been limited by islet cell death in the early post-transplant period, followed by a long-term decline in islet function and viability [2]. Significant challenges to islet transplantation remain, including preservation of islet cells during isolation, revascularization of islets after implantation, and prevention of inflammation and autoimmune destruction of the islet graft. An emerging therapeutic strategy involves the use of biomaterials to encapsulate islets and overcome these obstacles. Biomaterials may enhance islet function by providing a three-dimensional cellular support and delivering proteins, growth factors, and immunosuppressive agents [3], [4].
Current natural and synthetic materials for islet encapsulation have had limited success due to fibroblast overgrowth and mechanical or chemical instability. Some polymeric systems have shown reduced viability and functionality of the islet cells due to polymer biodegradation and limited permeability of the capsules [5]. Consequently, alternative encapsulation materials are needed for islet transplantation. In this study, a hydrogel based on self-assembling silk fibroin proteins from the Bombyx mori silkworm was investigated for islet encapsulation. Silk fibroin supports cell adhesion, proliferation and differentiation, and has good material properties such as biocompatibility, slow degradation rate, and strong mechanical integrity [6]. Although silk-based hydrogels have been investigated for encapsulation of a variety of cell types, they have not been used for islet encapsulation or transplantation. In the present study, silk was used to recreate the islet microenvironment necessary for long-term islet graft function.
Natural stimuli from the islet microenvironment arise from contact with ECM proteins and trophic factors secreted by the surrounding cells that promote islet survival and proliferation to maintain β-cell mass. In synthetic hydrogels, ECM proteins have been co-encapsulated with islets to restore the native microenvironment damaged during cell isolation as a way to enhance islet graft function and survival [7], [8], [9], [10]. Moreover co-encapsulation of islets with stromal cells may replenish the nutrients and growth factors critical for islet graft maintenance. Previously, mesenchymal stromal cells (MSCs) were shown to secrete regulatory islet growth factors that are angiogenic, anti-apoptotic, and proliferation-stimulating (i.e.: HGF, TGF-β1 and IL-6) [11], [12], [13]. Additionally, MSCs have immunomodulatory properties that reduce inflammatory cytokine production and suppress allo-immune responses [14], [15]. For these reasons, the co-encapsulation and transplantation of MSCs with islets can improve allograft survival and function [14], [15].
Although the co-transplantation of islets with MSCs has been gaining traction, to date, there has not been a hydrogel platform that has co-encapsulated MSCs and islets with ECM proteins as a means to enhance graft survival. In the present study, silk hydrogels are investigated in vitro as an islet encapsulation platform to co-encapsulate islets, ECM proteins and MSCs to maintain or enhance islet function.
Section snippets
Silk hydrogel formation and incorporation of ECM proteins
Silk fibroin solutions were supplied by Kaplan et al. and prepared as previously described [16]. Silk self-assembly and gelation was vortex-induced by mixing 375 μL of 8 wt% silk fibroin with 625 μL of sterile water in a glass vial and vortexing for 7 min at 3200 rpm (VWR International, Radnor, PA). After phase separation, the white solid-like material was removed and the remaining silk solution was diluted 2-fold with media and allowed to gel for 2 h at 37 °C. For some experiments, silk
Incorporation of ECM proteins in silk hydrogels
Silk hydrogels were formed in the presence of two ECM proteins (collagen IV and laminin) to investigate the influence of ECM signaling on encapsulated islet cell survival and function. ECM proteins were encapsulated at 100 μg/mL of gel, based on concentrations tested in previous studies [8]. Immunostaining using a primary polyclonal laminin-specific antibody and a secondary Alexa Fluor 488 fluorescent antibody showed uniform laminin distribution throughout the hydrogel (data not shown). The
Discussion
In this study, silk hydrogels were evaluated as a biomaterial to co-encapsulate ECM proteins and stromal cells to enhance islet function. Silk fibroin was formulated to have vortex-induced gelation, with the kinetics of self-assembly controllable by vortex time, assembly temperature, and protein concentration [16]. Vortex-induced silk hydrogels are a promising injectable islet delivery scaffold due to the rapid gelation time and ability to recover from shear-thinning after being injected
Conclusion
The work presented herein demonstrates the potential utility of silk-based hydrogels as islet encapsulation platforms for transplantation. Silk, despite being used as a biomaterial for centuries, has had minimal investigation as an islet encapsulation material. Our results demonstrate that silk hydrogels provide a permissive 3D environment for encapsulating islets in vitro, to maintain islet function and viability. Furthermore, the vortex-induced silk hydrogels allow for the co-encapsulation of
Acknowledgments
We thankfully acknowledge the NIH P41 Center Grant (PI Kaplan) which provided the silk material used in our experiments, the NIH P40RR017447 Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White for providing the mesenchymal cells, the Cell Science Imaging Facility at Stanford University for the use of their equipment. We thank the NIH/NIBIB (R01EB003806) for generous support of this research as well as the Department of Pathology at Stanford
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