A static glucose-stimulated insulin secretion (sGSIS) assay that is significantly predictive of time to diabetes reversal in the human islet bioassay.

INTRODUCTION: Static incubation (static glucose-stimulated insulin secretion, sGSIS) is a measure of islet secretory function. The Stimulation Index (SI; insulin produced in high glucose/insulin produced in low glucose) is currently used as a product release criterion of islet transplant potency. RESEARCH DESIGN AND METHODS: Our hypothesis was that the Delta, insulin secreted in high glucose minus insulin secreted in low glucose, would be more predictive. To evaluate this hypothesis, sGSIS was performed on 32 consecutive human islet preparations, immobilizing the islets in a slurry of Sepharose beads to minimize mechanical perturbation. Simultaneous full-mass subrenal capsular transplants were performed in chemically induced diabetic immunodeficient mice. Logistic regression analysis was used to determine optimal cut-points for diabetes reversal time and the Fisher Exact Test was used to assess the ability of the Delta and the SI to accurately classify transplant outcomes. Receiver operating characteristic curve analysis was performed on cut-point grouped data, assessing the predictive power and optimal cut-point for each sGSIS potency metric. Finally, standard Kaplan-Meier-type survival analysis was conducted. RESULTS: In the case of the sGSIS the Delta provided a superior islet potency metric relative to the SI.ConclusionsThe sGSIS Delta value is predicitive of time to diabetes reversal in the full mass human islet transplant bioassay.

3D printed elastomeric biomaterial mitigates compaction during in vitro vasculogenesis.

A key parameter for the success of most cellular implants is the formation of a complete and comprehensive intra-implant vessel network. Pre-vascularization, the generation of vessel structures in vitro prior to transplantation, provides accelerated implant perfusion via anastomosis, but scalability and ease of integration hinder clinical translation. For fibrin-based vasculogenesis approaches, the remodeling and degradation of the fragile, hydrogel matrix during the formation of vessel-like structures results in rapid, cell-mediated construct compaction leading to dense, capillary-like structures with ineffective network coverage. To resolve these challenges, vasculogenic hydrogels were embedded within a highly porous, biostable three-dimensional (3D) polydimethylsiloxane (PDMS) scaffold. Using reverse-casting of 3D-printed molds, scaffolds exhibited highly interconnected and reproducible pore structures. Pore size was optimized via in vivo screening of intra-device angiogenesis. The inclusion of the PDMS frame with vasculogenic hydrogels significantly reduced fibrin compaction in vitro, resulting in easily manipulated constructs with predictable dimensionality and increased surface area compared to fibrin hydrogel alone. Globally, vascular morphogenesis was altered by the PDMS frame, with significantly larger and less dense network structures. Vasculogenic proteomic evaluation showed a temporal impact of the addition of the PDMS frame, indicating altered cellular proliferation and migration signaling. This work establishes a platform for improving the generation of translational pre-vascularized networks for greater flexibility to meet the needs of clinically scaled, engineered tissues. STATEMENT OF SIGNIFICANCE: Competent intra-implant vascularization is a significant issue hindering the success of engineered tissues. Pre-vascularization approaches, whereby a vascular network is formed in vitro and subsequently implanted into the host to anastomose, is a promising approach but it is limited by the compacted, dense, and poorly functional microcapillary structures typically formed using soft hydrogels. Herein, we have uniquely addressed this challenge by adding a 3D printed PDMS-based open framework structure that serves to prevent hydrogel compaction. Globally, we observed distinct differences in overall construct geometry, vascular network density, compaction, and morphogenesis, indicating that this PDMS framework lead to elevated maturity of this in vitro network while retaining its global dimensions. Overall, this novel approach elevates the translational potential of pre-vascularized constructs.

Engineering Modular, Oxygen-Generating Microbeads for the In Situ Mitigation of Cellular Hypoxia.

Insufficient oxygenation is a key obstacle in the design of clinically scalable tissue-engineered grafts. In this work, an oxygen-generating composite material, termed OxySite, is created through the encapsulation of calcium peroxide (CaO2 ) within polydimethylsiloxane and formulated into microbeads for ease in tissue integration. Key material parameters of reactant loading, porogen addition, microbead size, and an outer rate-limiting layer are modulated to characterize oxygen generation kinetics and their suitability for cellular applications. In silico models are developed to predict the local impact of different OxySite microbead formulations on oxygen availability within an idealized cellular implant. Promising OxySite microbead variants are subsequently coencapsulated with murine ß-cells within macroencapsulation devices, resulting in improved cellular metabolic activity and function under hypoxic conditions when compared to controls. Additionally, the coinjection of optimized OxySite microbeads with murine pancreatic islets within a confined transplant site demonstrates ease of integration and improved primary cell function. These works highlight the broad translatability delivered by this new oxygen-generating biomaterial format, whereby the modularity of the material provides customization of the oxygen source to the specific needs of the cellular implant.

Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases: Workshop Proceedings.

The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

The Foundation for Engineering a Pancreatic Islet Niche.

Progress in diabetes research is hindered, in part, by deficiencies in current experimental systems to accurately model human pathophysiology and/or predict clinical outcomes. Engineering human-centric platforms that more closely mimic in vivo physiology, however, requires thoughtful and informed design. Summarizing our contemporary understanding of the unique and critical features of the pancreatic islet can inform engineering design criteria. Furthermore, a broad understanding of conventional experimental practices and their current advantages and limitations ensures that new models address key gaps. Improving beyond traditional cell culture, emerging platforms are combining diabetes-relevant cells within three-dimensional niches containing dynamic matrices and controlled fluidic flow. While highly promising, islet-on-a-chip prototypes must evolve their utility, adaptability, and adoptability to ensure broad and reproducible use. Here we propose a roadmap for engineers to craft biorelevant and accessible diabetes models. Concurrently, we seek to inspire biologists to leverage such tools to ask complex and nuanced questions. The progenies of such diabetes models should ultimately enable investigators to translate ambitious research expeditions from benchtop to the clinic.

Integrating Additive Manufacturing Techniques to Improve Cell-Based Implants for the Treatment of Type 1 Diabetes.

The increasing global prevalence of endocrine diseases like type 1 diabetes mellitus (T1DM) elevates the need for cellular replacement approaches, which can potentially enhance therapeutic durability and outcomes. Central to any cell therapy is the design of delivery systems that support cell survival and integration. In T1DM, well-established fabrication methods have created a wide range of implants, ranging from 3D macro-scale scaffolds to nano-scale coatings. These traditional methods, however, are often challenged by their inherent limitations in reproducible and discrete fabrication, particularly when scaling to the clinic. Additive manufacturing (AM) techniques provide a means to address these challenges by delivering improved control over construct geometry and microscale component placement. While still early in development in the context of T1DM cellular transplantation, the integration of AM approaches serves to improve nutrient material transport, vascularization efficiency, and the accuracy of cell, matrix, and local therapeutic placement. This review highlights current methods in T1DM cellular transplantation and the potential of AM approaches to overcome these limitations. In addition, emerging AM technologies and their broader application to cell-based therapy are discussed.

Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases: Workshop Proceedings.

ABSTRACT: The "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Engineering the Multi-Enzymatic Activity of Cerium Oxide Nanoparticle Coatings for the Antioxidant Protection of Implants.

Imbalance of oxidants is a universal contributor to the failure of implanted devices and tissues. A sustained oxidative environment leads to cytotoxicity, prolonged inflammation, and ultimately host rejection of implanted devices/grafts. The incorporation of antioxidant materials can inhibit this redox/inflammatory cycle and enhance implant efficacy. Cerium oxide nanoparticles (CONP) is a highly promising agent that exhibits potent, ubiquitous, and self-renewable antioxidant properties. Integrating CONP as surface coatings provides ease in translating antioxidant properties to various implants/grafts. Herein, we describe the formation of CONP coatings, generated via the sequential deposition of CONP and alginate, and the impact of coating properties, pH, and polymer molecular weight, on their resulting redox profile. Investigation of CONP deposition, layer formation, and coating uniformity/thickness on their resulting oxidant scavenging activity identified key parameters for customizing global antioxidant properties. Results found lower molecular weight alginates and physiological pH shift CONP activity to a higher H2O2 to O2 --scavenging capability. The antioxidant properties measured for these various coatings translated to distinct antioxidant protection to the underlying encapsulated cells. Information gained from this work can be leveraged to tailor coatings towards specific oxidant-scavenging applications and prolong the function of medical devices and cellular implants.

Strategies for durable ß cell replacement in type 1 diabetes.

Technological advancements in blood glucose monitoring and therapeutic insulin administration have improved the quality of life for people with type 1 diabetes. However, these efforts fall short of replicating the exquisite metabolic control provided by native islets. We examine the integrated advancements in islet cell replacement and immunomodulatory therapies that are coalescing to enable the restoration of endogenous glucose regulation. We highlight advances in stem cell biology and graft site design, which offer innovative sources of cellular material and improved engraftment. We also cover cutting-edge approaches for preventing allograft rejection and recurrent autoimmunity. These insights reflect a growing understanding of type 1 diabetes etiology, ß cell biology, and biomaterial design, together highlighting therapeutic opportunities to durably replace the ß cells destroyed in type 1 diabetes.

Immunosuppressive PLGA TGF-ß1 Microparticles Induce Polyclonal and Antigen-Specific Regulatory T Cells for Local Immunomodulation of Allogeneic Islet Transplants.

Allogeneic islet transplantation is a promising cell-based therapy for Type 1 Diabetes (T1D). The long-term efficacy of this approach, however, is impaired by allorejection. Current clinical practice relies on long-term systemic immunosuppression, leading to severe adverse events. To avoid these detrimental effects, poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were engineered for the localized and controlled release of immunomodulatory TGF-ß1. The in vitro co-incubation of TGF-ß1 releasing PLGA MPs with naïve CD4+ T cells resulted in the efficient generation of both polyclonal and antigen-specific induced regulatory T cells (iTregs) with robust immunosuppressive function. The co-transplantation of TGF-ß1 releasing PLGA MPs and Balb/c mouse islets within the extrahepatic epididymal fat pad (EFP) of diabetic C57BL/6J mice resulted in the prompt engraftment of the allogenic implants, supporting the compatibility of PLGA MPs and local TGF-ß1 release. The presence of the TGF-ß1-PLGA MPs, however, did not confer significant graft protection when compared to untreated controls, despite measurement of preserved insulin expression, reduced intra-islet CD3+ cells invasion, and elevated CD3+Foxp3+ T cells at the peri-transplantation site in long-term functioning grafts. Examination of the broader impacts of TGF-ß1/PLGA MPs on the host immune system implicated a localized nature of the immunomodulation with no observed systemic impacts. In summary, this approach establishes the feasibility of a local and modular microparticle delivery system for the immunomodulation of an extrahepatic implant site. This approach can be easily adapted to deliver larger doses or other agents, as well as multi-drug approaches, within the local graft microenvironment to prevent transplant rejection.

Designing biomaterials for the modulation of allogeneic and autoimmune responses to cellular implants in Type 1 Diabetes.

The effective suppression of adaptive immune responses is essential for the success of allogeneic cell therapies. In islet transplantation for Type 1 Diabetes, pre-existing autoimmunity provides an additional hurdle, as memory autoimmune T cells mediate both an autoantigen-specific attack on the donor beta cells and an alloantigen-specific attack on the donor graft cells. Immunosuppressive agents used for islet transplantation are generally successful in suppressing alloimmune responses, but dramatically hinder the widespread adoption of this therapeutic approach and fail to control memory T cell populations, which leaves the graft vulnerable to destruction. In this review, we highlight the capacity of biomaterials to provide local and nuanced instruction to suppress or alter immune pathways activated in response to an allogeneic islet transplant. Biomaterial immunoisolation is a common approach employed to block direct antigen recognition and downstream cell-mediated graft destruction; however, immunoisolation alone still permits shed donor antigens to escape into the host environment, resulting in indirect antigen recognition, immune cell activation, and the creation of a toxic graft site. Designing materials to decrease antigen escape, improve cell viability, and increase material compatibility are all approaches that can decrease the local release of antigen and danger signals into the implant microenvironment. Implant materials can be further enhanced through the local delivery of anti-inflammatory, suppressive, chemotactic, and/or tolerogenic agents, which serve to control both the innate and adaptive immune responses to the implant with a benefit of reduced systemic effects. Lessons learned from understanding how to manipulate allogeneic and autogenic immune responses to pancreatic islets can also be applied to other cell therapies to improve their efficacy and duration. STATEMENT OF SIGNIFICANCE: This review explores key immunologic concepts and critical pathways mediating graft rejection in Type 1 Diabetes, which can instruct the future purposeful design of immunomodulatory biomaterials for cell therapy. A summary of immunological pathways initiated following cellular implantation, as well as current systemic immunomodulatory agents used, is provided. We then outline the potential of biomaterials to modulate these responses. The capacity of polymeric encapsulation to block some powerful rejection pathways is covered. We also highlight the role of cellular health and biocompatibility in mitigating immune responses. Finally, we review the use of bioactive materials to proactively modulate local immune responses, focusing on key concepts of anti-inflammatory, suppressive, and tolerogenic agents.

Engineering a macroporous oxygen-generating scaffold for enhancing islet cell transplantation within an extrahepatic site.

Insufficient oxygenation is a serious issue arising within cell-based implants, as the hypoxic period between implantation and vascularization of the graft is largely unavoidable. In situ oxygen supplementation at the implant site should significantly mitigate hypoxia-induced cell death and dysfunction, as well as improve transplant efficacy, particularly for highly metabolically active cells such as pancreatic islets. One promising approach is the use of an oxygen generating material created through the encapsulation of calcium peroxide within polydimethylsiloxane (PDMS), termed OxySite. In this study, OxySite microbeads were incorporated within a macroporous PDMS scaffold to create a single, streamlined, oxygen generating macroporous scaffold. The resulting OxySite scaffold generated sufficient local oxygenation for up to 20 days, with nontoxic levels of reaction intermediates or by-products. The benefit of local oxygen release on transplant efficacy was investigated in a diabetic Lewis rat syngeneic transplantation model using a clinically relevant islet dosage (10,000 IEQ/kg BW) with different isolation purities (80%, 90%, and 99%). Impure islet preparations containing pancreatic non-islet cells, which are common in the clinical setting, permit examination of the effect of increased overall oxygen demand. Our transplantation outcomes showed that elevating the oxygen demand of the graft with decreasing isolation purity resulted in decreased graft efficacy for control implants, while the integration of OxySite significantly mitigated this impact and resulted in improved graft outcomes. Results highlight the superior clinical translational potential of these off-the-shelf OxySite scaffolds, where islet purity and the overall oxygen demands of implants are increased and highly variable. The oxygen-generating porous scaffold further provides a broad platform for enhancing the survival and efficacy of cellular implants for numerous other applications. STATEMENT OF SIGNIFICANCE: Hypoxia is a serious issue within tissue engineered implants. To address this challenge, we developed a distinct macroporous scaffold platform containing oxygen-generating microbeads. This oxygen-generating scaffold showed the potential to support clinically relevant cell dosages for islet transplantation, leading to improved treatment efficacy. This platform can also be used to mitigate hypoxia for other biomedical applications.

In vitro generation of peri-islet basement membrane-like structures.

The peri-islet extracellular matrix (ECM) is a key component of the microenvironmental niche surrounding pancreatic islets of Langerhans. The cell anchorage and signaling provided by the peri-islet ECM is critical for optimum beta cell glucose responsiveness, but islets lose this important native ECM when isolated for transplantation or in vitro studies. Here, we established a method to construct a peri-islet ECM on the surfaces of isolated rat and human islets by the co-assembly from solution of laminin, nidogen and collagen IV proteins. Successful deposition of contiguous peri-islet ECM networks was confirmed by immunofluorescence, western blot, and transmission electron microscopy. The ECM coatings were disrupted when assembly occurred in Ca2+/Mg2+-free conditions. As laminin network polymerization is divalent cation dependent, our data are consistent with receptor-driven ordered ECM network formation rather than passive protein adsorption. To further illustrate the utility of ECM coatings, we employed stem cell derived beta-like cell clusters (sBCs) as a renewable source of functional beta cells for cell replacement therapy. We observe that sBC pseudo-islets lack an endogenous peri-islet ECM, but successfully applied our approach to construct a de novo ECM coating on the surfaces of sBCs.

Controlled Release of Anti-Inflammatory and Proangiogenic Factors from Macroporous Scaffolds.

The simultaneous local delivery of anti-inflammatory and proangiogenic agents via biomaterial scaffolds presents a promising method for improving the engraftment of tissue-engineered implants while avoiding potentially detrimental systemic delivery. In this study, polydimethylsiloxane (PDMS) microbeads were loaded with either anti-inflammatory dexamethasone (Dex) or proangiogenic 17ß-estradiol (E2) and subsequently integrated into a single macroporous scaffold to create a controlled, dual-drug delivery platform. Compared to a standard monolithic drug dispersion scaffold, macroporous scaffolds containing drug-loaded microbeads exhibited reduced initial burst release and increased durability of drug release for both agents. The incubation of scaffolds with lipopolysaccharide (LPS)-stimulated M1 macrophages found that Dex suppressed the production of proinflammatory and proangiogenic factors when compared to drug-free control scaffolds; however, the coincubation of macrophages with Dex and E2 scaffolds restored their proangiogenic features. Following implantation, Dex-loaded microbead scaffolds (Dex-µBS) suppressed host cell infiltration and integration, when compared to controls. In contrast, the codelivery of dexamethasone with estrogen from the microbead scaffold (Dex+E2-µBS) dampened overall host cell infiltration, but restored graft vascularization. These results demonstrate the utility of a microbead scaffold approach for the controlled, tailored, and local release of multiple drugs from an open framework implant. It further highlights the complementary impacts of local Dex and E2 delivery to direct the healthy integration of implants, which has broad applications to the field of tissue engineering and regenerative medicine. Impact statement Inflammatory responses and vascularization are two significant challenges associated with the engraftment of tissue-engineered implants. To overcome these challenges, we developed a microbead scaffold platform for the local delivery of anti-inflammatory and proangiogenic agents. This drug delivery system showed the potential to simultaneously control the release of multiple agents, leading to a healthy integration of implants with host tissues. This multifunctional platform could be useful to numerous cellular transplants and engineered tissues.

In vitro platform establishes antigen-specific CD8+ T cell cytotoxicity to encapsulated cells via indirect antigen recognition.

The curative potential of non-autologous cellular therapy is hindered by the requirement of anti-rejection therapy. Cellular encapsulation within nondegradable biomaterials has the potential to inhibit immune rejection, but the efficacy of this approach in robust preclinical and clinical models remains poor. While the responses of innate immune cells to the encapsulating material have been characterized, little attention has been paid to the contributions of adaptive immunity in encapsulated graft destabilization. Avoiding the limitations of animal models, we established an efficient, antigen-specific in vitro platform capable of delineating direct and indirect host T cell recognition to microencapsulated cellular grafts and evaluated their consequential impacts. Using ovalbumin (OVA) as a model antigen, we determined that alginate microencapsulation abrogates direct CD8+ T cell activation by interrupting donor-host interaction; however, indirect T cell activation, mediated by host antigen presenting cells (APCs) primed with shed donor antigens, still occurs. These activated T cells imparted cytotoxicity on the encapsulated cells, likely via diffusion of cytotoxic solutes. Overall, this platform delivers unique mechanistic insight into the impacts of hydrogel encapsulation on host adaptive immune responses, comprehensively addressing a long-standing hypothesis of the field. Furthermore, it provides an efficient benchtop screening tool for the investigation of new encapsulation methods and/or synergistic immunomodulatory agents.

Nanotechnology Approaches to Modulate Immune Responses to Cell-based Therapies for Type 1 Diabetes.

Islet transplantation is a promising curative treatment option for type 1 diabetes (T1D) as it can provide physiological blood glucose control. The widespread utilization of islet transplantation is limited due to systemic immunosuppression requirements, persisting graft immunodestruction, and poor islet engraftment. Traditional macro- and micropolymeric encapsulation strategies can alleviate the need for antirejection immunosuppression, yet the increased graft volume and diffusional distances imparted by these coatings can be detrimental to graft viability and glucose control. Additionally, systemic administration of pro-engraftment and antirejection therapeutics leaves patients vulnerable to adverse off-target side effects. Nanoscale engineering techniques can be used to immunocamouflage islets, modulate the transplant microenvironment, and provide localized pro-engraftment cues. In this review, we discuss the applications of nanotechnology to advance the clinical potential of islet transplantation, with a focus on cell surface engineering, bioactive functionalization, and use of nanoparticles in T1D cell-based treatments.

Transplantation of PEGylated islets enhances therapeutic efficacy in a diabetic nonhuman primate model.

Islet cell transplantation can lead to insulin independence, reduced hypoglycemia, and amelioration of diabetes complications in patients with type 1 diabetes. The systemic delivery of anti-inflammatory agents, while considered crucial to limit the early loss of islets associated with intrahepatic infusion, increases the burden of immunosuppression. In an effort to decrease the pharmaceutical load to the patient, we modified the pancreatic islet surface with long-chain poly(ethylene glycol) (PEG) to mitigate detrimental host-implant interactions. The effect of PEGylation on islet engraftment and long-term survival was examined in a robust nonhuman primate model via three paired transplants of dosages 4300, 8300, and 10 000 islet equivalents per kg body weight. A reduced immunosuppressive regimen of anti-thymocyte globulin induction plus tacrolimus in the first posttransplant month followed by maintenance with sirolimus monotherapy was employed. To limit transplant variability, two of the three pairs were closely MHC-matched recipients and received MHC-disparate PEGylated or untreated islets isolated from the same donors. Recipients of PEGylated islets exhibited significantly improved early c-peptide levels, reduced exogenous insulin requirements, and superior glycemic control, as compared to recipients of untreated islets. These results indicate that this simple islet modification procedure may improve islet engraftment and survival in the setting of reduced immunosuppression.