Necrostatin-1 supplementation enhances young porcine islet maturation and in vitro function.

BACKGROUND: Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin-1 (Nec-1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre-weaned porcine islets (PPIs). METHODS: PPIs were isolated from pre-weaned Yorkshire piglets (8-15 days old) and either cultured in control islet culture media (n = 6) or supplemented with Nec-1 (100 µM, n = 5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate. RESULTS: Nec-1 supplementation induced a 2-fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec-1 treatment doubled the beta- and alpha-cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec-1-treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec-1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec-1. CONCLUSION: This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec-1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec-1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

Acetate and Butyrate Improve ß-cell Metabolism and Mitochondrial Respiration under Oxidative Stress.

Islet dysfunction mediated by oxidative and mitochondrial stress contributes to the development of type 1 and 2 diabetes. Acetate and butyrate, produced by gut microbiota via fermentation, have been shown to protect against oxidative and mitochondrial stress in many cell types, but their effect on pancreatic ß-cell metabolism has not been studied. Here, human islets and the mouse insulinoma cell line MIN6 were pre-incubated with 1, 2, and 4 mM of acetate or butyrate with and without exposure to the apoptosis inducer and metabolic stressor streptozotocin (STZ). Both short-chain fatty acids (SCFAs) enhanced the viability of islets and ß-cells, but the beneficial effects were more pronounced in the presence of STZ. Both SCFAs prevented STZ-induced cell apoptosis, viability reduction, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS) and nitric oxide (NO) at a concentration of 1 mM but not at higher concentrations. These rescue effects of SCFAs were accompanied by preventing reduction of the mitochondrial fusion genes MFN, MFN2, and OPA1. In addition, elevation of the fission genes DRP1 and FIS1 during STZ exposure was prevented. Acetate showed more efficiency in enhancing metabolism and inhibiting ROS, while butyrate had less effect but was stronger in inhibiting the SCFA receptor GPR41 and NO generation. Our data suggest that SCFAs play an essential role in supporting ß-cell metabolism and promoting survival under stressful conditions. It therewith provides a novel mechanism by which enhanced dietary fiber intake contributes to the reduction of Western diseases such as diabetes.

Polymer scaffolds for pancreatic islet transplantation - Progress and challenges.

Pancreatic-islet transplantation is a safe and noninvasive therapy for type 1 diabetes. However, the currently applied site for transplantation, ie, the liver, is not the optimal site for islet survival. Because the human body has shortcomings in providing an optimal site, artificial transplantation sites have been proposed. Such an artificial site could consist of a polymeric scaffold that mimics the pancreatic microenvironment and supports islet function. Recently, remarkable progress has been made in the technology of engineering scaffolds. The polymer-islet interactions, the site of implantation, and scaffold prevascularization are critical factors for success or failure of the scaffolds. This article critically reviews these factors while also discussing translation of experimental studies to human application as well as the steps required to create a clinically applicable prevascularized, retrievable scaffold for implantation of insulin-producing cells for treatment of type 1 diabetes mellitus.

Therapeutic Strategies for Modulating the Extracellular Matrix to Improve Pancreatic Islet Function and Survival After Transplantation.

PURPOSES OF REVIEW: Extracellular matrix (ECM) components modulate the interaction between pancreatic islet cells. During the islet isolation prior to transplantation as treatment for type 1 diabetes, the ECM is disrupted impacting functional graft survival. Recently, strategies for restoring ECM have shown to improve transplantation outcomes. This review discusses the current therapeutic strategies to modulate ECM components to improve islet engraftment. RECENT FINDINGS: Approaches applied are seeding islets in ECM of decellularized organs, supplementation of specific ECM components in polymeric scaffolds or immunoisolating capsules, and stimulating islet ECM production with specific growth factors or ECM-producing cells. These strategies have shown success in improving functional islet survival. However, the same experiments show that caution should be taken as some ECM components may negatively impact islet function and engraftment. ECM restoration resulted in improved transplantation outcomes, but careful selection of beneficial ECM components and strategies is warranted.

A Retrievable, Efficacious Polymeric Scaffold for Subcutaneous Transplantation of Rat Pancreatic Islets.

OBJECTIVE: We aim on developing a polymeric ectopic scaffold in a readily accessible site under the skin. SUMMARY BACKGROUND DATA: The liver as transplantation site for pancreatic islets is associated with significant loss of islets. Several extrahepatic sites were tested in experimental animals, but many have practical limitations in the clinical setting and do not have the benefit of easy accessibility. METHODS AND RESULTS: Functional survival of rat islets was tested during 7 days of culture in the presence of poly(D,L-lactide-co-ε-caprolactone) (PDLLCL), poly(ethylene oxide terephthalate)/polybutylene terephthalate (PEOT/PBT) block copolymer, and polysulfone. Tissue responses were studied in vivo after subcutaneous implantation in rats. Culture on PEOT/PBT and polysulfone profoundly disturbed function of islets, and induced severe tissue responses in vivo. Modification of their hydrophilicity did not change the suitability of the polymers. PDLLCL was the only polymer that promoted functional survival of rat islets in vitro and was associated with minor tissue reactions after 28 days. Rat islets were transplanted in the PDLLCL scaffold in a diabetic rat model. Before islet seeding, the scaffold was allowed to engraft for 28 days to allow the tissue response to dampen and to allow blood vessel growth into the device. Islet transplantation into the scaffold resulted in normoglycemia within 3 days and for the duration of the study period of 16 weeks. CONCLUSIONS: In conclusion, we found that some polymers such as PEOT/PBT and polysulfone interfere with islet function. PDLLCL is a suitable polymer to create an artificial islet transplantation site under the skin and supports islet survival.

Necrostatin-1 releasing nanoparticles: In vitro and in vivo efficacy for supporting immunoisolated islet transplantation outcomes.

Immunoisolation of pancreatic islets in alginate microcapsules allows for transplantation in the absence of immunosuppression but graft survival time is still limited. This limited graft survival is caused by a combination of tissue responses to the encapsulating biomaterial and islets. A significant loss of islet cells occurs in the immediate period after transplantation and is caused by a high susceptibility of islet cells to inflammatory stress during this period. Here we investigated whether necrostatin-1 (Nec-1), a necroptosis inhibitor, can reduce the loss of islet cells under stress in vitro and in vivo. To this end, we developed a Nec-1 controlled-release system using poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) as the application of Nec-1 in vivo is limited by low stability and possible side effects. The PLGA NPs stably released Nec-1 for 6 days in vitro and protected beta cells against hypoxia-induced cell death in vitro. Treatment with these Nec-1 NPs at days 0, 6, and 12 post-islet transplantation in streptozotocin-diabetic mice confirmed the absence of side effects as graft survival was similar in encapsulated islet grafts in the absence and presence of Nec-1. However, we found no further prolongation of graft survival of encapsulated grafts which might be explained by the high biocompatibility of the alginate encapsulation system that provoked a very mild tissue response. We expect that the Nec-1-releasing NPs could find application to immunoisolation systems that elicit stronger inflammatory responses, such as macrodevices and vasculogenic biomaterials.

Culturing Conditions Dictate the Composition and Pathways Enrichment of Human and Rat Perirenal Adipose-Derived Stromal Cells' Secretomes.

Understanding the impact of various culturing strategies on the secretome composition of adipose-derived stromal cells (ASC) enhances their therapeutic potential. This study investigated changes in the secretome of perirenal ASC (prASC) under different conditions: normoxia, cytokine exposure, high glucose, hypoxia, and hypoxia with high glucose. Using mass spectrometry and enrichment clustering analysis, we found that normoxia enriched pathways related to extracellular matrix (ECM) organization, platelet degranulation, and insulin-like growth factor (IGF) transport and uptake. Cytokine exposure influenced metabolism, vascular development, and protein processing pathways. High glucose affected the immune system, metabolic processes, and IGF transport and uptake. Hypoxia impacted immune and metabolic processes and protein processing. Combined hypoxia and high glucose influenced the immune system, IGF transport and uptake, and ECM organization. Our findings highlight the potential of manipulating culturing conditions to produce secretomes with distinct protein and functional profiles, tailoring therapeutic strategies accordingly.

Enhancing longevity of immunoisolated pancreatic islet grafts by modifying both the intracapsular and extracapsular environment.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease characterized by autoimmune destruction of pancreatic ß cells. Transplantation of immunoisolated pancreatic islets might treat T1DM in the absence of chronic immunosuppression. Important advances have been made in the past decade as capsules can be produced that provoke minimal to no foreign body response after implantation. However, graft survival is still limited as islet dysfunction may occur due to chronic damage to islets during islet isolation, immune responses induced by inflammatory cells, and nutritional issues for encapsulated cells. This review summarizes the current challenges for promoting longevity of grafts. Possible strategies for improving islet graft longevity are also discussed, including supplementation of the intracapsular milieu with essential survival factors, promotion of vascularization and oxygenation near capsules, modulation of biomaterials, and co-transplantation of accessory cells. Current insight is that both the intracapsular as well as the extracapsular properties should be improved to achieve long-term survival of islet-tissue. Some of these approaches reproducibly induce normoglycemia for more than a year in rodents. Further development of the technology requires collective research efforts in material science, immunology, and endocrinology. STATEMENT OF SIGNIFICANCE: Islet immunoisolation allows for transplantation of insulin producing cells in absence of immunosuppression and might facilitate the use of xenogeneic cell sources or grafting of cells obtained from replenishable cell sources. However, a major challenge to date is to create a microenvironment that supports long-term graft survival. This review provides a comprehensive overview of the currently identified factors that have been demonstrated to be involved in either stimulating or reducing islet graft survival in immunoisolating devices and discussed current strategies to enhance the longevity of encapsulated islet grafts as treatment for type 1 diabetes. Although significant challenges remain, interdisciplinary collaboration across fields may overcome obstacles and facilitate the translation of encapsulated cell therapy from the laboratory to clinical application.

Towards standardization of human adipose-derived stromal cells secretomes.

The secretome of adipose-derived stromal cells (ASC) is a heterogeneous mixture of components with a beneficial influence on cellular microenvironments. As such, it represents a cell-free alternative in regenerative medicine therapies. Pathophysiological conditions increase the therapeutic capacity of ASC and, with this, the benefits of the secretome. Such conditions can be partially mimicked in vitro by adjusting culturing conditions. Secretomics, the unbiased analysis of a cell secretome by mass spectrometry, is a powerful tool to describe the composition of ASC secretomes. In this proteomics databases review, we compared ASC secretomic studies to retrieve persistently reported proteins resulting from the most explored types of culturing conditions used in research, i.e., exposure to normoxia, hypoxia, or cytokines. Our comparisons identified only eight common proteins within ASC normoxic secretomes, no commonalities within hypoxic ASC secretomes, and only nine within secretomes of ASC exposed to proinflammatory cytokines. Within these, and regardless of the culturing condition that stimulated secretion, a consistent presence of extracellular matrix-related pathways associated with such proteins was identified. Confounders such as donors' age, sex, body mass index, the anatomical area where ASC were harvested, secretome collection method, data description, and how the data is shared with the scientific community are discussed as factors that might explain our outcomes. We conclude that standardization is imperative as the currently available ASC secretomic studies do not facilitate solid conclusions on the therapeutic value of different ASC secretomes.

Mechanisms of Foreign Body Giant Cell Formation in Response to Implantable Biomaterials.

Long term function of implantable biomaterials are determined by their integration with the host's body. Immune reactions against these implants could impair the function and integration of the implants. Some biomaterial-based implants lead to macrophage fusion and the formation of multinucleated giant cells, also known as foreign body giant cells (FBGCs). FBGCs may compromise the biomaterial performance and may lead to implant rejection and adverse events in some cases. Despite their critical role in response to implants, there is a limited understanding of cellular and molecular mechanisms involved in forming FBGCs. Here, we focused on better understanding the steps and mechanisms triggering macrophage fusion and FBGCs formation, specifically in response to biomaterials. These steps included macrophage adhesion to the biomaterial surface, fusion competency, mechanosensing and mechanotransduction-mediated migration, and the final fusion. We also described some of the key biomarkers and biomolecules involved in these steps. Understanding these steps on a molecular level would lead to enhance biomaterials design and improve their function in the context of cell transplantation, tissue engineering, and drug delivery.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Microfluidic Generation of Thin-Shelled Polyethylene Glycol-Tyramine Microgels for Non-Invasive Delivery of Immunoprotected ß-Cells.

Transplantation of microencapsulated pancreatic cells is emerging as a promising therapy to replenish ß-cell mass lost from auto-immune nature of type I diabetes mellitus (T1DM). This strategy intends to use micrometer-sized microgels to provide immunoprotection to transplanted cells to avoid chronic application of immunosuppression. Clinical application of encapsulation has remained elusive due to often limited production throughputs and body's immunological reactions to implanted materials. This article presents a high-throughput fabrication of monodisperse, non-immunogenic, non-degradable, immunoprotective, semi-permeable, enzymatically-crosslinkable polyethylene glycol-tyramine (PEG-TA) microgels for ß-cell microencapsulation. Monodisperse ß-cell laden microgels of ≈120 µm, with a shell thickness of 20 µm are produced using an outside-in crosslinking strategy. Microencapsulated ß-cells rapidly self-assemble into islet-sized spheroids. Immunoprotection of the microencapsulated is demonstrated by inability of FITC-IgG antibodies to diffuse into cell-laden microgels and NK-cell inability to kill microencapsulated ß-cells. Multiplexed ELISA analysis on live blood immune reactivity confirms limited immunogenicity. Microencapsulated MIN6ß1 spheroids remain glucose responsive for 28 days in vitro, and able to restore normoglycemia 5 days post-implantation in diabetic mice without notable amounts of cell death. In short, PEG-TA microgels effectively protect implanted cells from the host's immune system while being viable and functional, validating this strategy for the treatment of T1DM.

Successful Islet Transplantation Into a Subcutaneous Polycaprolactone Scaffold in Mice and Pigs.

Islet transplantation is a promising treatment for type 1 diabetes. It has the potential to improve glycemic control, particularly in patients suffering from hypoglycemic unawareness and glycemic instability. As most islet grafts do not function permanently, efforts are needed to create an accessible and replaceable site, for islet grafts or for insulin-producing cells obtained from replenishable sources. To this end, we designed and tested an artificial, polymeric subcutaneous transplantation site that allows repeated transplantation of islets. Methods: In this study, we developed and compared scaffolds made of poly(D,L,-lactide-co-ε-caprolactone) (PDLLCL) and polycaprolactone (PCL). Efficacy was first tested in mice' and then, as a proof of principle for application in a large animal model, the scaffolds were tested in pigs, as their skin structure is similar to that of humans. Results: In mice, islet transplantation in a PCL scaffold expedited return to normoglycemia in comparison to PDLLCL (7.7 ± 3.7 versus 16.8 ± 6.5 d), but it took longer than the kidney capsule control group. PCL also supported porcine functional islet survival in vitro. Subcutaneous implantation of PDLLCL and PCL scaffolds in pigs revealed that PCL scaffolds were more stable and was associated with less infiltration by immune cells than PDLLCL scaffolds. Prevascularized PCL scaffolds were therefore used to demonstrate the functional survival of allogenic islets under the skin of pigs. Conclusions: To conclude, a novel PCL scaffold shows efficacy as a readily accessible and replaceable, subcutaneous transplantation site for islets in mice and demonstrated islet survival after a month in pigs.

Applying Immunomodulation to Promote Longevity of Immunoisolated Pancreatic Islet Grafts.

Islet transplantation is a promising therapy for insulin-dependent diabetes, but large-scale application is hampered by the lack of a consistent source of insulin-producing cells and need for lifelong administration of immunosuppressive drugs, which are associated with severe side effects. To avoid chronic immunosuppression, islet grafts can be enveloped in immunoisolating polymeric membranes. These immunoisolating polymeric membranes protect islet grafts from cell-mediated rejection while allowing diffusion of oxygen, nutrients, and insulin. Although clinical trials have shown the safety and feasibility of encapsulated islets to control glucose homeostasis, the strategy does up till now not support long-term graft survival. This partly can be explained by a significant loss of insulin-producing cells in the immediate period after implantation. The loss can be prevented by combining immunoisolation with immunomodulation, such as combined administration of immunomodulating cytokines or coencapsulation of immunomodulating cell types such as regulatory T cells, mesenchymal stem cells, or Sertoli cells. Also, administration of specific antibodies or apoptotic donor leucocytes is considered to create a tolerant microenvironment around immunoisolated grafts. In this review, we describe the outcomes and limitations of these approaches, as well as the recent progress in immunoisolating devices. Impact statement Immunoisolation by enveloping islets in semipermeable membranes allows for successful transplantation of islet grafts in the absence of chronic immunosuppression, but the duration of graft survival is still not permanent. The reasons for long-term final graft failure is not fully understood, but combining immunoisolation with immunomodulation of tissues or host immune system has been proposed to enhance the longevity of grafts. This article reviews the recent progress and challenges of immunoisolation, as well as the benefits and feasibility of combining encapsulation approaches with immunomodulation to promote longevity of encapsulated grafts.

Inclusion of extracellular matrix molecules and necrostatin-1 in the intracapsular environment of alginate-based microcapsules synergistically protects pancreatic ß cells against cytokine-induced inflammatory stress.

Immunoisolation of pancreatic islets in alginate-based microcapsules is a promising approach for grafting of islets in absence of immunosuppression. However, loss and damage to the extracellular matrix (ECM) during islet isolation enhance susceptibility of islets for inflammatory stress. In this study, a combined strategy was applied to reduce this stress by incorporating ECM components (collagen type IV/RGD) and necroptosis inhibitor, necrostatin-1 (Nec-1) in alginate-based microcapsules in vitro. To demonstrate efficacy, viability and function of MIN6 ß-cells and human islets in capsules with collagen type IV/RGD and/or Nec-1 was investigated in presence and absence of IL-1ß, IFN-γ and TNF-α. The combination of collagen type IV/RGD and Nec-1 had higher protective effects than the molecules alone. Presence of collagen type IV/RGD and Nec-1 in the intracapsular environment reduced cytokine-induced overproduction of free radical species and unfavorable shifts in mitochondrial dynamics. In addition, the ECM components collagen type IV/RGD prevented a cytokine induced suppression of the FAK/Akt pathway. Our data indicate that the inclusion of collagen type IV/RGD and Nec-1 in the intracapsular environment prevents islet-cell loss when exposed to inflammatory stress, which might contribute to higher survival of ß-cells in the immediate period after transplantation. This approach of inclusion of stress reducing agents in the intracapsular environment of immunoisolating devices may be an effective way to enhance the longevity of encapsulated islet grafts. STATEMENT OF SIGNIFICANCE: Islet-cells in immunoisolated alginate-based microcapsules are very susceptible to inflammatory stress which impacts long-term survival of islet grafts. Here we show that incorporation of ECM components (collagen type IV/RGD) and necrostatin-1 (Nec-1) in the intracapsular environment of alginate-based capsules attenuates this susceptibility and promotes islet-cell survival. This effect induced by collagen type IV/RGD and Nec-1 was probably due to lowering free radical production, preventing mitochondrial dysfunction and by maintaining ECM/integrin/FAK/Akt signaling and Nec-1/RIP1/RIP3 signaling. Our study provides an effective strategy to extend longevity of islet grafts which might be of great potential for future clinical application of immunoisolated cells.

Species-dependent impact of immunosuppressive squalene-gusperimus nanoparticles and adipose-derived stem cells on isolated human and rat pancreatic islets.

Transplantation of pancreatic islets is a promising approach to controlling glucose levels in type 1 diabetes mellitus (T1DM), but islet survival is still limited. To overcome this, islet co-culture with mesenchymal stromal cells (MSCs) together with safe immunosuppressive agents like squalene-gusperimus nanoparticles (Sq-GusNPs) may be applied. This could support islet survival and engraftment. Here, we studied how Sq-GusNPs and adipose-derived stem cells (ASCs) influence islets response under pro-inflammatory conditions. Through qRT-PCR, we studied the expression of specific genes at 24 hours in human and rat islets and ASCs in co-culture under indirect contact with or without treatment with Sq-GusNPs. We characterized how the response of islets and ASCs starts at molecular level before impaired viability or function is observed and how this response differs between species. Human islets and ASCs responses showed to be principally influenced by NF-κB activation, whereas rat islet and ASCs responses showed to be principally mediated by nitrosative stress. Rat islets showed tolerance to inflammatory conditions due to IL-1Ra secretion which was also observed in rat ASCs. Human islets induced the expression of cytokines and chemokines with pro-angiogenic, tissue repair, and anti-apoptotic properties in human ASCs under basal conditions. This expression was not inhibited by Sq-GusNPs. Our results showed a clear difference in the response elicited by human and rat islets and ASCs in front of an inflammatory stimulus and Sq-GusNPs. Our data support the use of ASCs and Sq-GusNP to facilitate engraftment of islets for T1DM treatment.

In vivovascularization and islet function in a microwell device for pancreatic islet transplantation.

Islet encapsulation in membrane-based devices could allow for transplantation of donor islet tissue in the absence of immunosuppression. To achieve long-term survival of islets, the device should allow rapid exchange of essential nutrients and be vascularized to guarantee continued support of islet function. Recently, we have proposed a membrane-based macroencapsulation device consisting of a microwell membrane for islet separation covered by a micropatterned membrane lid. The device can prevent islet aggregation and support functional islet survivalin vitro. Here, based on previous modeling studies, we develop an improved device with smaller microwell dimensions, decreased spacing between the microwells and reduced membrane thickness and investigate its performancein vitroandin vivo. This improved device allows for encapsulating higher islet numbers without islet aggregation and by applying anin vivoimaging system we demonstrate very good perfusion of the device when implanted intraperitoneally in mice. Besides, when it is implanted subcutaneously in mice, islet viability is maintained and a vascular network in close proximity to the device is developed. All these important findings demonstrate the potential of this device for islet transplantation.

In vitro determination of the immunosuppressive effect, internalization, and release mechanism of squalene-gusperimus nanoparticles for managing inflammatory responses.

Gusperimus is an anti-inflammatory drug that has shown to be effective in managing autoimmunity and preventing graft rejection. This is unstable and easily broken down into cytotoxic components. We encapsulated gusperimus binding it covalently to squalene obtaining squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles enhanced the immunosuppressive effect of gusperimus in both mouse macrophages and T cells. The half-maximal inhibitory concentration in macrophages was 9-fold lower for Sq-GusNPs compared with the free drug. The anti-inflammatory effect of the Sq-GusNPs was maintained over time without cytotoxicity. By studying nanoparticles uptake by cells with flow cytometry, we demonstrated that Sq-GusNPs are endocytosed by macrophages after binding to low-density lipoprotein receptors (LDLR). In presence of cathepsin B or D release of gusperimus is increased demonstrating the participation of proteases in the release process. Our approach may allow the application of Sq-GusNPs for effective management of inflammatory disorders including autoimmunity and graft rejection.

In vitro degradation profiles and in vivo biomaterial-tissue interactions of microwell array delivery devices.

To effectively apply microwell array cell delivery devices their biodegradation rate must be tailored towards their intended use and implantation location. Two microwell array devices with distinct degradation profiles, either suitable for the fabrication of retrievable systems in the case of slow degradation, or cell delivery systems capable of extensive remodeling using a fast degrading polymer, were compared in this study. Thin films of a poly(ethylene glycol)-poly(butylene terephthalate) (PEOT-PBT) and a poly(ester urethane) were evaluated for their in vitro degradation profiles over 34 weeks incubation in PBS at different pH values. The PEOT-PBT films showed minimal in vitro degradation over time, while the poly(ester urethane) films showed extensive degradation and fragmentation over time. Subsequently, microwell array cell delivery devices were fabricated from these polymers and intraperitoneally implanted in Albino Oxford rats to study their biocompatibility over a 12-week period. The PEOT-PBT implants shown to be capable to maintain the microwell structure over time. Implants provoked a foreign body response resulting in multilayer fibrosis that integrated into the surrounding tissue. The poly(ester urethane) implants showed a loss of the microwell structures over time, as well as a fibrotic response until the onset of fragmentation, at least 4 weeks post implantation. It was concluded that the PEOT-PBT implants could be used as retrievable cell delivery devices while the poly(ester urethane) implants could be used for cell delivery devices that require remodeling within a 4-12 week period.

Toll-like receptor 2-modulating pectin-polymers in alginate-based microcapsules attenuate immune responses and support islet-xenograft survival.

Encapsulation of pancreatic islets in alginate-microcapsules is used to reduce or avoid the application of life-long immunosuppression in preventing rejection. Long-term graft function, however, is limited due to varying degrees of host tissue responses against the capsules. Major graft-longevity limiting responses include inflammatory responses provoked by biomaterials and islet-derived danger-associated molecular patterns (DAMPs). This paper reports on a novel strategy for engineering alginate microcapsules presenting immunomodulatory polymer pectin with varying degrees of methyl-esterification (DM) to reduce these host tissue responses. DM18-pectin/alginate microcapsules show a significant decrease of DAMP-induced Toll-Like Receptor-2 mediated immune activation in vitro, and reduce peri-capsular fibrosis in vivo in mice compared to higher DM-pectin/alginate microcapsules and conventional alginate microcapsules. By testing efficacy of DM18-pectin/alginate microcapsules in vivo, we demonstrate that low-DM pectin support long-term survival of xenotransplanted rat islets in diabetic mice. This study provides a novel strategy to attenuate host responses by creating immunomodulatory capsule surfaces that attenuate activation of specific pro-inflammatory immune receptors locally at the transplantation site.