Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation

Foreign body reaction (FBR) to implanted biomaterials and medical devices is common and can compromise the function of implants or cause complications. For example, in cell encapsulation, cellular overgrowth (CO) and fibrosis around the cellular constructs can reduce the mass transfer of oxygen, nutrients and metabolic wastes, undermining cell function and leading to transplant failure. Therefore, materials that mitigate FBR or CO will have broad applications in biomedicine. Here we report a group of zwitterionic, sulfobetaine (SB) and carboxybetaine (CB) modifications of alginates that reproducibly mitigate the CO of implanted alginate microcapsules in mice, dogs and pigs. Using the modified alginates (SB-alginates), we also demonstrate improved outcome of islet encapsulation in a chemically-induced diabetic mouse model. These zwitterion-modified alginates may contribute to the development of cell encapsulation therapies for type 1 diabetes and other hormone-deficient diseases

Altering β-cell number through stable alteration of miR-21 and miR-34a expression

Aim: An insufficient functional β-cell mass is a prerequisite to develop diabetes. Thus, means to protect or restore β-cell mass are important goals in diabetes research. Inflammation and proinflammatory cytokines play important roles in β-cell dysfunction and death, and recent data show that 2 miRNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed miR-21 and knocked down miR-34a, and investigated essential cellular processes.   Materials and Methods: miRNA expression was manipulated using Lentiviral transduction of the β-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were monitored in the absence or presence of cytokines. Results: Overexpression of miR-21 decreased net β-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a increased net β-cell number in the absence of cytokines, and reduced apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was decreased upon miR-34a knockdown. Conclusion: As overexpression of miR-21 increased proliferation, but also apoptosis and NO synthesis, the potential of miR-21 as a therapeutic agent to increase β-cell survival is doubtful. Knockdown of miR-34a slightly decreased proliferation, but as apoptosis and NO synthesis were highly reduced, miR-34a may be further investigated as a therapeutic target to reduce β-cell death and dysfunction

Mechanically reinforced hydrogel vehicle delivering angiogenic factor for beta cell therapy

Type 1 diabetes mellitus (T1DM) is a chronic disease affecting millions worldwide. Insulin therapy is currently the golden standard for treating T1DM; however, it does not restore the normal glycaemic balance entirely, which increases the risk of secondary complications. Beta-cell therapy may be a possible way of curing T1DM and has already shown promising results in the clinic. However, low retention rates, poor cell survival, and limited therapeutic potential are ongoing challenges, thus increasing the need for better cell encapsulation devices. This study aimed to develop a mechanically reinforced vascular endothelial growth factor (VEGF)-delivering encapsulation device suitable for beta cell encapsulation and transplantation. Poly(l-lactide-co-ε-caprolactone) (PLCL)/gelatin methacryloyl (GelMA)/alginate coaxial nanofibres were produced using electrospinning and embedded in an alginate hydrogel. The encapsulation device was physically and biologically characterised and was found to be suitable for INS-1E beta cell encapsulation, vascularization, and transplantation in terms of its biocompatibility, porosity, swelling ratio and mechanical properties. Lastly, VEGF was incorporated into the hydrogel and the release kinetics and functional studies revealed a sustained release of bioactive VEGF for at least 14 days, making the modified alginate system a promising candidate for improving the beta cell survival after transplantation.</p

Oxygen releasing hydrogels for beta cell assisted therapy

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Macrophage Contact Dependent and Independent TLR4 Mechanisms Induce β-Cell Dysfunction and Apoptosis in a Mouse Model of Type 2 Diabetes

<div><p>Type 2 diabetes (T2D) is evolving into a global disease and patients have a systemic low-grade inflammation, yet the role of this inflammation is still not established. One plausible mechanism is enhanced expression and activity of the innate immune system. Therefore, we evaluated the expression and the function of the toll-like receptor 4 (TLR4) on pancreatic β-cells in primary mouse islets and on the murine β-cell line MIN6 in the presence or absence of macrophages. Diabetic islets have 40% fewer TLR4 positive β-cells, but twice the number of TLR4 positive macrophages as compared to healthy islets. Healthy and diabetic islets respond to a TLR4 challenge with enhanced production of cytokines (5–10-fold), while the TLR4 negative β-cell line MIN6 fails to produce cytokines. TLR4 stimulation induces β-cell dysfunction in mouse islets, measured as reduced glucose stimulated insulin secretion. Diabetic macrophages from 4-months old mice have acquired a transient enhanced capacity to produce cytokines when stimulated with LPS. Interestingly, this is lost in 6-months old diabetic mice. TLR4 activation alone does not induce apoptosis in islets or MIN-6 cells. In contrast, macrophages mediate TLR4-dependent cell-contact dependent (3-fold) as well as cell-contact independent (2-fold) apoptosis of both islets and MIN-6 cells. Importantly, diabetic macrophages have a significantly enhanced capacity to induce β-cell apoptosis compared to healthy macrophages. Taken together, the TLR4 responsiveness is elevated in the diabetic islets and mainly mediated by newly recruited macrophages. The TLR4 positive macrophages, in both a cell-contact dependent and independent manner, induce apoptosis of β-cells in a TLR4 dependent fashion and TLR4 activation directly induces β-cell dysfunction. Thus, targeting either the TLR4 pathway or the macrophages provides a novel attractive treatment regime for T2D.</p></div

TLR4 induces β-cell apoptosis though release of soluble factors from macrophages.

<p>Db/db islets and MIN6 cells were stimulated with LPS or combined IL-1β and TNFα for 24 h and apoptosis was measured as cytosolic DNA-histone complexes (A). Media from 8-weeks (A), 16-weeks (B) or 24-weeks (C) old db+ or db/db TLR4 activated splenocytes were added to MIN6 cells for 24 h. MIN6 cell apoptosis was quantified as caspase3/7 activity. Data is shown as mean ±S.E.M. from three experiments. Statistical analysis by student T-test were *p<0.05; **p<0.01; ***p<0.001.</p

Diabetic splenocytes activated through TLR4 induce enhanced β-cell apoptosis.

<p>MIN6 cells were co-cultured with 8-week old db+ (A), 8-week old db/db (B), 24-week old db+ (C) or 24-week old db/db (D) for 24 h in media and then for an additional 24 h in the presence of absence of increasing concentration of LPS. Apoptosis was quantified as caspase3/7 activity. Data is shown as mean ±S.E.M. from three experiments. Statistical analysis by student T-test were *p<0.05; **p<0.01; ***p<0.001.</p