To achieve successful islet transplantation for treatment of T1D, the protocol must promote islet survival in the short term. Insulin secretory function in patients who receive intraportal islet transplantation averages only ~20% of that of nondiabetic persons despite the use of islets from multiple donors (35
)—a result suggesting that only a small proportion of transplanted islets successfully engraft. Reasons for this loss of islet function include (1) exposure to high concentrations of cytotoxic immunosuppressive drugs via portal blood, (2) proinflammatory cytokine release by intrahepatic endothelial cells activated by islet cell contact, (3) liver ischemia, focal necrosis, and inflammation induced by islet embolism, and (4) acute inflammatory reactions that involve platelet activation and binding at the islet surface, activation of coagulation and complement systems, and leukocyte infiltration of the islet mass. To address the shortcomings of the intrahepatic environment, we developed BIs for implantation in a nonhepatic site (the intestinal mesentery). We have demonstrated that these BIs can reliably reverse drug (STZ)-induced diabetes in mice.
Injection of islet suspensions under the kidney capsule (UKC) is the most frequently used model for studies of islet engraftment in mice. With the UKC model, normoglycemia can be established in STZ-treated diabetic mice within 24–48 h using 200 islets (45
). In contrast to the UKC model, we observed that engraftment of islets on the mouse mesentery in BIs required a larger number of islets (200 islets were insufficient, whereas 450–500 islets were effective) and required a longer time to achieve normoglycemia (10–13 days). Similar disparities in islet number and time to functionality between UKC and mesenteric (omental) graft sites in mice have been reported by Kim et al. (16
), who found that the marginal islet mass required for the omental site was twice that of the kidney site and that time to normoglycemia averaged 14 days for omental grafts, but only 3 days for UKC grafts. Notably, the omentum performed better than liver and muscle sites, which each required threefold more islets than the omentum and comparable or longer times to achieve normoglycemia (15 and 27 days for liver and muscle, respectively). The reason why engraftment of islets is less efficient in non-UKC sites relative to the UKC site is unclear but may relate to a lower availability of vasculature, particularly for muscle (16
Despite the efficiency of UKC transplant, the limited space within the kidney capsule cannot accommodate large numbers of injected islets or complex implants that include drug delivery devices, such as the one we describe. This limitation, among others (7
), suggests that the UKC site may be problematic for therapeutic islet transplant in human patients. In contrast, the human omentum should be able to accommodate relatively large, multicomponent BIs. Moreover, unlike the kidney, the omentum is not a critical organ and, therefore, could be excised with few negative consequences should post-transplantation complications arise.
In contrast to most other approaches in which islets are engrafted as dispersions, our BI retains the islets in a unified structure by means of a disk-shaped scaffold. The purpose of this scaffold is fourfold: (1) to keep the islets contained within a limited volume and in close proximity to the alginate delivery device in order to maximize the effects of the released cytokine; (2) to protect the relatively soft collagen hydrogel (which supports the islets directly) from physical disruption both before and after implantation; (3) to allow the BI, with all of its components, to be rapidly assembled and implanted easily without the additional complexity of a surgically produced omental/mesenteric pouch; (4) to allow the implant to be removed easily and maintained in a compact form that makes histological analysis straightforward. For the present experimental study (which has relatively short-term endpoints), nonbiodegradable PVA sponge works well as a scaffold. For therapeutic use in human patients, the PVA could be replaced by biodegradable materials that would be resorbed after the implanted islets become fully functional.
A beneficial effect of scaffolds on islet engraftment has been observed by others. Blomeier et al. (2
) reported that STZ-induced diabetic mice that received intraperitoneal implants of islets infused into cylindrical polylactide/glycolide (PLG) sponge scaffolds had higher conversions and shorter times to normoglycemia, greater weight gain, and improved response to intraperitoneal glucose tolerance tests compared to control mice that received islets not retained in scaffolds. These authors demonstrated that the islets transplanted in scaffolds remained localized at the original site of implantation, whereas the nonscaffolded islets tended to be more dispersed throughout the peritoneum. Therefore, the protective environment provided by the scaffold might have contributed to better performance of the graft. These results and our own observations that scaffolds facilitate the assembly and handling of multicomponent constructs argue for the continued development of scaffolds for islet transplantation.
In our BIs, direct physical support of the islets is accomplished by a fibrillar type I collagen hydrogel. The islets are dispersed in a single volume of monomeric collagen solution, which is infused into the protective PVA scaffold and polymerized in situ into a gel-like network of fibrils. Notably, neither the PVA scaffold, with its open structure, nor the collagen gel seem to impede the rate of vascularization from the mesentery, since dispersed islets implanted into mesenteric (omental) pouches also require the same time as our BIs to engraft and function (i.e., about 2 weeks) (16
The endocrine cells of islets in vivo are associated with a complex peri-insular and perivascular ECM, with components of the basement membrane (BM) (e.g., laminin, type IV collagen, fibronectin) predominating (38
). In this context, there is evidence that addition of specific BM components to islet graft sites improves islet graft performance. Salvay et al. (37
) demonstrated that islets implanted onto the epididymal fat pads of STZ-treated mice were more effective at reversing diabetes when the islets were infused into PLG sponge scaffolds preadsorbed with type IV collagen, compared with control scaffolds pretreated with serum proteins. Adsorbed fibronectin and laminin 5 were less effective than type IV collagen but were superior to the serum-treated controls. It was proposed that the adsorbed BM proteins might be improving islet function directly and/or promoting infiltration of beneficial cell types (e.g., endothelial cells) from the host, as vascularization of the ECM-treated grafts was better than vascularization of the serum-coated grafts. Collectively, these results suggest that incorporation of individual BM components (or perhaps more complex BM mixtures) into our BIs (e.g., via direct binding to or cogelation with the collagen hydrogel) might improve the overall performance of the grafts after implantation.
Reportedly, the level of vascularization of islets transplanted into the liver or kidney is lower than that of native islets in the pancreas (22
), but transfection of islets to express VEGF increases vascularization following transplantation (19
). Transfection-based approaches are therapeutically problematic; therefore, we incorporated a device (an alginate macrosphere) within the BI to achieve a local, sustained delivery of VEGF. In preliminary experiments, we produced BIs with macrospheres that incorporated high levels (160 ng) of VEGF. The VEGF in these constructs induced a very robust response from the host within 7 days of implantation in vivo, as indicated by high levels of angiogenesis, the presence of enlarged sinusoidal neovessels, and substantial vascular permeability (i.e., extravasated blood) within the BI. Although these results were a clear indication that the VEGF was biologically active, we considered this level of response to be excessive; therefore, we reduced the level of VEGF to approximately 20 ng per macrosphere for the subsequent experiments reported here. Inclusion of 20 ng of VEGF in the BI did not decrease the average time for transplanted mice to achieve normoglycemia (approximately 2 weeks), as compared to control mice with BIs that lacked VEGF. Although VEGF increased the percentage of mice that became normoglycemic compared to the controls lacking VEGF (100% vs. 62.5%), this increase was not statistically significant for our sample size (n
= 8 mice per group). These results suggest that exogenous VEGF may not have a major effect on implant performance when therapeutically “safe” (i.e., well above the minimum) numbers of islets are used.
We were particularly interested by the finding that, with the exception of one animal, the +VEGF/+Islet group did not exhibit the hypoglycemia that occurred within 24 h p-i in the −VEGF/+Islet group. Postoperative, transitory hypoglycemia has been observed in other models of islet transplantation and may be a consequence of an acute release of insulin from stressed or dying islets. Our observations suggest that the inclusion of VEGF within BIs mitigates this acute insulin release, perhaps via direct influences on the transplanted islets, as we found that exogenous VEGF suppresses glucose-stimulated release of insulin from isolated, cultured islets. This suppressive effect of exogenous VEGF on insulin release in vitro seems to support an earlier finding that islets isolated from RIP-CRE:VEGFfl/fl
mice, in which production of VEGF is prevented specifically in β-cells, had higher levels of insulin mRNA and secreted more insulin after glucose stimulation in vitro than did islets from control mice that expressed VEGF (15
). VEGF acts on endothelial cells to promote the maintenance and growth of vasculature, including the vasculature of islets, as illustrated by RIP-CRE:VEGFfl/fl
mice, which have deficiencies in their microvessels (14
). Although prosurvival, proangiogenic responses of intraislet endothelial cells to VEGF would be expected, additional responses by these cells might include the production of paracrine factors that promote the survival and function of islet endocrine cells.
In treatments of diabetic patients that involve transplantation of islets, controlling rejection is typically accomplished by systemic immunosuppressive compounds. Dosing of these compounds is a difficult balance—levels must be low enough to permit a reasonable degree of protective immunity against pathogenic organisms, but high enough to effectively suppress allo- and autoimmune activity directed against the transplant. In the case of SPK transplants, some current immunosuppression regimens are inadequate to control autoimmunity (18
). Moreover, no matter what the dose, systemic immunosuppression can be accompanied by a variety of undesirable side effects on tissue and organ systems that are not directly associated with the transplant. In light of the problems associated with systemic treatments, an alternative approach would be to confine the delivery of immunotherapy to the implant itself. In this way, immunomodulatory compounds could be delivered at relatively high concentrations, but within the limited volume of the implant, thereby minimizing side effects on tissues and organs outside the zone of delivery. To this end, the BI described here includes a mechanically supportive scaffold and collagen hydrogel that concentrates the islets in a small volume. Localized immunotherapy might be achieved by supplementing or replacing the collagen hydrogel with ECM components that have been shown to have immunosuppressive properties, such as hyaluronan (3
). This ECM-based approach could be augmented by sustained release of specific, immunomodulatory cytokines [e.g., interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)] from biocompatible storage media placed within the implant. We are currently investigating the use of alginate-based media for this purpose.
In the present study, we have developed a BI and associated protocols for its implantation that can effectively reverse STZ-induced diabetes in syngeneic mice. Our future studies will evaluate the effectiveness of the BI in the context of strains of mice that develop autoimmune diabetes. In this way, the BI will serve as a platform to evaluate the capability of a variety of immunomodulatory compounds and formulations, delivered locally, to prevent or reverse diabetes in the setting of autoimmune dysfunction.