Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets

Graft failure of alginate-polylysine microencapsulated islets is often interpreted as the consequence of a non-specific foreign body reaction against the microcapsules, initiated by impurities present in crude alginate. The aim of the present study was to investigate if purification of the alginate improves the biocompatibility of alginate-polylysine microcapsules. Alginate was purified by filtration, extraction and precipitation. Microcapsules prepared from crude or purified alginate were implanted in the peritoneal cavity of normoglycaemic AO-rats and retrieved at 1, 2, 3, 6, 9, and 12 months after implantation. With crude alginate, all capsules were overgrown within 1 month after implantation. With purified alginate, however, the portion of capsules overgrown was usually less than 10%, even at 12 months after implantation. All recipients of islet allografts in capsules prepared of purified alginate became normoglycaemic within 5 days after implantation, but hyperglycaemia reoccurred after 6 to 20 weeks. With intravenous and oral glucose tolerance test, all recipients had impaired glucose tolerance and insulin responses were virtually absent. After graft failure, capsules were retrieved (80-100%) by peritoneal lavage. Histologically, the percentage of overgrown capsules was usually less than 10% and maximally 31%. This small portion cannot explain the occurrence of graft failure. The immunoprotective properties of the capsules were confirmed by similar if not identical survival times of encapsulated islet allo- and isografts. Our results show that purification of the alginate improves the biocompatibility of alginate-polylysine microcapsules. Nevertheless, graft survival was still limited, most probably as a consequence of a lack of blood supply to the encapsulated islets

Characterisation of physico-mechanical properties and degradation potential of calcium alginate beads for use in embolisation

High molecular weight alginate beads with 59% mannuronic acid content or 68% guluronic acid were prepared using a droplet generator and crosslinked in calcium chloride. The alginate beads were compared to current embolisation microspheres for compressibility and monitored over 12 weeks for size and weight change at 37°C in low volumes of ringers solutions. A sheep uterine model was used to analyse bead degradation and inflammatory response over 12 weeks. Both the in vitro and in vivo data show good delivery, with a compressibility similar to current embolic beads. In vitro, swelling was noted almost immediately and after 12 weeks the first signs of degradation were noted. No difference was noted in vivo. This study has shown that high molecular weight alginate gel beads were well tolerated by the body, but beads associated with induced thrombi were susceptible to inflammatory cell infiltration. The beads were shown to be easy to handle and were still observable after 3 months in vivo. The beads were robust enough to be delivered through a 2.7 Fr microcatheter. This study has demonstrated that high molecular weight, high purity alginate bead can be considered as semi-permanent embolisation beads, with the potential to bioresorb over time

THE EFFECT OF GLUCOSE STIMULATION ON CA-45 UPTAKE OF RAT PANCREATIC-ISLETS AND THEIR TOTAL CALCIUM CONTENT AS MEASURED BY A FLUOROMETRIC MICROMETHOD

Glucose-stimulated 45calcium uptake and total calcium content of rat pancreatic islets has been studied, using a new fluorometric micro-method to estimate total calcium. Extracellular calcium was separated from incubated tissue by a rapid micro-filtration procedure. Islets incubated up to 60 min with calcium chloride 2.5 mmol/l and glucose 2.5 mmol/l maintained the same calcium content (670±7.5 pmol/μg DNA). When the glucose concentration was raised to 15 mmol/l no change in the total calcium content could be detected. On incubation with glucose 2.5 mmol/l in the absence of calcium, the calcium content decreased to 488±27 pmol/μg DNA. On incubation with 45calcium chloride 2.5 mmol/l for 5 or 30 min at 2.5 mmol/l glucose, islets exchanged 21 ±2 and 28±1% of their total calcium content and, at 15 mmol/l glucose, 30±3 and 45±2%, respectively. Thus, islet calcium has a high turn-over rate. Glucose stimulation results in an increase of the calcium uptake without enhancing the total calcium content and hence must increase the calcium-exchangeable pool

EFFECT OF ALGINATE-POLYLYSINE-ALGINATE MICROENCAPSULATION ON INVITRO INSULIN RELEASE FROM RAT PANCREATIC-ISLETS

We investigated the effect of alginate-polylysine-alginate microencapsulation on glucose-induced insulin secretion by rat islets. Applying the encapsulation method originally described by Lim, we found severely reduced in vitro insulin release (expressed as picomoles of insulin · 10 islets−1 · 45 min−1 when incubated in 16.5 mM glucose), because the insulin release with encapsulated islets was 1.42 ± 0.49 compared to 13.58 ± 0.80 with free control islets. This could not be explained by inadequate permeability of the capsule, because insulin release was also severely reduced (2.12 ± 0.61) when islets were subjected to the procedure but without the membrane-forming polylysine step. Therefore, islets were tested after having been subjected separately to each of the steps of the procedure. Insulin release was not affected by either alginate or CaCI2 but was severely reduced after prolonged suspension in saline or treatment with citrate. When saline and citrate were replaced by Ca2+-free Krebs-Ringer bicarbonate buffer (KRBB) and 1 mM EGTA, respectively, insulin release improved significantly both with complete and with incomplete (no polylysine step) encapsulation. This outcome was verified in a set of experiments run in parallel with islets derived from the same isolation procedure. Insulin release was 1.20 ± 0.23 from islets encapsulated with the method of Lim and 10.73 ± 1.04 from free control islets. With the modified procedure, insulin release was 9.17 ± 0.52 vs. 9.61 ± 1.27 for complete versus incomplete encapsulation, respectively. We conclude that Ca2+-free KRBB instead of saline and EGTA instead of citrate should be used to obtain an adequate insulin response from encapsulated islets and that the capsule membrane as such has no influence on glucose and insulin diffusion