Biocompatibility and function of microencapsulated pancreatic islets

Encapsulation of pancreatic islets in alginate is used to protect against xenogenic rejection in different animal models. In this study, several factors, including differences in alginate composition, the presence or absence of xenogenic islet tissue and a transient immunosuppression, were investigated in a model of bovine islet transplantation in rats. A pure alginate with predominantly guluronic acid (Manugel) and an ultrapure low viscosity guluronic acid alginate (UP-LVG) were used. When microcapsules of Manugel or UP-LVG containing 16,000 bovine islet equivalents were transplanted in diabetic rats, we observed normoglycemia for 8.3+/-0.7 (range 6-12 days) and 7.5+/-0.2 days (range 7-8 days) on average, respectively. To ameliorate immunoprotection of alginate microcapsules we repeated the same experiments using transient immunosuppressive therapy. Low doses of cyclosporin A (CyA) administered for 18 days after implantation increased the time in normoglycemia, which averaged 27+/-3 days (range 8-55 days) in Manugel capsules while in UP-LVG capsules it averaged 18+/-8 days (range 3-39 days). The surface of recovered capsules showed less capsules free of overgrowth in Manugel with respect to UP-LVG alginate. These data were comparable with those observed in empty microcapsules similarly implanted, indicating that the capsular overgrowth was not promoted by the presence of xenogenic islet tissue. In recovered Manugel capsules the percentage of capsules without fibrotic overgrowth was higher than that observed without CyA. The same observation was made in empty capsules. These observations indicate that a combination of a highly purified alginate and short-term immunosuppression prolong islet function in a model of xenotransplantation.     

Effect of cross-linked hemoglobin on functionality and viability of microencapsulated pancreatic islets

Of many obstacles involved in developing a bioartificial pancreas, which consists of encapsulated and physically immunoprotected islets, for long-term implantation in insulin-dependent diabetic patients, the impaired functionality and decreasing viability of encapsulated islets over time are critical factors in determining the size and longevity of the implant. These factors are closely associated with short oxygen supply to the encaged islets from the implant site. To facilitate oxygen transport to islets in the capsules, we coencapsulated hemoglobin cross-linked with difunctional polyethylene glycol (Hb-conjugate, Hb-C) which is large in size (>100 kDa), thus preventing diffusional loss through the immunoprotecting membrane. The coencapsulation of Hb-C with islets in alginate-poly-L-lysine microcapsules by dissolving Hb-C in an islet-suspended alginate solution at a concentration of 0.25 mM improved the insulin secretion and viability of the islets. At week 0, the islets, coencapsulated with Hb-C, cultured at P(O2) = 40 mmHg (assumed oxygen partial pressure in the most common implant site, the peritoneal cavity), secreted 200% more insulin compared with the control islets without Hb-C at glucose concentrations of both 100 and 300 mg/dL. The Hb-C effect became more significant with time at higher glucose concentrations. After culturing the islets for 8 weeks at 40 mmHg, the insulin secretion was enhanced 200 and 550% at glucose concentrations of 100 and 300 mg/dL as compared with the control, respectively. The results were closely associated with improved viability and suggest that the introduction of Hb-C is an effective approach to maintaining the oxygen supply to encapsulated islets. In addition, Hb-C coencapsulation with pancreatic islets may (1) provide a partial clue to reducing the large size of the biohybrid artificial pancreas, (2) lead to a reduced need for pancreas donation, and (3) prolong the longevity of the biohybrid artificial pancreas in the body.     

In vitro biocompatibility and bioactivity of microencapsulated heparan sulfate

The glycosaminoglycan sugar heparan sulfate (HS) is an attractive agent for the repair of bone defects due to its ability to regulate endogenous growth factors. The sustained delivery of HS to the localized wound site over the period of healing which can last for over 1 month may prove advantageous for its therapeutic use. In this study we investigated the encapsulation of HS by the water-in oil-in water (W(1)/O/W(2)) technique in polycaprolactone (PCL) microcapsules as a prolonged delivery device. Encapsulation efficiencies of 70% could be achieved by using a 1:1 mixture of dichloromethane (DCM) and acetone as the solvent in the organic phase, while DCM alone gave poor encapsulation. Although addition of polyvinyl alcohol (PVA) to the drug phase did not affect the size or drug loading of the microcapsules, it did however produce a large change in the morphology and drug distribution, which resulted in different release rates. Release from capsules made with PVA in the drug phase reached 60% after 40 days, while those made with water in the drug phase completed release after 20 days. In vitro biocompatibility studies were performed and detected no increase in cell death in human mesenchymal stem cells (hMSC) or induction of an inflammatory response in macrophages after exposure to release products from HS-loaded microcapsules. The released HS retained its ability to increase the proliferation of hMSC after the encapsulation process. These results indicate that encapsulation of HS by the W(1)/O/W(2) method creates a promising device for the repair of bone tissue.     

Transplantation of microencapsulated pancreatic human islets for therapy of diabetes mellitus. A preliminary report.

To circumvent immune destruction of pancreatic islet grafts, human islets were deposited, enveloped within algin-polyaminoacidic microcapsules, in artificial prostheses directly anastomosed to blood vessels. Three dogs and two patients with insulin-dependent diabetes received transplantations with microencapsulated human islets embodied in arterial or AV prosthesis bypasses without any pharmacologic immunosuppression. Graft function was documented in all recipients by significative production and sustenance of serum C-peptide levels, although insulin independence was consistently achieved in only one of three dogs and, transiently, in one of two patients. On retrieval of grafted prostheses, viable human islets were found in microcapsules, most of which were freely dispersed in the vascular chambers. Vascular prostheses may represent a suitable site of implantation for large numbers of human islets, immunoprotected in semipermeable and biocompatible microcapsules, and be a novel strategy for therapy of insulin-dependent diabetes mellitus (IDDM).     

肝前性门脉高压大鼠胰岛功能的变化

目的：探讨肝前性门脉高压大鼠胰岛α、β细胞功能及其与循环血液中胰岛素及胰高血糖素水平变化的关系。方法：采用部分结扎门静脉的方法复制肝前性门脉高压,假手术及正常鼠的胰岛进行分离和纯化,并对其体外生物学活性进行检测。结果：肝前性门脉高压组大鼠循环血液中胰岛素及腹高血糖素水平明显高于正常组大鼠（P＜0．01）。肝前性门脉高压级大鼠分离纯化的胰岛经体外培养48h或在含糖基质中孵育2h后释放胰岛素量明显低于正常,而陵高血糖素明显高于正常（P＜0．01）。结论：胰岛α细胞分泌功能增加可能有助于解释高胰高血糖素血症的发生;门脉高压时,循环血液中胰岛素水平增加,但哪胞功能低于正常。     

The development of alternative vitrification solutions for microencapsulated islets

Bioartificial pancreas in which islets of Langerhans (islets) are enclosed in a semipermeable membrane is one of the approaches to treat insulin-dependent diabetic patients. Although there are advantages in this method, one of the issues that still remains is the long-term storage of tissue engineering devices before transplantation. One of the possible routes to address this is through cryopreservation. In this study, a freezing solution, 2 m DMSO in RPMI-1640, a conventional vitrification solution, VS55, and the newly developed vitrification solution KYO-1 were examined to cryopreserve microencapsulated islets in agarose hydrogel. The insulin release ability, morphology of islets, and physico-chemical properties of the agarose gel membrane were examined after a cryopreservation and thawing process. Frozen and vitrified (by KYO-1) groups showed a similar insulin secretion. Frozen groups by 2 m DMSO, however, showed destruction of agarose capsules and some islets were out of the capsule. When KYO-1 was used, islets still maintained the ability to release insulin in response to glucose stimulation, and agarose capsule showed morphological integrity, and mechanical properties. In conclusion, vitrification using KYO-1 which is composed of 5.38 m ethylene glycol, 2 m DMSO, 0.1 m PEG 1000 and 0.00175 m PVP K10 in EuroCollins, is a suitable method for cryopreservation of microencapsulated islets.     

Amylin in pancreatic islets and pancreatic endocrine neoplasms

Amylin is a chief constituent of the amyloid present in insulinomas, and is colocalized in beta islet cells. By immunocytochemical staining, all four islet cells including insulin, glucagon, somatostatin (SRIF) and pancreatic polypeptide (PP) cells were positively stained for amylin. The strongly insulin-positive cells corresponded with the strongly amylin-positive cells, and glucagon cells appeared to be strongly positive for amylin, whereas SRIF and PP cells were weakly positive for amylin. Among 37 cases of pancreatic endocrine neoplasms, insulinomas were more stronger stained for amylin than other islet cell tumors; however, amylin staining was the same or weaker than insulin staining. Glucagonomas and PP-omas were weakly positive for amylin, whereas six of 11 gastrinomas were weakly positive for amylin. It is concluded that three orthoendocrine tumors including insulinomas, glucagonomas and PP-omas were all positive for amylin, whereas ectopic hormone secreting gastrinomas were positive for amylin in six of 11 cases (55%). This colocalization of amylin with insulin, glucagon and PP may support a structure-function relationship of amylin and pancreatic hormones. The lesser immunoreactive amylin in pancreatic endocrine neoplasms than in normal islet cells may contribute to autonomous hypersecretion of hormones by pancreatic endocrine neoplasms.     

Dual origin,development, and fate of bovine pancreatic islets

Endocrine cells are evident at an early stage in bovine pancreatic development when the pancreas still consists of primitive epithelial cords. At this stage, the endocrine cells are interspersed between the precursor cells destined to form the ductulo‐acinar trees of later exocrine lobules. We here demonstrate that, in bovine fetuses of crown rump length ≥ 11 cm, the endocrine cells become increasingly segregated from the developing exocrine pancreas by assembly into two units that differ in histogenesis, architecture, and fate. Small numbers of ‘perilobular giant islets’ are distinguishable from larger numbers of ‘intralobular small islets’. The two types of islets arise in parallel from the ends of the ductal tree. Aside from differences in number, location, and size, the giant and small islets differ in cellular composition (predominantly insulin‐synthesising cells vs. mixtures of endocrine cells), morphology (epithelial trabeculae with gyriform and rosette‐like appearance vs. compact circular arrangements of endocrine cells), and in their relationships to intrapancreatic ganglia and nerves. A further difference becomes apparent during the antenatal period; while the ‘interlobular small islets’ persist in the pancreata of calves and adult cattle, the perilobular giant islets are subject to regression, characterised by involution of the parenchyma, extensive haemorrhage, leukocyte infiltration (myeloid and T‐cells) and progressive fibrotic replacement. In conclusion, epithelial precursor cells of the ductolo‐acinar tree may give rise to populations of pancreatic islets with different histomorphology, cellular composition and fates. This should be taken into account when using these cells for the generation of pancreatic islets for transplantation therapy.     

Development of pancreatic islets in pancreatic polypeptide-overexpressing mice

Recently we produced pancreatic polypeptide transgenic (PPTG) mice and found that PP was overexpressed in pancreatic islets. The present study examines development of four islet hormones in PPTG mice at embryonic days (ED) 15, 17, and 19, and in adult animals. Adult PPTG mice showed massive aggregation of PP-positive cells and glucagon-positive cells seen at the central area of the islets. Confocal laser microscopic study showed that three islet hormones (insulin, glucagon and PP) were completely overlapped in islets of PPTG mice. Overlapping of somatostatin/glucagon and somatostatin/PP were also increased at the peripheral area of the islets in adult PPTG mice compared to wild-type mice. In prenatal development of pancreatic islets of PPTG mice, somatostatin/glucagon overlapping cells appeared at ED 15, two days earlier than in wild-type mice. Differentiation of these somatostatin/glucagon double-positive cells into single-positive cells was disturbed in the PPTG mice during perinatal to postnatal periods. Differentiation of glucagon/insulin-double positive cells into single-positive cells was disturbed remarkably in postnatal development of the islets of PPTG mice. The present results suggest that early and overexpression of PP may engender the early appearance of somatostatin producing cells; however, that may disturb differentiation of multihormonal immature endocrine cells into single hormonal mature endocrine cells.     

Long-term biocompatibility of implanted polymer-based intrafascicular electrodes

Polymer-based longitudinal intrafascicular electrodes (polyLIFEs) were chronically implanted into the sciatic nerve of white New Zealand rabbits (n=8) for a period of 6 months (hereafter referred to as the long-term group). The impact of the implantation procedure, as observed 6 months post surgery, was evaluated in a sham-treated control group (n=9). The contralateral sciatic nerve served as the control for each animal. Nerve-fiber counts, fiber diameters, and myelin thickness were estimated at the level of the implant site, 1.5 cm proximally, and 1.5 cm distally for both nerves in sham-treated and long-term groups. Implantation of polyLIFEs had no significant effect on fiber counts, nerve-fiber diameter, or myelin thickness. A slight increase in connective tissue in the vicinity of the implant site was evident in the long-term group, including a thin but dense capsule immediately surrounding the implanted electrode.     

Transthyretin, identified by proteomics, is overabundant in pancreatic juice from pancreatic carcinoma and originates from pancreatic islets

Analyses of pancreatic juice by proteomics have identified many proteins that are overabundant in pancreatic cancer (PC) juice. The mechanism by which secretion of these proteins occur remains unclear. Pancreatic juice was collected from patients with three pancreatic diseases: PC, chronic pancreatitis (CP), and simple choledocholithiasis (CDS), and analyzed by 2-DE, MALDI-TOF/MS, and Western blot. Five PC cell lines, 30 PC tissues and their corresponding adjacent pancreatic tissues were used to validate the expression of genes which code for overabundant proteins in PC juice. The mRNA and protein levels were measured by RT-PCR and immunohistochemistry, respectively. Using proteomics, it was demonstrated that the protein transthyretin (TTR) was upregulated more than 2-fold in PC juice compared with CP and CDS, while apolipoprotein A-I, lithostathine, and regenerating islet-derived 1 beta precursor were downregulated more than 2-fold. Western blots confirmed that TTR was overabundant in the PC juice. However, TTR mRNA was not detected in any of the five PC cell lines, and was only detected in islet cells. By microscopy, it was shown that islet architecture was almost completely destroyed, and the islet's maximum diameter appeared larger in PC tissues than in normal. Some overabundant proteins in PC juice, such as TTR expressed only in islets, leak into the pancreatic ductal system due to hyperplasia and architectural damage in PC tissues. The destruction of organ and tissue architecture by tumor growth may result in novel tumor markers even if the markers are not secreted directly by tumor cells.     

Subcutaneous xenotransplantation of bovine pancreatic islets

Transplantation of pancreatic islets in diabetes is currently limited by the need of immunosuppressive therapy. The present study was designed to test an immunoprotection planar device for subcutaneous xenotransplantation of pancreatic islets in the diabetic rat. We tested three different devices made of polyethersulfone hollow fibers. In all diabetic rats, implantation of islet-containing devices promptly normalized hyperglycemia. In vitro membrane permeability to glucose was correlated with implant function duration. These data confirm that bovine islets contained within devices and implanted subcutaneously remain functional for several days. Strategies to prolong islet function may allow achieving successful long-term islet implantation in this attractive site.     

Demonstration of calcium in pancreatic islets

Summary The GBHA [=glyoxal bis (2-hydroxyanil)] technique allows light microscopic demonstration of mobile calcium in some soft tissues.We used this method to compare granulation and calcium content of B cells of mice in the normal state and under conditions of suppression and of acute and chronic stimulation. For the suppression of B cells, hypoglycaemia was induced by the injection of bovine insulin. Acute stimulation of B cells by hyperglycaemia was achieved by a single injection of anti-insulin serum of guinea-pigs, and chronic stimulation by repeated injections of this antiserum for 4 days.In untreated animals the GBHA reaction indicated high amounts of calcium located selectively within the cytoplasm of B cells. In contrast to the degree of granulation, the calcium content showed slight variations from islet to islet, which may indicate differences in the secretory activity of the different islets at the time of sacrifice. Cellular calcium seemed to be loosely bound; it was possible to remove it with the aid of aqueous fixation fluids, while the addition of oxalate caused it to be retained. The suppressed B cells showed marked granulation and a uniformly high calcium content. After acute stimulation, there was a slight decrease of granulation in some B cells; the amount of calcium varied from cell to cell and was markedly elevated in some. The differences in the animal groups were only discrete and demand further evaluation. In contrast, after chronic stimulation the B cells showed signs of hypersecretory degeneration, marked degranulation and an almost total loss of stainable calcium. In addition we observed slight insulitis, which is thought not to be a consequence of chronic stimulation, however, but a response to the deposition of immune complexes within the islet.Our findings suggest that the high amounts of calcium detectable in the B cells by light microscopy depend predominantly on the degree of granulation. In addition, in cases of identical granulation an increased secretory activity seems to induce a slight elevation of cellular calcium.

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Zusammenfassung Mit GBHA [= Glyoxal bis (2-hydroxyanil)] ist in einigen Weichteilgeweben eine lichtmikroskopische Anfärbung des mobilen Calcium möglich.Der Calciumgehalt der Pankreasinseln von Mäusen mit akut und chronisch stimulierten, supprimierten und normalen B-Zellen wurde mit dieser Methode vergleichend untersucht und mit dem Gehalt an B-Zell-Granula in Beziehung gebracht. Die B-Zell-Suppression mit ausgeprägter Hypoglycaemie erfolgte durch einmalige Injektion von Rinderinsulin. Eine akute Stimulierung der B-Zellen mit Hyperglycaemie wurde durch einmalige Gabe von Anti-Insulinserum vom Meerschweinchen 90 bzw. 180 min vor der Tötung, eine chronische Stimulierung ebenfalls mit Hyperglycaemie durch zweimal tägliche, insgesamt viertägige Gabe des Antiserums erzielt.Mit der GBHA-Reaktion ließ sich bei den unbehandelten Tieren selektiv im Cytoplasma der B-Zellen ein hoher Calciumgehalt darstellen. Dieser variierte im Gegensatz zu dem Granulierungsgrad von Insel zu Insel etwas, was möglicherweise auf Unterschiede in der sekretorischen Aktivität der einzelnen Inseln zum Zeitpunkt der Tötung hinweist. Das celluläre Calcium ist locker gebunden, in wässerigen Fixierungsmitteln ausschwemmbar und durch Oxalat im Gewebe retinierbar. Die supprimierten B-Zellen zeigten bei starker Granulierung einen gleichmäßig hohen Calciumgehalt, während die akut stimulierten B-Zellen vereinzelt bereits eine geringe Abnahme der Granulierung und einen von Zelle zu Zelle unterschiedlichen, teils deutlich vermehrten Calciumgehalt aufwiesen. Die Unterschiede in den vorgenannten Tiergruppen waren insgesamt nur gering und erfordern eine weitere Überprüfung. Im Gegensatz dazu zeigten die chronisch stimulierten B-Zellen neben den Zeichen der hypersekretorischen Degeneration eine starke Degranulierung und einen fast vollständigen Verlust des nachweisbaren Calciums. Außerdem trat eine mäßige Insulitis auf, die jedoch nicht als Folge der chronischen Stimulierung, sondern als Antwort auf die Ablagerung von Immunkomplexen in der Insel aufgefaßt wird.Die Befunde zeigen, daß der lichtmikroskopisch nachweisbare hohe Calciumgehalt der B-Zelle in erster Linie von dem Granulierungsgrad abhängt. Bei gleicher Granulierung scheint darüber hinaus eine gewisse Zunahme des cellulären Calciumgehaltes bei steigender sekretorischer Aktivität aufzutreten.

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Supported by SFB 34, Hamburg.     

Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4±30.1×10⁶) compared with empty capsules (41.4±19.7×10⁶) and non-encapsulated porcine islets (18.1±3.3×10⁶). The percentage of CD 3 positive T-lymphocytes rose to 44.5±11.5% in case of microencapsulated xenografts compared with 19.2±8.2% for non-encapsulated xenografts and 4.9±2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2±8.9% vs 15.2±7.0% for free islets and 4.9±1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.     

Effect of secretory activity of the salivary glands on morphology and function of the pancreatic islets

Chronic experiments on dogs showed that the loss of saliva by the animals through exteriorized ducts of the parotid, submandibular, and sublingual salivary glands causes morphohistochemical changes in the pancreatic islets indicative of reduced functional activity of the cells.Departments of Histology and Embryology and of Normal Physiology, Tomsk Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR I. V. Toroptsev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1397–1399, November, 1976.     

The role of pancreatic islets in experimental pancreatic carcinogenicity.

Our previous studies have suggested that the presence of intact islets is essential for the induction of pancreatic exocrine tumors in the Syrian hamster model. To validate this, we investigated the effect of the carcinogen, N-nitrosobis(2-oxo-propyl)amine (BOP) in hamsters, in which homologous isolated intact islets were transplanted into the submandibular gland (SMG). Freshly isolated pure islets from hamster donors were transplanted into the left SMG of 20 female host hamsters. Ten of these hamsters (group 1) received BOP (40 mg/kg) weekly for 3 weeks. Another 10 hamsters (group 2) were kept untreated. In groups 3 and 4 (10 hamsters each) the salt solution or isolated pancreatic ductal cells, respectively, was injected into the gland. In other groups (10 hamsters each) islets were transplanted into the peri-SMG connective tissue (group 5) or into the renal subcapsular space (group 6). Hamsters of group 1 (40 mg/kg, weekly for 3 weeks) as were group 7 hamsters, which served as BOP-treated controls. All BOP-treated hamsters developed pancreatic lesions. Similar hyperplastic and atypical ductal/ductular proliferation and in situ carcinoma were found in the SMG of many group 1 hamsters. No such lesions were found in the SMG, peri-SMG, or renal subcapsular space of the other groups. Islets appear to be involved in carcinogenicity of BOP. The mechanism is obscure.     

Amyloidosis of the pancreatic islets and diabetes mellitus

Pancreas was examined in 136 patients who died at the age of 7 to 89 years of various diseases including 22 with diabetes mellitus. Amyloidosis of its islands was observed in 9 patients (aged 49 and over); 6 out of them suffered from diabetes mellitus. Number of islands with amyloidosis and amyloid quantity were determined morphometrically. Glucagon-producing A-cells and insulin-producing B-cells in the islands not involved in amyloidosis were counted in sections impregnated by Grimelius. It is found that the development of diabetes is determined not only by the islands amyloidosis but by the quantitative domination of A-cells over B-cells in the islands without amyloidosis as well being the manifestation of aging processes.