Denis Dufrane

Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept.

Background. Pig islets xenotransplantation remains associated with a strong humoral and cellular xenogeneic immune responses. The aim of this study was to assess the long-term biocompatibility of alginate encapsulated pig islets after transplantation in primates Methods. Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) were transplanted under the kidney capsule of nondiabetic Cynomolgus maccacus. Additional primates received empty capsules (n=1) and nonencapsulated pig islets (n=2) as controls. Capsule integrity, cellular overgrowth, pig islet survival, porcine C-peptide and anti-pig IgM/IgG antibodies were examined up to 6 months after implantation Results. Nonencapsulated islets and islets encapsulated in nonoptimal capsules were rapidly destroyed. In seven primates receiving perfectly encapsulated pig islets, part of the islets survived up to 6 months after implantation without immunosuppression. Porcine C-peptide was detected after 1 month in 71% of the animals. The majority of grafts (86%) were intact and completely free of cellular overgrowth or capsule fibrosis. Explanted capsules, after 135 (n=2/2) and 180 (n=2/3) days, demonstrated residual insulin content and responses to glucose challenge (stimulation index of 2.2). Partial islet survival was obtained despite an elicited anti-pig IgG humoral response Conclusions. Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.

Alginate Macroencapsulation of Pig Islets Allows Correction of Streptozotocin-Induced Diabetes in Primates up to 6 Months Without Immunosuppression.

Background. This study assessed the capacity of alginate-encapsulated islets to reverse diabetes in a pig-to-primate model. Methods. Adult pig islets were encapsulated in microcapsules implanted under the kidney capsule (n=4) or in a subcutaneous macrodevice (n=5) in diabetic primates. Fasting blood glucose (FBG), insulin, porcine C-peptide, glycosylated hemoglobin (HbA1C), and cellular and humoral responses were followed. Results. Nonencapsulated pig islets were rejected within 7 days. A transient decrease of FBG was observed only during the 2 weeks after microencapsulated pig islet implantation under the kidney capsule. After subcutaneous transplantation of a macrodevice, diabetes was corrected up to a maximum of 6 months in five animals: FBG less than 107 mg/dL and HbA1C at 8%±1.4%. Two of the five animals received a new macrodevice between 25 and 35 weeks after the first graft dysfunction (HbA1C ≥13), and diabetes was controlled for an additional 18 weeks in these animals. Although a strong humoral response was elicited after transplantation of encapsulated islets, a total impermeability of alginate 3% wt/vol to IgG was demonstrated before and up to 20 weeks after transplantation of the subcutaneous macrodevice. Conclusions. Pig islets encapsulated in a subcutaneous macrodevice can control diabetes up to 6 months without immunosuppression.

Low molecular weight dextran sulfate: a strong candidate drug to block IBMIR in clinical islet transplantation.

The instant blood‐mediated inflammatory reaction (IBMIR) is triggered in clinical islet transplantation when human pancreatic islets come in contact with blood and may explain the initial tissue loss associated with this procedure. Low molecular weight dextran sulfate (LMW‐DS; MM 5000), today available for clinical use, inhibits both complement and coagulation activation. In a tubing loop model, LMW‐DS at concentrations ranging from 0.01 to 1 g/L showed a dose‐dependent inhibition of IBMIR with an inhibition of coagulation and complement activation and less consumption of platelets and other blood cells. In blood or plasma APTT was demonstrated to be an excellent method for monitoring the LMW‐DS concentration both in vitro and in vivo in a nonhuman primate model. The toxicity was assessed using a glucose challenge test and the pharmacokinetics was tested in the nonhuman primate model. Here, we present a tentative protocol for using LMW‐DS in clinical islet transplantation.

Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation.

Abstract:  Background:  A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non‐human primates. The detrimental instant blood‐mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen.

Streptozotocin-induced diabetes in large animals (pigs/primates): role of GLUT2 transporter and beta-cell plasticity.

Background. To induce irreversible diabetes in large animals, the efficiency of streptozotocin (STZ) was evaluated in pigs, primates and compared to the gold standard model in rats. Methods. Low (50 mg/kg) and high (150 mg/kg) doses of STZ were tested. Hepatic/renal function, glucose metabolism (intravenous glucose tolerance tests, fasting blood glucose) and histomorphometry were evaluated prior to, 1, and 4 weeks after STZ treatment. Results. In rats and primates, expressing a high level of GLUT2 expression on &bgr; cells, a dose of 50 mg/kg STZ induced irreversible diabetes (due to the 97% destruction of beta cell mass) without provoking liver or renal failure. In pigs, despite the use of high STZ dose, partial correction of hyperglycaemia was observed four weeks after STZ injection (decreased fasting blood glucose and intravenous glucose tolerance tests; increased insulin production). The correction of hyperglycaemia was associated with significant hypertrophy of immature pig &bgr;-cell clusters (+30%, P<0.05), whereas no hypertrophy was observed in rats/primates. Conclusion. These results demonstrated that STZ might be used to induce irreversible diabetes in rats and primates. In contrast, the low STZ sensitivity in pigs related to a low expression of GLUT2, higher number of immature &bgr; cells and compensatory &bgr;-cell hypertrophy, renders STZ-induced diabetes inappropriate for studying islet allografts in swine.

The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells

Adipose tissue was only recently considered as a potential source of mesenchymal stem cells (MSCs) for bone tissue engineering. To improve the osteogenicity of acellular bone allografts, adipose MSCs (AMSCs) and bone marrow MSCs (BM-MSCs) at nondifferentiated and osteogenic-differentiated stages were investigated in vitro and in vivo. In vitro experiments demonstrated a superiority of AMSCs for proliferation (6.1±2.3 days vs. 9.0±1.9 days between each passage for BM-MSCs, respectively, P<0.001). A significantly higher T-cell depletion (revealed by mixed lymphocyte reaction, [MLR]) was found for AMSCs (vs. BM-MSCs) at both non- and differentiated stages. Although nondifferentiated AMSCs secreted a higher amount of vascular endothelial growth factor [VEGF] in vitro (between 24 and 72 h of incubation at 0.1-21% O(2)) than BM-MSCs (P<0.001), the osteogenic differentiation induced a significantly higher VEGF release by BM-MSCs at each condition (P<0.001). After implantation in the paraspinal muscles of nude rats, a significantly higher angiogenesis (histomorphometry for vessel development (P<0.005) and VEGF expression (P<0.001)) and osteogenesis (as revealed by osteocalcin expression (P<0.001) and micro-CT imagery for newly formed bone tissue (P<0.05)) were found for osteogenic-differentiated AMSCs in comparison to BM-MSCs after 30 days of implantation. Osteogenic-differentiated AMSCs are the best candidate to improve the angio-/osteogenicity of decellularized bone allografts.

Impact of porcine islet size on cellular structure and engraftment after transplantation: adult versus young pigs.

Objectives: To study the impact of porcine islet size on structural properties and cellular engraftment. Methods: The endocrine structure and collagen/vascular localization in pig islets were studied before and after enzymatic isolation on the pancreas from 6 young and 6 adult Landrace pigs. Isolated islets from both pig types were transplanted under the kidney capsula of diabetic nude rats to assess cellular engraftment. Results: In comparison with adult pig pancreata, a significantly greater number of small beta cells (<100 μm) were observed before and after isolation (82% vs. 32%, respectively, P < 0.005) from young pig pancreata. Small islets (<100 μm) showed a peripheral vascular structure, whereas large islets showed a more centralized vascular organization, thereby providing protection during the enzymatic digestion procedure. The islet endocrine structure was not affected by the islet size, but a loss of glucagon cells (−7.9%, P < 0.005) was observed in large isolated islets. The purity of islet preparation was better with pancreata from adult than young donors (86% vs. 64%, respectively, P < 0.05). A lack of engraftment was observed for small islets from young pig donors as compared with large islets from adult donors. Conclusions: Large and well-structured islets, mainly found in adult pig pancreata, probably possess a better potential for cellular engraftment due to centralized vascularization and collagen distribution.

Pig islet xenotransplantation into non-human primate model.

Allogeneic islet transplantation faces difficulties because (1) organ shortage is recurrent; (2) several pancreas donors are often needed to treat one diabetic recipient; and (3) the intrahepatic site of islet implantation may not be the most appropriate site. Another source of insulin-producing cells, therefore, would be of major interest, and pigs represent a possible and serious source for obtaining such cells. Pig islet grafts may seem difficult because of the species barrier, but recent reports demonstrate that pig islets may function in primates for at least 6 months. Pig islet xenotransplantation, however, must still overcome several hurdles before becoming clinically applicable. The actual consensus is to produce more preclinical data in the pig-to-primate model as a necessary requirement to envisage any pig-to-human transplantation of islets; therefore, a summary of the actual acquired knowledge of pig islet transplantation in primates seemed useful and is summarized in this overview.

Macro- or microencapsulation of pig islets to cure type 1 diabetes.

Although allogeneic islet transplantation can successfully cure type 1 diabetes, it has limited applicability. For example, organs are in short supply; several human pancreas donors are often needed to treat one diabetic recipient; the intrahepatic site may not be the most appropriate site for islet implantation; and immunosuppressive regimens, which are associated with side effects, are often required to prolong survival of the islet graft. An alternative source of insulin-producing cells would therefore be of major interest. Pigs represent a possible alternative source of beta cells. Grafting of pig islets may appear difficult because of the immunologic species barrier, but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression. Therefore, a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation. Although several groups have shown that encapsulated pig islets are functional in small-animal models, less is known about the use of bioartificial pancreases in large-animal models. In this review, we summarize current knowledge of encapsulated pig islets, to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.

Indirect cytotoxicity evaluation of pseudowollastonite.

This study aimed to evaluate the cytotoxicity of substances leached by pseudowollastonite (CaSiO3). It has been previously shown that calcium (Ca2+) and silicate (SiO3−) ions are released from pseudowollastonite into biological solutions. Both of these ions are known to influence the biological metabolism of osteoblastic cells essential in the mineralization process and bone-bonding mechanism. The indirect toxicity evaluation was performed by extraction method, according to International Standard Organization (ISO). Pseudowollastonite pellets obtained by solid-state reaction were incubated, in culture medium, during 24, 48, 72 or 168 h at different concentrations (5, 10, 15, 50, 100, 200 mg/ml). The cytotoxicity of each extract in presence of human osteoblastic cell line (SaOS-2) was quantitatively assessed by measuring the viability (succinate dehydrogenase activity, MTT), the membrane integrity (the uptake of the neutral red by viable cells, NR) as well as the cell necrosis by measuring the lactate dehydrogenase (LDH) released in the culture medium. No significant alteration of membrane integrity or cell suffering was detectable. However, increased cell metabolism was observed for cells exposed to pseudowollastonite extract with longest extraction time (168 h). In conclusion, mineral elements leached by pseudowollastonite do not significantly affect the metabolism of osteoblastic cells.