Intravital microscopic analysis of vascularization of transplanted pancreatic islets, pseudoislets and modified pseudoislets

Abstract

Background: During the last years, the transplantation of isolated pancreatic islets has been proposed as an ideal therapeutic strategy for the treatment of type 1 diabetes mellitus. However, the applicability of this therapy is limited, because it requires the transplantation of a large number of islets in order to induce normoglycemia. In order to overcome this problem, generation of modified islets by combining islet cells with bone marrow derived cells may represent a promising approach to increase the amount of transplantable tissue. Moreover, a major prerequisite for the success of this therapy is a rapid and sufficient vascularization of the grafts in order to guarantee their survival and long-term function. The aim of the present study was therefore to analyze whether the addition of bone marrow cells to islet cells accelerates the vascularization after transplantation. Materials and Methods: Langerhans islets of Syrian golden hamsters were isolated by collagenase digestion of the pancreas. The isolated islets were dispersed into single cells by trypsin treatment and then cultured for 3 days to generate pseudoislets by reaggregation of the cells. Furthermore, the bone marrow of the femur and tibia of donor hamsters was removed and co-cultured with monolayer cultures of islet cells to create modified pseudoislets. Subsequently, 7–8 pancreatic islets, pseudoislets (without bone marrow cells) and modified pseudoislets (with bone marrow cells) were transplanted into the dorsal skinfold chamber of recipient animals. Using the technique of intravital fluorescence microscopy we then analyzed angiogenesis, vascularization and microhemodynamics of the grafts over a 14-day observation period. Results: The islet grafts exhibited a size of 0.06–0.09mm2 after transplantation into the dorsal skinfold chamber without any significant differences between the groups. First signs of angiogenesis, i.e. capillary sprouts, could be observed in the grafts of all three groups at day 3. Interestingly, modified pseudoislets exhibited an improved vascularization during the further time course, as indicated by a significantly increased functional capillary density of 365±18 cm/cm2 (p<0.05) at day 6 when compared to islets (223±20 cm/cm2) and pseudoislets (211±37 cm/cm2). Moreover, newly formed blood vessels in modified pseudoislets presented with increased diameters (day 6:7.9±0.1 μm; p<0.05) and volumetric blood flow (day 6:6.7±0.4pl/s; p<0.05) when compared to those of islets (6.8±0.2 μm and 4.0±0.6 pl/s) and pseudoislets (7.0±0.1 μm and 4.5±0.8 pl/s). Conclusions: In the present study, we could demonstrate that modified pseudoislets created by the combination of pancreatic islet cells and bone marrow derived cells exhibit an improved vascularization after transplantation when compared to normal islets. This may be most probably due to the angiogenic potential of stem cells included in the bone marrow. Thus, generation of modified pseudoislets may represent a promising approach in order to improve the success rates of islet transplantation for the treatment of diabetes mellitus.