The Optimal Timing for Pancreatic Islet Transplantation into Subcutaneous Scaffolds Assessed by Multimodal Imaging

Abstract

Subcutaneously implanted polymeric scaffolds represent an alternative transplantation site for pancreatic islets (PIs) with the option of vascularisation enhancement by mesenchymal stem cells (MSC). Nevertheless, a proper timing of the transplantation steps is crucial. In this study, scaffolds supplemented with plastic rods were implanted into diabetic rats and two timing schemes for subsequent transplantation of bioluminescent PIs (4 or 7 days after rod removal) were examined by multimodal imaging. The cavities were left to heal spontaneously or with 10 million injected MSCs. Morphological and vascularisation changes were examined by MRI, while the localisation and viability of transplanted islets were monitored by bioluminescence imaging. The results show that PIs transplanted 4 days after rod removal showed the higher optical signal and vascularisation compared to transplantation after 7 days. MSCs slightly improved vascularisation of the graft but hindered therapeutic efficiency of PIs. Long-term glycaemia normalisation (4 months) was attained in 80% of animals. In summary, multimodal imaging confirmed the long-term survival and function of transplanted PIs in the devices. The best outcome was reached with PIs transplanted on day 4 after rod removal and therefore the suggested protocol holds a potential for further applications.

Figure 1

Design of the experiment. Scaffolds were divided into 6 experimental groups (A–F) according to the day of transplantation (Tx) of mesenchymal stem cells (MSCs) or pancreatic islets (PIs). Phosphate buffered saline (PBS) was added to the control scaffolds. The star indicates a photograph of a scaffold supplemented with a long polytetrafluorethylene rod.

Figure 2

In vitro characteristics of isolated MSCs and PIs. Optical images of isolated PIs (a). The numbers below the images represent the amount of PIs in the well. Representative images of adipocytes (b), osteocytes (c), and chondrocytes (d) differentiated from isolated MSCs. The scale bar size is 200 μm (b, c) and 400 μm (d).

Figure 3

Glycaemia and body weight of animals during the examination. Blood glucose levels (BGL) of animals during examination show normalisation of glycaemia in groups A-D and diabetes reversal in groups B and D (a). The arrows show the day of pancreatic islet transplantation (Tx PIs) and the day of insulin pellet removal. Body weight changes of animals with functional and nonfunctional grafts (b). Nonfunctional grafts are represented by a dotted line.

Figure 4

Representative MR images of scaffolds. Representative DCE-MR image of an animal with an implanted scaffold with MSCs and PIs (a) after contrast agent administration. The arrows indicate the kidneys (K), the scaffold, and a reference tube. Representative T₂-weighted anatomical MR images of the scaffolds of different groups on day 7 after scaffold implantation (b).

Figure 5

DCE-MR analysis related to vascularisation. Differences in AUC between scaffolds with and without MSCs and controls in animal groups with pancreatic islets transplanted on days 4 (a) and 7 (b) after rod removal. Comparison of pancreatic islet transplantation on days 4 and 7 after rod removal according to AUC values in scaffolds without (c) and with MSCs (d). ^∗p < 0.05,   ^∗∗p < 0.01.

Figure 6

In vivo bioluminescence imaging. Representative in vivo bioluminescence images of pancreatic islets transplanted into scaffolds without (a) and with MSCs (b). Images show the scaffolds on day 7 after PI transplantation. Differences between optical signals originating from pancreatic islets transplanted on day 4 and day 7 after rod removal without (c) and with MSC support (d). D4 and D7 refer to the day after rod removal. ^∗p < 0.05.

Figure 7

Histology analysis. Representative images of transplanted pancreatic islets in scaffolds stained with haematoxylin/eosin (H&E) and immunohistochemically with primary antibodies anti-insulin and CD31. Viable islets containing insulin positive cells were present in the scaffolds (H&E) in all groups A-D. Endothelial structures (CD31) were found in close proximity to the islets. Scale bars correspond to 100 µm.

Figure 8

Microvascular density analysis. Representative images of CD-31 stained tissue sections from scaffolds. The arrows point vascular structures that were counted for MVD analysis in groups A (a), B (b), C (c), and D (d). Scale bars correspond to 50 μm.