A Gelatin Hydrogel Nonwoven Fabric Combined With Adipose Tissue-Derived Stem Cells Enhances Subcutaneous Islet Engraftment

Abstract

Subcutaneous islet transplantation is a promising treatment for severe diabetes; however, poor engraftment hinders its prevalence. We previously revealed that a gelatin hydrogel nonwoven fabric (GHNF) markedly improved subcutaneous islet engraftment. We herein investigated whether the addition of adipose tissue-derived stem cells (ADSCs) to GHNF affected the outcome. A silicone spacer sandwiched between two GHNFs with (AG group) or without (GHNF group) ADSCs, or a silicone spacer alone (Silicone group) was implanted into the subcutaneous space of healthy mice at 6 weeks before transplantation, then diabetes was induced 7 days before transplantation. Syngeneic islets were transplanted into the pretreated space. Intraportal transplantation (IPO group) was also performed to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and inflammatory mediators were evaluated. The results in the subcutaneous transplantation were compared using the Silicone group as a control. The results of the IPO group were also compared with those of the AG group. The AG group showed significantly better blood glucose changes than the Silicone and the IPO groups. The cure rate of AG group (72.7%) was the highest among the groups (GHNF; 40.0%, IPO; 40.0%, Silicone; 0%). The number of vWF-positive vessels in the subcutaneous space of the AG group was significantly higher than that in other groups before transplantation (P < 0.01). Lectin angiography also showed that the same results (P < 0.05). According to the results of the ADSCs tracing, ADSCs did not exist at the transplant site (6 weeks after implantation). The positive rates for laminin and collagen III constructed around the transplanted islets did not differ among groups. Inflammatory mediators were higher in the Silicone group, followed by the AG and GHNF groups. Pretreatment using bioabsorbable scaffolds combined with ADSCs enhanced neovascularization in subcutaneous space, and subcutaneous islet transplantation using GHNF with ADSCs was superior to intraportal islet transplantation.

Keywords: adipose tissue–derived stem cells; extracellular matrix; gelatin hydrogel nonwoven fabrics; islet; neovascularization; subcutaneous transplantation.

Figure 1.

Characterization of ADSCs. Flow cytometry revealed that ADSCs were negative for CD31 (2.5%) and CD45 (0.1%), whereas were positive for the Sca-1 in 99.0%, CD44 in 86.8%, and CD90 in 99.4%.

Figure 2.

The outcome of islet engraftment after marginal islet mass transplantation (180 IEQs). (A) Changes in blood glucose levels after islet transplantation in the AG (filled square, n = 11), GHNF (filled rhombus, n = 15), and Silicone (open circle n = 10) groups. The AG group significantly showed significantly better glucose changes than the Silicone group (**; P < 0.01). (B) Changes in blood glucose levels after islet transplantation in the AG the IPO (open triangle, n = 10) groups. The AG group showed significantly better glucose changes than the IPO group (**; P < 0.01). (C) The cure rate curve of diabetic mice after islet transplantation in each group. The cure rate at 60 days after islet transplantation in the AG group (72.7%) was significantly higher than that in the Silicone (0%) group (**AG vs. Silicone; P < 0.01, *GHNF vs. Silicone; P < 0.05). (D) The cure rate curve in the AG and IPO groups. The cure rate of the AG group (72.7%) was higher than that of the IPO group (40.0%) (P = 0.329), but the difference did not reach statistical significance.

Figure 3.

The glucose tolerance profiles of the AG, GHNF, IPO, and Silicone groups. (A) The results of the intraperitoneal glucose tolerance test (IPGTT) in the AG (filled square, n = 11), GHNF (filled rhombus, n = 12), and Silicone (open circle n = 9) groups at approximately 60 days after islet transplantation. The AG group showed significantly better glucose changes than the Silicone group (*, P < 0.05). (B) The results of the IPGTT in the AG and IPO (open triangle, n = 9) groups. (C) The area under the curve (AUC) of the IPGTT in each group is shown. Although the difference did not reach statistical significance, the AUC of the AG group was lower than that of the other groups (P = 0.202). (D) The AUC of the IPGTT in the AG and IPO groups. No significant difference was observed between the two groups (P = 0.283).

Figure 4.

Immunohistochemical analyses of von Willebrand factor (vWF)-positive vessels. (A) Photomicrographs of vWF staining before islet transplantation. The vWF-positive vessels (black arrows) in the capsule around the silicone spacer were counted. Magnification: ×100. Calibration bars: 200 µm. (B) The number of vWF-positive vessels in the AG group was significantly higher than that in the other groups (**, P < 0.01). Black arrows represent the vWF-positive vessels.

Figure 5.

Immunohistochemical analyses of extracellular matrix (ECM). (A) Representative photomicrographs of laminin, collagen III, and collagen IV staining. Black arrows represent the transplanted islets. “Positive” for laminin, collagen III, and collagen IV indicates that distinct immunopositivity was detectable in the fibrous capsule around the islets. Magnification: ×200. Calibration bars: 200 µm. (B) The rates of laminin, collagen III, and collagen IV immunopositivity in the AG (white box), GHNF (gray box), and Silicone (black box). Although the rate of collagen IV positivity in the GHNF group was significantly lower than that in the other groups (*, P < 0.05), there was no significant difference in the rates of collagen III or laminin positivity among the groups.

Figure 6.

Immunohistochemical analyses of insulin-positive islets at 1 week after transplantation (A). Photomicrographs of islets. Magnification: ×100. Calibration bars: 200 µm. Insulin-positive islets were categorized to Grade 1 to 3 according to their shape and uniformity of insulin staining. (B) The percentage of Grade 2 and 3 islets in the AG and GHNF groups was higher than that in the Silicone group (AG: 96.4%, GHNF: 94.4%, Silicone: 77.7%, P = 0.097). (C) The average size of islets in the AG group was significantly bigger than that in the Silicone group (AG: 32,578 ± 40,763 vs. GHNF: 30,979 ± 40,998 vs. Silicone: 12,921 ± 9,947 μm², respectively. *, P < 0.05).

Figure 7.

Immunohistochemical analyses of GFP-positive ADSCs. (A) Photomicrographs of GFP staining in the GHNF. Magnification: ×100 (upper row) and ×200 (lower row). Calibration bars: 100 µm. Black arrows represent the GHNF. (B) The mean number of GFP-positive ADSCs in the GHNF. The number of GFP-positive ADSCs at 0 or 1 week after implantation was significantly higher than that at 2 or 6 weeks after implantation (P < 0.01). The number of GFP-positive ADSCs decreased in a time-dependent manner, and only a limited number of them were detected at 6 weeks after implantation (**, P < 0.01).

Figure 8.

Quantification of the vascular volume using lectin angiography in the capsule at the transplant site of the AG, GHNF, and Silicone groups. (A) Image of lectin angiography. The yellow structure on the left side of the picture shows the vascular volume constructed in the capsule, and the yellow structure on the right side of the picture shows the total capsular volume. (B) Summary of vascular density among the groups. The results represent the vascular volume (μm³)/capsular volume (μm³), and the density of vessels in the AG group was significantly higher than that in the GHNF and Silicone groups (*, P < 0.05, **, P < 0.01).

Figure 9.

Upregulated and downregulated genes at subcutaneous capsules in the AG group (n = 5) in comparison to the GHNF group (n = 5). RNA was extracted from the recipient subcutaneous capsules surrounding the silicone spacer 6 weeks after pretreatment. Values represent the mean log2 relative quantification (RQ). Error bars represent the standard error on a log2 RQ-based scale. The +1 or −1 values represent a two-fold increase or decrease threshold in the gene expression. The gene expression analyses showed that seven target genes in the AG group were significantly upregulated in comparison to the GHNF group (*, P < 0.05).

Figure 10.

The concentration of inflammatory mediators and islet protective growth factor in the supernatant of the homogenized subcutaneous fibrous capsule surrounding the silicone spacer in each group (AG, n = 5; GHNF, n = 5; Silicone, n = 5). The concentrations of (A) interleukin-1α (IL-1α), (B) interleukin-1β (IL-1β), (C) interleukin-6 (IL-6), (D) granulocyte colony stimulating factor (G-CSF), (E) interferon-γ (IFN-γ), (F) tumor necrosis factor-α (TNF-α), and (G) insulin-like growth factor-2 (IGF-2).