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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 in comparison with intraportal islet transplantation. We herein investigated whether the duration of pretreatment using GHNF affected the outcome of subcutaneous islet transplantation. A silicone spacer with GHNF was implanted into the subcutaneous space of healthy mice at 2, 4, 6, or 8 weeks before transplantation, and then diabetes was induced 7 days before transplantation. Syngeneic islets were transplanted into the pretreated space. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, inflammatory mediators, and gene expression were evaluated. The 6-week group showed significantly better blood glucose changes than the other groups (P < 0.05). The cure rate of the 6-week group (60.0%) was the highest among the groups (2-week = 0%, 4-week = 50.0%, 8-week = 15.4%). The number of von Willebrand factor (vWF)–positive vessels in the 6-week group was significantly higher than in the other groups at pre-islet and post-islet transplantation (P < 0.01 [vs 2-and 4-week groups] and P < 0.05 [vs all other groups], respectively). Notably, this beneficial effect was also observed when GHNF was implanted into diabetic mice injected with streptozotocin 7 days before GHNF implantation. The positive rates for laminin, collagen III, and collagen IV increased as the duration of pretreatment became longer and were significantly higher in the 8-week group (P < 0.01). Inflammatory mediators, including interleukin (IL)-1b, granulocyte colony-stimulating factor (G-CSF), and interferon (IFN)-γ, were gradually downregulated according to the duration of GHNF pretreatment and re-elevated in the 8-week group. Taken together, the duration of GHNF pretreatment apparently had an impact on the outcomes of subcutaneous islet transplantation, and 6 weeks appeared to be the ideal duration. Islet graft revascularization, extracellular matrix compensation of the islet capsule, and the inflammatory status at the subcutaneous space would be crucial factors for successful subcutaneous islet transplantation.

Keywords

islet transplantation gelatin hydrogel nonwoven fabrics subcutaneous extracellular matrix neovascularization

Introduction

Pancreatic islet transplantation, in which isolated islet grafts are commonly injected into the portal vein, has become an effective treatment option for severe type 1 diabetes¹. However, problems remain to be solved in this procedure^2,3, including graft loss due to innate immune reactions^4,5, technical complications of bleeding and/or thrombosis^6,7, portal hypertension^8,9, need for multiple transplant procedures to achieve insulin independence¹, and difficulty in monitoring islet grafts once implanted. Therefore, alternative extrahepatic sites for transplantation, including kidney subcapsule, liver surface, subcutaneous and intramuscular spaces, have been investigated^10,11.

The subcutaneous space is an attractive candidate for such an alternative extrahepatic site and has many advantages¹². It is less invasive than intraportal transplantation, easily accessible for implantation and/or removal of islet grafts if necessary, and grafts can be easily monitored using ultrasound examination and/or biopsy^13,14. Furthermore, when induced pluripotent stem cell–derived islets or xenografts are tested¹⁵, subcutaneous space is ideal because it is easy to monitor and remove them when any graft-related complications occur, such as infection and tumorigenesis. On the contrary, the subcutaneous space is a severe environment for graft survival due to hypovascularity and hypoxia, which lead to poor transplant efficacy¹⁶. Thus, optimization of the subcutaneous environment with extracellular matrix (ECM) and prevascularization prior to islet transplantation is necessary to improve engraftment.

We previously reported that a recombinant peptide (RCP: alpha-1 sequence of recombinant collagen type I supplemented with 12 RGD [Arg-Gly-Asp] motifs in 1 molecule) was effective for improving islet engraftment in subcutaneous transplantation¹⁷. In that study, the RCP device was implanted 10 or 28 days before islet transplantation. Interestingly, the duration of RCP pretreatment apparently affected the cure rate of diabetes and islet graft function. One drawback of the RCP device was that it must be removed when islets are implanted. In this process, compensated ECM and/or newly constructed vessels surrounding the islet grafts might be destroyed.

Thus, we next focused on a gelatin hydrogel nonwoven fabric (GHNF)¹⁸, which is a biodegradable scaffold made from gelatin. Gelatin hydrogel nonwoven fabric is produced by the solution-blow method using gelatin solution^19,20. When used for cell culture, it was shown to be gradually degraded and replaced with ECM and host cells. Therefore, there is no need for its removal once implanted into the subcutaneous tissue²¹. Gelatin itself was reported to enhance the engraftment of transplanted cells and promote angiogenesis, together with cellular proliferation by ECM²². We also reported that GHNF pretreatment markedly improved the outcomes of subcutaneous islet transplantation in comparison with the current standard procedure (ie, intraportal transplantation), most likely due to ECM compensation and/or the increase of various growth factors, including insulin-like growth factor 2 (IGF-2) and hepatocyte growth factor (HGF) (manuscript submitted). However, the optimal duration of GHNF pretreatment remains unknown.

In this study, we investigated whether the duration of GHNF pretreatment affects the outcomes of subcutaneous islet transplantation and sought to find the optimal duration. Through this process, we also tried to clarify the key factors for successful subcutaneous islet transplantation.

Materials and Methods

Animals

All animals used in this study were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health²³. All experimental protocols of this study (protocol ID: 2020 MdA-022) were approved by the animal experimental committee at Tohoku University. Male C57BL/6J mice (age: 6–14 weeks) (Japan SLC Inc, Shizuoka, Japan) were used as both donors and recipients. All surgical operations were performed under general anesthesia using isoflurane (Viatris Inc, Tokyo, Japan), and all efforts were made to minimize suffering.

The Induction and Diagnosis of Diabetes in the Recipients

Diabetes was induced by the intravenous injection of streptozotocin (STZ; 170 mg/kg) (Sigma-Aldrich, Inc, St. Louis, MO, USA) 7 days before islet transplantation. Mice whose nonfasting blood glucose levels were ≥400 mg/dL on two consecutive measurements were considered diabetic. Serial blood glucose levels were determined, and recipients whose nonfasting blood glucose levels were <200 mg/dL on two consecutive measurements were considered to be cured.

Islet Isolation

Islet isolation and culturing were performed as previously described²⁴. Islets were cultured in Roswell Park Memorial Institute-1640 medium containing 5.5 mmol/L glucose and 10% fetal bovine serum at 37°C in 5% CO₂ and humidified air overnight before transplantation.

Implantation of GHNF and Islet Transplantation

Gelatin hydrogel nonwoven fabric (NIKKE MEDICAL Co, Ltd, Osaka, Japan) was prepared using a previously reported method¹⁸, and processed into a circular sheet type (diameter: 11 mm; thickness: 0.5 mm). After swelling with physiological saline, two GHNF sheets sandwiching a silicone spacer (diameter: 11 mm; thickness: 0.5 mm) were placed into the left dorsal subcutaneous space. To compare the impact of pretreatment duration, four groups were designed according to the duration of GHNF placement (2 weeks [2W, n = 11], 4 weeks [4W, n = 16], 6 weeks [6W, n = 15], and 8 weeks [8W, n = 13] groups). Gelatin hydrogel nonwoven fabrics were implanted into healthy mice, and then mice were injected with STZ, 7 days before islet transplantation. To examine the influence of diabetic conditions on the impact of GHNF pretreatment, a 6 week-diabetes mellitus (6WDM) group (n = 10), in which STZ injection was performed 7 days before GHNF implantation and islet transplantation was performed 6 weeks after GHNF implantation, was designed. During 6 weeks of GHNF pretreatment, subcutaneous injection of insulin degludec (Novo Nordisk A/S, Bagsvaerd, Denmark) was performed to keep mice alive (two insulin units/week).

In each group, after removal of the silicone spacer, 270 islet equivalents (IEQs) of syngeneic mouse islets were transplanted into the pretreated space using a gastight syringe (Hamilton Co, Reno, NV, USA)¹⁶. Recipients were followed by measuring nonfasting blood glucose levels every 3 to 4 days throughout the study period (60 days post-transplantation).

Intraperitoneal Glucose Tolerance Test

An intraperitoneal glucose tolerance test (IPGTT) was performed 60 to 64 days post-transplantation, as described previously²⁵. The blood glucose curve was generated, and the area under the curve (AUC) was used for comparison.

Immunohistochemical Analyses

After 2, 4, 6 and 8 weeks of subcutaneous implantation of GHNF before islet transplantation, recipient tissue at the site of subcutaneous implantation was procured, fixed with 4% paraformaldehyde, and embedded in paraffin for immunohistochemical staining. In addition, at 60 days post-transplantation, tissue at the site of subcutaneous islet transplantation was procured. Immunohistochemical staining was performed using anti-von Willebrand factor (vWF) (ab6994; Abcam, Cambridge, UK), anti-collagen III (ab7778; Abcam), anti-collagen IV (ab6586; Abcam), anti-laminin (ab11575; Abcam), and anti-CD206 (24595; Cell Signaling Technology, Danvers, MA, USA) antibodies. EnVision+ System- HRP-labeled polymer anti-rabbit (4003; DAKO, Glostrup, Denmark) was used as a secondary antibody. For the evaluation of neovascularization, the vWF-positive vessels in the interstitial area were counted before and after islet transplantation²⁶. CD206-positive cells were counted in the GHNF and capsule around the GHNF. In collagen III²⁷, collagen IV, and laminin staining²⁸, “positive” was defined as marked immunopositivity that was detectable in the fibrous capsule around the islets. Six sections from each experimental group (2W, n = 5; 4W, n = 5; 6W, n = 5; 8W, n = 5) before islet transplantation and an average of six sections from each group (2W, n = 5; 4W, n = 8; 6W, n = 6; 8W, n = 4) after islet transplantation were evaluated by a pathologist using a blind method.

Real-Time PCR Using TaqMan Arrays

RNA was extracted from the recipient subcutaneous fibrous capsules surrounding the silicone spacer at 2, 4, 6 and 8 weeks after pretreatment²⁹ (2W, n = 5; 4W, n = 5; 6W, n = 5; 8W, n = 5). The relative gene expression was determined using a TaqMan array 96-well FAST plate (4413257; Applied Biosystems, Bedford, MA, USA). A TaqMan array plate contains 46 target genes and two assays for candidate endogenous control genes (Table 1). The samples were analyzed using a StepOnePlus Real-Time polymerase chain reaction (PCR) System (Applied Biosystems) under the following amplification conditions: 50°C for 2 min and 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The results were analyzed using ExpressionSuite Software (ver. 1.3; Applied Biosystems). Relative quantification (RQ) was calculated using the comparative CT method. To determine the relative gene expression in the 6W group, the samples in the 2W group were designated as a calibrator. 18S was used as a housekeeping gene.

Table 1.

List of Analyzed Target Genes.

Assay ID	Gene symbol	Gene name
Hs99999901_s1	18S	18S ribosomal RNA
Mm01277161_m1	Cd44	CD44 antigen
Mm01247357_m1	Cdh1	Cadherin 1
Mm01162497_m1	Cdh2	Cadherin 2
Mm01249209_m1	Cdh3	Cadherin 3
Mm05751874_s1	Chsy1	Chondroitin sulfate synthase 1
Mm00801666_g1	Col1a1	Collagen, type I, alpha 1
Mm01309565_m1	Col2a1	Collagen, type II, alpha 1
Mm00802300_m1	Col3a1	Collagen, type III, alpha 1
Mm01210125_m1	Col4a1	Collagen, type IV, alpha 1
Mm00489299_m1	Col5a1	Collagen, type V, alpha 1
Mm00487160_m1	Col6a1	Collagen, type VI, alpha 1
Mm01192933_g1	Ctgf	Connective tissue growth factor
Mm00438696_m1	Egf	Epidermal growth factor
Mm00679872_m1	Fgf12	Fibroblast growth factor 12
Mm01285715_m1	Fgf2	Fibroblast growth factor 2
Mm00438980_m1	Flt1	FMS-like tyrosine kinase 1
Mm01256744_m1	Fn1	Fibronectin 1
Mm99999915_g1	Gapdh	Glyceraldehyde-3-phosphate dehydrogenase
Mm00433590_g1	Gh	Growth hormone
Mm03048195_m1	Has1	Hyaluronan synthase 1
Mm01135184_m1	Hgf	Hepatocyte growth factor
Mm00468869_m1	Hif1a	Hypoxia inducible factor 1, alpha subunit
Mm00780907_s1	Hs3st3a1	Heparan sulfate (glucosamine)
Mm03649146_m1	Hs3st4	Heparan sulfate (glucosamine) 3-O-sulfotransferase 4
Mm01181173_g1	Hspg2	Perlecan (heparan sulfate proteoglycan 2)
Mm00516023_m1	Icam1	Intercellular adhesion molecule 1
Mm00439560_m1	Igf1	Insulin-like growth factor 1
Mm00439564_m1	Igf2	Insulin-like growth factor 2
Mm01222421_m1	Kdr	Kinase insert domain protein receptor
Mm01226102_m1	Lama1	Laminin alpha 1
Mm01156972_m1	Met	Met proto-oncogene
Mm01205760_m1	Pdgfa	Platelet derived growth factor, alpha
Mm00440677_m1	Pdgfb	Platelet derived growth factor, B polypeptide
Mm00480205_m1	Pdgfc	Platelet derived growth factor, C polypeptide
Mm01242576_m1	Pecam1	Platelet/endothelial cell adhesion molecule 1
Mm01178820_m1	Tgfb1	Transforming growth factor beta 1
Mm00436955_m1	Tgfb2	Transforming growth factor beta 2
Mm00436960_m1	Tgfb3	Transforming growth factor beta 3
Mm00449032_g1	Thbs1	Thrombospondin 1
Mm01279240_m1	Thbs2	Thrombospondin 2
Mm00616592_m1	Vash1	Vasohibin 1
Mm00523123_m1	Vash2	Vasohibin 2
Mm01320970_m1	Vcam1	Vascular cell adhesion molecule 1
Mm01283063_m1	Vcan	Versican
Mm00437306_m1	Vegfa	Vascular endothelial growth factor A
Mm00495976_m1	Vtn	Vitronectin
Mm00550376_m1	Vwf	Von Willebrand factor homolog

Immunoassay for the Protein Expression of Inflammatory Mediators and Islet-Protective Factors

The recipient subcutaneous fibrous capsule surrounding the silicone spacer at 2, 4, 6, and 8 weeks after pretreatment was procured (2W, n = 5; 4W, n = 5; 6W, n = 5; 8W, n = 5). Recipient subcutaneous fibrous capsules were homogenized using SONICS Vibra-cell (SONICS & MATERIALS, Inc, Newtown, CT, USA) in five volumes of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 1 mM phenylmethylsulfonyl fluoride (P7626; Sigma-Aldrich, Inc)³⁰ and 1.0% Protease Inhibitor Cocktail Set V (162-26031; FUJIFILM Wako Pure Chemical Corporation). The extract was centrifuged at 10,000×g for 20 min, and then the supernatant was recovered, diluted, aliquoted, and frozen at −20 to −80°C. Both IGF-2 and HGF concentrations in the supernatant were measured using a Quantikine ELISA Mouse/Rat/Porcine/Canine IGF-2 Immunoassay Kit and Quantikine ELISA Mouse/Rat HGF (R&D Systems, Minneapolis, MN, USA). The total protein concentrations in the supernatant were measured using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Cytokines in the supernatant were measured using a Bio-Plex Pro Mouse Cytokine 23-plex Assay (#M60009RDPD; Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.

Statistical Analyses

All data except for body weight are expressed as the mean ± standard error. Body weight was expressed as the mean ± standard deviation. All statistical analyses were performed using JMP Pro 16 (SAS Institute Inc, Cary, NC, USA). Changes in blood glucose levels and IPGTT were analyzed by mixed effect model analysis, and Tukey–Kramer test was used for post hoc comparisons between the groups. The AUC of the IPGTT was analyzed by one-way analysis of variance (ANOVA) (Fig. 2B) and the Mann–Whitney U test (Fig. 8D). The numbers of vWF-positive vessels and CD206-positive cells were analyzed by ANOVA, and Tukey–Kramer test was used for post hoc comparisons among the groups. The number of vWF-positive vessels in Fig. 8E was analyzed by the Mann–Whitney U test. The immunopositive rate in ECM staining was analyzed using Pearson’s chi-square test with a Bonferroni post hoc test. Kaplan–Meier curves were compared using a log-rank test with a Bonferroni post hoc test. P values of <0.05 were considered to indicate statistical significance.

Results

Comparison of Islet Engraftment After Marginal Islet Mass Transplantation Among the 2W, 4W, 6W, and 8W Groups

Blood glucose changes after islet transplantation in the 6W group (n = 15) were significantly better than those in the other groups (P < 0.05) (Fig. 1A). The cure rate of diabetic mice at 60 days post-transplantation in the 6W group was higher than that in the 2W, 4W, and 8W groups (6W, 60.0% [9/15] vs 2W, 0% [0/11] vs 4W, 50.0% [8/16] vs 8W, 15.4% [2/13], 6W vs 2W: P < 0.01) (Fig. 1B).

Figure 1.

The outcome of islet engraftment after marginal islet mass transplantation (270 IEQs). (A) The changes in the blood glucose levels after islet transplantation in the 2W (open circle, n = 11), 4W (open triangle, n = 16), 6W (filled square, n = 15), and 8W groups (filled rhombus, n = 13). The 6W group showed significantly better glucose changes than the other groups (**P < 0.01. *P < 0.05). (B) The cure rate curve of diabetic mice after islet transplantation in each group. The cure rate at 60 days after islet transplantation in the 6W group (60.0%) was higher than that in the 2W (0%), 4W (50.0%), and 8W (15.4%) groups (*P < 0.05). IEQ: islet equivalents; W: week.

Intraperitoneal Glucose Tolerance Test

In the IPGTT, the blood glucose changes in the 6W group were better than those in the other groups (6W vs 2 and 4Ws, 2W vs 4 and 8Ws: P < 0.01) (Fig. 2A). Although the difference did not reach statistical significance, the AUC in the 6W group was lower than that in the other groups (6W, 34,969 ± 3,443 vs 2W, 45,976 ± 5,808 vs 4W, 36,693 ± 3,881 vs 8W, 43,630 ± 2,267, P = 0.15) (Fig. 2B).

Figure 2.

The glucose tolerance profiles of the 2W, 4W, 6W, and 8W groups. (A) The results of the intraperitoneal glucose tolerance test (IPGTT) in the 2W (open circle, n = 4), 4W (open triangle, n = 8), 6W (filled square, n = 7), and 8W (filled rhombus, n = 8) groups at approximately 60 days after islet transplantation. The 6W group showed better glucose changes in comparison with the other groups (6W vs 2 and 4Ws, 2W vs 4 and 8Ws: **P < 0.01). (B) The area under the curve of the IPGTT in each group is shown. Although the difference did not reach statistical significance, the area under the curve of the 6W group was lower than that of the other groups (P = 0.15). W: week.

Time-Course Changes of GHNF Absorption Under the Subcutaneous Space

After pretreatment using GHNF, subcutaneous tissues, including GHNF, were removed and observed with hematoxylin eosin staining. Gelatin hydrogel nonwoven fabric was absorbed, and its volume gradually decreased over time. Autologous cells and fiber tissues were observed in the gap of GHNF (Fig. 3).

Figure 3.

Microscopic pathology. Representative photos of the microscopic pathology of subcutaneous tissues, including GHNF, in diabetic mice at 2, 4, 6 and 8 weeks after pretreatment are shown (hematoxylin eosin staining). Magnification: ×50 and ×200. Calibration bars: 200 µm. Implanted GHNF (white arrows). Muscle (black arrows). Skin (gray arrows). The GHNF was absorbed, and its volume decreased over time. At higher magnification, autologous cells and fiber tissues were observed in the gap of the GHNF. GHNF: gelatin hydrogel nonwoven fabric.

Immunohistochemical Analyses

The number of vWF-positive vessels in the interstitial area was counted to examine neovascularization before (Fig. 4A) and after (Fig. 4B) islet transplantation. The number of vWF-positive vessels in the 6W group was higher than that in the other groups before islet transplantation (6W, 46.1 ± 3.9 vs 2W, 10.7 ± 0.9 vs 4W, 27.5 ± 1.4 vs 8W, 43.3 ± 3.2 number/5 fields, 2W vs 4, 6, and 8Ws, 4W vs 6 and 8Ws: P < 0.01) (Fig. 4C). Notably, this trend was also observed after islet transplantation and the number of vessels in the 6W group was significantly higher than that in the other groups (6W, 28.8 ± 2.7 vs 2W, 15.8 ± 3.6 vs 4W, 18.2 ± 2.4 vs 8W, 15.8 ± 3.6 number/mm², P < 0.05) (Fig. 4D).

Figure 4.

Immunohistochemical analyses of von Willebrand factor (vWF)-positive vessels. (A) Photomicrographs of vWF before islet transplantation. (B) Photomicrographs of vWF after islet transplantation. The vWF-positive vessels (black arrows) in the capsule around the gelatin hydrogel nonwoven fabric (GHNF) were counted before transplantation, and vessels in the interstitial area around the islets were counted after transplantation. Implanted GHNF (white arrows). Magnification: ×200. Calibration bars: 200 µm. (C) The mean number of new vessels in the capsule around the GHNF. The number of vWF-positive vessels in the 6W group was higher than that in the other groups (2W vs 4, 6, and 8Ws, 4W vs 6 and 8Ws: **P < 0.01). (D) In the interstitial area around islet grafts, the number of vWF-positive vessels in the 6W group was significantly higher than that in the other groups (*P < 0.05). W: week.

Previous studies have reported that ECM surrounding islet grafts was lost during islet isolation and shown that compensation of the ECM surrounding islets substantially improves islet function^31–34. To examine the effect of ECM on subcutaneous islet engraftment, collagen III, collagen IV, and laminin at the fibrous capsule around the islet grafts were evaluated. “Positive” for laminin, collagen III, and collagen IV indicates that distinct immunopositivity was detectable in the fibrous capsule around the islets. “Negative” indicates that immunopositivity was undetectable (Fig. 5A). The rate of collagen III, collagen IV, and laminin positivity in the 8W group was significantly higher than that in the other groups (P < 0.01) and tended to increase according to the duration of GHNF placement (Fig. 5B).

Figure 5.

Immunohistochemical analyses of extracellular matrix. (A) Representative photomicrographs of laminin, collagen III, and collagen IV staining. “Positive” for laminin, collagen III, and collagen IV indicates that distinct immunopositivity was detectable in the fibrous capsule around the islets. “Negative” indicates that immunopositivity was undetectable. Magnification: ×200. Calibration bars: 200 µm. (B) The rates of laminin, collagen III, and collagen IV positivity in the 2W (black-dot box), 4W (white box), 6W (gray box) and 8W groups (black box). The immunopositivity rate of the 8W group was significantly higher than that of the other groups (**P < 0.01). The rates of laminin, collagen III, and collagen IV positivity tended to increase as the duration of gelatin hydrogel nonwoven fabric pretreatment became longer. W: week.

CD206 has been suggested to be useful marker for the M2-like macrophage phenotype, which is responsible for the tissue-reparative phase, and are widely accepted as an anti-inflammatory subtype^35–37.CD206-positive cells were evaluated in the GHNF and capsule around the GHNF (Fig. 6A). The number of CD206-positive cells in the GHNF in the 2W group was significantly higher than that in the other groups, and decreased over time, and was extremely low in the 8W group (2W, 220.2 ± 11.5 vs 4W, 142.4 ± 16.0 vs 6W, 82.2 ± 5.5 vs 8W, 26.6 ± 6.6 number/5 fields, P < 0.01) (Fig. 6B). A similar trend was observed in the capsule around the GHNF (2W, 233.1 ± 15.3 vs 4W, 122.4 ± 13.7 vs 6W, 117.6 ± 5.5 vs 8W, 72.0 ± 8.3 number/5 fields, P < 0.01) (Fig. 6C).

Figure 6.

Immunohistochemical analyses of CD206-positive cells. (A) Photomicrographs of CD206 in the gelatin hydrogel nonwoven fabric (GHNF) and the capsule around the GHNF. Capsule around the GHNF (black arrows). Implanted GHNF (white arrows). Magnification: ×100 and ×200. Calibration bars: 200 µm. (B) The mean number of CD206-positive cells in GHNF. The number of CD206-positive cells in the GHNF was significantly higher in the 2W group than in the other groups, decreased over time, and was extremely low in the 8W group (*P < 0.05. **P < 0.01). (C) The mean number of CD206-positive cells in the capsule around the GHNF. In the capsule around the GHNF, a similar trend was observed (*P < 0.05. **P < 0.01). W: week.

Relative Gene Expression

To identify genes that were upregulated in subcutaneous capsules in the 6W group in comparison with the 2W group, 46 target genes were analyzed using TaqMan Arrays (Table 1). The relative gene expression in the 6W group is shown in Fig. 7. The gene expression analyses revealed that 21 target genes (met proto-oncogene, vitronectin, vascular cell adhesion molecule 1, fibroblast growth factor 2, hyaluronan synthase 1, FMS-like tyrosine kinase 1, transforming growth factor–β3, Von Willebrand factor homolog, platelet derived growth factor C, vascular endothelial growth factor A, transforming growth factor–β2, thrombospondin 2, cadherin 2, fibronectin 1, hepatocyte growth factor, connective tissue growth factor, heparan sulfate [glucosamine], epidermal growth factor, kinase insert domain protein receptor, transforming growth factor–β1, and hypoxia inducible factor 1–α subunit) were significantly upregulated in comparison with the 2W group (P < 0.05).

Figure 7.

Upregulated and downregulated genes in the 6W group (n = 5) in comparison with the 2W group (n = 5). RNA was extracted from the recipient subcutaneous capsules surrounding the silicone spacer after pretreatment. Values represent the mean log2 relative quantification (RQ). Error bars represent the standard error on a log2 RQ-based scale. The +1 and –1 values represent a twofold increase or decrease threshold in the gene expression. The gene expression analyses showed that 21 target genes in the 6W group were significantly upregulated in comparison with the 2W group (*P < 0.05. **P < 0.01). W: week.

Protein Expression of Inflammatory Mediators and Islet-Protective Factors

To evaluate the inflammatory status in each group, 23 inflammatory mediators in the subcutaneous capsules were measured (Table 2). Interestingly, the expression of proinflammatory mediators, including interleukin (IL)-1a, IL-1b, IL-6, granulocyte colony-stimulating factor (G-CSF), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α), was elevated in the 2W group and decreased over time; however, it increased again in the 8W group. Regarding islet-protective factors, HGF expression in the 2W group was significantly higher than that in the other groups (2W, 824.2 ± 79.0 vs 4W, 284.4 ± 21.5 vs 6W, 209.3 ± 18.0 vs 8W, 282.8 ± 68.9 pg/mg, P < 0.01), while IGF-2 expression in the 6W group was higher than that in the other groups (6W, 20.8 ± 3.3 vs 2W, 18.2 ± 6.4 vs 4W, 12.5 ± 2.2 vs 8W, 6.23 ± 1.0 pg/mg, 6W vs 2 and 8Ws: P < 0.05).

Table 2.

The Analysis of Inflammatory Mediators by Multiplex.

Inflammatory mediators	2W	4W	6W	8W	P value
IL-1α	8.1 ± 0.9	5.4 ± 0.6	4.8 ± 1.0	9.2 ± 2.0	0.067
IL-1β	3.8 ± 0.6	2.5 ± 0.3	2.4 ± 0.4	2.8 ± 0.6	0.21
IL-2	16.4 ± 2.6	13.6 ± 0.9	8.7 ± 2.0	14.9 ± 4.1	0.23
IL-3	0.9 ± 0.2	1.4 ± 0.2	1.2 ± 0.2	2.2 ± 0.6	0.14
IL-4	1.4 ± 0.3	1.2 ± 0.1	0.9 ± 0.3	2.1 ± 0.6	0.17
IL-5	2.1 ± 0.3	3.2 ± 0.4	2.5 ± 0.3	5.7 ± 1.4	0.025
IL-6	3.6 ± 1.5	3.6 ± 1.0	2.0 ± 0.6	4.3 ± 0.9	0.64
IL-9	9.3 ± 1.7	71 ± 1.1	4.6 ± 0.7	10.1 ± 2.9	0.25
IL-10	16.2 ± 1.8	13.1 ± 2.1	8.6 ± 0.8	20.5 ± 4.9	0.086
IL-12 (p40)	74.2 ± 13.4	46.8 ± 3.1	43.9 ± 2.1	43.0 ± 4.8	0.024
IL-12 (p70)	74.6 ± 4.4	74.0 ± 7.3	44.5 ± 7.7	56.8 ± 7.0	0.016
IL-13	24.9 ± 4.4	30.1 ± 3.7	17.6 ± 2.4	45.6 ± 11.7	0.075
IL-17A	2.7 ± 0.2	2.5 ± 0.1	1.5 ± 0.4	2.4 ± 0.6	0.17
Eotaxin	778.8 ± 181.4	539.3 ± 58.1	556.5 ± 233.8	241.9 ± 157.1.	0.10
G-CSF	9.6 ± 1.4	7.0 ± 0.5	4.1 ± 0.8	7.90 ± 1.8	0.043
GM-CSF	17.6 ± 1.8	21.9 ± 1.6	16.4 ± 3.6	28.0 ± 6.0	0.16
IFN-γ	15.1 ± 4.0	11.1 ± 1.5	5.8 ± 1.2	14.4 ± 3.0	0.099
KC	14.9 ± 2.8	10.2 ± 0.9	7.55 ± 1.5	13.5 ± 3.2	0.15
MCP-1	339.8 ± 44.1	239.1 ± 24.2	272.0 ± 38.5	242.0 ± 34.6	0.21
MIP-1α	58.2 ± 17.0	26.1 ± 5.3	26.6 ± 4.1	8.5 ± 1.2	0.011
MIP-1β	187.1 ± 59.8	42.3 ± 5.9	44.5 ± 11.7	30.6 ± 8.2	0.0071
RANTES	167.5 ± 49.4	102.4 ± 18.4	128.3 ± 24.3	59.1 ± 9.3	0.11
TNF-α	32.8 ± 4.5	35.6 ± 4.0	20.1 ± 4.1	83.6 ± 25.0	0.017

W: week; IL: interleukin; G-CSF: granulocyte colony stimulating factor; GM-CSF: granulocyte macrophage colony stimulating factor; IFN-γ: interferon-γ; KC: keratinocyte-derived chemokine; MCP-1: monocyte chemotactic protein–1; MIP-1α: macrophage inflammatory protein–1α; MIP-1β: macrophage inflammatory protein–1β; RANTES: regulated upon activation, normal T-cell expressed and secreted; TNF-α: tumor necrosis factor–α.

The Influence of Diabetic Conditions on the Impact of GHNF Pretreatment

To examine the influence of diabetic conditions on the impact of GHNF pretreatment, 6WDM group (n = 10), in which STZ injection was performed 7 days before GHNF implantation and islet transplantation was performed 6 weeks after GHNF implantation, was designed and compared with 6W group (n = 15), in which GHNF implantation was performed 6 weeks before transplantation and STZ injection was performed 7 days before transplantation. Blood glucose changes, cure rate of recipient mice, IPGTT, and the number of vWF-positive vessels at pre-islet transplantation were compared between the 6WDM and 6W groups. The average body weight of the 6WDM group was significantly lighter than that of the 6W group (6WDM, 24.2 ± 0.63 vs 6W, 25.6 ± 1.35 g, P < 0.01). The average blood glucose level on the day of islet transplantation in the 6WDM group was also significantly lower than that in the 6W group (6WDM, 451.8 ± 18.6 vs 6W, 488.9 ± 6.4 mg/dL, P < 0.05). Although blood glucose changes after islet transplantation in the 6WDM group were significantly better than those in the 6W group (P < 0.01) (Fig. 8A), no significant difference was detected in the cure rate (6WDM, 60% [6/10] vs 6W, 60.0% [9/15], P = 0.78) (Fig. 8B). In addition, the IPGTT results of the groups did not differ to a statistically significant extent (P = 0.92) (Fig. 8C and 8D). Likewise, there was no significant difference in the vWF-positive vessels in the capsule around the GHNF between the two groups (P = 0.35) (Fig. 8E).

Figure 8.

Comparison of transplantation outcomes between the 6 week-diabetes mellitus (6WDM) and 6W groups. (A) Blood glucose changes after islet transplantation in the 6WDM (open circle, n = 10) and 6W (filled square, n = 15) groups. The 6WDM group showed significantly better glucose changes than the 6W group (**P < 0.01). (B) The cure rate curve of diabetic mice after islet transplantation in each group. No significant difference was observed between the groups (6WDM, 60% [6/10] vs 6W, 60.0% [9/15], P = 0.78). (C) The results of the intraperitoneal glucose tolerance test (IPGTT) in the 6WDM (open triangle, n = 7) and 6W (filled square, n = 7) groups at approximately 60 days after islet transplantation. The 6WDM group showed slightly better glucose changes than the 6W group, but the difference did not reach statistical significance (P = 0.92). (D) The area under the curve of the IPGTT between the two groups is shown. No significant difference was detected between the groups (P = 0.56). (E) The mean number of new vessels in the capsule around the GHNF before islet transplantation. There was no significant difference in the number of new vessels between the two groups (P = 0.35). W: week.

Discussion

The present study showed that the duration of implantation of GHNF apparently affected the outcomes of subcutaneous islet transplantation. Although the cure rate of diabetic mice in the 6W group (60.0%) was comparable with that of the 4W group (50.0%), it was markedly better than that of the 2W (0%) and 8W (15.4%) groups (Fig. 1B). Furthermore, blood glucose changes during the observation period in the 6W group were significantly superior to those in the other groups (Fig. 1A). Corroborating these observations, the same tendency was detected in the glucose tolerance of the four groups (Fig. 2A, B). Taken together, 6 weeks of pre-implantation of GHNF into the subcutaneous space appeared to be ideal in the murine model.

According to microscopic pathology, the persistence of GHNF at the transplant site appeared to be dramatically reduced in the 8W group (Fig. 3). Given that the transplant outcome of the 8W group was significantly worse than that of the 4W and 6W groups (Fig. 1A), GHNF may be required at the transplant site when islets are implanted, at least in part to serve as an effective scaffold for islet grafts.

Although subcutaneous space is suitable for testing alternative β-cell sources, the main drawback of subcutaneous islet transplantation is the poor revascularization of islet grafts^16,38. It reportedly takes approximately 10 to 14 days for the construction of new vessels that nourish islet grafts^39,40. Therefore, pre-vascularization of the subcutaneous space prior to transplantation is essential. Various devices, including nylon, stainless-steel mesh, and porous polymer matrix combined with or without growth factors, have been implanted in subcutaneous sites aiming for neovascularization^17,41,42. The duration of pretreatment ranged from 14 to 28 days in each report. However, no studies have focused on the optimal duration of each pretreatment. In the presence of GHNF, the immunohistochemical analyses in this study showed that the number of vessels under the subcutaneous space gradually increased until 6 weeks after implantation, and then decreased at 8 weeks (Fig. 4C). The reason for this finding is uncertain, but it may be related to the downregulation of angiogenic factors derived from indwelling host cells in GHNF, according to the disappearance of GHNF at the transplant site (Fig. 3). Corroborating the immunohistochemical findings, the gene analyses also revealed that angiogenic genes, including vascular endothelial growth factor A (VEGFA), HGF, and fibroblast growth factor^43,44, were significantly upregulated in the 6W group in comparison with the 2W group (Fig. 7). These findings suggest that neovascularization under subcutaneous tissues may be an explanation for the beneficial implantation period (6W) of GHNF. Notably, the neovascularization effect induced by GHNF prior to islet transplantation was demonstrated to be long-lasting (≥60 days) after islet transplantation (Fig. 4D). This long-lasting effect may be partially based on angiogenic factors (eg, VEGF and HGF) derived from islet grafts^45,46. However, considering that an extremely small number of islets (270 IEQs) were transplanted as a marginal mass in this study, this beneficial effect may largely depend on GHNF pretreatment at 6 weeks before transplantation.

Another key factor for the optimal GHNF implantation period is compensation of ECM. Previous studies have reported that ECM surrounding islet grafts was lost during islet isolation^32,33. Furthermore, several reports have shown that compensation of the ECM surrounding islets substantially improves islet function^31–34. In fact, on immunohistochemistry, the expression of collagen III and IV in the fibrous capsule surrounding the islet grafts was not detected, and laminin expression was also quite low in the 2W group, which was subsequently supported by the poor outcome of islet transplantation in the 2W group. The present study demonstrated that ECM compensation in the fibrous capsule around islet grafts tended to increase over time (Fig. 5B). Accordingly, the genes on ECM itself and growth factors that are supposed to stimulate the increase of ECM were significantly upregulated in the 6W group in comparison with the 2W group (Fig. 7). In this study, maximal ECM compensation was observed in the 8W group, whereas the peak of neovascularization under subcutaneous tissues was detected in the 6W group. Although both factors are highly important for improving the outcomes of subcutaneous islet transplantation, considering the sharp difference in transplant outcomes between the 6W and 8W groups, neovascularization rather than ECM compensation may play a more critical role in subcutaneous islet transplantation. The reason for the marked elevation of the positive rate of laminin, collagen III, and collagen IV in the 8W group is uncertain. One possible explanation is that re-elevated subcutaneous inflammation in the 8W group induced the synthesis of laminin, collagen III, and collagen IV, which was also reported in the skin scarring, hypertrophic scars, and keloid disease^47,48.

Indwelling macrophages in the GHNF would also be key factors for determining the optimal period for implantation of GHNF⁴⁹. Macrophages play an important role in tissue repair and are usually divided into M1 and M2 phenotypes. M1 macrophages are responsible for proinflammatory responses and produce proinflammatory factors that mainly mediate the tissue-destructive phase^50,51. In contrast, M2 macrophages have potent phagocytosis capacity and produce ECM components and/or angiogenic factors. They are responsible for the tissue-reparative phase, and are widely accepted as an anti-inflammatory subtype^37,50–52. CD206 and CD163 have been suggested to be useful markers for the M2-like macrophage phenotype^35–37. In this study, the number of CD206-positive cells in the GHNF in the 2W group was significantly higher than that in the other groups, decreased over time, and was extremely low in the 8W group (Fig. 6B). A similar trend was observed in the capsule around the GHNF, where islet grafts were supposed to have direct contact with macrophages (Fig. 6C). In accordance with our findings, Lucke et al.⁵³ previously reported that the percentage of CD163-positive area was highest at 7 days after implantation of porcine collagen matrix in subcutaneous site and maintained high levels during 28 days, and then dramatically dropped at 56 days. Given that the unique characteristics of GHNF (fibrous and porous structure) are well known to attract host cells into the gap of GHNF^18,49, the decrease of CD206-positive cells (anti-inflammatory macrophages) in this study seemed to occur in parallel with the disappearance of GHNF per se at the transplant site. This could explain why the expression of several inflammatory mediators increased again at 8 weeks after implantation of GHNF. In this context, the persistence of a marginal volume of GHNF at the transplant site might play crucial roles as an effective scaffold against islet grafts and in regulating inflammatory reactions around islets.

In this study, we could not classify M1 and M2 macrophages because Iba-1 staining did not work sufficiently in the murine model (data not shown). In support of our staining results, it was previously reported that, unlike the rat model⁵⁴, it is difficult to strictly categorize macrophages into M1 and M2 phenotypes in a mouse model^52,55. Of note, macrophage inflammatory protein-1 (MIP-1) proteins are most likely produced and secreted by activated M1 macrophages to attract other proinflammatory cells^56,57. Considering that the concentrations of MIP-1α and 1β were remarkably high in the 2W group, dramatically decreased in the 4W group, and were maintained at low levels until 8 weeks after implantation (Table 2), we hypothesized that M1 macrophages stayed at the transplant site until 2 weeks after GHNF implantation, and then left as inflammation resolved.

Finally, the release of islet protective growth factors from the indwelling host cells would also be a key factor for determining the ideal duration of GHNF pretreatment. Notably, IGF-2 protein expression in the subcutaneous fibrous capsule was highest in the 6W group. Likewise, IGF-2 gene expression in the 6W group was considerably upregulated in comparison with the 2W group. We have also found that the marked elevation of IGF-2 in the subcutaneous site may greatly contribute to the beneficial effects of GHNF pretreatment (manuscript submitted). Considering that IGF-2 has been reported to maintain islet viability during the culture period and to improve islet engraftment due to anti-apoptotic effects⁵⁸, this factor may—at least in part—play crucial roles in determining the optimal period of GHNF implantation.

Although we clearly demonstrated that GHNF pretreatment is effective for improving the outcomes of subcutaneous islet transplantation and 6 weeks of pre-implantation of GHNF into the subcutaneous space seemed to be preferable, a question may be raised as to whether this finding is truly applicable even in diabetic patients, as GHNF was implanted into the subcutaneous space of healthy mice before STZ injection due to the technical limitations in the present study. In fact, it was previously reported that blood vessel growth was impaired under diabetic conditions⁵⁹. Therefore, we compared the transplant outcomes between the 6WDM and 6W groups to examine the influence of diabetic conditions on the impact of GHNF pretreatment. As a result, no significant difference was observed in vWF-positive vessels in the capsule around the GHNF between the two groups (Fig. 8E). Corroborating this observation, no detrimental influences of diabetic conditions on the impact of GHNF pretreatment were detected in the blood glucose changes and IPGTT (Fig. 8A–D). Of particular note, the results of blood glucose changes after islet transplantation were unexpectedly better in the 6WDM group than in the 6W group (Fig. 8A). One possible explanation for this unexpected result is that the average body weight of the 6WDM group was significantly lighter than that of the 6W group, subsequently resulting in the situation that the amount of transplanted islets/body weight in the 6WDM group was greater than that in the 6W group. Another possible explanation is that the influence of glucose toxicity in the 6WDM group might have been lower than that in the 6W group due to the continuous insulin injection during GHNF pretreatment. Although it is difficult to completely mimic the clinical situation using a murine model due to technical limitations, GHNF pretreatment may be effective for improving the outcomes of subcutaneous islet transplantation irrespective of diabetic conditions during pretreatment.

Although the GHNF does not have lot-to-lot variation problems, the indwelling period of this material may vary according to animal species. Through this study, it was hypothesized that GHNF should be properly absorbed to some extent but simultaneously should persist as a scaffold for islet grafts and as a saucer for the attraction of autologous cells. Therefore, detection of the remnant volume of GHNF by hematoxylin and eosin staining and/or the anti-inflammatory macrophages by CD206 staining could be useful when GHNF is applied in other animal models. Further studies using large animal models are warranted as next steps.

In conclusion, this study revealed that the duration of GHNF pretreatment apparently affected the outcomes of subcutaneous islet transplantation. The revascularization of islet grafts, extracellular matrix-compensation of the islet capsule, the inflammatory status at the subcutaneous space, the persistence of GHNF at the transplant site as a scaffold, and the release of islet protective growth factors from indwelling host cells would be crucial factors for successful subcutaneous islet transplantation. The optimal duration of GHNF pretreatment could be determined by the balance of these complex components, and a 6-week pre-implantation period appeared to be ideal in our murine model.

Footnotes

Acknowledgements

The authors thank Kozue Maya and Megumi Goto (Division of Transplantation and Regenerative Medicine, Tohoku University) for their excellent technical assistance. The authors also acknowledge the support of the Biomedical Research Core of Tohoku University, Graduate School of Medicine, and TAMRIC (Tohoku Advanced Medical Research and Incubation Center).

Author Contributions

R.S. participated in the research design, performance of the research, and writing of the paper. A.I. participated in the performance of the research and the writing of the paper. Y.N. participated in pathological analysis in the research. T.I. participated in the performance of the research. N.K. participated in the performance of the research. H.M. participated in the performance of the research. Y.E. participated in the performance of the research. T.K. participated in the performance of the research. S.S. participated in the performance of the research. K.T. participated in the performance of the research. T.K. participated in the writing of the paper. M.U. participated in the writing of the paper. K.W. participated in the writing of the paper. Y.T. participated in technological advice on gelatin hydrogel nonwoven fabrics. M.G. participated in the research design, the performance of the research, and the writing of the paper.

Availability of Data and Materials

All data generated or analyzed in the present study are included in this published manuscript.

Ethical Approval

This study was approved by our institutional review board.

Statement of Human and Animal Rights

This article contains studies with animal subjects, and all animal experiments were conducted in accordance with animal ethics and safety regulations. This article does not contain any studies with human subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Research Ethics and Patient Consent

All animal experiments in this research had been carried out by the guideline established by Tohoku University Animal Experiment Facility. All experiments were conducted in accordance with animal ethics and safety regulations. We prepared the experimental plan and submitted it to the host research unit, and then obtained the permission for all experiments. Efforts had been made to reduce the number of animals and to shorten the duration of the experiment. All surgical procedures were performed under the adequate anesthesia. Patient content was not applicable because handling of personal information was not required in this research.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article, although this study was performed according to the patent application agreement with NIKKE MEDICAL Co., Ltd.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a Japanese Grant-in-Aid for Scientific Research (A) (Grant Number 18H04056) and Research (B) (Grant Number 22H03133) from the Japan Society for the Promotion of Science, and by AMED under Grant Number JP19bm0404043. The funders played no role in the study design, the collection and analysis of the data, the decision to publish, or the preparation of the manuscript.

ORCID iDs

Ryusuke Saito

Takumi Katano

References

Shapiro

Lakey

Ryan

Korbutt

Toth

Warnock

Kneteman

Rajotte

. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343(4):230–38.

Anazawa

Saito

Goto

Kenmochi

Uemoto

Itoh

Yasunami

Kenjo

Kimura

Ise

Tsuchiya

, et al. Long-term outcomes of clinical transplantation of pancreatic islets with uncontrolled donors after cardiac death: a multicenter experience in Japan. Transplant Proc. 2014;46(6):1980–84.

Goto

Johansson

Eich

Lundgrem

Engkvist

Felldin

Foss

Kallen

Salmela

Tibell

Tufveson

, et al. Key factors for human islet isolation and clinical transplantation. Transplant Proc. 2005;37(2):1315–16.

Goto

Johansson

Maeda

Elgue

Korsgren

Nilsson

. Low molecular weight dextran sulfate prevents the instant blood-mediated inflammatory reaction induced by adult porcine islets. Transplantation. 2004;77(5):741–47.

Tokodai

Goto

Inagaki

Nakanishi

Okada

Satomi

. C5a-inhibitory peptide combined with gabexate mesilate prevents the instant blood-mediated inflammatory reaction in a rat model of islet transplantation. Transplant Proc. 2010;42(6):2102–2103.

Ryan

Lakey

Paty

Imes

Korbutt

Kneteman

Bigam

Rajotte

Shapiro

. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes. 2002;51(7):2148–57.

Yin

Ding

Shen

Hara

Chong

. Liver ischemia contributes to early islet failure following intraportal transplantation: benefits of liver ischemic-preconditioning. Am J Transplant. 2006;6(1):60–68.

Kawahara

Kin

Shapiro

. A comparison of islet autotransplantation with allotransplantation and factors elevating acute portal pressure in clinical islet transplantation. J Hepatobiliary Pancreat Sci. 2012;19(3):281–88.

Sakata

Goto

Motoi

Hayashi

Nakagawa

Mizuma

Yamaya

Hasegawa

Yamaguchi

Sawada

Ottomo

, et al. Clinical experiences in the treatment of pancreatic arteriovenous malformation by total pancreatectomy with islet autotransplantation. Transplantation. 2013;96(5):e38–40.

10.

Inagaki

Imura

Nakamura

Ohashi

Goto

. The liver surface is an attractive transplant site for pancreatic islet transplantation. J Clin Med. 2021;10(4):724.

11.

Rajab

. Islet transplantation: alternative sites. Curr Diab Rep. 2010;10(5):332–37.

12.

Yasunami

Nakafusa

Nitta

Nakamura

Goto

Ono

Taniguchi

. A novel subcutaneous site of islet transplantation superior to the liver. Transplantation. 2018;102(6):945–52.

13.

Sakata

Aoki

Yoshimatsu

Tsuchiya

Hata

Katayose

Egawa

Unno

. Strategy for clinical setting in intramuscular and subcutaneous islet transplantation. Diabetes Metab Res Rev. 2014;30(1):1–10.

14.

Sakata

Goto

Gumpei

Mizuma

Motoi

Satomi

Unno

. Intraoperative ultrasound examination is useful for monitoring transplanted islets: a case report. Islets. 2012;4(5):339–42.

15.

Maxwell

Millman

. Applications of iPSC-derived beta cells from patients with diabetes. Cell Rep Med. 2021;2(4):100238.

16.

Mitsugashira

Imura

Inagaki

Endo

Katano

Saito

Miyagi

Watanabe

Kamei

Unno

Goto

. Development of a novel method for measuring tissue oxygen pressure to improve the hypoxic condition in subcutaneous islet transplantation. Sci Rep. 2022;12(1):1–11.

17.

Uematsu

Inagaki

Nakamura

Imura

Igarashi

Fathi

Miyagi

Ohuchi

Satomi

Goto

. The optimization of the prevascularization procedures for improving subcutaneous islet engraftment. Transplantation. 2018;102(3):387–95.

18.

Nakamura

Saotome

Shimada

Matsuno

Tabata

. A gelatin hydrogel nonwoven fabric facilitates metabolic activity of multilayered cell sheets. Tissue Eng Part C Methods. 2019;25(6):344–52.

19.

Matsuno

Saotome

Shimada

Nakamura

Tabata

. Effect of cell seeding methods on the distribution of cells into the gelatin hydrogel nonwoven fabric. Regen Ther. 2020;14:160–64.

20.

Medeiros

Glenn

Klamczynski

Orts

Mattoso

. Solution blow spinning: a new method to produce micro- and nanofibers from polymer solutions. J Appl Polym Sci. 2009;113(4):2322–30.

21.

Nakajima

Fujita

Matsui

Tohyama

Tamura

Kanazawa

Seki

Kishino

Hirano

Okada

Tabei

, et al. Gelatin hydrogel enhances the engraftment of transplanted cardiomyocytes and angiogenesis to ameliorate cardiac function after myocardial infarction. PLoS ONE. 2015;10(7):e0133308. doi:10.1371/journal.pone.0133308

22.

Dou

Liang

Wen

Qin

. Fabrication and characterization of PVA/CS-PCL/gel multi-scale electrospun scaffold: simulating extracellular matrix for enhanced cellular infiltration and proliferation. J Biomater Sci Polym Ed. 2020;31(6):729–46.

23.

Bayne

. Revised guide for the care and use of laboratory animals available. Physiologist. 1996;39(4):199208–11.

24.

Asami

Inagaki

Imura

Sekiguchi

Fujimori

Masutani

Yodoi

Satomi

Ohuchi

Goto

. Thioredoxin-1 attenuates early graft loss after intraportal islet transplantation in mice. PLoS ONE. 2013;8(8):e70259. doi:10.1371/journal.pone.0070259

25.

Fathi

Nishimura

Imura

Inagaki

Kanai

Ushiyama

Kikuchi

Maekawa

Yamaguchi

Goto

. KRP-203 is a desirable immunomodulator for islet allotransplantation. Transplantation. 2022;106(5):963–72.

26.

Jimbo

Inagaki

Imura

Sekiguchi

Nakamura

Fujimori

Miyagawa

Ohuchi

Satomi

Goto

. A novel resting strategy for improving islet engraftment in the liver. Transplantation. 2014;97(3):280–86.

27.

Fujio

Murayama

Yamagata

Watanabe

Imura

Inagaki

Ohbayashi

Shima

Sekiguchi

Fujimori

Igarashi

, et al. Collagenase H is crucial for isolation of rat pancreatic islets. Cell Transplant. 2014;23(10):1187–98.

28.

Dendo

Maeda

Yamagata

Murayama

Watanabe

Imura

Inagaki

Igarashi

Katoh

Ebina

Fujimori

, et al. Synergistic effect of neutral protease and clostripain on rat pancreatic islet isolation. Transplantation. 2015;99(7):1349–55.

29.

Schmidt

Krook

Goto

Korsgren

. MyD88-dependent toll-like receptor signalling is not a requirement for fetal islet xenograft rejection in mice. Xenotransplantation. 2004;11(4):347–52.

30.

Miyazaki

Murayama

Fathi

Imura

Yamagata

Watanabe

Maeda

Inagaki

Igarashi

Miyagi

Shima

, et al. Strategy toward tailored donor tissue-specific pancreatic islet isolation. PLoS ONE. 2019;14(5):e0216136. doi:10.1371/journal.pone.0216136

31.

Choong

Freeman

Parish

Simeonovic

. Islet heparan sulfate but not heparan sulfate proteoglycan core protein is lost during islet isolation and undergoes recovery post-islet transplantation. Am J Transpl. 2015;15(11):2851–64.

32.

Cross

Vaughan

Willcox

McBride

Abraham

Han

Johnson

Maillard

Bateman

Ramracheya

Rorsman

, et al. Key matrix proteins within the pancreatic islet basement membrane are differentially digested during human islet isolation. Am J Transplant. 2017;17(2):451–61.

33.

Daoud

Petropavlovskaia

Rosenberg

Tabrizian

. The effect of extracellular matrix components on the preservation of human islet function in vitro. Biomaterials. 2010;31(7):1676–82.

34.

Olaniru

Pingitore

Giera

Piao

Castañera González

Jones

Persaud

. The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve β-cell function. Cell Mol Life Sci. 2018;75(21):4007–19.

35.

Klopfleisch

. Macrophage reaction against biomaterials in the mouse model—phenotypes, functions and markers. Acta Biomater. 2016;43:3–13.

36.

Yang

Gao

Xiao

Zhao

Liu

. Naringin improves sepsis-induced intestinal injury by modulating macrophage polarization via PPARγ/miR-21 axis. Mol Ther Nucleic Acids. 2021;25:502–14.

37.

Nakagawa

Ohnishi

Kosaki

Saito

Horlad

Fujiwara

Takeya

Komohara

. Optimum immunohistochemical procedures for analysis of macrophages in human and mouse formalin fixed paraffin-embedded tissue samples. J Clin Exp Hematop. 2017;57(1):31–36.

38.

Juang

Bonner-Weir

Ogawa

Vacanti

Weir

. Outcome of subcutaneous islet transplantation improved by polymer device. Transplantation. 1996;61(11):1557–61.

39.

Nishimura

Nishioka

Fujisawa

Shiku

Shimada

Sekiguchi

Fujimori

Ushiyama

Matsue

Ohuchi

Satomi

, et al. Tacrolimus inhibits the revascularization of isolated pancreatic islets. PLoS ONE. 2013;8(4):e56799. doi:10.1371/journal.pone.0056799

40.

Song

Chiu

Wang

L-H

Schwartz

Bouklas

Bowers

Cheong

Flanders

Pardo

, et al. Engineering transferrable microvascular meshes for subcutaneous islet transplantation. Nature Commun. 2019;10(1):1–12.

41.

Pepper

Gala-Lopez

Pawlick

Merani

Kin

Shapiro

. A prevascularized subcutaneous device-less site for islet and cellular transplantation. Nat Biotechnol. 2015;33(5):518–23.

42.

Peters

Polverini

Mooney

. Engineering vascular networks in porous polymer matrices. J Biomed Mater Res. 2002;60(4):668–78.

43.

Cross

Claesson-Welsh

. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci. 2001;22(4):201–207.

44.

Golocheikine

Tiriveedhi

Angaswamy

Benshoff

Sabarinathan

Mohanakumar

. Cooperative signaling for angiogenesis and neovascularization by VEGF and HGF following islet transplantation. Transplantation. 2010;90(7):725–31.

45.

Saitoh

Inagaki

Fathi

Imura

Nishimaki

Ogasawara

Matsumura

Miyagi

Yasunami

Unno

Kamei

, et al. Improvement of hepatocyte engraftment by co-transplantation with pancreatic islets in hepatocyte transplantation. J Tissue Eng Regen Med. 2021;15(4):361–74.

46.

Sakata

Goto

Yoshimatsu

Egawa

Unno

. Utility of co-transplanting mesenchymal stem cells in islet transplantation. World J Gastroenterol. 2011;17(47):5150–55.

47.

Naitoh

Hosokawa

Kubota

, et al. Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid. Biochem Biophys Res Commun. 2001;280(5):1316–22.

48.

Sidgwick

Bayat

. Extracellular matrix molecules implicated in hypertrophic and keloid scarring. J Eur Acad Dermatol Venereol. 2012;26(2):141–52.

49.

Sawaragi

Sakamoto

Nakano

Yamanaka

Tsuge

Matsuno

Tabata

Morimoto

. Development of gelatin hydrogel nonwoven fabrics (Genocel®) as a novel skin substitute in murine skin defects. Regen Ther. 2022;21:96–103.

50.

Shapouri-Moghaddam

Mohammadian

Vazini

Taghadosi

Esmaeili

Mardani

Seifi

Mohammadi

Afshari

Sahebkar

. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol. 2018;233(9):6425–40.

51.

Yunna

Mengru

Lei

Weidong

. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020;877:173090. doi:10.1016/j.ejphar.2020.173090

52.

Rőszer

. Understanding the mysterious M2 macrophage through activation markers and effector mechanisms. Mediators Inflamm. 2015;2015:816460. doi:10.1155/2015/816460

53.

Lucke

Hoene

Walschus

Kob

Pissarek

Schlosser

. Acute and chronic local inflammatory reaction after implantation of different extracellular porcine dermis collagen matrices in rats. Biomed Res Int. 2015;2015:938059. doi:10.1155/2015/938059

54.

Maeda

Goto

Zhang

, et al. Immunosuppression with FTY720 and cyclosporine A inhibits rejection of adult porcine islet xenografts in rats. Transplantation. 2003;75(8):1409–14.

55.

Luttikhuizen

Harmsen

Van Luyn

. Cellular and molecular dynamics in the foreign body reaction. Tissue Eng. 2006;12(7):1955–70.

56.

Maurer

Von Stebut

. Macrophage inflammatory protein-1. Int J Biochem Cell Biol. 2004;36(10):1882–86.

57.

Menten

Wuyts

Van Damme

. Macrophage inflammatory protein-1. Cytok Growth Factor Rev. 2002;13(6):455–81.

58.

Hughes

Rojas-Canales

Drogemuller

Voelcker

Grey

Coates

. IGF2: an endocrine hormone to improve islet transplant survival. J Endocrinol. 2014;221(2):R41–18.

59.

Costa

Soares

. Neovascularization in diabetes and its complications. Unraveling the angiogenic paradox. Life Sci. 2013;92(22):1037–45.

Ideal Duration of Pretreatment Using a Gelatin Hydrogel Nonwoven Fabric Prior to Subcutaneous Islet Transplantation

Abstract

Keywords

Introduction

Materials and Methods

Animals

The Induction and Diagnosis of Diabetes in the Recipients

Islet Isolation

Implantation of GHNF and Islet Transplantation

Intraperitoneal Glucose Tolerance Test

Immunohistochemical Analyses

Real-Time PCR Using TaqMan Arrays

Immunoassay for the Protein Expression of Inflammatory Mediators and Islet-Protective Factors

Statistical Analyses

Results

Comparison of Islet Engraftment After Marginal Islet Mass Transplantation Among the 2W, 4W, 6W, and 8W Groups

Intraperitoneal Glucose Tolerance Test

Time-Course Changes of GHNF Absorption Under the Subcutaneous Space

Immunohistochemical Analyses

Relative Gene Expression

Protein Expression of Inflammatory Mediators and Islet-Protective Factors

The Influence of Diabetic Conditions on the Impact of GHNF Pretreatment

Discussion

Footnotes

Acknowledgements

Author Contributions

Availability of Data and Materials

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

Research Ethics and Patient Consent

Declaration of Conflicting Interests

Funding

ORCID iDs

References