{"paper_id":"35a56f55-e7aa-47ae-b368-5ae820445cc2","body_text":"Improving Pancreatic Islet Transplantation Using Fibrin Hydrogel Containing Microvascular Fragments in Subcutaneous Tissue of Type I Diabetic Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improving Pancreatic Islet Transplantation Using Fibrin Hydrogel Containing Microvascular Fragments in Subcutaneous Tissue of Type I Diabetic Rats Saba Behzadifard, Mona Navaei-Nigjeh, Mahin Behzadifard, Ensiyeh Hajizadeh-Saffar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6626455/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective(s): Islet transplantation offers a promising treatment for Type 1 diabetes mellitus (T1DM). Subcutaneous tissue is a non-invasive site, but it has poor blood supply and requires more islets to achieve normoglycemia. We assessed the impact of fibrin hydrogel containing microvascular fragments (MVF) on the vascularization of subcutaneous tissue using two approaches: prevascularization (prior to islet transplantation) or co-transplantation (simultaneously with islet transplantation). Materials and methods: Wistar rats were prevascularized subcutaneously for 7 days with fibrin (H), 5000 MVF, or fibrin + 5000 MVF (HMVF). After streptozotocin injection to induce T1DM, 1500 islet equivalents (IEQ) were transplanted into prevascularized groups. A co-transplantation group received 1500 IEQ and HMVF (Co-HMVF 1500 ), and a control group received 3000 IEQ alone (Islet only 3000 ). Graft function was evaluated through blood glucose monitoring, glucose tolerance tests, immunostaining, and plasma insulin concentration over 28 days. Results: HMVF-prevascularized group presented significantly more CD31-positive cells compared with those from H- or MVF-prevascularized groups (p<0.05). Prevascularization and co-transplantation approaches using HMVF resulted in normoglycemia with a reduced islet mass (1500 IEQ) compared with those in the Islet only 3000 , H 1500 , and MVF 1500 groups. In addition, both the HMVF 1500 and Co-HMVF 1500 rats indicated superior islet survival and function, compared with the H 1500 , MVF 1500 , and Islet only 3000 groups, as evidenced by increased CD31-positive cells and insulin-positive cells, improved glucose tolerance, and elevated plasma insulin concentrations (p<0.05). Conclusion: Fibrin hydrogel containing MVF could significantly increase the survival and function of subcutaneously transplanted islets, allowing for effective glycemic control with a reduced islet mass. Fibrin Islet transplantation Microvascular Fragments Subcutaneous tissue Type 1 diabetes mellitus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Type 1 Diabetes Mellitus (T1DM) is an autoimmune disorder in which the immune system damages insulin-producing beta cells, necessitating lifelong insulin therapy (Quattrin, Mastrandrea et al. 2023 ). The standard treatment, intrahepatic islet transplantation, involves significant challenges, such as an instant blood-mediated inflammatory response (IBMIR), limited blood supply, and ischemic injury to the islets, leading to poor engraftment and function (Addison, Fatakhova et al. 2020 ). To overcome these issues, researchers are exploring extrahepatic transplantation sites, such as subcutaneous tissue, which offer benefits such as easier access, noninvasive procedures, and reduced IBMIR risk (Pellegrini 2020 ). However, the hypoxic nature of the subcutaneous space negatively affects islet survival and function, making enhanced vascularization a crucial step for improving outcomes (Cayabyab, Nih et al. 2021 ). Revascularization is crucial for the survival of transplanted beta cells; poor vascular integration results in hypoxia, impaired function, or cell death (Pepper, Gala-Lopez et al. 2015 ). Strategies to improve vascularization, including co-transplantation with vascular endothelial cells (ECs) and the use of proangiogenic factors, are being investigated to increase therapeutic efficacy (Dubiel, Lakey et al. 2014 , Bowers, Song et al. 2019 ). Experimental studies indicate that vascularization occurs with a delay between the graft and the host, initiating within 1–3 days and peaking approximately 14 days after transplantation, with complete vascularization typically achieved by day 28 (Mateus Gonçalves and Almaça 2021 ). To increase the success of subcutaneous islet transplantation, prevascularization techniques have been developed (Luan and Iwata 2014 , Später, Frueh et al. 2018 ). These methods aim to establish a suitable vascular bed prior to transplantation, thereby improving the integration and functionality of the transplanted islets. A notable approach is device-free prevascularization, which minimizes the risks associated with surgical tools, such as infection or mechanical damage (Vlahos, Talior-Volodarsky et al. 2021 , Zhou, Xu et al. 2023 ). In this study, we used fibrin hydrogel containing microvascular fragments (MVF) the prevascularization and co-transplantation methods for subcutaneous islet transplantation in T1DM rats and compared. MVF are preformed vascular segments that are easily isolated from adipose tissue and maintain a well-defined vascular structure with endothelial cells and pericytes (Frueh, Später et al. 2017 ). They serve as suitable vascularization units and are rich in proangiogenic factors (Laschke and Menger 2015 ). Compared with individual endothelial cells, MVF can rapidly reassemble into functional microvascular networks, which are crucial for the survival of islets during the critical initial post-transplant phase (Nalbach, Müller et al. 2021 ). Studies have shown that MVF demonstrate improved engraftment efficiency and vasculogenic activity in vivo, making them valuable in regenerative medicine and tissue engineering (Später, Marschall et al. 2021 ). FDA-approved fibrin is highly biodegradable, biocompatible, and biologically safe (Ahmed, Dare et al. 2008 ). Its structure facilitates interactions between cells and the extracellular matrix (ECM) (Riopel, Stuart et al. 2013 ). Studies indicate that fibrin-based scaffolds can effectively integrate grafted islets into host tissue, enhancing diabetes management outcomes (Najjar, Manzoli et al. 2015 ). When used as a carrier for pancreatic islets in subcutaneous grafts, fibrin stimulates local vascularization and prevents islet dispersion, making it an effective biomaterial for vascularizing pancreatic islets (Kim, Lim et al. 2012 ). We aimed to evaluate the potential of fibrin hydrogel containing MVF for subcutaneous prevascularization and co-transplantation. We further hypothesized that the fibrin-MVF composite would reduce the marginal islet mass and improve glycemic control compared with those of naked islets transplanted into unmodified subcutaneous tissue. Methods Chemical reagents and Antibodies Most of the chemical reagents used in this study were obtained from Sigma‒Aldrich (St. Louis, MO, USA) unless otherwise noted. The cell culture materials were sourced from Gibco (Gaithersburg, MD, USA). The antibodies used included CD31 (orb10314; 1:100) and αSMA (orb195993; 1:100) from Biorbyt (Durham, North Carolina, USA). The anti-insulin antibody (GTX34797; 1:100) was purchased from GeneTex (Alton Pkwy Irvine, CA, USA). Additionally, secondary antibodies, including those for rabbits (orb688925) and mice (orb688924), were also obtained from Biorbyt (Durham, North Carolina, USA). Animals Forty adult male Wistar rats weighing 250-300g were purchased from Pasteur Institute (Tehran, Iran). The animals were acclimated to the laboratory environment for one week. All rats were housed under constant conditions conditions: 23 ± 2°C, humidity (55% ± 5%), 12 h/12 h dark/light cycle, and free access to food and water. All research procedures were approved by the ethics committee of Tehran University of Medical Sciences (Ethics approval number: IR.TUMS.AEC.1402.066). The care and use of all animals were conducted in adherence to the guidelines outlined in the National Institute of Health Guide for the Care and Use of Laboratory Animals (8th edition). Isolation and characterization of MVF MVF were freshly isolated from the epididymal fat tissue of donor rats via established protocols (Shepherd, Chen et al. 2004 ). Briefly, the rats were anesthetized via intraperitoneal (IP) injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). Under aseptic conditions, epididymal fat pads were excised, minced, and subjected to enzymatic digestion using 2 mg/mL collagenase Type I with agitation for 7 minutes. The digestion process was stopped by the addition of Dulbecco's modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS). The suspension was then filtered sequentially through 500-µm and 30-µm nylon meshes and then washed twice by centrifugation at 600 × g for 5 minutes at room temperature. The final MVF pellet was resuspended in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin and incubated at 37°C with 5% CO2 for subsequent experiments. Research Design and Experimental Groups This study consisted of two phases. The first phase (PI) consisted of prevascularization for one week, from day − 7 to day 0. The second phase (PII) was the transplantation of islets performed on day 0 and took 28 days (Fig. 1 ). The experimental groups were as follows. Each group contained four rats: Normal group Diabetic group Diabetic rats prevascularized with fibrin hydrogel receiving 1500 islets (H 1500 ) Diabetic rats prevascularized with 5000 MVF receiving 1500 islets (MVF 1500 ) Diabetic rats prevascularized with fibrin hydrogel containing 5000 MVF receiving 1500 islets (HMVF 1500 ) Diabetic rats receiving co-transplantation of fibrin hydrogel containing 5000 MVF and 1500 islets (Co-HMVF 1500 ) Diabetic rats receiving 3000 islets (Islet only 3000 ) Hydrogel Preparation The prevascularization in PI involved the preparation of 100 µl of a mixture of 0.5% fibrinogen and 1 U/ml thrombin combined with 5000 MVF (25). This mixture was injected into the flanks of rats and allowed to polymerize for 10 minutes before the incision was closed via nonabsorbable sutures. Isolation and characterization of islets Pancreatic islets were extracted from donor rat pancreata via a collagenase digestion method described in previous studies (Moazenchi, Nejad et al. 2022 ). After digestion, the islets were separated from the digested tissue through centrifugation via a stepwise Ficoll density gradient (Pan-Biotech, Germany). The isolated islets were subsequently cultured overnight in RPMI-1640 medium (Roswell Park Memorial Institute Medium) supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin to facilitate recovery from the digestion procedure. The identity of the isolated islets was verified via dithizone (DTZ) staining, which specifically marks insulin-containing beta cells within the islets. Islet viability was evaluated via acridine orange/ethidium bromide (AO/EB) staining, which distinguishes live cells (green in color) from dead cells (red in color). Diabetes induction and islet transplantation Three days prior to islet transplantation, the recipient rats received a single IP injection of 50 mg/kg streptozotocin (STZ) dissolved in 0.1 mol/L sodium citrate buffer (pH = 4.5). Diabetic rats were selected for transplantation if their blood glucose levels consistently ranged from 250–350 mg/dL over two consecutive days following STZ injection. Diabetic animals were anesthetized through an IP injection of ketamine at a dosage of 100 mg/kg and xylazine at 10 mg/kg. In the prevascularized groups and the Co-HMVF group, 1500 islets were transplanted, whereas the positive control group received 3000 free islets subcutaneously. Recipient monitoring Post transplantation, non-fasting blood glucose was monitored twice a week during the first week and then weekly. Blood samples were collected from the tail vein, and glucose levels were measured via a glucometer (Roche Diagnostics, USA). Additionally, body weight was recorded weekly. Normoglycemia was defined as a blood glucose level ˂200 mg/dL. In addition, blood samples were collected from different groups via heart puncture at the end of PII. Plasma insulin levels were measured via a rat-specific ELISA kit (RK09278, Abclonal, USA). To validate graft-dependent normoglycemia, the HMVF 1500 and Co-HMVF 1500 groups underwent glycemic control for 2 days after graft removal. Assessment of graft function To assess the capacity of the graft to respond to a glucose bolus, the animals were fasted for 4 hours on day 21 before being administered a glucose bolus of 20% glucose solution (2 g/kg) via IP injection. Blood glucose levels were measured at each time point (before the test and after 30, 60, 90, and 120 minutes). Immunohistochemical analysis The subcutaneous implants were removed on day 28 for histological analysis. The subcutaneous tissue blocks were fixed in 4% paraformaldehyde at 4°C for 24 hours. Following dehydration, the samples were embedded in paraffin, and 5 µm-thick sections were prepared. The sections were permeabilized with 2% Triton X-100 for 10 minutes and blocked with goat serum. Primary antibodies, including anti-insulin (1:100), anti-CD31 (1:100), and anti-αSMA (1:100), were applied, and the sections were incubated overnight at 4°C. Secondary antibodies were then used for detection. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Analysis was conducted via a fluorescence microscope (BX60 microscope; Olympus, Japan). Every tenth section of the tissue sample was subjected to immunofluorescent (IF) staining. Positively stained cells relative to the total graft area in each section were quantified at 40x magnification via ImageJ software. The percentage of vascular density was acquired from 10 micrographs per rat. Statistical analysis Statistical analysis was performed via Prism software version 8 (GraphPad, USA, California). For comparisons among multiple groups, one-way ANOVA was employed, followed by the Tukey post hoc test. The results are presented as the means ± SDs, and P < 0.05 was considered statistically significant. Results Characterization of isolated MVF and islets The isolated MVF were characterized by CD31 and α-SMA surface markers, revealing a well-defined luminal structure (Fig. 2 a,b). Additionally, islets stained with DTZ were red in color. Islet viability assessment via AO/EB staining indicated high viability (93%) of the isolated islets. (Fig. 2 c,d). Fibrin hydrogel containing MVF improve subcutaneous vascularization Immunofluorescence analysis of graft-bearing subcutaneous tissue at the end of the PI revealed that the expression of CD31 in the HMVF group (37/94 ± 2/32%) was significantly greater than that in the H group (16/07 ± 1/08%, p < 0.0001) and MVF group (27/01 ± 2/39%, p < 0.01). Additionally, the MVF group had significantly greater numbers of CD31-positive cells than did the H group (p < 0.01) (Fig. 3 ). Immunofluorescence staining of graft-bearing subcutaneous tissue at the end of phase II revealed a significant difference in CD31 expression between the HMVF 1500 (34/68 ± 2/27%) and MVF 1500 groups (26/82 ± 3/17%, p < 0.05), but it was not statistically significant compared with the H 1500 group (31/32 ± 0/81%, p > 0.05) or the Co-HMVF 1500 group (31/12 ± 1/98%, p > 0.05). As expected, the Islet only 3000 group had lower CD31 expression than the other groups did (20/99 ± 2/437%) (Fig. 4 ). Fibrin hydrogel containing MVF supports subcutaneous islet engraftment and function Insulin expression in the HMVF 1500 group was significantly greater than that in the H 1500 and MVF 1500 groups (56/41 ± 0/79% vs. 41/32 ± 1/24% and 43/99 ± 2/66%, respectively, P < 0.001). In addition, there was no significant difference in insulin expression between the HMVF 1500 and Co-HMVF 1500 groups (52/08 ± 1/36%, P > 0.05). Insulin expression in the Co-HMVF 1500 group was significantly different from that in the H 1500 and MVF 1500 groups (p < 0.001 and p < 0.001, respectively) (Fig. 5 ). In the second phase, Islet only 3000 was not successful in reversing diabetes (Fig. 6 a). HMVF 1500 was superior to H 1500 and MVF 1500 in reducing blood glucose levels (Fig. 6 a, ✱✱ p < 0.01, ✱✱✱✱✱ p < 0.0001). The efficacy of MVF 1500 alone in lowering blood glucose levels was not comparable to that of the co-transplantation strategy (Fig. 6 a, ✱✱ p < 0.01). Co-delivery of the hydrogel and MVF in both the prevascularization and co-transplantation methods led to euglycemia over 28 days after islet transplantation. Following graft removal, blood glucose in the HMVF 1500 and Co-HMVF 1500 groups increased within 2 days and showed graft-dependent insulin dependency (Fig. 6 a). Among the different transplanted groups, the body weights of the MVF 1500 and Islet-only 3000 groups tended to decrease (Fig. 6 b). Moreover, no significant differences were observed in the body weights of the rats in either of the transplanted groups over the 28-day study period. The results of the IPGTTs revealed that both the HMVF 1500 and Co-HMVF 1500 groups were able to respond to the glucose bolus and reached normoglycemia within 120 minutes (Fig. 6 c, purple and green lines). The related area under the curve (AUC) for the HMVF 1500 and Co-HMVF 1500 groups was smaller than that for the Islet only 3000 group (Fig. 6 d). There was no significant difference in the AUC between the HMVF 1500 or Co-HMVF 1500 group and the normal group (P > 0.05) (Fig. 6 d). In the H 1500 and MVF 1500 groups, the blood glucose levels remained above 200 mg/dl (Fig. 6 c, blue and red lines). Additionally, animals that received only islet transplantation in the unmodified subcutaneous space were unable to respond to the glucose challenge and remained hyperglycemic (Fig. 6 c, gray line), as confirmed by the increased AUC of blood glucose (Fig. 6 d). The same pattern was observed for the plasma insulin concentration, which was significantly greater in the HMVF 1500 and Co-HMVF 1500 groups (9.435 ± 0.24 µU and 9.258 ± 0.55 µU, respectively) than in the Islet only 3000 group (6.686 ± 0.30 µU, p < 0.0001; Fig. 5 e). However, the insulin concentration in these groups was not significantly different from that in the normal group (10.27 ± 0.30 µU, p > 0.5; Fig. 6 e). Additionally, the HMVF 1500 and Co-HMVF 1500 groups presented superior results to those of the H1500 and MVF1500 groups (Fig. 6 e). Overall, this co-delivery approach led to a 50% reduction in the marginal islet mass required to reverse diabetes in rats. Discussion Subcutaneous tissue is gaining attention as an alternative for extrahepatic islet transplantation because of its accessibility and reduced risk of IBMIR (Smink, Li et al. 2017 ). However, subcutaneous tissue requires a significant number of islets to maintain normal glucose levels because of its low oxygen pressure and inadequate blood supply (Zhou, Xu et al. 2023 ). Additionally, the process of isolating islets, particularly through collagenase digestion, can adversely affect the intra islet microvasculature (Santini-González, Simonovich et al. 2021 ). Therefore, it is essential to employ techniques for regenerating the ECM and enhancing islet vascularization. To address these challenges, we investigated improvements in subcutaneous islet transplantation via the vasculogenic potential of fibrin hydrogels containing MVF in diabetic rat models. To the best of our knowledge, this study is the first to demonstrate that the combination of fibrin hydrogel and MVF synergistically enhances vascularization for subcutaneous islet transplantation. Fibrin hydrogels not only provide structural support by delivering arginine-glycine-aspartic acid (RGD) sequences that bind to integrins (αvβ1), maintaining the spatial organization of islets but also degrade within 28 days without eliciting foreign body reactions (Kim, Lim et al. 2012 , Kuehn, Lakey et al. 2013 ). Studies conducted in porcine models indicate that fibrin alone can induce angiogenesis (Salama, Seeberger et al. 2020 ), although its effects are enhanced when additional vasculogenic factors are included. Nalbach demonstrated that, compared with the use of single fat-derived cells, prevascularized islets with MVF before transplantation into diabetic mice improved vessel fusion, blood perfusion, islet viability, and function (Nalbach, Roma et al. 2021). We hypothesized that MVF could further enhance vasculogenic stimulation within the fibrin hydrogel. Embedding islets with MVF in collagen hydrogels promotes in vitro islet vascularization. Within 8 days, MVF-derived ECs form capillary sprouts connecting to the islets, which maintain viability, function, and glucose-stimulated insulin secretion (Salamone, Rigogliuso et al. 2021 ). Previous studies have shown that MVF-derived ECs can establish functional capillaries within just 7 days, resulting in a 2.8-fold increase in capillary density, a 42% increase in blood flow velocity during the early post-transplant phase, and a 65% increase in the revascularized area, which reduces the marginal islet mass requirement from 600 to 250 islets in mouse models (Nalbach, Roma et al. 2021). Our results indicated that using MVF combined with fibrin results in more favorable engraftment, which decreases the marginal islet mass by 50% in diabetic rats. The vascular density also significantly improved, with 34% and 31% in the HMVF groups compared with 26% in the MVF group. Interestingly, we observed no significant difference in islet function between the HMVF1500 (prevascularized) and Co-HMVF 1500 (co-transplantation) groups. Both groups benefited from the combined use of fibrin hydrogel and MVF, with fibrin providing a scaffold for vascular network formation while also supporting islet viability by mimicking the extracellular matrix. MVF secrete proangiogenic factors such as growth factor (HGF) and vascular endothelial growth factor (VEGF), enhancing both vascularization and islet survival (Aghazadeh, Poon et al. 2021 , Wrublewsky, Weinzierl et al. 2022 ). This synergy likely created a sufficiently supportive microenvironment regardless of the timing of application. Moreover, the initial days post transplantation represents a critical and vulnerable period for islet survival because of factors such as hypoxia and inflammation. Perhaps both our prevascularization and co-transplantation strategies provided sufficient microvascular support to meet the metabolic demands of the islets, leading to similar outcomes at the 28-day endpoint through MVF-derived endothelial cell sprouting, reduced apoptosis via HGF-mediated survival signals, and enhanced oxygen and nutrient diffusion through the porous structure of fibrin. Finally, we must acknowledge that the lack of a statistically significant difference could be due to limitations in our study design, such as the sample size and the sensitivity of our methods for assessing microvascular architecture. Future research could benefit from extended observation periods of more than three months to assess durability and more sophisticated imaging techniques. Conclusion In summary, our study demonstrates that the inherent pro-angiogenic properties of fibrin, combined with the vascularization potential of microvascular fragments, create a synergistic environment that promotes vascularization, improved islet survival and function. This approach addresses the critical challenge of inadequate vascularization in subcutaneous islet transplantation and shows potential for reducing the quantity of donor islets required for successful outcomes. Declarations The authors report no conflict of interest. Author Contribution SB, MNN, and BMZ designed the experiments; SB and AA performed experiments and collected data; MB, EHS, and FMZ discussed the results and strategy; BMZ Supervised, directed and managed the study; SB prepared the original draft. All authors reviewed the manuscript and approved the final version. Acknowledgment This work was fully supported by a by a grant from the Tehran University of Medical Sciences (grant number:1402.3.410.68148). 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Sefton (2021). \"A scalable device-less biomaterial approach for subcutaneous islet transplantation.\" Biomaterials 269: 120499. https://doi.org/10.1016/j.biomaterials.2020.120499 Wrublewsky, S., A. Weinzierl, I. Hornung, L. Prates-Roma, M. D. Menger, M. W. Laschke and E. Ampofo (2022). \"Co-transplantation of pancreatic islets and microvascular fragments effectively restores normoglycemia in diabetic mice.\" NPJ Regenerative Medicine 7(1): 67. https://doi.org/10.1038/s41536-022-00262-3 Zhou, X., Z. Xu, Y. You, W. Yang, B. Feng, Y. Yang, F. Li, J. Chen and H. Gao (2023). \"Subcutaneous device-free islet transplantation.\" Frontiers in Immunology 14: 1287182. https://doi.org/10.3389/fimmu.2023.1287182 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6626455\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":457036877,\"identity\":\"fb8ab5f2-d9bd-4f72-89ba-4bc09cc819bc\",\"order_by\":0,\"name\":\"Saba Behzadifard\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tehran University of Medical Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Saba\",\"middleName\":\"\",\"lastName\":\"Behzadifard\",\"suffix\":\"\"},{\"id\":457036878,\"identity\":\"af45c9fd-159d-4909-bec5-e8af8485153c\",\"order_by\":1,\"name\":\"Mona Navaei-Nigjeh\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tehran University of Medical Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mona\",\"middleName\":\"\",\"lastName\":\"Navaei-Nigjeh\",\"suffix\":\"\"},{\"id\":457036879,\"identity\":\"ddd05bd3-d93c-4fcc-8a14-d40746ec0935\",\"order_by\":2,\"name\":\"Mahin Behzadifard\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Dezful university of medical sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mahin\",\"middleName\":\"\",\"lastName\":\"Behzadifard\",\"suffix\":\"\"},{\"id\":457036880,\"identity\":\"be8b4fd0-1ae7-4449-bc43-8a153318f196\",\"order_by\":3,\"name\":\"Ensiyeh Hajizadeh-Saffar\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Royan Institute\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ensiyeh\",\"middleName\":\"\",\"lastName\":\"Hajizadeh-Saffar\",\"suffix\":\"\"},{\"id\":457036881,\"identity\":\"2ad4c5eb-3fdd-4f68-b006-3b14d02a270c\",\"order_by\":4,\"name\":\"Fatemeh Minaei-Zangi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tehran University of Medical Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fatemeh\",\"middleName\":\"\",\"lastName\":\"Minaei-Zangi\",\"suffix\":\"\"},{\"id\":457036882,\"identity\":\"b9a4ec94-2df1-431d-a0e4-16b1b3e092ce\",\"order_by\":5,\"name\":\"Arefeh Aryan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Lorestan University of Medical Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Arefeh\",\"middleName\":\"\",\"lastName\":\"Aryan\",\"suffix\":\"\"},{\"id\":457036883,\"identity\":\"e9fe0491-33b8-4860-94ae-e91338dc69f1\",\"order_by\":6,\"name\":\"Bagher Minaei-Zangi\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYDACZjB5QA4uwEasFmMe4rVAwIHEHqLdpdvOe/DDhz930vdLZCd/YKixY+CTPoBfi9lhvmTJGTzPcnskcrdJMBxLZmDjSyCkhcdAmkfiMFgL0CMHGNh48OsAaTH+/cfgcDqPRO7mDwz/iNNiJs2QcDgBqGWDBGMbkVosew48M+w583abRGJfMg9hLefPGN/48eeOPHs70GEfvtnJyRMf3CCQwMBAyI5RMApGwSgYBcQAAAu0O+Pr8xxOAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Tehran University of Medical Sciences\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Bagher\",\"middleName\":\"\",\"lastName\":\"Minaei-Zangi\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-05-09 08:08:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6626455/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6626455/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":83035340,\"identity\":\"c94b0588-52d8-4863-81d5-9e22634051b5\",\"added_by\":\"auto\",\"created_at\":\"2025-05-19 09:48:48\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":243954,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTimeline of transplant phases. \\u003c/strong\\u003eThe HMVF used by two approches: prevascularization for one-week (Phase I, days -7 to 0) followed by a 28-day post islet transplantation ((Phase II, starting on day 0), or Co-transplantation approach on day 0 followed by a 28-day post islet transplantation\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6626455/v1/a163ef9f339f11b5fdb6469e.png\"},{\"id\":83035338,\"identity\":\"5e464cce-c058-4140-a32d-49200848e25b\",\"added_by\":\"auto\",\"created_at\":\"2025-05-19 09:48:48\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":206229,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of isolated MVF and islets.\\u003c/strong\\u003e A, B) Immunofluorescence staining MVF for the CD31 and α-SMA markers, respectively (scale bar= 20 μm). C) DTZ staining of islets (scale bar= 100 μm). D) AO/EB staining of islets (scale bar= 200 μm)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6626455/v1/ff1cd06ec8e924b988835b9f.png\"},{\"id\":83036468,\"identity\":\"57ce9a92-6408-4e4f-be39-3e65f553d437\",\"added_by\":\"auto\",\"created_at\":\"2025-05-19 09:56:48\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":369829,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunofluorescence staining of CD31 in the prevascularized groups at the end of the PI.\\u003c/strong\\u003e\\u0026nbsp; Fibrin hydrogel (H), microvascular fragment (MVF), and fibrin hydrogel containing MVF (HMVF). DAPI (blue): nucleus; anti-CD31 (green). The values represent the means ± SDs. (n=4 per group, \\u003csup\\u003e✱✱\\u003c/sup\\u003ep\\u0026lt;0.01, \\u003csup\\u003e✱✱✱✱\\u003c/sup\\u003e p\\u0026lt;0.0001, scale bar= 20 μm)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6626455/v1/d9c8161db941663e27170f3d.png\"},{\"id\":83037593,\"identity\":\"9dd056a9-96ed-4345-9ca8-2c336257172f\",\"added_by\":\"auto\",\"created_at\":\"2025-05-19 10:12:48\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":457944,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemical analysis of graft-bearing subcutaneous tissue at the end of phase II.\\u003c/strong\\u003e Immunofluorescence staining of CD31 in the islet-receiving groups on day 28 post islet transplantation: H1500 (fibrin hydrogel+1500 islets), MVF1500 (microvascular fragment+1500 islets), HMVF1500 (fibrin hydrogel containing MVF+1500 islets), co-HMVF1500 (co-transplantation of fibrin hydrogel containing MVF+1500 islets), and islet-only 3000. DAPI (blue): nucleus; anti-CD31 (green). The values represent the means ± SDs. (n=4 per group, \\u003csup\\u003e✱\\u003c/sup\\u003ep\\u0026lt;0.05, ns= non-significant, scale bar= 20 μm)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6626455/v1/60f3e65c49383246f0db2872.png\"},{\"id\":83036643,\"identity\":\"b1840dae-e512-4350-81f1-9d3ed3930b48\",\"added_by\":\"auto\",\"created_at\":\"2025-05-19 10:04:48\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":411868,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemical analysis of graft-bearing subcutaneous tissue at the end of phase II. \\u003c/strong\\u003eImmunofluorescence staining of insulin in the islet-receiving groups on day 28 post islet transplantation: H\\u003csup\\u003e1500\\u003c/sup\\u003e (fibrin hydrogel+1500 islets), MVF\\u003csup\\u003e1500\\u003c/sup\\u003e (microvascular fragment+1500 islets), HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e (fibrin hydrogel containing MVF+1500 islets), co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e (co-transplantation of fibrin hydrogel containing MVF+1500 islets), and islets only \\u003csup\\u003e3000\\u003c/sup\\u003e. DAPI (blue): nucleus; anti-insulin (green). The values represent the means ± SDs. (n=4 per group, \\u003csup\\u003e✱✱✱\\u003c/sup\\u003e p\\u0026lt;0.001, ns= nonsignificant, scale bar= 20 μm)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6626455/v1/88003c2a9b01b2f7ba6439f1.png\"},{\"id\":83035343,\"identity\":\"e4a64c3d-304b-46a2-bc3f-ce38d58cd334\",\"added_by\":\"auto\",\"created_at\":\"2025-05-19 09:48:48\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":457177,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eGraft function assessment.\\u003c/strong\\u003e (a) Blood glucose levels, (b) body weights, (c) IPGTT (intraperitoneal glucose tolerance test), (d) AUC (area under the curve) of the IPGTT and (e) plasma insulin concentration (n=4 per group, Tx: transplantation, \\u003csup\\u003e✱\\u003c/sup\\u003ep \\u0026lt; 0.05, \\u003csup\\u003e✱✱\\u003c/sup\\u003ep\\u0026lt;0.01, \\u003csup\\u003e✱✱✱\\u003c/sup\\u003e p\\u0026lt;0.001, \\u003csup\\u003e✱✱✱✱ \\u003c/sup\\u003ep\\u0026lt;0.0001, ns= non-significant)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6626455/v1/4d5887644fa250ef3183d3ea.png\"},{\"id\":97215484,\"identity\":\"a9517a01-858b-4543-8682-1af00fea8b9c\",\"added_by\":\"auto\",\"created_at\":\"2025-12-02 06:09:01\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3042459,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6626455/v1/cec94043-4121-47d8-a424-16c07ed4ac1c.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Improving Pancreatic Islet Transplantation Using Fibrin Hydrogel Containing Microvascular Fragments in Subcutaneous Tissue of Type I Diabetic Rats\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eType 1 Diabetes Mellitus (T1DM) is an autoimmune disorder in which the immune system damages insulin-producing beta cells, necessitating lifelong insulin therapy (Quattrin, Mastrandrea et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). The standard treatment, intrahepatic islet transplantation, involves significant challenges, such as an instant blood-mediated inflammatory response (IBMIR), limited blood supply, and ischemic injury to the islets, leading to poor engraftment and function (Addison, Fatakhova et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). To overcome these issues, researchers are exploring extrahepatic transplantation sites, such as subcutaneous tissue, which offer benefits such as easier access, noninvasive procedures, and reduced IBMIR risk (Pellegrini \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). However, the hypoxic nature of the subcutaneous space negatively affects islet survival and function, making enhanced vascularization a crucial step for improving outcomes (Cayabyab, Nih et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eRevascularization is crucial for the survival of transplanted beta cells; poor vascular integration results in hypoxia, impaired function, or cell death (Pepper, Gala-Lopez et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Strategies to improve vascularization, including co-transplantation with vascular endothelial cells (ECs) and the use of proangiogenic factors, are being investigated to increase therapeutic efficacy (Dubiel, Lakey et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e, Bowers, Song et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Experimental studies indicate that vascularization occurs with a delay between the graft and the host, initiating within 1\\u0026ndash;3 days and peaking approximately 14 days after transplantation, with complete vascularization typically achieved by day 28 (Mateus Gon\\u0026ccedil;alves and Alma\\u0026ccedil;a \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). To increase the success of subcutaneous islet transplantation, prevascularization techniques have been developed (Luan and Iwata \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e, Sp\\u0026auml;ter, Frueh et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). These methods aim to establish a suitable vascular bed prior to transplantation, thereby improving the integration and functionality of the transplanted islets. A notable approach is device-free prevascularization, which minimizes the risks associated with surgical tools, such as infection or mechanical damage (Vlahos, Talior-Volodarsky et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e, Zhou, Xu et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). In this study, we used fibrin hydrogel containing microvascular fragments (MVF) the prevascularization and co-transplantation methods for subcutaneous islet transplantation in T1DM rats and compared.\\u003c/p\\u003e \\u003cp\\u003eMVF are preformed vascular segments that are easily isolated from adipose tissue and maintain a well-defined vascular structure with endothelial cells and pericytes (Frueh, Sp\\u0026auml;ter et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). They serve as suitable vascularization units and are rich in proangiogenic factors (Laschke and Menger \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Compared with individual endothelial cells, MVF can rapidly reassemble into functional microvascular networks, which are crucial for the survival of islets during the critical initial post-transplant phase (Nalbach, M\\u0026uuml;ller et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Studies have shown that MVF demonstrate improved engraftment efficiency and vasculogenic activity in vivo, making them valuable in regenerative medicine and tissue engineering (Sp\\u0026auml;ter, Marschall et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e FDA-approved fibrin is highly biodegradable, biocompatible, and biologically safe (Ahmed, Dare et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). Its structure facilitates interactions between cells and the extracellular matrix (ECM) (Riopel, Stuart et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). Studies indicate that fibrin-based scaffolds can effectively integrate grafted islets into host tissue, enhancing diabetes management outcomes (Najjar, Manzoli et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). When used as a carrier for pancreatic islets in subcutaneous grafts, fibrin stimulates local vascularization and prevents islet dispersion, making it an effective biomaterial for vascularizing pancreatic islets (Kim, Lim et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). We aimed to evaluate the potential of fibrin hydrogel containing MVF for subcutaneous prevascularization and co-transplantation. We further hypothesized that the fibrin-MVF composite would reduce the marginal islet mass and improve glycemic control compared with those of naked islets transplanted into unmodified subcutaneous tissue.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eChemical reagents and Antibodies\\u003c/h2\\u003e \\u003cp\\u003eMost of the chemical reagents used in this study were obtained from Sigma‒Aldrich (St. Louis, MO, USA) unless otherwise noted. The cell culture materials were sourced from Gibco (Gaithersburg, MD, USA). The antibodies used included CD31 (orb10314; 1:100) and αSMA (orb195993; 1:100) from Biorbyt (Durham, North Carolina, USA). The anti-insulin antibody (GTX34797; 1:100) was purchased from GeneTex (Alton Pkwy Irvine, CA, USA). Additionally, secondary antibodies, including those for rabbits (orb688925) and mice (orb688924), were also obtained from Biorbyt (Durham, North Carolina, USA).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eAnimals\\u003c/h3\\u003e\\n\\u003cp\\u003eForty adult male Wistar rats weighing 250-300g were purchased from Pasteur Institute (Tehran, Iran). The animals were acclimated to the laboratory environment for one week. All rats were housed under constant conditions conditions: 23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2\\u0026deg;C, humidity (55% \\u0026plusmn; 5%), 12 h/12 h dark/light cycle, and free access to food and water. All research procedures were approved by the ethics committee of Tehran University of Medical Sciences (Ethics approval number: IR.TUMS.AEC.1402.066). The care and use of all animals were conducted in adherence to the guidelines outlined in the National Institute of Health Guide for the Care and Use of Laboratory Animals (8th edition).\\u003c/p\\u003e\\n\\u003ch3\\u003eIsolation and characterization of MVF\\u003c/h3\\u003e\\n\\u003cp\\u003eMVF were freshly isolated from the epididymal fat tissue of donor rats via established protocols (Shepherd, Chen et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). Briefly, the rats were anesthetized via intraperitoneal (IP) injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). Under aseptic conditions, epididymal fat pads were excised, minced, and subjected to enzymatic digestion using 2 mg/mL collagenase Type I with agitation for 7 minutes. The digestion process was stopped by the addition of Dulbecco's modified Eagle\\u0026rsquo;s medium (DMEM) supplemented with 20% fetal bovine serum (FBS). The suspension was then filtered sequentially through 500-\\u0026micro;m and 30-\\u0026micro;m nylon meshes and then washed twice by centrifugation at 600 \\u0026times; g for 5 minutes at room temperature. The final MVF pellet was resuspended in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin and incubated at 37\\u0026deg;C with 5% CO2 for subsequent experiments.\\u003c/p\\u003e\\n\\u003ch3\\u003eResearch Design and Experimental Groups\\u003c/h3\\u003e\\n\\u003cp\\u003eThis study consisted of two phases. The first phase (PI) consisted of prevascularization for one week, from day \\u0026minus;\\u0026thinsp;7 to day 0. The second phase (PII) was the transplantation of islets performed on day 0 and took 28 days (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The experimental groups were as follows. Each group contained four rats:\\u003c/p\\u003e \\u003cp\\u003e \\u003col\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eNormal group\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eDiabetic group\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eDiabetic rats prevascularized with fibrin hydrogel receiving 1500 islets (H\\u003csup\\u003e1500\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eDiabetic rats prevascularized with 5000 MVF receiving 1500 islets (MVF\\u003csup\\u003e1500\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eDiabetic rats prevascularized with fibrin hydrogel containing 5000 MVF receiving 1500 islets (HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eDiabetic rats receiving co-transplantation of fibrin hydrogel containing 5000 MVF and 1500 islets (Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eDiabetic rats receiving 3000 islets (Islet only \\u003csup\\u003e3000\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003c/ol\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eHydrogel Preparation\\u003c/h3\\u003e\\n\\u003cp\\u003eThe prevascularization in PI involved the preparation of 100 \\u0026micro;l of a mixture of 0.5% fibrinogen and 1 U/ml thrombin combined with 5000 MVF (25). This mixture was injected into the flanks of rats and allowed to polymerize for 10 minutes before the incision was closed via nonabsorbable sutures.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIsolation and characterization of islets\\u003c/h2\\u003e \\u003cp\\u003ePancreatic islets were extracted from donor rat pancreata via a collagenase digestion method described in previous studies (Moazenchi, Nejad et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). After digestion, the islets were separated from the digested tissue through centrifugation via a stepwise Ficoll density gradient (Pan-Biotech, Germany). The isolated islets were subsequently cultured overnight in RPMI-1640 medium (Roswell Park Memorial Institute Medium) supplemented with 10% FBS, 100 units/mL penicillin, and 100 \\u0026micro;g/mL streptomycin to facilitate recovery from the digestion procedure. The identity of the isolated islets was verified via dithizone (DTZ) staining, which specifically marks insulin-containing beta cells within the islets. Islet viability was evaluated via acridine orange/ethidium bromide (AO/EB) staining, which distinguishes live cells (green in color) from dead cells (red in color).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eDiabetes induction and islet transplantation\\u003c/h3\\u003e\\n\\u003cp\\u003eThree days prior to islet transplantation, the recipient rats received a single IP injection of 50 mg/kg streptozotocin (STZ) dissolved in 0.1 mol/L sodium citrate buffer (pH\\u0026thinsp;=\\u0026thinsp;4.5). Diabetic rats were selected for transplantation if their blood glucose levels consistently ranged from 250\\u0026ndash;350 mg/dL over two consecutive days following STZ injection. Diabetic animals were anesthetized through an IP injection of ketamine at a dosage of 100 mg/kg and xylazine at 10 mg/kg. In the prevascularized groups and the Co-HMVF group, 1500 islets were transplanted, whereas the positive control group received 3000 free islets subcutaneously.\\u003c/p\\u003e\\n\\u003ch3\\u003eRecipient monitoring\\u003c/h3\\u003e\\n\\u003cp\\u003ePost transplantation, non-fasting blood glucose was monitored twice a week during the first week and then weekly. Blood samples were collected from the tail vein, and glucose levels were measured via a glucometer (Roche Diagnostics, USA). Additionally, body weight was recorded weekly. Normoglycemia was defined as a blood glucose level ˂200 mg/dL. In addition, blood samples were collected from different groups via heart puncture at the end of PII. Plasma insulin levels were measured via a rat-specific ELISA kit (RK09278, Abclonal, USA). To validate graft-dependent normoglycemia, the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups underwent glycemic control for 2 days after graft removal.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAssessment of graft function\\u003c/h2\\u003e \\u003cp\\u003eTo assess the capacity of the graft to respond to a glucose bolus, the animals were fasted for 4 hours on day 21 before being administered a glucose bolus of 20% glucose solution (2 g/kg) via IP injection. Blood glucose levels were measured at each time point (before the test and after 30, 60, 90, and 120 minutes).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunohistochemical analysis\\u003c/h2\\u003e \\u003cp\\u003eThe subcutaneous implants were removed on day 28 for histological analysis. The subcutaneous tissue blocks were fixed in 4% paraformaldehyde at 4\\u0026deg;C for 24 hours. Following dehydration, the samples were embedded in paraffin, and 5 \\u0026micro;m-thick sections were prepared. The sections were permeabilized with 2% Triton X-100 for 10 minutes and blocked with goat serum. Primary antibodies, including anti-insulin (1:100), anti-CD31 (1:100), and anti-αSMA (1:100), were applied, and the sections were incubated overnight at 4\\u0026deg;C. Secondary antibodies were then used for detection. Nuclei were counterstained with 4\\u0026prime;,6-diamidino-2-phenylindole dihydrochloride (DAPI). Analysis was conducted via a fluorescence microscope (BX60 microscope; Olympus, Japan). Every tenth section of the tissue sample was subjected to immunofluorescent (IF) staining. Positively stained cells relative to the total graft area in each section were quantified at 40x magnification via ImageJ software. The percentage of vascular density was acquired from 10 micrographs per rat.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eStatistical analysis was performed via Prism software version 8 (GraphPad, USA, California). For comparisons among multiple groups, one-way ANOVA was employed, followed by the Tukey post hoc test. The results are presented as the means\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SDs, and P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCharacterization of isolated MVF and islets\\u003c/h2\\u003e \\u003cp\\u003eThe isolated MVF were characterized by CD31 and α-SMA surface markers, revealing a well-defined luminal structure (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea,b). Additionally, islets stained with DTZ were red in color. Islet viability assessment via AO/EB staining indicated high viability (93%) of the isolated islets. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec,d).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFibrin hydrogel containing MVF improve subcutaneous vascularization\\u003c/h2\\u003e \\u003cp\\u003eImmunofluorescence analysis of graft-bearing subcutaneous tissue at the end of the PI revealed that the expression of CD31 in the HMVF group (37/94\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2/32%) was significantly greater than that in the H group (16/07\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1/08%, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001) and MVF group (27/01\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2/39%, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). Additionally, the MVF group had significantly greater numbers of CD31-positive cells than did the H group (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eImmunofluorescence staining of graft-bearing subcutaneous tissue at the end of phase II revealed a significant difference in CD31 expression between the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e (34/68\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2/27%) and MVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups (26/82\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3/17%, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), but it was not statistically significant compared with the H\\u003csup\\u003e1500\\u003c/sup\\u003e group (31/32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0/81%, p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05) or the Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e group (31/12\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1/98%, p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05). As expected, the Islet only\\u003csup\\u003e3000\\u003c/sup\\u003e group had lower CD31 expression than the other groups did (20/99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2/437%) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFibrin hydrogel containing MVF supports subcutaneous islet engraftment and function\\u003c/h2\\u003e \\u003cp\\u003eInsulin expression in the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e group was significantly greater than that in the H\\u003csup\\u003e1500\\u003c/sup\\u003e and MVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups (56/41\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0/79% vs. 41/32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1/24% and 43/99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2/66%, respectively, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001). In addition, there was no significant difference in insulin expression between the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups (52/08\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1/36%, P\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05). Insulin expression in the Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e group was significantly different from that in the H\\u003csup\\u003e1500\\u003c/sup\\u003e and MVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 and p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001, respectively) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the second phase, Islet only\\u003csup\\u003e3000\\u003c/sup\\u003e was not successful in reversing diabetes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea). HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e was superior to H\\u003csup\\u003e1500\\u003c/sup\\u003e and MVF\\u003csup\\u003e1500\\u003c/sup\\u003e in reducing blood glucose levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea, \\u003csup\\u003e✱✱\\u003c/sup\\u003ep\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, \\u003csup\\u003e✱✱✱✱✱\\u003c/sup\\u003e p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001). The efficacy of MVF\\u003csup\\u003e1500\\u003c/sup\\u003e alone in lowering blood glucose levels was not comparable to that of the co-transplantation strategy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea, \\u003csup\\u003e✱✱\\u003c/sup\\u003ep\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). Co-delivery of the hydrogel and MVF in both the prevascularization and co-transplantation methods led to euglycemia over 28 days after islet transplantation. Following graft removal, blood glucose in the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups increased within 2 days and showed graft-dependent insulin dependency (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea). Among the different transplanted groups, the body weights of the MVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Islet-only\\u003csup\\u003e3000\\u003c/sup\\u003e groups tended to decrease (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb). Moreover, no significant differences were observed in the body weights of the rats in either of the transplanted groups over the 28-day study period.\\u003c/p\\u003e \\u003cp\\u003eThe results of the IPGTTs revealed that both the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups were able to respond to the glucose bolus and reached normoglycemia within 120 minutes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec, purple and green lines). The related area under the curve (AUC) for the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups was smaller than that for the Islet only\\u003csup\\u003e3000\\u003c/sup\\u003e group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ed). There was no significant difference in the AUC between the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e or Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e group and the normal group (P\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ed). In the H\\u003csup\\u003e1500\\u003c/sup\\u003e and MVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups, the blood glucose levels remained above 200 mg/dl (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec, blue and red lines). Additionally, animals that received only islet transplantation in the unmodified subcutaneous space were unable to respond to the glucose challenge and remained hyperglycemic (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec, gray line), as confirmed by the increased AUC of blood glucose (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ed).\\u003c/p\\u003e \\u003cp\\u003eThe same pattern was observed for the plasma insulin concentration, which was significantly greater in the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups (9.435\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.24 \\u0026micro;U and 9.258\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.55 \\u0026micro;U, respectively) than in the Islet only \\u003csup\\u003e3000\\u003c/sup\\u003e group (6.686\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.30 \\u0026micro;U, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ee). However, the insulin concentration in these groups was not significantly different from that in the normal group (10.27\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.30 \\u0026micro;U, p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.5; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ee). Additionally, the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e groups presented superior results to those of the H1500 and MVF1500 groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ee). Overall, this co-delivery approach led to a 50% reduction in the marginal islet mass required to reverse diabetes in rats.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eSubcutaneous tissue is gaining attention as an alternative for extrahepatic islet transplantation because of its accessibility and reduced risk of IBMIR (Smink, Li et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). However, subcutaneous tissue requires a significant number of islets to maintain normal glucose levels because of its low oxygen pressure and inadequate blood supply (Zhou, Xu et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Additionally, the process of isolating islets, particularly through collagenase digestion, can adversely affect the intra islet microvasculature (Santini-Gonz\\u0026aacute;lez, Simonovich et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Therefore, it is essential to employ techniques for regenerating the ECM and enhancing islet vascularization.\\u003c/p\\u003e \\u003cp\\u003eTo address these challenges, we investigated improvements in subcutaneous islet transplantation via the vasculogenic potential of fibrin hydrogels containing MVF in diabetic rat models. To the best of our knowledge, this study is the first to demonstrate that the combination of fibrin hydrogel and MVF synergistically enhances vascularization for subcutaneous islet transplantation.\\u003c/p\\u003e \\u003cp\\u003eFibrin hydrogels not only provide structural support by delivering arginine-glycine-aspartic acid (RGD) sequences that bind to integrins (αvβ1), maintaining the spatial organization of islets but also degrade within 28 days without eliciting foreign body reactions (Kim, Lim et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e, Kuehn, Lakey et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). Studies conducted in porcine models indicate that fibrin alone can induce angiogenesis (Salama, Seeberger et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e), although its effects are enhanced when additional vasculogenic factors are included. Nalbach demonstrated that, compared with the use of single fat-derived cells, prevascularized islets with MVF before transplantation into diabetic mice improved vessel fusion, blood perfusion, islet viability, and function (Nalbach, Roma et al. 2021). We hypothesized that MVF could further enhance vasculogenic stimulation within the fibrin hydrogel.\\u003c/p\\u003e \\u003cp\\u003eEmbedding islets with MVF in collagen hydrogels promotes in vitro islet vascularization. Within 8 days, MVF-derived ECs form capillary sprouts connecting to the islets, which maintain viability, function, and glucose-stimulated insulin secretion (Salamone, Rigogliuso et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Previous studies have shown that MVF-derived ECs can establish functional capillaries within just 7 days, resulting in a 2.8-fold increase in capillary density, a 42% increase in blood flow velocity during the early post-transplant phase, and a 65% increase in the revascularized area, which reduces the marginal islet mass requirement from 600 to 250 islets in mouse models (Nalbach, Roma et al. 2021). Our results indicated that using MVF combined with fibrin results in more favorable engraftment, which decreases the marginal islet mass by 50% in diabetic rats. The vascular density also significantly improved, with 34% and 31% in the HMVF groups compared with 26% in the MVF group.\\u003c/p\\u003e \\u003cp\\u003eInterestingly, we observed no significant difference in islet function between the HMVF1500 (prevascularized) and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e (co-transplantation) groups. Both groups benefited from the combined use of fibrin hydrogel and MVF, with fibrin providing a scaffold for vascular network formation while also supporting islet viability by mimicking the extracellular matrix. MVF secrete proangiogenic factors such as growth factor (HGF) and vascular endothelial growth factor (VEGF), enhancing both vascularization and islet survival (Aghazadeh, Poon et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e, Wrublewsky, Weinzierl et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). This synergy likely created a sufficiently supportive microenvironment regardless of the timing of application. Moreover, the initial days post transplantation represents a critical and vulnerable period for islet survival because of factors such as hypoxia and inflammation. Perhaps both our prevascularization and co-transplantation strategies provided sufficient microvascular support to meet the metabolic demands of the islets, leading to similar outcomes at the 28-day endpoint through MVF-derived endothelial cell sprouting, reduced apoptosis via HGF-mediated survival signals, and enhanced oxygen and nutrient diffusion through the porous structure of fibrin. Finally, we must acknowledge that the lack of a statistically significant difference could be due to limitations in our study design, such as the sample size and the sensitivity of our methods for assessing microvascular architecture. Future research could benefit from extended observation periods of more than three months to assess durability and more sophisticated imaging techniques.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn summary, our study demonstrates that the inherent pro-angiogenic properties of fibrin, combined with the vascularization potential of microvascular fragments, create a synergistic environment that promotes vascularization, improved islet survival and function. This approach addresses the critical challenge of inadequate vascularization in subcutaneous islet transplantation and shows potential for reducing the quantity of donor islets required for successful outcomes.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eThe authors report no conflict of interest.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eSB, MNN, and BMZ designed the experiments; SB and AA performed experiments and collected data; MB, EHS, and FMZ discussed the results and strategy; BMZ Supervised, directed and managed the study; SB prepared the original draft. All authors reviewed the manuscript and approved the final version.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgment\\u003c/h2\\u003e \\u003cp\\u003eThis work was fully supported by a by a grant from the Tehran University of Medical Sciences (grant number:1402.3.410.68148).\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eData is provided within the manuscript or supplementary information files\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAddison, P., K. Fatakhova and H. L. 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Ampofo (2022). \\\"Co-transplantation of pancreatic islets and microvascular fragments effectively restores normoglycemia in diabetic mice.\\\" NPJ Regenerative Medicine 7(1): 67. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1038/s41536-022-00262-3\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/s41536-022-00262-3\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhou, X., Z. Xu, Y. You, W. Yang, B. Feng, Y. Yang, F. Li, J. Chen and H. Gao (2023). \\\"Subcutaneous device-free islet transplantation.\\\" Frontiers in Immunology 14: 1287182. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3389/fimmu.2023.1287182\\u003c/span\\u003e\\u003cspan address=\\\"10.3389/fimmu.2023.1287182\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Fibrin, Islet transplantation, Microvascular Fragments, Subcutaneous tissue, Type 1 diabetes mellitus\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6626455/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6626455/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eObjective(s): \\u003c/strong\\u003eIslet transplantation offers a promising treatment for Type 1 diabetes mellitus\\u003cstrong\\u003e \\u003c/strong\\u003e(T1DM). Subcutaneous tissue is a non-invasive site, but it has poor blood supply and requires more islets to achieve normoglycemia. We assessed the impact of fibrin hydrogel containing microvascular fragments (MVF) on the vascularization of subcutaneous tissue using two approaches: prevascularization (prior to islet transplantation) or co-transplantation (simultaneously with islet transplantation).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMaterials and methods: \\u003c/strong\\u003eWistar rats were prevascularized subcutaneously for 7 days with fibrin (H), 5000 MVF, or fibrin + 5000 MVF (HMVF). After streptozotocin injection to induce T1DM, 1500 islet equivalents (IEQ) were transplanted into prevascularized groups. A co-transplantation group received 1500 IEQ and HMVF (Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e), and a control group received 3000 IEQ alone (Islet only \\u003csup\\u003e3000\\u003c/sup\\u003e). Graft function was evaluated through blood glucose monitoring, glucose tolerance tests, immunostaining, and plasma insulin concentration over 28 days.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults: \\u003c/strong\\u003eHMVF-prevascularized group presented significantly more CD31-positive cells compared with those from H- or MVF-prevascularized groups (p\\u0026lt;0.05). Prevascularization and co-transplantation approaches using HMVF resulted in normoglycemia with a reduced islet mass (1500 IEQ) compared with those in the Islet only\\u003csup\\u003e3000\\u003c/sup\\u003e, H\\u003csup\\u003e1500\\u003c/sup\\u003e, and MVF\\u003csup\\u003e1500 \\u003c/sup\\u003egroups. In addition, both the HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e and Co-HMVF\\u003csup\\u003e1500\\u003c/sup\\u003e rats indicated superior islet survival and function, compared with the H\\u003csup\\u003e1500\\u003c/sup\\u003e, MVF\\u003csup\\u003e1500\\u003c/sup\\u003e, and Islet only\\u003csup\\u003e3000\\u003c/sup\\u003e groups, as evidenced by increased CD31-positive cells and insulin-positive cells, improved glucose tolerance, and elevated plasma insulin concentrations (p\\u0026lt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion: \\u003c/strong\\u003eFibrin hydrogel containing MVF could significantly increase the survival and function of subcutaneously transplanted islets, allowing for effective glycemic control with a reduced islet mass.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Improving Pancreatic Islet Transplantation Using Fibrin Hydrogel Containing Microvascular Fragments in Subcutaneous Tissue of Type I Diabetic Rats\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-05-19 09:48:43\",\"doi\":\"10.21203/rs.3.rs-6626455/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"1c5e453f-212b-4391-8f2f-73e86076a65d\",\"owner\":[],\"postedDate\":\"May 19th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-12-02T06:08:27+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-05-19 09:48:43\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6626455\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6626455\",\"identity\":\"rs-6626455\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}