A macroporous heparin-releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival

Accepted Manuscript A macroporous heparin-releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival Duo Mao, Meifeng Zhu, Xiuyuan Zhang, Rong Ma, Xiaoqing Yang, Tingyu Ke, Lianyong Wang, Zongjin Li, Deling Kong, Chen Li PII: DOI: Reference:

S1742-7061(17)30421-X http://dx.doi.org/10.1016/j.actbio.2017.06.039 ACTBIO 4960

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Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

28 February 2017 21 June 2017 26 June 2017

Please cite this article as: Mao, D., Zhu, M., Zhang, X., Ma, R., Yang, X., Ke, T., Wang, L., Li, Z., Kong, D., Li, C., A macroporous heparin-releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio.2017.06.039

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A macroporous heparin-releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival Duo Mao1#, Meifeng Zhu 1#, Xiuyuan Zhang2, Rong Ma3, Xiaoqing Yang3, Tingyu Ke3, Lianyong Wang1, Zongjin Li1,4*, Deling Kong1,2, Chen Li2 * 1

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials of Ministry of Education, Collaborative Innovation Centre of Chemical Science and Engineering (Tianjin), College of Life Science, Nankai University, Tianjin 300071, China 2 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, 300192, China 3 Department of Endocrinology, the Second Affiliated Hospital, Kunming Medical University, Kunming 650101, Yunnan, China 4 School of Medicine, Nankai University, Tianjin 300071, China Running title: A silk fibroin scaffold for islet grafts Keywords: islet transplantation; silk fibroin scaffold; heparin; revascularisation, graft survival # these authors contributed equally to this work Corresponding authors Chen Li, PhD Institute of Biomedical Engineering, BaiDi Road 236, Tianjin 300192, China Tel. 0086 22 87893696; Fax 0086 22 87893696 e-mail: [email protected] Zongjin Li, PhD School of Medicine, Nankai University, Tianjin 300071, China Tel. 0086 22 23509332; Fax 0086 22 23498775 e-mail: [email protected]

Abstract Islet transplantation is considered the most promising therapeutic option with the potential to cure diabetes. However, efficacy of current clinical islet transplantation is limited by long-term graft dysfunction and attrition. We have investigated the therapeutic potential of a silk fibroin macroporous (SF) scaffold for syngeneic islet transplantation in diabetic mice. The SF scaffold was prepared via lyophilisation, which enables incorporation of active compounds including cytokines, peptide and growth factors without compromising their biological activity. For the present study, a heparin-releasing SF scaffold (H-SF) in order to evaluate the versatility of the SF scaffold for biological functionalisation. Islets were then co-transplanted with H-SF or SF scaffolds in the epididymal fat pad of diabetic mice. Mice from both H-SF and SF groups achieved 100% euglycaemia, which was maintained for 1 year. More importantly, the H-SF-islets co-transplantation led to more rapid reversal of hyperglycaemia, complete normalisation of glucose responsiveness and lower long-term blood glucose levels. This superior transplantation outcome is attributable to H-SF-facilitated islet revascularisation and cell proliferation since significant increase of islet endocrine and endothelial cells proliferation was shown in grafts retrieved from H-SF-islets co-transplanted mice. Better intra-islet vascular reformation was also evident, accompanied by VEGF upregulation. In addition, when H-SF was co-transplanted with islets extracted from vegfr2-luc transgenic mice in vivo, sustained elevation of bioluminescent signal that corresponds to vegfr2 expression was collected, implicating a role of heparin-dependent activation of

endogenous VEGF/VEGFR2 pathway in promoting islet revascularisation and proliferation. In summary, the SF scaffolds provide an open platform as scaffold development for islet transplantation. Furthermore, given the pro-angiogenic, pro-survival and minimal post-transplantation inflammatory reactions of H-SF, our data also support the feasibility of clinical implementation of H-SF to improve islet transplantation outcome.

Introduction Type 1 diabetes is an autoimmune disorder caused by destruction of insulin-producing β-cells within the pancreatic islets [1]. Allogeneic islet transplantation is considered a viable therapeutic approach for diabetic patients with the potential to cure the disease. Despite remarkable improvements that have been made with the development of the “Edmonton protocol”, long-term insulin independence remains to be desired as majority of recipients eventually reversed to insulin replacement [2, 3]. The lack of sustained therapeutic efficacy of islet transplantation

is

mainly

due

to

early

depletion

of

functional

islets

post-transplantation followed by a gradual decline of islet function and viability. Islets are highly vascularised requiring 10-20% of the pancreatic blood supply while accounting for merely 2-3% of the total pancreatic mass [4-8]. The highly enriched vascularisation within each islet allows rapid exchange of oxygen, nutrients, islet hormones and other cellular effectors [5, 7]. During the isolation procedure prior to clinical islets transplantation, the extracellular matrix (ECM) that is essential to

maintain islet vascularisation and innervations is often damaged due to enzymatic digestion, resulting in disrupted islet microvasculature and impaired islet function and survival [4, 5]. Given that accessibility to nutrition, adequate physical support and vascular regeneration are essential factors for successful engraftment, the possibility of incorporating biomaterials to co-deliver islets during transplantation have been explored. Macroporous scaffolds have been extensively used as temporary artificial ECM for cell therapy by providing an optimal in situ site for cell accommodation, subsequent proliferation and differentiation. Due to their three-dimensional (3D) and highly porous structures, they also facilitate nutrients exchange, cell infiltration and revascularisation in vivo [4-6, 8-19], which are important factors for improving post-transplantation islet survival and function. Multiple scaffolds have been engineered with surface-coated ECM proteins, growth factors and anti-inflammatory cytokines to enhance cell survival and neovascularisation during islet transplantation [14, 15, 18, 20, 21]. Previous studies reported reversal of diabetes after extrahepatic islets transplantation using a poly(lactic-co-glycolide) (PLGA)-based scaffold in syngeneic and tolerance-inducing allogeneic models [9, 12, 15]. However, despite the promising potential shown by the PLGA-based scaffolds, degradation of the polymer releases acidic by-products causing substantial pro-inflammatory responses in vivo [16]. The hydrophobic nature of PLGA also interferes with cell infiltration, and would be detrimental to post-transplantation vascular regeneration when lacking porosity [13]. Recent reports also demonstrated excellent outcome post islet transplantation

using poly(dimethylsiloxane) (PDMS)-based and polyurethane (PU) scaffolds [17, 19], although PU lacks suppleness and flexibility while PDMS tends to become hydrophobic following polymerisation and require extra processes for surface adaptation. In addition, porous hydrogel-based scaffolds fabricated using fibrin and PEG have all been employed to assist islet transplantation. However, the challenge of reproducibility and graft retrieval compared to conventional 3D scaffolds impedes their potential for clinical implementation. Silk fibroin (SF) is a natural structural protein derived from Bombyx mori. Known for its excellent biodegradability and low immunogenicity, SF-based scaffolds have demonstrated exceptional advantages over conventional synthetic and other natural biomaterials

in

various

aspects

of

tissue

engineering

[22-26].

Indeed,

co-encapsulation of islets with the mesenchymal stem cells (MSCs) and two components of ECM (laminin and collagen IV) by an SF-based hydrogel was reported in which prolonged islet survival and enhanced function were observed in vitro [10]. Moreover, an SF matrix incorporated with an endothelial cell binding motif was also proven to be facilitative in “pseudoislets” formation using either MIN6 β-cell or human endocrine cells [27], although in vivo potential of using silk fibroin scaffold for islet transplantation has never been reported. We have previously reported fabrication of an interconnected macroporous SF scaffold via lyophilisation, which enables incorporation of cellular mediators such as cytokines, growth factors and proteins without compromising their biological activity. [28]. Since heparin has been shown to enhance islet angiogenesis by stabilising VEGF and other growth factors [29]

while dampen local inflammatory reactions [30], we have also prepared a heparin-releasing SF scaffold to evaluate the versatility of the SF scaffold for biological functionalisation in islet transplantation. Thus, a “proof-of-concept” study was conducted by investigating the in vivo therapeutic efficacy of the SF scaffolds (heparinised and non-heparinised) in syngeneic islet transplantation. Moreover, islets were also obtained from vegfr2-luc and β-actin-luc transgenic mice for bioluminescence and imaging analysis to further illustrate the mechanistic role of heparin in islet revascularisation and survival after co-transplantation with the heparin-SF scaffold.

Materials and Methods Scaffold preparation and characterisation Fabrication of the macroporous SF scaffold was carried out as previously detailed [28]. Briefly, SF solution was concentrated to 20 wt.% by dialysis against 15 wt.% poly (ethylene oxide) (Mw = 20,000) aqueous solution. Ethanol was added to adjust SF concentration. The solution was then poured into a cylinder-shaped polytetrafluoroethylene mold（height 1 cm, diameter 1.5 cm）and frozen at 20, -70 or -196 °C, followed by lypophilisation to form a porous SF scaffold. The scaffolds were immersed in 90% methanol for 30 min to induce SF crystallization, and dried at room temperature in vacuum for 2 days. All scaffolds were cut into cylinder-shaped slices (height 0.5 mm, diameter 5 mm) for islet transplantation. The scaffolds were examined by scanning electronic microscope (SEM). For Heparin-SF scaffolds, 2 wt.%

heparin sodium salt extracted from porcine intestinal (Mw 14,000 Da, Pierce) was added to the SF solution. Heparin-releasing kinetics was determined as previously described by toluidine blue assay [28].

Animals and induction of T1 Male C57BL/6 mice and GFP transgenic C57BL/6 mice (8 weeks, 20 g) were purchased from the Laboratory Animal Centre of the Academy of Military Medical Sciences (Beijing, China). The β-actin-luc transgenic FVB mice, which constitutively express firefly luciferase and the vegfr2-luc transgenic C57BL/6 mice, which express luciferase under the promoter of vascular endothelial growth factor receptor 2 (VEGFR2) were obtained from Xenogen Corporation (Hopkinto, MA, USA)[31]. Diabetes was induced by intraperitoneal (i.p.) injection of 220 mg/kg STZ (Sigma-Aldrich, St. Louis, MO). Non-fasting blood glucose level was measured by using tail blood samples with an OneTouch Ultra glucometer (Lifescan, Johnson & Johnson, CA). Animals were considered diabetic with random blood glucose level >11.1 mmol/L. All animal procedures were performed under the Regulations for the Administration of Affairs Concerning Experimental Animals (Tianjin, revised in June 2004), which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised in 1996).

Islet isolation and transplantation

Islet isolation was performed as previously described via collagenase (type XI; Sigma Chemical Company, St Louis, MO) digestion and Histopaque gradient purification (Sigma-Aldrich, Shanghai, China) [32]. Islets were maintained in RPMI 1640 media supplemented with 10% foetal bovine serum (FBS) in 95% air/5% CO2 at 37°C. For quality assessment, islets were stained with dithizone (DTZ) and examined under a light microscope. Prior to islet transplantation, the scaffolds were immersed in 70% ethanol for 30 s and rinsed in sterile RPMI 1640 containing 10% FBS. Approximately 300 islets were handpicked using a P200 pipette under a dissection microscope and seeded in each scaffold in minimal volume of media (~50 µL) and incubated for 30 min in 6-well plate before additional media (~2 mL) was added. It is worth noting that the number of islets used for transplantation for the present study was selected based on “pre-screening” data from our lab as well as previously published studies [17, 33-35]. To examine islet distribution within the scaffold in vitro, islets isolated from GFP transgenic mice were seeded, maintained in culture for up to 14 days and examined under a con-focal microscope (Leica SP8, Germany). Islet viability was also assessed by pre-seeding islets in each scaffold in 50 µL serum-supplemented RPMI 1640 and maintained in culture in a 6-well plate for 30 min followed by addition of 2 mL of the same culturing media. A live/dead assay kit (Molecular Probes, Eugene, OR, USA) was then used on Day 0 and Day 14. Transplantation was performed as reported in earlier studies [9, 36]. After being anaesthetised with isoflurane 2%, mouse abdomen was sterilised, shaved and a

midline incision was made to locate the epididymal fat pad (EFP), which was then spread on the abdominal surface. Once the islet-seeded scaffold was wrapped in the EFP, the EFP tissues were then folded as previously reported to contain the implanted scaffolds. In parallel, for the control group, free islets were similarly wrapped into the folds of the EFP tissues. The EFP was then returned to the abdominal cavity followed by wound closure [11, 17, 37].

Assessment of graft function Non-fasting blood glucose levels and body weight of all treatment groups (H-SF: islets co-transplanted with heparin-SF scaffold; SF: islets co-transplanted with SF scaffold; Ctrl: islets alone; NT: non-treated, n=20 per group) were measured everyday during for the first week after transplantation, followed by every other day during week 2 to 4 post-transplantation, weekly during week 5 to 24 and monthly until the end of the study. All measurements were taken between 12:00 and 17:00. Intraperitoneal glucose tolerance tests (IPGTTs) were performed as previously described at day 30 and 180 post-transplantation. Reversal of diabetes is determined if random glucose levels were stably maintained below 11.1 mmol/L with no reoccurrence of hyperglycaemia. At the end of day 360 post-transplantation, grafts were removed and random blood glucose levels was continuously monitored for another 72 hours to observe any changes in random blood glucose levels.

Examination of islet revascularisation by bioluminescence imaging and western

blotting For in vitro assessment, approximately 300 islets from vegfr2-luc transgenic mice were seeded into the heparin-SF scaffolds (H-SF) and maintained in RPMI1640 media supplemented with 10% FBS in 95% air/5% CO2 at 37°C for 14 days. Islets seeded in SF scaffolds without heparin (SF) or cultured in suspension with no scaffolds (Ctrl) were also included in parallel. At day 0, 7 and 14, the reporter probe D-Luciferin (0.01mg/L, Biosynth International, Naperville, IL, USA) was added into the culturing medium and the bioluminescence signals were measured using the Xenogen IVIS 200 system. For in vivo assessment of islet survival and local neoangiogenesis, islets obtained from the vegfr2-luc or β-actin-luc transgenic were transplanted with heparin SF scaffolds (H-SF), SF scaffold without surface heparin (SF) or without scaffolds (Ctrl). On day 0, 14 or 30 post-transplantation, the recipients were i.p. injected with D-Luciferin (150 mg/kg). Bioluminescence imaging was then performed using the Xenogen IVIS 200 system. Imaging signals were quantified as maximum photons per second per square centimeter per steridian (p/s/cm2/sr). In addition, grafts retrieved 7 days following islet transplantation with or without SF/H-SF. Protein expression of VEGF was analysed by western blotting and quantified using densitometry [32].

Histological and immunohistochemical analyses Four weeks post-transplantation, mice of each treatment groups (n=5) were anaesthetized and infused with 200 µl of biotinylated tomato-lectin (2 mg/ml, Vector

Labs, Burlingame, CA) via the femoral vein. After 15 min, grafts were removed, paraffin-embedded and cut into 5 µm sections, which were used for hematoxylin and eosin (H&E), Masson’s trichrome and co-immunofluorescence staining using antibodies raised against insulin (guinea pig anti-insulin; 1:800, Abcam, Shanghai, China), lectin by a fluorescein-conjugated goat anti-biotin (1:200, Vector Labs), proliferating cell nuclear antigen (PCNA; 1:1000), CD68 (1:100), Glucagon (1:100) and collagen IV (1:200; Abcam, Shanghai, China). Sections were examined under light or con-focal microscope respectively. For estimation of microvascular density and islet cell proliferation, quantification of individual microvascular structures (lectin+ immunofluorescence signals that correspond to circular vascular vessels) or proliferating cells (PCNA+ immunoreactivity) in every microscopic view was performed and analysed using the Image J software (Wayne Rasband, NIH, USA).

Quantitative real-time PCR analysis of inflammatory cytokines Graft samples were obtained 7 days post-transplantation from mice of all groups (n=5). Total RNA was extracted with TranZol Reagent (TransGen Biotechnology, Beijing, China). The qPCRs were performed in triplicate using the FastStart Universal SYBR Green Master (ROX; Roche, Mannheim, Germany) on an iCycler iQ5 2.0 Standard Edition Optical System (Bio-Rad, Hercules, CA, USA). Messenger RNA expression of inflammatory cytokines including the tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and interleukin 6 (IL-6; primer sequence as

previously detailed and listed in Supplementary Table 1) [38] were estimated by ∆∆Ct. GAPDH was used as reference gene.

Statistics Results are presented as standard error of the mean (SEM). The student’s t-test, Pearson chi-square test or one-way ANOVA analysis of variance with post-hoc Bonferroni’s test was used to determine statistical significance as appropriate. Differences in the number of days for diabetes reversal were compared using the Kaplan-Meier survival analysis (Prism Software, GraphPad, CA). Data was considered statistically significant with P<0.05.

Results Heparin-SF scaffolds as suitable co-delivery vehicles for islet transplantation SF scaffolds were prepared via phase separation method as previously reported [28]. They showed highly interconnected and uniformed pores (porosity >90%) with pore size ranging between 200-300 µm as examined by scanned electron microscopy (Figure 1A). Since average size of an islet is ~150 µm, these macroporous SF scaffolds could provide adequate physical space and support for implanted islets while facilitate cell infiltration for revascularisation. Furthermore, considering that heparin binds to an array of growth factors and is known for its anti-coagulant and anti-inflammatory properties, a heparinised SF scaffold (H-SF) was also fabricated

which releases heparin in a continuous manner for 7 days under physiological condition (Figure 1B). To examine the potential of scaffold as islet delivery vehicle, isolated GFP+ islets were seeded with a density of 300 islets/scaffold (Figure 1D I-II) or 50 islets/scaffold (Figure 1D III). It could be seen that regardless of density, islets were well-housed within the scaffolds with no excessive clumping (Figure 1D). Co-culture of islets with H-SF or SF scaffolds also improved islet cell viability as compared to islets cultured without scaffold (Figure 2A & Supplementary Figure 1). Also shown in Figure 2B, the average amounts of PI+ cells per islet from the H-SF and SF co-cultured groups were significantly lower than the control group (20±3% [H-SF] and 52±7% [SF] over controls, P<0.001). In parallel, islets from vegfr2-luc transgenic mice were also co-cultured with scaffolds in vitro. Significant elevation of vascular endothelial growth factor receptor-2 (VEGFR2) expression could be detected from the H-SF group after 7 days (230±23% [H-SF] and 161±15% [SF] over Control, P<0.05 H-SF vs SF) and 14 days (285±78% [H-SF] and 135±64% [SF] over Control, P<0.05 H-SF vs SF), suggesting a pro-angiogenic and pro-survival role of H-SF for in vitro islet culturing.

Effects of co-transplantation of islets with scaffolds on glycaemic control in diabetic mice To assess the therapeutic efficacy of SF scaffolds for islet transplantation, STZ-induced diabetic mice were stratified by blood glucose levels and randomly

assigned to 3 treatment groups (n>10 each). Approximately 300 islets were transplanted by injection into the EFP (Ctrl), via H-SF (H-SF) or SF scaffolds (SF). Diabetic mice that were untreated were subjected to sham operations and included in parallel (NT, n=5). Long-term performance of graft function was followed for 1 year by measuring random blood glucose levels at designated times (see Methods). Thus, all mice from the H-SF and SF groups achieved euglycaemia after an average of 4.7±1.5 and 10.1±2.2 days post-transplantation (Figure 3A, P<0.01 H-SF vs SF) with average blood glucose levels of 7.34±0.6 and 9.01±0.58 mmol/L (Figure 3B, P<0.01 H-SF vs SF), respectively. Euglycaemia was maintained for 1 year for both H-SF and SF groups (Figure 3A-D), after which islet grafts were removed and all mice reversed back to being hyperglycaemic (Figure 3B, red arrow), indicating that the transplanted islets were solely responsible for maintaining euglycaemia. Glucose tolerance was also completely normalised in mice from the H-SF group 30 days post-transplantation, significantly better than the SF and islets-only groups (AUC: 126±56% over non-diabetic [Normal], P<0.05 H-SF vs SF/Control; Figure 3C&E). By day 180 post-transplantation, better glucose responsiveness was still evident from the H-SF group (AUC: 109±49% over Normal, P<0.01 H-SF vs SF/Control; Figure 3D&F). In addition, average serum insulin levels of mice from the H-SF and SF groups were also comparable to non-diabetics (84±20% and 71±15% over Normal, P<0.05 vs Control; Figure 3G).

H-SF co-delivery maintained islet morphology post-transplantation

Histological analysis of retrieved grafts from mice 30 days after islets and scaffolds co-transplantation revealed relatively intact islet structure (Figure 4 & 2A-B). Adequate amount of cell infiltration was seen adjacent to the implanted islets. Microvascular structures were also visible within the H-SF-delivered islets, demonstrating evident islet revascularisation post-transplantation (Figure 4A). Masson’s staining revealed no fibrotic tissues surrounding scaffolds regardless of heparin presence (Figure 4B & Supplementary Figure 2A), showing minimal inflammatory responses elicited by scaffold implantation. In contrast, viable islets were scarce in grafts retrieved from controls (Supplementary Figure 2C). Islet necrosis, shown by tissue calcification and a lack of DAPI+ nuclei, was also observable (Supplementary Figure 2D), likely attributable to ill-maintained islet morphology as a result of excessive clumping and poor post-transplantation revascularisation (Supplementary Figure 2C-F).

H-SF co-delivery improves islet revascularisation post-transplantation Islet revascularisation is a crucial process that determines post-transplantation islet survival and function, and consequently the therapeutic efficacy and clinical outcome of islet transplantation. To investigate the effect of scaffold-assisted islet transplantation on islet revascularisation, FITC-conjugated tomato lectin, a compound that binds to endothelial cells for visualising functional microvasculatures was infused to animals of all groups 1 month after islet transplantation. Thus, average intra-islet microvasculature density was significantly elevated in the H-SF group as compared to

SF and control groups (139±11% over SF, P<0.05). It was also evident that the lectin+ immunoreactive signals were of circular patterns (Figure 5A, white arrow), which are absence from the SF groups. In parallel, pancreatic islets of age-matched euglycaemic mice were shown as positive control and the capillary density of which is similar to those from the H-SF group, further illustrating a positive impact of H-SF on post-transplantation intra-islet vascular reformation. In addition, a moderate increase of lectin+/PCNA+ cells was also detectable in islets from the H-SF group in comparison to the SF group (Supplementary Figure 4, white arrows), emphasising a major

role

of

heparin in promoting

islet

revascularisation.

Furthermore,

immunostaining and western blotting analyses also revealed significant upregulation of VEGF expression in grafts retrieved from islet-H-SF co-transplanted animals 7 days post-transplantation (Figure 6A-C), partly responsible for H-SF-enhanced neo-capillary formation and vascular regeneration of the H-SF-delivered islets. To further investigate the pro-angiogenic role of the H-SF on post-transplantation islet revascularisation, islets were extracted from the vegfr2-luc transgenic mice, in which production of luciferase is triggered upon expression of vegfr2 [31]. Given the correlation between VEGFR2 upregulation and capillary regeneration, we co-transplanted vegfr2-luc islets with H-SF, SF or alone into EFP and luminescence signals of all mice were monitored by an in vivo imaging system at day 7 and 14 post-transplantation. Consistent with the immunofluorescence results, highest luciferase signal that corresponds to upregulation of VEGFR2 was recorded from mice that received co-transplantation of islets with H-SF (Figure 6 D&E). In fact,

subcutaneous implantation of H-SF alone elicited significant elevation of VEGFR2 expression by day 14 after implantation compared to SF and sham groups (Supplementary Figure 3), confirming a facilitative impact of heparin on neoangiogenesis.

H-SF co-delivery improves islet survival post-transplantation We also assessed the effect of scaffold co-transplantation on islet survival and regeneration. Thus, PCNA staining revealed significantly increased number of PCNA+ islet cells (shown as PCNA+ cells within the insulin+ intra-islet area) in grafts retrieved from the H-SF and SF groups compared to islets-only controls 14 and 30 days post-transplantation (Figure 7A, white arrows). Substantial amount of PCNA+ cells were also found in non-islet area around the implanted scaffolds (Figure 7A, yellow arrows), consistent with a previously reported facilitative role of scaffolds on cell infiltration and tissue regeneration [28]. In addition, collagen IV has been shown to suppress early islet cell apoptosis and maintain islet function post-transplantation [10, 15]. Elevated expression of islet collagen IV was observed from islets co-transplanted with H-SF and SF scaffolds (Supplementary Figure 5), suggesting a potential role of the scaffolds on collagen IV deposition. Moreover, when we performed syngeneic islet transplantation using islets isolated from β-actin-luc transgenic mice, sustained bioluminescent signals could be detected 1 month post-transplantation from both H-SF and SF groups (Figure 8). To the contrary, a gradual drop of luciferase signals was observed from the control animals,

indicating continuous post-transplantation islet loss that was absence in the H-SF or SF co-transplanted groups.

Inflammatory reactions of scaffold implantation In line with the Masson’s staining results, implantation of scaffolds elicited minimal inflammatory reactions as shown by a limited number of local CD68 + macrophages observed in grafts (Supplementary Figure 6A). Messenger RNA expressions of pro-inflammatory cytokines including TNF-α, IFN-γ and IL-6 were also assessed and no changes in mRNA expression level were detected following islets transplantation 7 days post-transplantation (Supplementary Figure 6B-D). Thus, minimal inflammatory reactions were elicited by scaffold implantation, suggesting that the scaffold is a suitable candidate to assist in vivo islet transplantation.

Discussion We report here the use of an interconnected macroporous silk fibroin scaffold for syngeneic islet transplantation in a murine model of diabetes. Traditionally, clinical islet transplantation was conducted by intrahepatic islet infusion, although this approach

is

limited

by

the

scarcity

of

islet

material

and

significant

post-transplantation islet loss. In order to improve transplantation outcome, alternative extrahepatic sites including the renal capsule, omental pouch, intraperitoneum and subcutis have all been evaluated albeit with limited clinical success [33, 37, 39, 40]. Therapeutic strategies employing biomaterials have also been implemented to create

or engineer optimal local environment for cell and islet transplantation [9-14, 37]. Disappointingly though, despite significant progress that has been made to assist islet transplantation, post-transplantation islet revascularisation remains a major challenge and primary concern for long-term graft function and survival. The ECM is a complex multi-component structure that is essential to maintain physical cohesiveness, cell interaction, vascularisation and innervations of the pancreatic islets. Disruption of ECM during islet isolation significantly hampers post-transplantation vascular reformation [33]. We have developed a silk fibroin-based macroporous scaffold and we expected that this three-dimensional SF scaffold may provide adequate structural support and bioactivity for rapid vascular regeneration of the implanted islets. Furthermore, the SF scaffold was prepared by lyophilisation and could potentially be biologically functionalised with active compounds without compromising their in vivo activities. Since heparin, which is clinically safe to use, is present in the ECM and able to recruit angiogenic factors such as the basic fibroblast growth factor, platelet-derived growth factor, hepatocyte growth factor and VEGF via electrostatic interaction [41], we have also assessed the functions of SF scaffold with incorporated heparin that could be continuously released in vivo for islet transplantation. Our results showed that euglycaemia was achieved in all H-SF and SF recipients and maintained for over 1 year, confirming that maintenance of islet morphology during islet transplantation is beneficial to engraftment and transplantation outcome [42]. Superior transplantation outcome was evident from the H-SF group, which

exhibited significantly better rate of recovery, glucose responsiveness and markedly lower long-term blood glucose levels. The difference regarding transplantation efficacy between H-SF and SF is due to enhanced post-transplantation survival of islets co-transplanted with H-SF. Indeed, robust islet cell viability was shown in islets co-cultured with H-SF in vitro. Transplantation of β-actin-luc islets also demonstrated better survival of islets that were co-delivered with scaffolds. Enhanced islet cell proliferation and intra-islet vascular reformation are both accountable for better post-transplantation islets survival from the H-SF group. Indeed, small circular-shaped lectin+ immunoreactive signals that represent intra-islet microvascular vessels were clearly discernable in grafts retrieved from mice of the H-SF but not SF or control groups. Significantly elevated proliferation of intra-islet endothelial cells shown as lectin+/PCNA+ cells and PCNA+ cells within islets was also identified in the H-SF group 14 days post-transplantation. Moreover, the level of intra-islet vasculature within the H-SF co-transplanted islets was comparable to pancreatic islets of non-diabetic animals, demonstrating better vascularisation of the H-SF co-transplanted islets. Typically, islet vascular reformation starts to take place within the first few weeks post-transplantation [33]. We also observed upregulation of VEGF expression in grafts of the H-SF group 7 days post-transplantation, considerably more than the SF and control groups. Co-transplantation of vegf2-luc islets with H-SF further revealed a heparin-dependent role in promoting islet revascularisation and consequently islet survival via recruitment of endogenous VEGF and perhaps subsequent activation of

the bona fide VEGF/VEGFR2 cascade. Moreover, expression of collagen IV, a major component of the ECM that has been shown to inhibit early apoptosis in transplanted islets [10, 15], was upregulated, indicating successful reestablishment of an ECM-like supporting structure of islets co-transplanted with H-SF and is in turn accountable for better islet revascularisation of the H-SF group. Thus, it could be speculated that heparin released by H-SF plays a crucial role in facilitating post-transplantation islet engraftment by creating a pro-angiogenic and regenerative microenvironment to facilitate post-transplantation islet survival and function. We were also concerned about the inflammatory responses elicited by the SF scaffolds since long-term implantation of biomaterials may initiate local inflammatory reactions that often led to eventual graft failure [33]. In contrast to synthetic polymers that have been used clinically as catheter materials, no apparent changes in the mRNA expression pattern of pro-inflammatory cytokines were detectable following islets-H-SF, islets-SF and islets-only implantation. No formation of avascular fibrotic tissues was observed 30 days post-transplantation, demonstrating excellent biological compatibility and clinical safety of the SF scaffolds and therapeutic potential of H-SF implementation for clinical islet transplantation. Some earlier studies reported the use of SF-based hydrogels for in vitro islet culturing and encapsulation [10, 27], although none has been evaluated in vivo since it has been traditionally difficult to fabricate SF-based scaffolds with uniformed pore structures suitable for islets transplantation. To overcome limitations of conventional SF fabrication techniques, we have previously reported an approach that enables

tailoring of pore structure [28]. Considering the evident limitations posed by intrahepatic islet transplantation, our “easy-to-adopt” scaffold fabrication method and transplantation procedure could be considered a feasible alternative for the current clinical islet transplantation protocol. Moreover, the fabrication process of the SF scaffold also enables incorporation of heparin, as reported in the present study, or any other growth factors and/or cytokines without compromising their bioactivities, and thus provides an open platform for SF-based bio-regulatory scaffold development not limited to islet transplantation.

Contribution statement This study was conceived and designed by Z. L and C. L. Data were collected and analysed by D. M., M. Z., X. Z., R. M., X. Y., T. K. and L. W. The manuscript was drafted by C. L. and edited by D. K. and Z. L. All authors revised the article critically and gave their final approval of the current version to be published.

Acknowledgement We are grateful for the generous financial support of the Tianjin Research Programme of Application Foundation and Advanced Technology (No. 17JCZDJC33400), the Programme for Changjiang Scholars and Innovative Research Team in University (No. IRT13023), the National Natural Science Foundation of China (project Nos. 81220108015, 31260223, 81371620, 81601625), China Postdoctoral Science Foundation (2016MS590197) and Peking Union Medical College Youth Fund & the Fundamental Research Funds for the Central Universities (project No. 3332016102).

Conflict of interest statement No conflicts of interest are declared by the authors.

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Figure legends Figure 1. Physical characterisation of silk fibroin scaffold. (A) Representative images of SF scaffold viewed via light microscopy (upper right) and scanning electron microscopy revealed average pore size between 200-400 µm in diameter. (B) Heparin releasing curve. Data presented as mean±SEM, n=5. (C) Dithizone (DTZ) staining of isolated islets. Scale bar=100 µm. (D) Stereo fluorescence microscopic images showing GFP+ islets distribution within the SF scaffold. Scale bar=200 µm. Figure 2. Co-culturing with heparin-silk fibroin scaffolds enhanced vascular regenerating activities of islets in vitro. (A-B) Islets were co-cultured with heparin-silk fibroin scaffold (H-SF), non-heparin scaffold (SF) or alone (Ctrl) for 14 days. Live/dead staining revealed significantly reduced islet cell death after H-SF/SF co-culturing. Living cells were shown in green and dead cells in red. Scale bar=50 µm. Data presented as mean±SEM, n=30 islets per group. *P<0.05, ***P<0.001 vs Control; †P<0.05 H-SF vs SF, one-way ANOVA with post-hoc Bonferroni’s test. (C-D) Elevated bioluminescence signals were also observed in islets co-cultured with H-SF compared to SF or alone (Ctrl) on Day 7 and 14. Data are presented as mean±SEM , *P<0.05 vs Control; †P<0.05 H-SF vs SF, one-way ANOVA with post-hoc Bonferroni’s test. Figure 3. Long-term function of syngeneic islet grafts. (A) Proportion of animals that achieved euglycaemia following transplantation of islets with heparin-silk fibroin (H-SF), silk fibroin scaffolds (SF), alone (Ctrl). Non-treated diabetic mice (NT) were also included. (B) Random blood glucose levels of recipient mice up to 1 year post-transplantation, n=10 per treatment group (C-F) IPGTT of all mice on day 30 (C) and 180 (D) post-transplantation. Area under curve of corresponding IPGTT values of all mice on day 30 (E) and 180 (F). Nor/Normal: non-diabetic mice, n=4-5 per treatment group. (G) Serum insulin level of all mice on day 30 post-transplantation, n=10 per treatment group. (H) Percentage changes of body weight after islet transplantation, n=10 per treatment group. Data are presented as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 vs Control; †P<0.05, ††P<0.01 H-SF vs SF; #P<0.05, ##P<0.01, ###P<0.001 vs Nor/Normal, one-way ANOVA with post-hoc Bonferroni’s test. Figure 4. Histological analysis of islets co-transplanted with heparin-silk fibroin scaffold. Representative images of H&E (A) Masson’s trichrome (B) immunohistochemistry (C) and immunofluorescence staining (D) of grafts retrieved from islet-H-SF transplantation group. Tissue: infiltrated tissues; Islet: islets; Scaf: H-SF scaffold; EFP: epididymal fat pad. In immunihistochemistry staining images (C), insulin was stained in brown and nuclei in blue. Immunofluorescence images (D), areas within dashed lines represent implanted H-SF. Images are representative of 5 animals. Scale bar=100 µm.

Figure 5. H-SF co-transplanted islets exhibited enhanced revascularisation in vivo. Animals were infused with tomato-lectin (green) and islet vasculatures were visualised. (A) Representative images of lectin (green)/insulin (red) double staining. White arrows indicate circular shaped intra-islet microvasculatures. Nuclei were visualised by DAPI (blue). Scale bar=100 µm. (B, C) Vascular microstructure densities (B) and numbers (C) are quantified. Images are representative of 5 animals. Data are presented as mean±SEM, *P<0.05, one-way ANOVA with post-hoc Bonferroni’s test. Figure 6. H-SF enhanced islet revascularisation by promoting local VEGF expression and activation of VEGFR2. (A) Double staining of insulin and VEGF. Scale bar=200 µm. (B, C) Expression of VEGF and β-actin in grafts 7 days post-transplantation by western blotting and level of protein expression was quantified by densitometry. (D) Vegfr2-luc islets were transplanted with H-SF, SF or alone, bioluminescence signals were collected on day 0, 7 and 14 and quantified (E). Data are presented as mean±SEM, n=4, *P<0.05 vs Control. Figure 7. H-SF enhanced islet cell proliferation. (A) Double staining of insulin and PCNA. White arrows indicate insulin+PCNA+ cells. Scale bar=200 µm. (B-C) Numbers of insulin+ PCNA+ (B) and PCNA+ (C) cells were counted and plotted accordingly. Images are representative of 5 animals. Data are presented as mean±SEM, *P<0.05, one-way ANOVA with post-hoc Bonferroni’s test. Figure 8. H-SF enhanced post-transplantation islet survival. (A) Representative bioluminescence images on day 0, 14 and 30 following transplantation of β-actin-luc islets with H-SF, SF or alone. (B) Bioluminescence intensity was measured for each mice. Data presented as mean±SEM, n=5, *P<0.05 vs Control, one-way ANOVA with post-hoc Bonferroni’s test.

Statement of Significance 1) The silk fibroin scaffold presented in the present study provides an open platform for scaffold development in islet transplantation, with heparinisation as an example. 2) Both heparin and silk fibroin have been used clinically. The excellent in vivo therapeutic outcome reported here may therefore be clinically relevant and provide valuable insights for bench to bed translation. 3) Compared to conventional clinical islet transplantation, during which islets are injected via the hepatic portal vein, the physical/mechanical properties of silk fibroin scaffolds create a more accessible transplantation site (i.e., within fat pad), which significantly reduces discomfort. 4) Islet implantation into the fat pad also avoids an instant blood mediated inflammatory response, which occurs upon contact of islet with recipient’s blood during intraportal injection, and prolongs survival and function of implanted islets.