Therapeutic polymers such as methacrylic acid (MAA)-based biomaterials generate 86 vessels when subcutaneously transplanted in a variety of different mouse models (CD1 10,11 , 87 SCID/bg 3 and C57BL6 12 ), without the use of exogenous growth factors or cells. In a separate 88 study, we showed that in coating a polypropylene mesh chamber with this copolymer 12 , we 89 could restore diabetic C56Bl/6 mice to normoglycemia with syngeneic islets 13 ; uncoated mesh 90 chambers were ineffective in this model.
In this study, we adapted a method used by Pepper et al 2 . A silicone catheter was 92 coated with a copolymer of MAA and isodecyl acrylate (IDA) and used to pre-vascularized the 93 subcutaneous space in SCID/bg mice and Sprague Dawley (SD) rats. Following pre-94 vascularization, the catheter was removed, and pancreatic islets were delivered into the pre-95 vascularized cavity (Fig. 1) . First, we determined when the MAA-coated tubes generated the 96 maximal number of vessels after subcutaneously implanted in immune-compromised SCID/bg 97 mice. We confirmed that the pre-vascularized site could support the engraftment of a marginal 98 dosage of pancreatic islets. Finally, we showed that this MAA-based approach could be scaled 99 up into a larger rat model and support the long-term engraftment of allogeneic islets in 100 immunosuppressed recipients. This study highlights the potential of using a biomaterial-based 101 approach to pre-vascularize the subcutaneous space for islet transplantation, with emphasis on 102 its ability to be scaled up into larger animal models. 137 SCID/bg mice were made diabetic using a single i.p. injection of streptozotocin (180 138 mg/kg, Sigma Aldrich) one week before islet transplantation (Supplementary Figure 1A) .
Sprague Daley rats were made diabetic using a single i.v. injection of streptozotocin (40 mg/kg) 140 via the tail vein one week before islet transplantation. Animals were monitored daily (OneTouch 141 UltraStar2) and only animals with non-fasting blood glucose levels above 20 mM for two 142 consecutive days were used for transplantation studies.
Prior to transplantation, the pancreatic islets were washed three times with PBS and 144 then mixed with neutralized Type I collagen (3.0 mg/mL -PureCol, Advanced Biomatrix).
Depending on the recipient species, different numbers of IEQs were mixed with various volumes 146 of collagen as outlined in Table 1 
which was used as a guide to inject the islet/collagen mixture into the pre-vascularized site. The
larger tube was partly removed during the injection (Fig. 1) . Following the injection, both tubes 162 were removed, and the incision was then sutured using PolysorbTM 3-0 sutures (Covidien).
Age-matched mice were used as no pre-vascularization controls, delivering the islet/collagen 164 mixture (with HUVEC) using the PE90 tubing into the unmodified subcutaneous space.
Xenogeneic islet transplantations into the SCID/bg mouse did not require any intra-islet vessels. The average vessel density was highest in the grafts retrieved from the MAA-249 coated tube group (105 ± 15 vessels/mm 2 ), which was significantly higher than the unmodified 250 SQ space group (54 ± 11 vessels/mm 2 ). This value was also higher than the uncoated silicone 251 tube group (60 ± 10 vessels/mm 2 ) but this was not significant (Supplementary Figure 2B) . 
The majority of rats in this study had their grafts assessed for up to 21 days, but some 283 Surprisingly, animals from each of the three groups responded to a glucose bolus when 284 challenged with an OGTT at day 14, returning to normoglycemia within 120 minutes post-285 administration (Fig. 5D ). There were no significant differences among the three groups in 286 response to the glucose bolus (Fig. 5E ).
Similar to the SCID/bg mice, retrieving the grafts for histological analysis was more 288 challenging than anticipated. Islet grafts from the unmodified subcutaneous group did not have 289 any insulin positive staining, consistent with the poor functional outcome observed on 290 normalizing blood glucose levels. At day 21, we were able to retrieve 4 of 8 grafts from animals 291 within the MAA-coated pre-vascularization group. Grafts that were retrieved at day 21 showed 292 extensive insulin staining with intact pancreatic islets and intra-islet vasculature (Fig. 6B) . Both 293 intra-islet vessels and the vessels throughout the surrounding area were mature (CD31 + and 294 SMA + ). The average CD31 + vessel density of the explanted grafts (islet and surrounding tissue)
295 was 178 ± 8 vessels per mm 2 (Fig. 6C) . Grafts at day 90 were unable to be reliably retrieved 296 since they were completely integrated with the host's subcutaneous space. 
To reduce the therapeutic dose required to achieve normoglycemia in the subcutaneous 309 space, pre-vascularization is a suitable alternative where vessels are generated before islet 310 transplantation to provide the necessary oxygen and nutrients to support islet engraftment and 311 survival 5,7,9 . We hypothesized that a purely biomaterial-based approach for pre-vascularization 312 would be simpler to translate than an approach based on using cells to generate vessels 1 
Consistent with these results, we observed that uncoated silicone tubes generated many 324 vessels in the subcutaneous space of both mice and rats, presumably by generating a foreign 325 body response that was halted by removing the tube at day 14 (Fig. 2B, 4B) .
We followed a protocol similar to that used by Pepper 
there was a significant delay in the time it took to achieve normoglycemia (Fig. 3B) . Pancreatic 344 islets that survive the immediate hypoxic conditions secrete VEGF-A 22 to recruit neutrophils 23 345 and drive re-vascularization once transplanted, which may explain the delay observed in the no 346 pre-vascularization group relative to the groups with pre-vascularization (Fig. 3A, B) .
The benefits of MAA-driven pre-vascularization were most apparent in the larger SD rat 348 model (Fig. 5A) . MAA-coated tubes generated the greatest number of vessels (Fig. 4B) and
showed the most consistent return to normoglycemia (Fig. 5A) , compared to the less 350 vascularized site associated with the uncoated silicone tube group (Fig. 4B) . Due to the 351 avascular nature of the subcutaneous space, 4000 IEQ did not have any therapeutic effect 352 when transplanted into the unmodified subcutaneous space (Fig. 5A -red line) . Interestingly, 353 all groups could respond to an OGTT at day 14 (Fig. 5D) , suggesting that although islet grafts 354 could not regulate non-fasting blood glucose levels, some residual islet function remained 355 sufficient to respond in a glucose challenge test 24 .
The co-delivery of HUVEC with the islets in SCID/bg (but not the rat) may have also 357 contributed to generating a more robust vascularization response after transplantation into the 358 pre-vascularized site. In previous studies, the addition of HUVEC with islet collagen 359 microtissues generated a greater number of vessels at early time points and prevented collagen 360 degradation compared to grafts without HUVEC following subcutaneous islet transplantation 1 .
HUVEC were included to enable a comparison with these previous studies where 750 IEQ was 362 needed to restore normoglycemia without pre-vascularization. HUVEC help drive re-363 vascularization of transplanted pancreatic islets by secreting VEGF 25 (in addition to that 364 produced by islets) and other pro-angiogenic growth factors 26 . However, by co-delivering 365 HUVEC, the perceived therapeutic benefit of having more vessels before islet transplantation 366 ( Fig. 2A, B) may have been obscured.
Retrieval of the islet grafts was more challenging than anticipated in the SD rat model,
unlike what was seen in the SCID/bg mouse with or without 1 pre-vascularization. In the rat, 369 pancreatic islets were delivered within a collagen gel to support their engraftment and 370 function 27-29 , however collagen degrades over time when subcutaneously transplanted without 371 endothelial cells 1 . In our explants, we could only find intact islets (insulin + ) in implants that were harvested at day 21 in the MAA-coated tube group (Fig. 6B) . The area surrounding the 373 embedded islets was well vascularized (CD31 + and SMA + ), and the presence of intra-islet 374 vessels was also observed (Fig. 6B, C) . Explanted grafts from the uncoated tube or unmodified 375 subcutaneous groups could not be distinguished from the surrounding subcutaneous space.
There was little or no insulin staining in the explanted tissue and may be due to poor islet 377 survival, since the uncoated tube and unmodified subcutaneous space groups had significantly 378 higher non-fasting blood glucose levels compared to the MAA-coated tube group (Fig. 5A) .
At day 90, grafts had completely integrated with the host's tissue and could not be 380 distinguished from the surrounding subcutaneous space making retrievability not possible.
Although we did not directly confirm islet integration and re-vascularization using CLARITY as in 382 previous studies 1,4 , we expect that the vessel islet integration seen before 1 was critical for their 383 therapeutic efficacy. Long-term integration of the islet graft was possible because the collagen 384 delivery vehicle was remodeled, but this integration can make retrieval more difficult, unlike in the subcutaneous space but not islet transplants. In our study, we found a combination of 399 ALS, fingolimod and MMF was necessary to prevent rapid graft rejection (Fig. 5A) .
Tacrolimus is a calcineurin inhibitor, which inhibits T-cell function, but its 401 immunosuppressive effect can be variable and is dependent on its bioavailability 37 , 832  833  834  835  836  837  838  839  840  841  842  843  844  845  846 