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Abstract

Treatment of type 1 diabetes is based on exogenous insulin injections and has drastically improved diabetes mortality and morbidity. Nevertheless, this therapy failed to control effectively and safely the glycaemic levels in some patients suffering from brittle diabetes. For these patients, morbidity and mortality remain high despite improved metabolic results, but transplantation development of pancreatic islets allowed to achieve optimal glycaemic control and insulin-independency (with an acceptable safety profile in comparison to pancreas organ transplantation).

Human islets are mainly transplanted by infusion in the portal vein. However, numerous factors limit the full development of this technique, such as: immune rejection, inflammation, and lack of oxygen and blood supply during the first days post-transplantation.

Although islets represent a small fraction of the pancreatic volume, they receive an abundant vascularization. The islet isolation process disrupts all the vascular connections and a delayed revascularization process occurs after grafting. During this period of revascularization, hypoxia and ischemia impair islet viability as well as functionality. During the early post-transplantation period, islets undergo central necrosis which leads to a huge loss of islet mass during this early post-transplantation period. Improving islet vascularization and oxygenation during this period remains a key challenge to enhance islet transplantation outcomes.

It is widely accepted that vascularization and oxygenation are two essential axes to address, but only few innovative strategies were clinically tested to optimise islet transplantation. We believe that improving at least one of these axes will greatly benefit the outcome of the pancreatic islets. In this review, we shed light on the current advances regarding islets’ vascularization and oxygenation, in an attempt to identify the most clinically relevant ones. We briefly review the concept of immunoisolation before developing the main axes regarding vascularization and oxygenation. We also mention some developments in bioengineering.

Introduction

According to the World Health Organization, diabetes was in the top ten causes of death worldwide in 2016 [1]. In 2019, the International Diabetes Foundation estimates that 463 million people lived with diabetes [2] and that type 1 diabetes (T1D) represented 7% to 12% of the diabetic population [3]. This number is expected to rise to 700 million people by 2045 [2]. T1D is an auto-immune disease, characterized by a β-cell dysfunction. Physiopathology of T1D is complex and implies a cross-talk between injured or stressed β-cells and immune cells (macrophages and T cells) that lead to pancreas inflammation (insulitis) and β-cell apoptosis [4].

It provokes an absolute insulin deficiency which is responsible for chronic hyperglycaemia. It is well known that insulin deficiency leads to multiple complications such as retinopathy [5], nephropathy [6], neuropathy [7] and a major increase in cardiovascular risk [8]. Treatment of T1D is based on insulin replacement therapy through multi-daily injections or insulin pump. Optimal glycaemic control is necessary to reduce microvascular complications [9,10]. However, despite modern diabetes care strategies and treatment, patients with T1D fail to achieve optimal glycaemic control [11]. Moreover, a subpopulation develops a severe form of diabetes known as brittle diabetes [12]. Brittle diabetes is responsible for frequent, unpredictable severe metabolic events (keto-acidosis and/or severe hypoglycaemia) which dramatically impact the quality of life and vital prognosis [12]. For these patients, β-cells replacement (pancreas organ or islets transplantation) may serve as an alternative.

Data from the International Pancreas Transplant Registry showed improvement of pancreas transplantation over time: one year insulin independence rate is now of 80% for pancreas transplanted alone (PTA, i.e. without kidney graft association), 86% for simultaneous pancreas-kidney graft and 80% for pancreas after kidney transplantation (PAK) [13]. After five years of graft survival, insulin independence increases from 55 to 58% for PTA and 55% to 65% for PAK [13,14]. Despite these good metabolic results, whole PTA requires a complex surgery associated with a higher risk of complications such as pancreas thrombosis (10-35% according to literature) [15] responsible for early graft loss. Bleeding, graft pancreatitis, sepsis, and fistula may also occur [15].

To isolate pancreatic islets, pancreas from brain-dead donors are removed and undergo a two-step dissociation: an enzymatic dissociation followed by a mechanical dissociation [16]. Islets are then purified, using a density gradient centrifugation, and collected. The islet suspension is infused in the portal vein by percutaneous transhepatic approach. The complexity of the islet isolation procedure is multifaceted considering ischemia, the double dissociation and the density purification. However the islet transplant infusion procedure per say is less complex than PTA. Over the past two decades, results of islet transplantation have also improved: between 2007 and 2010, the graft survival (C-peptide ≥0.3 ng/mL) was close to PTA, with 92% at 1 year [17]. Moreover, 66% of patients transplanted were insulin independent at 1 year. Results from the GRAGIL Network showed a 74% graft survival 5 years after islet transplantation, transplanted alone (ITA) or after kidney graft (IAK) [18]. In 2016, a prospective multicentric clinical trial confirmed the efficacy of islet transplantation on metabolic results [19]. Later, the first randomised controlled trial on islet transplantation showed an improvement of metabolic results at 6 months compared to intensive insulin therapy [20]. In addition, islet transplantation reduced severe hypoglycemic events [18–20] and improved Clarke and HYPO scores [19]. Taken together, these results demonstrate that islet transplantation is an efficient and rather lasting therapy to restore glycaemic control and to prevent severe hypoglycaemia in patients with severe glycaemic lability. Moreover, islet transplantation reduced retinopathy progression and did not deteriorate renal function [21].

Despite these convincing clinical results, major obstacles need to be overcome to further improve islet transplantation [22] including: shortage of donor pancreas, long-term toxicity of immunosuppressant therapy, poor engraftment linked to lack of oxygen, poor blood supply, inflammation, long-term immune rejection. At early stage post-intrahepatic transplantation, between 50 to 60% of the graft is lost [23,24] due to inflammation [25,26], cytokines exposure, hyperglycaemia [24] and hypoxia [27] impacting islet outcomes. Moreover, immunosuppressant therapy can expose recipients to severe complications (infection, cancer or renal function impairment) [28], which have led the way to the development of new research strategies such as immunoisolation. Islet immunoisolation currently implies using either macroencapsulation in which the whole islet preparation is encapsulated in a single device, or microencapsulation in which a single islet or a duet of islets are encapsulated in microcapsules. Historically, the first transplantation devices were intravascular hollow fibers [29–33] which were developed to allow islets to be close to arterial rich oxygenated blood (or in arteriovenous shunt). These macrodevices were successfully implanted in large animal models [34]. However, these devices provoked cloating [31] and preventive-heparin injection was often associated with massive haemorrhage. This approach was abandoned in the late 90’s and meanwhile, clinical intraportal transplantations of free islets were increasing [16,35], encouraging the development of macro and microencapsulation. Macroencaspulation often involves alginate sheets or other planar devices, now developed for extravascular use [36-39]. Microencapsulation [40-44] consists of several capsules containing islets, often composed of alginate formed with an air-jet syringe pump extrusion [45-48].

However, encapsulation leaves unsolved the problems of delayed graft vascularization and oxygenation, and of central necrosis [49]. Indeed, if macroencapsulation offers several advantages (islets are in a single device, which can be easily and totally removed if needed), vascularization and oxygen supply to islets within macrodevices remain a challenge [50]. To provide sufficient nutrient and allow oxygen diffusion, the total volume of islets in a sheet cannot exceed 40% of the size of the device [36]. Clinical trials using such devices and stem derived β-cells are currently ongoing (NCT02239354, NCT02939118, NCT03163511, NCT03162926) and preliminary results presented at conferences did not show restoration of insulin secretion [51].This could be due to the foreign body response and also certainly due to central islet necrosis triggered by low oxygen diffusion through the device, partially due to fibrotic overgrowth surrounding the macrodevices [51]. The few published clinical studies on macrodevices showed disappointing metabolic results [52,53]. Microencapsulation could theoretically help to enhance oxygen diffusion by reducing the size between the oxygenated environment and the islet core: however, data on animal models showed that microencapsulated islets are also prone to central necrosis and oxygenation problems [54,55]. Clinical trials with human microencapsulated islets demonstrated a good feasibility and safety but metabolic outcomes were contrasted depending on the study [56–59].

Consequently, strategies have been developed to improve engraftment of islets and encapsulated islets outcome. Recently, a review by Papas, et al. nicely summarised the oxygenation strategies [60], but in this review, we wish to take this reflexion a step further by focusing on the current advances regarding both islets’ vascularization and oxygenation. We believe both these aspects should be considered in order to try to identify the most clinically relevant strategies. We aim at summarizing and comparing the various strategies that have been developed so far to enhance vascularization and oxygen diffusion regarding islets or encapsulated islets transplantation. First, we briefly mention islet physiology and then we will summarize the existing devices, and the various transplantation sites, and their different issues focusing on vascularization and oxygenation.

Oxygenation and vascularization of pancreatic islets in physiological conditions and response of islets to hypoxia

Previously, Jansson and Carlsson have reviewed precisely the physiologic vascularization of pancreatic islets and the process of revascularization after graft [61]. In fact, islets are surrounded by a “glomerular-like” capillary network, that derive either from capillaries of the exocrine pancreas (for small islets) or directly from proper arterioles [61]. While islets represent 1 to 2% of pancreas, they receive between 10% to 20% of pancreatic blood flow, depending on species and age [62], thus maintaining adequate oxygen supply, to keep an oxygen tension around 30-40mmHg in pancreas [63,64]. Indeed, islets have one of the highest blood supplies in the human body [65]. During islet preparation, vascular connections are disrupted.  After islet transplantation, oxygenation of islets during the early first days depends on the diffusion of oxygen according to a gradient between the core (less oxygenated) and the periphery (better oxygenated) of the islet [64]. It is estimated that after transplantation, 70% of islets are hypoxic [63], whichever the site of transplantation, with an oxygen tension around 5mmHg while it is around 40mmHg in normal pancreas [64,66]. The revascularization process starts a few days after islet transplantation, with an increase of vessels diameter from day 7 to day 14 [65]. During this period, oxygen and nutrient deprivation may contribute to the mass loss of islets partly because of central islet necrosis.

The effect of hypoxia on rat islets has been studied in normoxic conditions (pO2 = 160mmHg, reflecting atmospheric conditions): after 24h, the pO2 decreased in culture medium. After 24h of culture in hypoxic conditions (pO2 = 15mmHg), viability was reduced, insulin secretion was impaired and levels of Hypoxia inducible factor-1α (HIF-1α) mRNA increased [67]. De Groot, et al. showed that the viability of encapsulated rat islets decreased in hypoxic conditions, mainly due to an increase of necrosis [68]. High density seeding decreased viability of free islets and impaired secretion by inducing local hypoxia [69].

Muthyala, et al. [70] compared effects of 2-5% hypoxia on viability and on metabolic activity of free pig islets versus encapsulated islets. Under normoxia, no viability changes were observed over time, both in free and in encapsulated islets. Interestingly, viability of encapsulated islet was maintained in hypoxic conditions, compared to normoxic conditions and decreased only at day 6 [70]. However, the encapsulation did not improve the insulin secretion nor the metabolic activity. Thus, even if the study of Muthyala, et al. was performed in pig islets (which have a different metabolic profile than human islets), encapsulation in these conditions seems to confer a protection to hypoxia, giving hope for future explorations in rodent and in human islets.

Hals, et al. compared the effect of an 8h-hypoxia followed by a 14h-18h reoxygenation period on human encapsulated islets: they showed that viability decreased in hypoxic conditions versus normoxic conditions but interestingly, they did not find differences in viability between encapsulated islets and free islets in hypoxic conditions [71].

Alternative transplantation sites to enhance islet oxygenation and vascularization

The ideal site for islet transplantation is described as requiring a rich arterial and venous vascularization in order to provide nutrients oxygen supply and to normalise blood glucose after release of insulin, respectively [72]. The ideal site should also be easily accessible with a minimally invasive procedure [72]. An easy access to islets would enable the monitoring of islets’ viability and allow to retrieve the graft if necessary [73,74] without triggering early inflammation, thrombosis and instant blood mediated inflammatory reaction (IBMIR).

Finding a transplantation site satisfying all these criterions simultaneously remains impossible so far. We will detail several sites of transplantation which are still competing as “the ideal site”. Transplantation sites [75,76] and oxygen partial pressure in various potential transplantation sites [60] have been previously reviewed, here we will discuss the interest of each transplantation site in terms of oxygenation and vascularization.

Nowadays, 90% of clinical islet transplantations in human take place in the intraportal site [77], due to the arterial supply and venous drainage, and the good accessibility to inject islets being minimally-invasive. However, drawbacks are numerous [73] as islets are exposed in the portal vein to high concentrations of glucose, as well as high toxic drug concentrations compared to the whole pancreas and the direct contact with the intravascular system triggering IBMIR [63,73]. Finally, the hepatic oxygen tension (5 -10 mmHg) is lower than that of the pancreas (40mmHg), but to date it remains the site with the best clinical results in human [78].

Despite a low oxygen tension [79], the kidney capsule is often used in rodents, since it enables morphological and histological follow up of the transplant by biopsy, restores normoglycemia and allows better engraftment than the liver [75]. In human, the risk of altering the kidney in a population of patients who are susceptible to renal impairment makes the kidney capsule inappropriate for clinical islet transplantation [75].

The pancreas site has surprisingly been less evaluated than other sites, despite being the native place for islets, providing ideal conditions of oxygenation, microenvironment, and exposure to glucose. This is probably due to the invasive surgery required to implant islets, the poor knowledge regarding the risk of recurrence of auto-immunity and the destruction of the graft [74,75].

The peritoneum cavity and the omentum represent an attractive alternative to the intraportal site. Indeed, the accessibility in surgery without an invasive technique and the size allow large infusion of islets [75]. The rich vascularization of this site, the physiological function as fibrotic inhibition makes it a good candidate to compete with the intraportal site [80]. Indeed, the omentum seems to provide a faster revascularization and oxygenation compared with intraportal route: vascular density was better in human islets transplanted in mice omentum and the number of hypoxic β-cells decreased during a 7 day-period, while it remained unchanged for islets transplanted in the intraportal site [81]. Other preclinical studies validated the omentum as a safe transplantation site for microencapsulated islets or seeded islets in biologic scaffold, with faster revascularization [82], better differentiation [83], lower stress-markers [83] and good glycaemic results [82,84]. In 2017, encouraging results were obtained through the transplant of islets within a bioengineered omental pouch in a type 1 diabetic patient [85]. In 2018, Stice, et al. published four cases of patients that underwent a total pancreatectomy and islet autotransplantation and who could not have a complete intraportal islet transplantation (12 to 36% of islets have been transplanted in an omental pouch): the glycaemic results were equivalent to a control population (i.e. receiving all the transplant by intraportal route). No patient had intraoperative complications. The postoperative complications reported (anemia, nosocomial pneumonia, etc.) were not specifically related to the omental pouch technique.

A clinical trial involving the omental site for islet transplantation was recently completed (NCT02821026) but no results were published so far.

The subcutaneous site is often used in rodents, mostly for implantation of macrodevices [75]. The main advantage is the easy and rapid access, performed under local anesthesia, authorizing full graft removal but also the possibility to reload the device if necessary [74]. However, the engraftment is limited by the low vascularization [75,86] and in order to overcome this, prevascularization strategies have been developed such as the preimplantation of an empty microdevice showing encouraging results [87–89]. More recently, the inguinal subcutaneous white adipose tissue have shown promising results in mice, since the site is correctly vascularized by the epigastric inferior artery [90]. However, most subcutaneous tissues lack rich vascularization, thus inadequate for both the good engraftment and the development in human. Also, the volume of islets to inject seems to be too important regarding the capacity of this site: a recent clinical trial showed the safety of macroencapsulation in subcutaneous space in human but failed to restore metabolic control [53].

The splenic site would expose islets to the same environment as the pancreas, with a good vascularization and delivery of insulin in the splenic vein, thus in the portal venous system [77].  Advantages are similar to the intraportal site, but the major drawback is the risk of massive haemorrhage [77] although surgical procedure advances could minimize the risk of haemorrhage [91].

Gastric submucosal space [92], bone marrow [93], epididymal [94] and mammary fat pads [95], muscle [96,97] have the advantage of good vascularization, but further studies need to confirm the potential of these sites. Epididymal and mammary fat pads being murine specific tissues, they have no human relevance. Two recent clinical trials showed the feasibility and safety of islet transplantation in the bone marrow, but the graft survival was less than 4 months [98]. More recently, it has been established that lymph nodes allowed survival of functional pancreatic islets  in mice [99] and rats [100] but the site has not been explored further.

It seems to us that the omental site pouch is the most promising alternative to the hepatic site due to its richer vascularization. The results of the recent clinical trial NCT02821026 are not yet published, but they will enable to determine if these favorable vascularization conditions offered by the omental site clearly improve islet transplantation outcomes.

Enhancing oxygen delivery to islets

Oxygenation of islets has been an issue for a long time and particularly since the 90’s. In 1991, Dionne, et al. presented a perifusion system to assess effect of pO2 on insulin secretion [101]. Then, Küthreiber, et al. determined pO2 in vivo in diffusion chambers seeded with islets [102], Schrezenmeir, et al. assessed the pO2 of free versus microencapsulated islets [103], Wu, et al. developed an electrolyzer for generating oxygen in situ by electrolysis of water, which can be associated with a diffusion chamber [104]. Carlsson, et al. evaluated the oxygen tension in transplanted rats and human islets [66,105,106].

Two methods have been identified to improve islet oxygenation: to supply O2 or to improve O2 diffusion. The first can be achieved either by O2 carriers or using abilities of membranes to carry O2, or even by direct perfusion of O2 in the site of islet transplantation. The second, by changing the macrodevice conformation, opting for a planar conformation or using small islets. The use of medium supplemented with oxygen-precharged perfluorocarbons (PFC) or directly into the encapsulation material to increase its oxygen permeability have been widely explored [107]. Some strategies with enriched culture medium, bioengineered capsules or O2 have been tested but only in vitro so far. For example: the use of a marine haemoglobin able to bind about 150 oxygen molecules [67], the enrichment of medium with micro/nanobubbles filled with pure O2 [108], co-encapsulation with haemoglobin [109], co-encapsulation with algae as photosynthetic generator [110]. Improving conditions of shipment by gas-permeable culture bags [111] or culture islets in gas-permable silicone rubber membrane vessels [112–114] have shown promising results on oxygen consumption rate (OCR)  in vitro. Islet culture in bioreactors, allowing remodeling of islets with the formation of channels with external openings that could allow a better oxygen diffusion were tested. These studies showed that islets had a preserved viability and function, but so far none have assessed oxygen parameters [115,116]. In this review, we focus on oxygen supply techniques that have already been tested in vivo ((Table 1 and Figure 1).

Figure 1. Current developments to enhance the oxygen supply of isolated islets

Four different strategies to improve transplanted islets’ oxygenation are illustrated.

Table 1. Strategies to improve oxygen delivery to islets

Pre-transplantation step: enriched culture medium or bioengineered capsule

Adjonction of Deferoxamine (DFO, iron chelator activating HIF-1α) in islet culture medium prior to transplantation increased the β-cell mass at day 28 compared to control, in a HIF-1α-dependent mechanism, suggesting that increase of HIF-1α is a protective factor for islets [117]. HIF-1α seems to be a protective factor in islet transplantation, required for successful islet transplant outcomes [117]. Despite this protective aspect, some studies have also showed negative effects regarding transplant outcome, for example in hematopoietic stem cell transplantation [118]. These contradictory results could be due to the antecedent of islet stress induction, which then activate HIF-1α [119,120].

Fraker, et al. pre-cultured islets in a PFC/ Polydimethylsiloxane (PDMS) culture dish allowing islets to receive O2 from both the top and the bottom: in vitro, they showed an increase of O2 diffusivity, a better OCR rate, a better glucose stimulated insulin release index, and protection against hypoxia. In vivo data showed that the percentage of diabetes reversal in mice transplanted with human islets cultured in standard dishes or in the PFC/PDMS dishes was improved and seemed to appear sooner [121].

In encapsulated islets, the use of 20% Perfluorodecalin enriched microcapsules increased islets viability for 2 days, limited the Reactive Oxygen Species (ROS) production and maintained longer euglycemia in rats compared to alginate microcapsules and naked islets [122].

Co-transplantation or co-encapsulation of islets with others cellular types or with metalloproteins to improve oxygenation of islets.

Islets can be transplanted along with another cell type, so that the latter encourages the development of a vascular network. However, current transplantation strategies require a retrievable transplant for patients. This major limitation has encouraged macrodevices developments. In fact, macrodevices implanted in subcutaneous site and seeded with sole adipocyte mesenchymal stem cells (aMSCs) were significantly better oxygenated than those seeded with bone marrow mesenchymal stem cells bone marrow mesenchymal stem cells (bmMSCs) [123]. This proof of concept has been supported by subsequent results regarding co-encapsulation with islets [124]: in rats, pig islets co-encapsulated with aMSCs had a better oxygenation. The benefit of the MSCs is certainly due to the secreting capacity of these cells.

In the meantime, microdevices were developed with a different strategy, trying to maintain proximity of co-culture and limit the fibrosis which often occurs with larger devices. MSCs have been encapsulated by microfluidics and have proven to improve islet survival [125], but no O2 results were published so far. Development of a new generation of Haemoglobin Based Oxygen Carrier is currently in progress [126–128], but in vitro application and co-encapsulation with cells have not been tested so far. More recently, promising data on co-encapsulation of rat islets with human amniotic epithelial cells showed an improvement of viability and maintained glucose response under hypoxic conditions [129]. Hemarina, a French company, has developed a universal oxygen carrier which has been used for therapeutic purposes including Covid-19 [130,131]. We believe this new oxygen carrier could be of great interest for future co-encapsulation approaches and look forward to further investigations.

Use of external sources of oxygen supply

Hydrolytic activation dissociates calcium peroxide and therefore provides an O2 supply [132]. However, this reaction is extremely rapid and leads to hyperoxide condition, with the formation of hydroxyl radical, a ROS, toxic for islets [132]. Encapsulation of solid calcium peroxide (CaO2) within a polydimethylsiloxane (PDMS) disk tempers the hydrolitic activity, enabling slow water infiltration to dissociate the calcium peroxide [132]. This device, called “OxySite”, increases pO2 in culture as long as in contact with water, benefiting non-encapsulated rat islets. It also significantly increased the metabolic activity compared to control hypoxic islets and control normoxic islets [133]. Implantation of a macrodevice containing islets and an OxySite disk in immunocompetent diabetic mice allowed to restore normoglycemia [134]. Recently, OxySite has managed to miniaturised microbeads from 10mm disks to 220µm, directly integrated into a PDMS scaffold [135]. The OxySite scaffold was able to release oxygen during 20 days in vitro, suggesting that it should theoretically maintain islets oxygenation during the 14-day period of transplant revascularisation. Moreover, the OxySite maintained graft efficacy of lower purity islets in vivo: with 99%-purity islets, all diabetic recipients reached normoglycemia, whether they were transplanted with OxySite or control scaffold; but with 80%-purity islets, only recipients with the OxySite scaffold reached normoglycemia [135].

A similar device was developed by another team, with PDMS+CaO2, which improved viability of MIN-6 cells and of pig Neonatal Pancreatic Cell Clusters (NPCCs) particularly in hypoxic conditions [136]. During hypoxia, levels of caspase 3 and 7 were reduced compared to control and ROS production was decreased in PDMS+CaO2 device compared with PDMS alone and control group, while insulin secretion was significantly higher for NPCCs in PDMS+CaO2 group compared to PDMS alone [136]. More recently, a study demonstrated the effect of oxygenation for islet transplantation, as well as O2 toxicity by excessive oxygenation [137]. In addition to developing a new oxygen–generating bioscaffold facilitating islet survival and function, they also showed the appropriate O2 range for islets which may be narrower than expected.

Evron, et al. reported a successful implantation of a macrodevice made of several layers, in which several immobilized Synechococcus lividus (active thermophilic cyanobacteria) produce oxygen by photosynthesis when illuminated by a light source integrated to the device [138]. However, pO2 furnished by this device was under the 50mmHg required and the authors estimated that for the application of this device in human, regarding the number of islets requested, the size would have to approach 450 cm².

A macrodevice with an oxygen chamber with daily refuelling (βAir device) showed good results in a proof-of-concept study [139]: explantation of the device at day 13-post transplantation showed no fibrosis and no inflammatory reaction around the device, and morphologically intact islets and OCR increased from 0.86 ± 0.16 pmol/min/IEQ before transplantation to 1.85 ± 0.06 at day 13 [139]. Study on mini-pigs was successful with a reversal of diabetes [140] ; moreover, the oxygen concentration in the gas chambers was constantly > 300mmHg, allowing the constitution of an oxygen gradient between the gas-chamber and the islet module and thus ensuring an adequate oxygen supply for the islets [140]. However, even if first phase 1 studies demonstrated a good safety profile of the βAir device, the clinical outcomes were disappointing, probably due to the low number of IEQ transplanted [52,53]. Implantable Electrochemical Oxygen Generator is currently in development, in the proof-of-concept state, and should be able to continuously generate and supply oxygen, compared to the βAir device that needs daily refuelling [60].

More recently, simple oxygen inhalation with 50% O2 in rats increased the subcutaneous pO2 from 45mmHg to 140mmHg, both in prevascularized subcutaneous site and in native subcutaneous site [141]. It led to a functional graft reversing diabetes and maintaining viability of islets > 150µm compared to rats in normoxia conditions [141]. In 2003, a study demonstrated that hyperoxia improved the survival of intraportally transplanted syngeneic pancreatic islets [142]. This finding suggests that O2 inhalation could be rather simple and clinically applicable. However, this technique should be adjusted since hyperoxia could also cause brain and lung damage in human. Indeed, hyperoxia may aggravate oxidative stress and exacerbate inflammatory response. Hyperoxia increases the production of peroxynitrite and apoptosis (mediated by caspases), impact gene regulation implied in cell death (such as c-myc or bax) and can be responsible of vasoconstriction, leading to abnormalities in microcirculation [143]. In patients, high O2 concentration can be used in intensive care unit: conflicting clinical data exist on hyperoxia, but for stroke, there does not seem to be any benefit from hyperoxia or even possible adverse effects [143]. Indeed, hyperoxia can reduce the blood flow for acute myocardial infarction and thus increase myocardial ischemia; in sepsis a study showed an association between hyperoxia and mortality [143].

Improving neovascularization after transplantation

Beside the enhancement of oxygen delivery, strategies to support and enhance islet or encapsulation device vascularization have been developed. Historically, a significant part of research on vascularization concentrated on biomaterials, such as synthetic scaffolding or membrane [144], with research on ideal pore size [145]. More recent approaches evaluated the potential of extra-cellular matrix [146–149], prevascularization of transplantation site [89,150,151], delivery of pro-angiogenic factors [152–154] and bioengineering of macro or microcapsule to improve neovascularization [155,156]. Bowers, et al. recently reviewed the physical properties of devices such as pore size, surface roughness and stiffness of devices, extracellular matrix scaffolding, and the prevascularization of transplanting sites allowing better vascularization [157]. For example, pre-implantation of the macrodevice increases vascular profiles, with a more homogeneous distribution of the vessels [158] and increases endocrine tissue volume within the microdevice [89]. Design of a macrodevice appears particularly important since micropatterned lid with MSCs seems to facilitate prevascularization in vivo [159] whereas meshes [160] encourage cells to organize and create new vessels. Bowers, et al. also reviewed the way to release pro-angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF) [157]. In this review, we will focus mainly on two ways improving vascularization: co-injection with other cell types and systemic injection of pharmacological agents (Table 2 and Figure 2).

Figure 2. Current developments to enhance the vascularization of isolated islets

Six different strategies to improve transplanted islets’ vascularization are shown.

Table 2. Strategies to improve vascularization of transplant

Co-transplantation with other cell types to improve vascularization.

We previously mentioned the benefit of co-transplantation with other cell types regarding oxygenation, and in this part we will discuss the capacity of other cell types to provide a supportive network and the secretion of pro-angiogenic factors.

Mesenchymal stem cells: MSCs promote angiogenesis by secreting pro-angiogenic factors [161]. Several studies investigated their benefit in islet graft revascularization. Vériter, et al. [123] evaluated the sole impact on vascularization of MSCs derived either from bone marrow or from adipocytes, without islets, seeded in a macrodevice and then implanted subcutaneously in Wistar rats. No differences were observed in terms of number of vessels surrounding the macrodevice, but a higher number of VEGF-positive cells was observed for the macrodevice seeded with adipocyte derived MSCs [123]. Interestingly, MSCs seem to improve graft function when co-transplanted with islets, partly by promoting revascularization [162,163]: co-transplantation of MSCs and naked islets under the renal capsules in Lewis rats significantly increased by two folds the capillaries in the group MSC+ [162].

A study showed that bone marrow MSCs pre-cultured in hypoxic conditions (hMSCs) [164] have adapted by secreting pro-angiogenic and anti-apoptotic factors such as VEGFA, IL-6, MCP-1 and MMP9: when co-cultured with islets in hypoxic conditions, MSCs improved viability compared to islets alone and MSCs pre-cultured in normoxic conditions (nMSCs) [164]. This observation was confirmed in vivo: mice transplanted with hMSCs had better glycemic control and less apoptotic rate than nMSCs group and control islet group, for non-encapsulated islets [164] or for islets encapsulated in a PLGA macrodevice previously coated with hMSCs [165]. MSCs derived from human umbilical cord also promoted neovascularization and transplant function [166].

Interestingly, Kogawa, et al. tried to combine micro and macroencapsulation to benefit from both techniques and to overcome their respective drawbacks: mice were co-transplanted subcutaneously with microencapsulateds islets and MSC-CellSaic (a bioresorbable scaffold seeded with MSCs), into a mesh bag [167]. Microencapsulated islets + MSC-CellSaic had better metabolic outcomes than microencapsulated islets alone, increased the vascular area and the number of vessels. When seeded without islets, MSC-CellSaic significantly improved the vascular area and the number of vessels compared to MSCs spheroids and controls [167].

Development of 3D-culture allows better interaction between cells and the formation of spheroid that could be used as a support for a vascularization network [168]. Intraportal infusion of islets co-transplanted with bone marrow mononuclear cell derived spheroid (BM-spheroid) in mice succeeded to reverse diabetes and improved vascularization compared to islets transplanted alone: vascular density was almost 3 times higher than in the control group, without increasing thrombogenicity [169]. MSC-spheroid, derived from mouse pancreatic MSCs, caused thrombosis, leading to liver necrosis but co-transplantation with islets was not tested in the study.

Fibroblasts: Co-transplantation of islets with fibroblasts is of great interest considering their capacity to secrete pro-angiogenic factors such as VEGF, HIF-1α or FGF. Fibroblasts seeded in a scaffold and grafted in the subcutaneous site significantly improved the vascularization of islets [170].  At day 1 and 3, revascularization (considering the number of vessels)  was faster with fibroblasts, but on day 7 and 10 the rate of revascularization was similar between the two groups [170]. In this model, the fibrotic process allowed the creation of a new ECM with increased vascularization. This new organised and vascularised ECM improved islet engraftment as well as the long-term survival and function of the graft [170]. More recently, another study confirmed the importance of fibroblast-like population in the revascularisation process by helping reconstitute and organise ECM [171]. This highlights the importance of fibroblasts in future developments.

Endothelial cells and endothelial progenitors: Co-transplantation of naked islets under the kidney capsule with vascular endothelial cells transfected to increase the production of VEGF showed promising results in rats since the vessels density was 6-fold higher than the control groups [172]. Human islets coated with endothelial cell progenitors from human umbilical cord had a significantly higher vascular density, a higher blood oxygen tension and a higher blood perfusion compared with control at 1 month post transplantation in normoglycemic mice [173]. Glycaemic control was not assessed in this study. Interestingly, coating a collagen macrodevice containing rat islets with endothelial cells significantly increased the number of vessels after transplantation [174]. Even if the number of vessels was similar at day 21 between the module coated with endothelial cells and the control module, the difference at day 7 and 14 suggests a higher revascularization process which seems to limit ischemic effects [174].

Use of systemic pharmacologic agent

The use of GLP1 agonist or DPP4 inhibitor could be of great interest in future clinical practice in islet transplantation. For example, sitagliptin, a DPP4 inhibitor, significantly increased VEGFR-2 expression in islets transplanted under the kidney capsule in mice, but also increased the phosphorylation of cAMP response element-binding, finally leading to increased VEGF secretion [175]. Consequently, vascularization of islets increased, as shown by the increased proliferation of endothelial cells in the sitagliptin group in comparison with the control group. Sitagliptin also increased mTOR expression in transplanted β-cells, explaining the increase of expression of VEGFR-2 [175].

Mice transplanted with human islets under the kidney capsule and injected with either Exenatide, GLP1 or the oral administration of sitagliptin showed increased secretion of human C-peptide and improved post-prandial glycemia during an oral glucose tolerance test [176]. Exandin-4, a GLP1 receptor agonist, improved vascular proliferation around a macrovascular device transplanted under the kidney capsule in mice, in comparison with controls [177]. Authors showed that Exandin-4 significantly enhanced mRNA expression of Bcl-2 (antiapoptotic gene), Pdx-1 (gene encoding for proliferation and differentiation of β-cells) and HIF-1α (encoding for vessels proliferation) [177]. Exenatide, another GLP1 agonist, increased insulin secretion and glycaemic outcomes in patients transplanted in the intraportal site [178]. Langlois, et al. [179] demonstrated the angiogenic properties of liraglutide in vitro, with increased number of CD31 positive cells and increased staining intensity compared to control islets. The effect was at least partly mediated by HIF-1α since liraglutide induced a transient overexpression of HIF 1α after 12h in culture, before turning back to the control expression level, leading to the overexpression of VEGF gene, compared to control islets. VEGF gene overexpression is confirmed by elevated VEGF release at 48h. In vitro, viability was preserved at 48h compared to control, and insulin secretion was higher with liraglutide [179]. In vivo, liraglutide improved body weight gain and fasting glycaemia and immunohistological analysis of islets showed a significant higher number of endothelial cells around as well as inside transplanted islets when treated with liraglutide compared to the control group [179].

This encouraging effect of liraglutide on vascularization was not found in a previous study: Nishimura, et al. [180] transplanted mice islets into a dorsal skinfold chamber of recipient mice that were divided into a control group, a group with islets pre-cultured with liraglutide for 24h before transplantation and a group with subcutaneous injection of liraglutide for 8 days. There was no increase in the rate of newly formed vessels in any group. However, details are not given regarding the metabolic control of this transplantation.

Interestingly, in mice, the pre-treatment of mice islets for 10 min with liraglutide prior transplantation was more efficient to restore normoglycemia than systemic treatment with liraglutide [181]. In human, effect of liraglutide as an adjunctive treatment of type 1 diabetes remains disappointing [182–184]. A clinical trial on sitagliptin + pantoprazole in islet transplanted patients, with early graft insufficiency showed that sitagliptin allowed 2/8 patients to reached insulin independence (defined as: no insulin use for at least 1 week + HbA1c < 6.0% + fasting plasma glucose < 7mmol/L + C-peptide > 0.5nmol/L) [185]. Three months after stopping sitagliptin and pantoprazole, no patient reached the primary endpoint.

Other substances have been tested such as prolactin. Prolactin was used in vitro in a 24h pre-treatment in isolated islets and in vivo as injections during the 7 first days post transplantation under the renal capsule in mice. In both cases the blood flow increased by 40% at 1-month post transplantation [186]. The vascular density was markedly increased when islets were treated with prolactin compared with control mice and the O2 tension was increased in treated mice [186]. It also decreased the level of thrombospondin-1 mRNA, implied as a negative regulator of angiogenesis [186].

Pharmacological stimulation of VEGF production by DFO iron chelator led to VEGF overexpression for 3 days by inducing HIF-1α factor, whereas VEGF overexpression with an adenoviral transduction was maintained 14 days [187]. However, transduction with VEGF-adenovirus led to loss of functionality of islets,  as opposed to the use of DFO [187]. Even if DFO had adverse effects (mimicking iron deficiency), this study constitutes an interesting proof of concept for pharmacological stimulation with a more specific drug [187].

Hydrogen sulfide (H2S) was previously described as an effective angiogenic stimulator. It has been tested in mice: animals implanted with a poly(D, L-lactide-co-ε-caprolactone) scaffold were intraperitoneally injected with sodium hydroxysulfide [188]. Unexpectedly, authors described a lower vascularization of the device and a lower CD31 immunostaining in mice with H2S treatment compared to control [188].

Resveratrol (RSV) has been tested in diabetic mice: animals were transplanted with 200 IEQ under the left kidney capsule and treated orally with RSV [189]: RSV increased glycaemic control, β-cell and vascular density after transplantation compared to control transplanted mice. Oxidative stress was reduced in RSV group. In hypoxic conditions, RSV pre-treatment of isolated islets prevented cell death and ROS increase.

A recent study with Tocilizumab, an IL6 blocker, expected to reduce initial inflammatory response and thus improve vessels formation, succeeded to avoid C-reactive protein peak after transplantation but lowered the revascularization in monkeys intraportally transplanted [190].

The protein tyrosine phosphatase 1B (PTP1B) regulates phosphotyrosine signaling in several pathways implied in differentiation, cell growth, metabolism or apoptosis. PTP1B inhibits phosphorylation of VEGFR2 and VE-Cadherin, and impairs stimulation of angiogenesis by VEGF-A [191]. In a recent proof-of-concept and mechanistic study, PTP1B-KO mice islets showed excellent results after transplantation in rodent recipients [191] allowing diabetes reversion, increasing the vascular density by 3 times compared to control mice and doubling vessels area at day 30 and drastically decreasing Caspase-3 positive cells. The PTP1B-KO mice islets increased VEGF-A in an independent HIF1α way. The development of pharmacological inhibitors of PTP1B and its effect on islet transplant would be interesting to test in the future.

Pre-treatment of diabetic mice with intraperitoneal injection of erythropoietin (EPO) [192] improved engraftment of islets, neovascularization and shortens the return to normoglycemia. Since pre-treatment with EPO might not be transferred to human subjects, a long-lasting analogue of EPO called darbepoetin-α treatment was tested but failed to accelerate revascularization process [193].

Conclusion

Despite its safety and efficacy, islet transplantation struggles to overcome oxygenation and vascularization challenges to fully develop. As we summarised in this review, limiting factors are well identified and several solutions have been proposed for each issue, but the pieces of the puzzle remain to be put together.

Concerning oxygenation, studies lack consensus on the main judgment criterion. For example, some studies evaluate the improvement in pO2, while others evaluate the impact on the viability and the insulinosecretion of islets. A variety of ways to evaluate oxygenation is found in the literature: some authors use pO2 while others refer to the oxygen consumption rate, or to the consumption of O2 per week. Recently, non-invasive method of oxygen measurement by Fluorine-19 magnetic resonance has been described allowing to measure pO2 [194] or dissolved oxygen concentration [195] in non-human primates.  Besides, only few studies have managed to evaluate the effect of improved oxygenation in hypoxia condition. This achievement seems essential as improving the oxygenation of islets in normoxic conditions in vitro does not presume of an improvement in hypoxia in vivo. If improving oxygenation is a crucial aspect of islet transplantation, some concerns have been raised regarding the potential O2 toxicity, especially regarding oxidative metabolism. Some studies have nicely highlighted the paradoxical positive finding of the attenuation of hypoxia-induced effects which could be secondary to a protective effect of the hyperoxia induced reduction of oxidative metabolism [196,197.]

Concerning vascularization, several strategies of improvement are appearing especially co-transplantation with other cells types, use of a pharmacological agent, injection of a proangiogenic factor and bioengineering devices. However, studies lack consensus on the priority of evaluation criteria, either islet viability and functionality or the proliferation of vessels, with a common endpoint based on glycaemic control. In most studies, quantification of vessels is based on histological sections, with CD31 staining, and more recently lectin [106]. All these methods require explantation of islets or of the device, implying there is no dynamic study of the revascularization process in vivo. In 2016, Jansson, et al. reviewed the different techniques that can be used to measure pancreatic islets blood flow [65] to adapt to transplanted islets. Recently, Weaver, et al. performed an elegant quantitative approach to evaluate the vascularization of leading islet transplant extrahepatic tissue sites, with lectin, and nicely showed the integration of islets into the host vasculature [86]. Use of laser-scanning microscopy that have been previously reported to monitor in vivo islet graft in the anterior chamber of the eye appears as a nice tool to visualise vascularization process [171,198].

Clearly, vascularization and oxygenation are linked but authors often evaluate either one or the other criterion, while it seems that a global evaluation of both parameters would be interesting. We believe that recent advances in this field are extremely promising, and much remains to be done before giving a single direction regarding further developing islet transplantation with or without encapsulation. Interestingly, both oxygenation and vascularization issues remain a common concern regarding islet transplantation. As developed in this review, co-encapsulation with MSCs or oxygen nano-carriers seems especially promising, as well as the potential of some pharmacological agents, once further evaluated. The results of clinical trials regarding the omentum transplantation site will undeniably modify the future of islet transplantation. The idea of combining macro and microencaspulation in order to co-transplant islets with other cell types [167] is very original but is a double-edged sword. It allows to remove microencapsulated islets graft but it can dramatically deteriorate the functionality and viability of the islets: the encouraging results obtained in that study need to be further confirmed.

Other strategies diverging from islet transplantation are now rising such as injection of exosomes derived from MIN-6 cells (mouse insulinoma cell line) [199], development of in vitro model of spheroid and islets-on-chip to help create 3D-vascularization [200], reconstitution of pseudo-islets (disaggregated islets embedded into endothelialized collagen rods) [201] and changing the islet source by using of induced pluripotent stem cells [202] that are able to created viable vascularized transplants. Very recently, an important study reported the creation of organoids derived from the fusion of islet cells and functional blood vessels, with positive results on vascularization and impressive results on the numbers of islets needed to normalize glycemia [203]. All these new strategies give further hope for the improvement of islet transplantation technique. In particular, the organoids technology is very promising for regenerative and personalized medicine, but requires technical [204] and ethical [205,206] consensus.

Altogether, the future of islet transplantation remains an exciting perspective for type 1 diabetic patients in regards to the possible improvements in vascularization and oxygenation. Both short and long-term islet transplantation success will be improved and will further open-up this treatment strategy to more diabetic patients.

Authors' contributions

JC wrote the manuscript. ET, PYB and SL completed, discussed and reviewed the manuscript.

Disclosures and funding sources

The authors declare that they have no competing interests. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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