A Case for Material Stiffness as a Design Parameter in Encapsulated Islet Transplantation

Abstract

Diabetes is a disease that plagues over 463 million people globally. Approximately 40 million of these patients have type 1 diabetes mellitus (T1DM), and the global incidence is increasing by up to 5% per year. T1DM is where the body's immune system attacks the pancreas, specifically the pancreatic beta cells, with antibodies to prevent insulin production. Although current treatments such as exogenous insulin injections have been successful, exorbitant insulin costs and meticulous administration present the need for alternative long-term solutions to glucose dysregulation caused by diabetes. Encapsulated islet transplantation (EIT) is a tissue-engineered solution to diabetes. Donor islets are encapsulated in a semipermeable hydrogel, allowing the diffusion of oxygen, glucose, and insulin but preventing leukocyte infiltration and antibody access to the transplanted cells. Although successful in small animal models, EIT is still far from commercial use owing to necessary long-term systemic immunosuppressants and consistent immune rejection. Most published research has focused on tailoring the characteristics of the capsule material to promote clinical viability. However, most studies have been limited in scope to biochemical changes. Current mechanobiology studies on the effect of substrate stiffness on the function of leukocytes, especially macrophages—primary foreign body response (FBR) orchestrators, show promise in tailoring a favorable response to tissue-engineered therapies such as EIT. In this review, we explore strategies to improve the clinical viability of EIT. A brief overview of the immune system, the FBR, and current biochemical approaches will be elucidated throughout this exploration. Furthermore, an argument for using substrate stiffness as a capsule design parameter to increase EIT efficacy and clinical viability will be posed.

Impact statement

This review makes a case for the use of substrate stiffness as a critical design parameter for the development of encapsulated islet transplantation. At present, biochemical strategies have been emphasized to improve clinical viability. However, mechanobiology literature is beginning to elucidate the role of stiffness in immune function and could be used as a strategy for tissue engineering therapies against foreign body response.

Introduction

Diabetes is a leading cause of death and affects >463 million people globally.¹ Categorized into two main types, type 1 and type 2, diabetes is the loss of function in pancreatic islets to produce insulin and regulate blood glucose levels. Although type 2, the most common of the two, refers to the body's ineffective use of produced insulin,² type 1 diabetes mellitus (T1DM) is an autoimmune disease associated with immune cells, leukocytes, attacking pancreatic islets, specifically the insulin-secreting β cell component, owing to a misrecognition of self-antigens.³ Treatment of diabetes consists of a routine schedule of exogenous insulin injected into the patient to regulate their blood glucose levels.

However, as the prevalence of people with diabetes expands, estimated to be >578 million people by 2035, the financial burden on health care institutions will exponentially grow.^1,4 Global spending on diabetes treatment was estimated at $673 billion in 2015, with a projection of $802 billion in 2040.⁵ Individuals are also negatively impacted financially. According to the American Diabetes Association, patients diagnosed with diabetes, on average, have medical expenses 2.3 times higher than patients without the disease.⁶ Financial costs of insulin have also hindered accessibility to treatment long term.⁷ This burden has increased the demand for therapies that provide long-term solutions to exogenous insulin dependence.

To address exogenous insulin dependence, particularly by people with T1DM, tissue engineers have developed encapsulated islet transplantation (EIT).⁸ This tissue engineering-based therapy consists of donor islets being encapsulated in a semipermeable polymeric membrane. The capsule protects the cells from the leukocytes of the patient's immune system while promoting islet function and viability.⁹ The permeability of the capsule allows the transport of oxygen, glucose, insulin, and waste products while blocking leukocyte migration into the capsule. Structurally, ideal pore size is needed for maximum diffusion of oxygen (∼300 pm in diameter), glucose (∼1 nm), and insulin (2.7 nm)¹⁰ but restrictive to antibody (10 nm) and leukocyte invasion (∼0.67 μm).^11–14 Larger pore sizes allow leukocytes to traverse entirely through the material, interacting with the transplanted cells and increasing the chances of rejection. Moreover, poor diffusion of oxygen and nutrients can hinder insulin production by the islets and cell death.

Encapsulation can range in size and geometry, categorized by its islet capacity and transplantation site in connection with the bloodstream. Macroencapsulation specifies the ability to encapsulate a large mass of islets within the capsule. Macrocapsules are usually tubular or planar in geometry, with a lower surface area/volume ratio than the smaller capsules. Developed to enhance oxygen diffusion to the encapsulated islets, microcapsules are typically spherical with a lower islet capacity and higher surface area/volume ratio compared with macrocapsules. Nanoencapsulation, also known as conformal coating or layer-by-layer coating, drastically minimizes the thickness of the capsule, creating a polymeric layer on the surface of the islet to maximize the diffusion of oxygen and nutrients from the host to the islet. Once constructed, capsules will be transplanted intravascularly, attached directly to an artery,¹⁵ or extravascularly, in locations such as peritoneal cavity,^16,17 subcutaneous space,^18,19 omentum,²⁰ and striated muscle.²¹

Each EIT technique has its advantages and disadvantages for islet vitality and functionality and has been extensively reviewed by Wu et al.²² While achieving results in autoimmune diabetic animal models, EIT therapies are not a long-term solution to insulin dependence. They have consistently encountered the major challenge of overcoming the body's natural defenses long term. For the continued success of EIT procedures, patients must continuously ingest harmful immunosuppressant drugs to weaken the immune system and prevent immune rejection systemically.²³ Furthermore, EIT therapies do not remain clinically viable long term, even with consistent immunosuppressant usage. Patients often still need exogenous insulin owing to loss of function by the transplanted islets over time.²⁴ As a result, a more effective strategy for sustained localized immune suppression is necessary to prevent EIT rejection by foreign body response (FBR).

Methods to tailor the immune system's response have become increasingly sought in islet transplantation. EIT strategies have generally emphasized the use of biochemical changes to the capsule's design by using various ligand incorporations,^25,26 material changes,^20,27,28 and cellular additions^29,30 to increase the viability of the therapy. However, recent literature has shown that a strong case can be made to include mechanical parameters in capsule design owing to advancements in immune cell mechanobiology. Mechanobiology can be described as the study of the essential roles physical factors, such as force,³¹ geometry,³² and matrix viscoelasticity,³³ play on cell and tissue function and morphology, human physiology, and pathology.³⁴ This form of immune engineering focuses on changing the material's mechanical properties to promote a more favorable response to the therapy.

Substrate stiffness has not been a parameter of focus in EIT design. However, there has been a multitude of evidence displaying the effect of extracellular matrix (ECM) stiffness on cellular function. Examples from literature include ECM stiffness's ability to induce stem cell differentiation into osteoblasts,³⁵ assist in stem cell differentiation into insulin-secreting β cells,^36,37 influence motility and migration,³⁸ actin cytoskeletal reorganization,³⁹ as well as regulate T cell activation and proliferation.⁴⁰ Recent findings on the effect of substrate stiffness on the function of the immune system, particularly macrophages, could be promising for encapsulated islet technology. Macrophages attach to the material using integrins and focal adhesion proteins, discussed further in the review. Through these adhesion proteins, the macrophages sense the stiffness of the substrate leading to the functional changes that could be utilized to improve EIT design against immune rejection.

Through this review, we will detail current strategies to increase the clinical viability of EIT with an argument to add substrate stiffness as a parameter of interest. We will briefly overview the FBR concerning EIT, discuss material changes to promote EIT viability, and provide substrate stiffness findings that would prove advantageous to include in capsule design. To focus the argument better, stiffness findings will be discussed primarily on macrophages, a significant contributor to the FBR and immune rejection.

FBR to EIT

As stated previously, the goal of EIT is to transplant viable insulin-secreting cells with minimal invasiveness into a vascularized area of a patient's body. A successful transplant should promote glycemic control without using long-term exogenous insulin or destruction from the patient's immune system (Fig. 1). FBR toward EIT spans the innate and adaptive immune systems as the phagocytes, primarily macrophages, cannot fully degrade the transplanted biomaterial. However, specific response and severity depend on the material's physical and biochemical composition and its effect on the activation of immune cells. As severity is variable based on a myriad of parameters, the timeframe of FBR can span from months to years.¹⁶ FBR can be organized into four phases: ignition, acute inflammation, chronic inflammation, and fibrosis.^41,42

FIG. 1.

A schematic of the overall goal of encapsulated islet therapy. Insulin-secreting cells—islets, beta cells, or differentiated stem cells—are encapsulated in a semipermeable biomaterial intended to block leukocyte interaction and their secreted antibodies. The biomaterial also allows the diffusion of glucose, oxygen, nutrients, insulin, and waste to maintain cellular function.

Ignition

FBR is initiated at the transplantation site, similar to other biomaterial implants. Owing to tissue injury and material–blood interactions, complement proteins from endothelial cells and blood coagulants are adsorbed to the capsule surface within seconds in an unrecognizable conformation through the Vroman effect.^43,44 This nonspecific protein adsorption forms a 2–5 nm thick transient provisional matrix coated at the interface between the material and the native tissue.^45,46 These proteins can include fibrinogen,⁴⁷ albumin, fibronectin,⁴⁸ gamma globulins, thrombospondin, and vitronectin.⁴⁹ Whereas fibrinogen, gamma globulins, and albumin modulate the immune response, fibronectin and vitronectin enhance cellular adhesion and play a role in the later chronic stage of FBR.^50–52

Simultaneously, cell membrane disruption of neighboring cells and injured tissue results in the release of chemokines CXCL1, CXCL2, CXCL8, and CCL2, as well as a diverse pool of endogenous proteins known as damage-associated molecular pattern molecules.^53,54 These proteins induce chemotaxis of neutrophils, monocytes, and immature dendritic cells (DCs) from the bloodstream and surrounding tissue to the transplantation site to elicit an immune response.^55,56

Acute inflammation

As neutrophils and monocytes infiltrate the transplantation site through ignition, proinflammatory cytokines, interleukin (IL)-1, and IL-8, among others, are released at the site, beginning acute inflammation.^55,57,58 Monocytes differentiate into macrophages accompanied by the attraction and stimulation of other innate immune cells. These cells are essential in the initiation, duration, and outcome of the response against the biomaterial.^59,60 Using integrins, macrophages attach to the provisional matrix and differentiate into their inflammatory M1 phenotype through pattern recognition receptors.⁶¹ Integrins are transmembrane glycoproteins that initiate cytoskeletal changes by actin polymerization and mechanotransduction of the focal adhesion kinase pathway.^62,63 Substrate stiffness is essential in this integrin engagement as it alters actin distribution in macrophages leading to cell morphology changes. On soft stiffnesses, macrophages tend to be more circular in morphology.

However, as substrate stiffness becomes more rigid, F-actin expands, creating more structural protrusions that grow the cell area and reduce circularity into a more spindle morphology.^64–66 Morphological changes have been linked to macrophage functional changes. Rounder cell morphology has been associated with proinflammatory functionality characteristic of M1 macrophages with the ability to secrete proinflammatory cytokines such as tumor necrosis factor (TNF-α), IL-1, and IL-6, as well as degrading enzymes.⁶⁷ In contrast, spindle-shaped cell morphology has corresponded to anti-inflammatory behavior associated with M2 macrophages.⁶⁸

M1 macrophages increase inflammation at the EIT site releasing reactive oxygen species, cytokines, and lysosomal enzymes, such as nitric oxide synthase, to phagocytose and dispose of the transplant.⁶¹ Substrate stiffness has also been demonstrated to affect proinflammatory gene expression in macrophages. Using lipopolysaccharide (LPS), a bacterial endotoxin, as a stimulant, substrate stiffness was investigated on the expression of genes along the LPS/Toll-like receptor 4 (TLR4) pathway⁶⁹ leading to proinflammatory cytokine secretion. Soft (0.2 kPa) stiffness polyacrylamide gels were shown to upregulate downstream TNF-α, IL-6, and IL-1β proinflammatory cytokine genes compared with more rigid (33.1 kPa) gels, illustrating stiffness can be used to reduce inflammation by macrophages.⁷⁰ Furthermore, cytokine and degradation enzyme secretion can be regulated by stiffness. Markov et al displayed acute inflammation increased with hardness, Young's modulus, and compressive strength in vivo.⁷¹ Soft pectin (14 kPa) hydrogel scaffolds implanted in Wistar albino rats held a lower proinflammatory potential.

In contrast, hard (106 kPa) pectin was overpopulated with collagen fibers and contributed to the formation of new connective tissue.⁷¹ Of interest, this outcome was the opposite of findings described in vitro, where soft stiffnesses promoted increased proinflammatory cytokine production.^70,72,73 However, this alternative result in vivo could be attributed to the biodegradable nature of pectin. Soft pectin degraded more rapidly in vivo than hard pectin, unquantifiable by day 3 compared with day 11. Using a 2D polyacrylamide model, Chen et al⁷⁴ determined that TNF-α and IL-1β secretion was reduced as substrate stiffness increased. These findings align with previous studies by Irwin et al,⁷⁵ Patel et al,⁷³ and Gruber et al⁶⁵ using a 2D polyacrylamide–polyethylene glycol (PEG) model and similar polyacrylamide models, suggesting a linear trend in key proinflammatory markers based on stiffness, which can assist in acute inflammation reduction.

Chronic inflammation

After 1–2 weeks of frustrated phagocytosis, the adaptive immune system begins the chronic inflammation phase owing to the EIT being too large to internalize. This phase should last no more than a couple of weeks as more extended periods can indicate infection.⁷⁶ DCs, now mature through activation at the transplantation site, migrate through the lymphatic vessels to the lymph nodes to activate and recruit T cells.⁷⁷ Once at the transplant site, T cells assist in prompting integration or exacerbating the destruction of EIT during this process, essentially moving the process along in returning to homeostasis. These cells secrete anti-inflammatory cytokines, IL-4 and IL-13, to begin tissue remodeling, inducing fibroblast migration and proliferation. While this occurs, macrophages begin shifting between M1 and their anti-inflammatory M2 phenotype.

Simultaneously, to increase their phagocytic capability and response to the presence of IL-4, adhered macrophages fuse to form foreign body giant cells (FBGCs)—a signature of chronic inflammation.⁷⁸ Recently, there has been evidence that the mechanosensitive cation channel transient receptor potential vanilloid 4 (TRPV4) is crucial in the formation of the FBGCs, which increases the role of substrate stiffness in chronic inflammation implanted materials.^79–81 FBGCs form a barrier around the implant, working to deteriorate it and participate in tissue regeneration around the implant.⁷⁸ Profibrogenic factors, such as vascular endothelial growth factor, transforming growth factor, platelet-derived growth factor, collagenase and matrix metalloproteinases, are released as a result of both innate and adaptive immune cells to begin the recruitment of myofibroblasts.^82,83

Fibrosis

The fibrosis step orchestrates the regeneration of damaged tissues and is the final step in FBR. M2 macrophages contribute significantly to the regeneration process through crosstalk with T cells, stimulation of fibroblasts and angiogenesis, and direct the production and remodeling of ECM.^84,85 Recruited fibroblasts secrete collagen I and III, further encapsulating the EIT, compromising its function owing to lack of protein diffusion. This secludes the implant from the body's vasculature, leading to hypoxic stress and apoptosis as the encapsulated cells cannot receive oxygen and nutrients from the patient's blood supply.

The duration of chronic inflammation and the anti-inflammatory response of the leukocytes in the area are critical determinants of implant integration with restored functional tissue or its seclusion by forming fibrotic tissue around the implant. The construction of fibrotic tissue depends on the ratio of collagen I to collagen III.⁸⁶ Greater collagen I to collagen III results in elevated fibrotic tissue formation. In the case of EIT, the fibrotic tissue formation results in pericapsular fibrotic overgrowth (PFO), a significant signal of rejection. PFO further leads to hypoxia of encapsulated islets.⁸ This is currently an expected outcome of the therapy where most patients receiving EIT clinically return to insulin dependence over time (Fig. 2).¹⁶

FIG. 2.

A time course of FBR to EIT. When encapsulated islets are transplanted into the body, instantaneously complement proteins adsorb to the material interface. Simultaneously, damaged endothelial cells release DAMP signals and chemokines to recruit innate leukocytes to the transplant site to restore homeostasis, initiating the FBR. Macrophages adhere to the material surface, secreting ROS and proinflammatory cytokines to degrade and phagocytose the encapsulated islets signaling acute inflammation. DCs, activated from proinflammatory antigens, navigate to the lymph nodes to activate and recruit T cells to the site for assistance in the response. After several days to a couple of weeks, frustrated macrophages, owing to ineffective phagocytosis, merge to FBGCs, a hallmark of chronic inflammation. Through the secretion of anti-inflammatory cytokines and profibrogenic factors, myofibroblasts are recruited to begin fibrosis surrounding the EIT. The FBR concludes with increased collagen production and fibrotic tissue secluding the EIT from receiving oxygen, glucose, and nutrients. DAMP, damage-associated molecular pattern; DCs, dendritic cells; EIT, encapsulated islet transplantation; FBGCs, form foreign body giant cells; FBR, foreign body response; ROS, reactive oxygen species.

Substrate stiffness has been shown to reduce PFO in vivo. Acellularly, Ibrahim et al studied FBR by subcutaneously implanting in mice five commonly used biomaterials: polyvinyl alcohol (PVA), silicone, expanded PTFE (ePTFE), polypropylene, and cotton.⁸⁷ Over 180 days, silicone held the thinnest fibrotic capsule. In contrast, the thickest fibrotic capsule varied per time point and material although PVA had the thickest fibrotic capsule from day 60 onward, indicating a need to consider the biomaterial carefully. Although the study did not quantify the stiffness of each material used, it can be inferred that Young's modulus varied between materials. There was also a geometric difference between the samples with PVA, silicone, and cotton constructed into 8 mm diameter disks and ePTFE and the polypropylene sectioned in 2 cm long tubes.

However, this physical difference can be ruled out as a factor for FBR changes in this study as this did not prove significant in changing fibrotic capsule thickness or leukocyte population. Elucidating the effect of stiffness on fibrotic capsule formation, Blakney et al implanted PEG-based hydrogels of different stiffnesses in C57BL/6 mice for 28 days, showing a decrease in fibrous capsule thickness in soft stiffnesses.⁷² More studies need to be conducted to determine which moduli would be most appropriate for EIT.

EIT Biomaterial Approaches

There is promising evidence to support using other mechanical properties, such as substrate stiffness, as another parameter to explore to increase EIT viability. Physiologically, Young's modulus of the human body ranges vastly and is tissue dependent (Fig. 3). Common physiological stiffness values relevant to EIT include the liver (2–8 kPa),^88,89 kidney capsule (5–10 kPa),⁹⁰ and hypodermis, referred to as the subcutaneous cavity (15–34 kPa),^91,92 as standard locations for islet transplantation.

FIG. 3.

Physiological and material elastic moduli range relevant to EIT. Adapted from Handorf et al,¹⁰⁰ Young's modulus has been measured for commonly known organs, such as adipose tissue,¹⁰¹ brain,^102,103 heart,¹⁰⁴ and bone.^105,106 Also, organ stiffnesses relevant to macrophages and EIT including the bone marrow,¹⁰⁷ thymus,¹⁰⁸ liver,^88,89 pancreas,^109,110 kidney,⁹⁰ and subcutaneous cavity (hypodermis).^91,92 Although substrate stiffness is not a primary parameter in EIT capsule design, Young's moduli of materials used, such as alginate,^37,111,112 agarose,¹¹³ chitosan,^114–116 PEG,^117–119 and polyurethane^120,121 are included. Although transplantation sites for EIT are secluded to the liver portal vein, kidney capsule, or subcutaneous cavity, it is essential to note macrophages engage with the entire physiological and material stiffness range as tissue-resident and migratory cells. PEG, polyethylene glycol.

Hydrogels are commonly used materials for encapsulation. Owing to their hydrophilic nature, high water content, tailorable three-dimensional structure, and transparency, hydrogels provide a robust, highly biocompatible material that prevents damage to surrounding tissues and easily visualizes encapsulated cells. As natural and synthetic polymers, the elastic moduli of hydrogels can be tuned to mimic the entire physiological range by increasing crosslinker concentration or co-polymerization with other materials during synthesis (Fig. 3). Several natural and synthetic polymers have been investigated to improve efficacy and clinical viability since the inception of encapsulated islet transplants. This includes alginate,⁹³ agarose,⁹⁴ chitosan,^95,96 PEG,^25,97 and polyurethane.^98,99

Alginate

The most widely used and studied hydrogel for microencapsulation-based therapies is alginate. For a more detailed review, see Vaithilingam et al.⁸ Characteristically, alginate is robust, topographically smooth, nonbiodegradable over prolonged periods, hydrophilic, readily available, and generally inert. The versatility of alginate has been displayed through various methods of microcapsule development to promote increased biocompatibility and islet viability.¹²² To make microcapsules for EIT, alginate is typically crosslinked with Ca²⁺ or Ba²⁺ with concentrations ranging from 10 to 100 mM.³⁷ The stiffness of these concentrations range from 1 to 100 kPa. However, the stiffness of alginate microcapsules can be increased to ∼600 kPa with changes in alginate and crosslinking concentrations.¹¹² Permeability can also be manipulated to vary diffusion in and out of the capsule.¹²³ These tailorable and beneficial features led alginate to be used clinically for EIT therapy.¹⁷

However, although widely used, alginate has several drawbacks that prevent commercial success. Alginate composition varies from sample to sample.¹²⁴ This variability makes it challenging to reproduce previous studies for comparable results. In addition, owing to environmental factors, alginate can contain various impurities, such as heavy metals, proteins, and endotoxins.¹²⁵ These contaminants are significant factors leading to fibrotic overgrowth and EIT failure. Although purification methods have been developed to remove these impurities, these methods are costly. They have a limited scope, purifying mainly endotoxins such as LPS, the most common endotoxin.¹²⁶ Even commercially sold “ultra-pure” alginate has been shown to induce a proinflammatory response by macrophages, promoting a thick fibrotic layer around the microcapsule, which must be considered for long-term transplantation.¹²⁷ However, owing to its chemical structure, alginate can bind with other polymers such as poly-l-lysine, poly-l-ornithine, and PEG to overcome challenges.

Although researchers have begun to study the effect of alginate microcapsule stiffness on EIT, the literature has been limited to islet functionality and capsule stability, not FBR.^37,110 For example, Richardson et al³⁷ investigated the effect of Ba²⁺ crosslinked alginate microcapsule stiffness ranging from 1 to 100 kPa, on human embryonic stem cell (hESC) differentiation and growth to pancreatic progenitor cells. The stiffness range of 4–7 kPa was more favorable for differentiating hESCs to pancreatic progenitor cells. Low stiffness also promotes cell growth, which was stunted at higher stiffnesses. Moreover, Enck et al¹¹⁰ found that the incorporation of pancreatic ECM fragments into the alginate matrix will significantly improve the durability of the capsule while slightly improving islet functionality at 7 days. The scalability of alginate-based microencapsulation techniques needs to be investigated in more extensive studies with larger animals. Still, alginate will remain an attractive material for EIT owing to its versatility.

Agarose

Similar to alginate, agarose is a seaweed-derived polysaccharide used for cell encapsulation. Agarose gel is temperature dependent, curing at a temperature <25°C and being able to withstand temperatures up to 60°C before degradation begins. This robust temperature range makes the hydrogel a promising candidate for islet encapsulation and has been in consideration since the 1980s.^128,129 Islets encapsulated in alginate were demonstrated to maintain comparable gene expression of insulin signaling pathways compared with free islets after 8 weeks of culture.¹³⁰ In addition, through incorporating a coculture of regulatory T cells, agarose encapsulated islets maintained viability and functionality in vivo for up to 100 days.¹³¹

The Young's modulus of agarose can range from 1.5 to 2600 kPa correlating with agarose weight percentage.¹¹³ This increase in agarose weight percentage leads to smaller pore sizes as well to assist with permeability.¹³² Holdcraft et al⁹⁴ investigated the effect of varying agarose type and concentration on islet viability and insulin production. Using 1.5% SeaKem Gold (SG) agarose, 0.8% SG agarose, and 0.8% Litex agarose, insulin production was significantly improved by the islets encapsulated in 0.8% agarose compared with 1.5% agarose. Although the team did not measure the modulus of the investigated agarose concentrations, it can be inferred that insulin production by islets are impacted by substrate stiffness and should be investigated further.

Chitosan

As an alternative to alginate microcapsules, chitosan has been proposed to increase the clinical viability of EIT.¹³³ Chitosan is a cationic polysaccharide synthesized from chitin shells of crustaceans with a stiffer and more robust modulus range than alginate ranging from 200 kPa to 1.4 GPa.^95,96 Chitosan has been demonstrated to reduce the production of inflammatory cytokines, IL-6 and TNF-α, and inhibit T cell proliferation.¹³⁴ It has been suggested to improve the mechanical stability of alginate long term. Chitosan-alginate microcapsules have decreased PFO, leading to a more biocompatible implant long term.^27,133,135 Yang et al's xenogeneic and allogeneic transplanted chitosan-coated alginate capsules demonstrated significantly reduced cell adhesion compared with alginate capsule control up to a year post-transplantation.¹³³

Although stiffness was not measured, we can infer the stiffness to be ∼5 kPa owing to the use of 10 mM barium crosslinked alginate, characterized by Richardson et al³⁷ and findings by Williams et al¹³⁶ demonstrating chitosan coatings slightly stiffens alginate microcapsules. Despite promising results, a low pH of at least four is needed to dissolve the polymer, which can be detrimental in islet applications. However, several groups are modifying chitosan for microcapsule development under physiological pH conditions to promote a more viable encapsulation method. For example, Kim et al used chitosan in conjunction with dexamethasone 21-phosphate and alginate to produce microcapsules for EIT.²⁷

Although stiffness was not characterized, similar to the previous study, the inclusion of dexa-chitosan in the alginate microcapsule synthesis can be inferred to increase the stiffness of the microcapsule. Moreover, the permeability remained comparable among the dexa-chitosan and alginate microcapsules. Dexa-chitosan microencapsulated islets promoted better glucose control and reduced PFO and inflammatory cell infiltration after 231 days in vivo compared with alginate controls.

Polyethylene glycol

A synthetic polymer, PEG, can be used with alginate-based microcapsules to increase mechanical stability by hindering swelling and rupture.^137,138 PEG hydrogels are nanoporous, preventing the infiltration of leukocytes into the material but allowing the transport or diffusion of oxygen, nutrients, and insulin. In addition, PEG removes the immunogenic variability seen with alginate by being synthetic, promoting immunoprotective properties, bio-inertness, and exceptional biocompatibility.^139,140 PEG can also be tailored through functionalization to assist with the engraftment of the implant.¹⁴¹ Owing to these factors, many labs have used PEG as a material of interest in EIT.^138,142,143 Weaver et al demonstrated that RGD-functionalized PEG gels produced smaller diameter capsules (310 ± 14 μm) compared with alginate (≥500 μm), resulting in increased insulin responsiveness and islet viability.¹⁴³ Although the stiffness of the microcapsules was not characterized by this study, it can be estimated using the concentrations provided.

The PEG microcapsules were synthesized using 5% PEG crosslinked with 30 mg/mL dithiothreitol; it can be inferred that the stiffness of the capsule would be ∼10 kPa.¹⁴⁴ Although the alginate microcapsules were synthesized using 1.6% alginate and 50 mM Ba²⁺, the stiffness can be estimated at ∼20 kPa.³⁷ This reduction in stiffness may also be associated with improved insulin responsiveness seen in the PEG microcapsules as it is closer to the native stiffness of the pancreas. Haque et al developed a layer-by-layer hyperbranched PEG and heparin (hb-PEG/heparin) nanoencapsulation for EIT.¹⁴² Nonhuman primate islets were encapsulated and transplanted into the subcapsular kidney space of diabetic C57BL/6 mice. Compared with nonencapsulated islets, the approach allowed the transplanted islet protection from leukocytes while promoting engraftment and better function over 4 weeks.

However, an immunosuppressive drug protocol was needed to achieve the intended results. Current limitations of PEG include the elicitation of inflammation owing to complement activation. Minimally invasive methods have been introduced as a prevention strategy against complement activation. One of which demonstrated the ability of an injectable PEG hydrogel as a minimally invasive way to transplant islets to a secure location subcutaneously and retrieve them if adverse events occur.¹⁴⁵ The team showed PEG encapsulated islet injections promoted reduced blood glucose levels 2 days after transplantation into a B6D2F1 mouse model compared with a nonencapsulated islet control. Although the work did not completely alleviate diabetes in the model compared with the tubular alginate implant under the same timeframe, the approach demonstrated some feasibility and warrants future investigations.

Polyurethane

Since the early 1990s, polyurethane has been a material of interest in EIT.⁹⁸ As polyurethane held wide success in other implant therapies, its biocompatibility and versatility provided great promise for EIT. This biocompatibility was tested in early EIT studies by Zondervan et al⁹⁸ and Ward et al¹⁴⁶; however, it held limited success due to permeability constraints. More recent studies showed that altering the chemical structure and scaffold development process of polyurethane could assist in overcoming these constraints.^99,147,148 As a proof-of-concept study, Liu et al recently developed a zwitterionic polyurethane implant for EIT.¹⁴⁸ This electrospun implant provides tunable nanoporous structures that prevent cell escape while allowing the diffusion of insulin and nutrients. When implanted intraperitoneally in C57BL/6 mice for 6 months, lower FBR and fibrotic overgrowth were induced compared to unmodified polyurethane implants.

Polyurethane has also been demonstrated to promote localized immunosuppression by adding rapamycin. Francis et al loaded 8 × 2 mm polyurethane disks for EIT with 2 nM of rapamycin.⁹⁹ The disks exhibited a sustained release of rapamycin over 7 days with no adverse effects on the viability of murine and human islets. T cell proliferation was also suppressed during in vitro studies. When implanted subcutaneously in mice, signs of engraftment could be detected from blood vessel formation. Although promising, more work is needed to strengthen the mechanical properties and reduce hydrogel swelling for clinical success.^149,150

Conclusion

Encapsulated islet therapy is a novel and minimally invasive strategy to treat T1DM. Although EIT has successfully cured most small animals of diabetes, returning hosts to normoglycemic conditions for extended periods without the use of injected insulin, it has been challenging to replicate this success with humans and nonhuman primates without systemic immunosuppressive drug assistance. A long-lasting functioning therapy must utilize biochemical and mechanical approaches to tailor the immune system to integrate the transplant.

This review detailed current methods to increase EIT viability and made a case for using substrate stiffness to assist in EIT success based on macrophage findings. Although these findings suggest stiffness could be a beneficial factor in immune rejection prevention, it is fair to note these studies have not incorporated glycemic condition as a parameter of study to mimic the mechanosensitivity of macrophages in healthy patients versus diabetic patients. Nor do these studies mimic the glucose dysregulation seen in diabetic patients alone as it varies from hypoglycemia (<70 mg/dL glucose), normoglycemia (70–120 mg/dL), and hyperglycemia (>120 mg/dL). As macrophages are glycolytic cells, changes in glycemic conditions have been shown to alter the function of macrophages.¹⁵¹ It is currently unknown how changes in glucose will alter mechanosensitivity in macrophages. It should be assessed as it is crucial for the movement of substrate stiffness as a parameter of interest for EIT capsule design.

Authors' Contributions

C.J. conceptualized the review, prepared the article, and generated the figures. H.A.E. and J.P.F. oversaw the project and reviewed the article.

Disclosure Statement

No competing financial interests exist.

Funding Information

The authors thank A. James Clark School of Engineering, NIBIB/NIH Center for Engineering Complex Tissues (P41EB023833), NSF (CBET1856350), and NSF (1507730) for funding this project.