ABSTRACT
2.1. Introduction
Diabetes is a metabolic disorder affecting millions of people globally and a leading cause of morbidity and mortality (Daneman, 2006; Muthyala et al., 2011). The global incidence of diabetes is increasing exponentially as suggested by recent statistical data (National Diabetes Statistics Reports, 2014). Within the total diabetic population, type 1 diabetes represents around 10% of all diabetic patients, worldwide (Shalaly et al., 2016). Clinically, this form of diabetes is associated with hyperglycemia due to defects in insulin secretion, insulin production or both. The resultant symptomatic hyperglycemia is associated with eventual failure of several vital organs (Association, 2006). Islet transplantation is considered a promising treatment to restore normoglycemia in type 1 diabetic patients; more successful than insulin injections because the islets can function both as detectors of glucose concentration and as a source of new insulin. However, a shortage of donors, a high rate of implant rejection and the associated complicated procedures restrict widespread utility (Niknamasl et al., 2014; Shapiro et al., 2000).
To rein in the “diabetes epidemic”, tissue engineering and regenerative medicine present a novel, effective solution for the replacement of diseased pancreas with engineered implants (Vacanti and Langer, 1999). Islets removed from natural extracellular matrices (ECM) and maintained in 2D culture become fragile and senescent, show decreased insulin expression (Shalaly et al., 2016) and tend to clump together leading to limited nutrient transfer (Beck et al., 2007). To address these issues, researchers have resorted to tissue engineering wherein, islets maintain structure and function when cultured in 3D porous scaffolds (Burdick and Vunjak-Novakovic, 2008).
Different biopolymers have been investigated for these needs, including alginate, gelatin, collagen, chitosan, and agarose. Several devices such as hollow fibers, membrane chambers, and intervascular devices have also been designed to maintain 3D cultures (O'Sullivan et al., 2011).
The additional and vital requirement in type 1 diabetes treatment is combating the autoimmune aspect which demands immunoisolation of the islets from the host’s immune system. For this, islets have been encapsulated within selectively permeable polymeric membranes that permit the diffusion of glucose, nutrients, oxygen and insulin while restricting the influx of immune cells and their molecular components (Lim and Sun, 1980; Narang and Mahato, 2006). This strategy circumvented the use of immunosuppressive drugs and their multiple side-effects that encompass systemic
immune suppression and secondary complications (O'Sullivan et al., 2011). The general immunoisolation techniques include both micro and macroencapsulation. Much of the work for bio-artificial pancreas (BAP) development has focused on microencapsulation of single or small groups of islets. This technique had inherent detrimental limitations like low mechanical strength of the capsules, poor retrieval if necessary and short lifespan in vivo (O'Sullivan et al., 2011). These limitations establish the portal for exploiting “macroencapsulation”, endowed with multiple advantages including concepts such as reloadable scaffolds with cells (O'Sullivan et al., 2011; Qi et al., 2004). Currently, tissue engineering using silk protein as a biomaterial has been invigorated due to its biocompatibility, enhanced mechanical strength, easy sterilization, controlled rate of degradation and minimal inflammation in vivo (Thurber et al., 2015; Vepari and Kaplan, 2007). Silk has been processed into various formats such as gels, scaffolds or films for tissue engineering and drug delivery (Rockwood et al., 2011). These formats have been widely used for the development of range of engineered tissues with promising outcomes in vivo (Kasoju and Bora, 2012; Kundu et al., 2013). Pancreatic cell culture has been conducted with different materials and recent work with silk has shown promising results both in vitro (Davis et al., 2012) and in vivo (Hamilton et al., 2015) systems.
Alginate, a linear copolymer extracted from brown seaweed, has been extensively used in encapsulating proteins and cells under mild conditions of room temperature, physiological pH, isotonic solutions, and without harmful cross-linkers (de Vos et al., 2006). As an encapsulating agent, alginate has shown major advantages over other materials as it does not hinder islet cellular functions. Earlier, sodium alginate was used with calcium chloride (CaCl2) (Pathak et al., 2010) for capsule formation, but these capsules had inferior mechanical strength. To improve upon this, poly-L-lysine (PLL) membranes have been used for making the capsules stronger and more selectively permeable (O'Sullivan et al., 2011); unfortunately, PLL is associated with enhanced foreign body responses (Strand et al., 2001). In the same mode, barium chloride (BaCl2) was used to make efficient capsules with appropriate robustness and selectivity but without using polycationic PLL-like membranes (Zimmermann et al., 2007). Furthermore, silk-alginate blends promoted cell proliferation and functional outcomes in different tissue engineering and drug delivery applications (Mandal and Kundu, 2009; Ziv et al., 2014). Similar to alginate, agarose has also been extensively
Figure 2.1. Principle of silk-based bio-artificial pancreas (BAP) with three different attributes of spheroid formation, local immunomodulation and immunoisolation.
used for islet encapsulation. Agarose is a linear polysaccharide and forms a thermo- reversible hydrogel. It is amenable to tissue engineering applications because of its biocompatibility, interconnected porous microstructure, tunable mechanical properties and long-term stability in vivo (Iwata et al., 1994; Iwata et al., 1992). In the present work, the objective was to exploit alginate and agarose as cell macroencapsulating agents along with silk as substrates for insulin-producing-cell adherence and proliferation with immunoisolatory attributes.
Despite of biological cues presented by 3D matrices, tissue damage due to surgery and implantation evoked inflammation and altered the immune environment of the implants (Liu et al., 2016). This process generated additional challenges for islet transplantation where stressed islets secrete their own pro-inflammatory factors, amplify local inflammation (Piemonti et al., 2002) and culminating in considerable islet tissue loss and dysfunction (McDaniel et al., 1996; Weaver et al., 2015; Wilson and Chaikof, 2008). Localized delivery of immunomodulatory factors with enhanced efficacy, reduced systemic side effects and toxicity has emerged as a strategy to control the immune response at implant site (Weaver et al., 2015). Thus, we hypothesized that IL-4 (anti-inflammatory cytokine) and dexamethasone (immunosuppressive drug) released from cell encapsulating scaffolds might decrease inflammation and delay immune rejection of the transplanted cells in vivo. This approach suggests that
macrocapsules primed with cytokines and drug could be used for local immunomodulation. Therefore, the developed macroencapsulating scaffolds were evaluated for three different attributes, (a) spheroid formation (b) local immunomodulation and (c) immunoisolation (Figure 2.1). Use of protein (silk) and saccharide (alginate/agarose) molecules as scaffolding matrix ensured minimal immunogenic response. Further, mild encapsulation conditions for both, alginate and agarose minimized islet loss during the process. Porous scaffold architecture was assumed to prevent islet aggregation and necrosis while facilitating good mass transport ability related to oxygen and glucose. It was also envisaged that the semi-permeable encapsulation membrane (alginate/agarose) would safeguard against direct blood contact and blood-mediated inflammation reactions (BMIR), while enhancing implant viability (Liao et al., 2013). We finally report the performance of these new silk-based macrocapsules with cultured islets/ islet-like spheroids with potential for bio-artificial pancreas.