Introduction and Literature Review
1.1. 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 exemplified by recent statistical data mined from two geographically different locations where >30.3 million Americans (National Diabetes Statistics Reports, 2017) and ~66.8 million Indians (Indian Council of Medical Research (ICMR) Report, 2016) were diabetic. Within this cohort, 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. Type 1 diabetes is an autoimmune disease characterized by destruction of insulin-producing β-cells by the body’s own immune system, resulting in hyperglycemia and accumulation of toxic acids in blood and urine (Foster and García, 2017b; Go et al., 2014; Mathis et al., 2001). The resultant symptomatic hyperglycemia is associated with eventual failure of several vital organs (Association, 2006) and the patients suffer from higher rates of morbidity and mortality (Go et al., 2014). The diabetic patients are at a risk of severe secondary complications like, neuropathy, retinopathy, chronic kidney disease, cardiovascular complications and infections (Foster and García, 2017b; Kort et al., 2011; Miller et al., 2016; Opara et al., 2010).
Till date insulin therapy has remained the ideal choice for treating type 1 diabetes and pancreatic transplantation is performed in severe hyperglycaemic conditions (Shapiro et al., 2000). However, fluctuations in blood glucose level and complications associated with surgical procedure have posed limitations in these traditional approaches so far. In order to address these challenges, islet transplantation might be a promising approach for therapeutic intervention in type 1 diabetes as it is minimally invasive and restores normoglycemia in the patients. Moreover, islets can function as a glucose sensor as well as a source of new insulin thereby making it more preferable than insulin injections (Niknamasl et al., 2014; Shapiro et al., 2000).
However, the application of this technique has been limited due to shortage of donors, high rate of implant rejection, long term immunosuppression and islet dysfunction in post-transplantation period (Hirshberg et al., 2003). Furthermore, the considerable loss
of islets (~60%) during islet infusion to portal vein demands multiple donors (2-4) for one diabetic patient (Del Toro-Arreola et al., 2016; Vlahos et al., 2017) which needs to be reviewed.
To rein in the “Type 1 diabetes epidemic”, tissue engineering and regenerative medicine present a novel and effective solution for the replacement of diseased pancreas with bioengineered implants (Vacanti and Langer, 1999). Biomaterial-based islet encapsulation approach provides an attractive alternative proposition for islet delivery in vivo by providing protective three-dimensional (3D) support for islet function (Liao et al., 2013). In the native pancreas, islets are surrounded by a capsule of extracellular matrix (ECM) comprising of different matrix components which bind to integrins on the islet surface to mediate islet adhesion, proliferation, and insulin secretion (Daoud et al., 2010). During islet isolation, due to enzymatic digestion, majority of islet microvasculature and islet-matrix connections are disrupted resulting in islet fragmentation, and glucose irresponsiveness (Brendel et al., 1994; Ilieva et al., 1999). Upon encapsulation in 3D biomimetic microenvironment with ECM-like biochemical cues, the islet functions are preserved as the 3D matrix compensates for the native ECM (Daoud et al., 2011; Davis et al., 2012; Shalaly et al., 2016).
In this reference, earlier several natural and synthetic biomaterials have been utilized with success to a certain extent (Borg and Bonifacio, 2011). Natural biopolymers (collagen, heparin, gelatin) often suffer from drawbacks like batch-to- batch variation, pathogen contamination, lack of tunability, and immunogenicity (Liao et al., 2013). On the other hand, synthetic polymers (poly (lactic acid) (PLA) and poly (lactic-co-glycolic acid) (PLGA)) demand pre-fabricated scaffolds with additional ECM components. Also, the biodegradation of these synthetic polymers release acidic by-products leading to pro-inflammatory responses in vivo (Blomeier et al., 2006; Liao et al., 2013; Salvay et al., 2008). Furthermore, the application of poly (vinyl alcohol) (PVA) for islet encapsulation necessitates sub-zero temperature to form hydrogels resulting in islet dysfunction and glucose irresponsiveness (Liao et al., 2013; Saudek et al., 1999). Polyethylene glycol (PEG) has been explored extensively for islet encapsulation, however, despite numerous favorable features; PEG has many restrictions including poor islet viability, non-degradable backbone and lack of functionality (Liao et al., 2013; Lin and Anseth, 2009). To overcome these limitations, herein, we explored silk protein polymer as a potential biomaterial for islet encapsulation and bio-artificial pancreas development.
Silk, a structural protein, is biocompatible, non-immunogenic, mechanically robust biomaterial with tunable degradation rates (Kundu et al., 2013). Silk protein is easy to process (under physiological-like conditions), and its degradation products are amino acids which can easily be metabolized in vivo (Davis et al., 2012; Vepari and Kaplan, 2007b). Silk resources are available in abundance and have demonstrated exceptional advantages over conventional synthetic and natural biomaterials (Thurber et al., 2015; Vepari and Kaplan, 2007a). The Food and Drug Administration (FDA) approved Mulberry Bombyx mori silk-based scaffolding matrices have been widely explored for the regeneration of a plethora of tissues including bone, cartilage, tendon, kidney, skin, liver, trachea, nerve, cornea, meniscus, eardrum, and urinary bladder (Kundu et al., 2013; Rockwood et al., 2011). The exciting prospect of exploring silk- based matrices as new biomaterial for islet encapsulation has received impetus with emerging reports that demonstrate their potential in in vitro islet culture maintenance and diabetes reversal in murine model (Davis et al., 2012; Hamilton et al., 2017; Mao et al., 2017). To harness the true potential of silk-based matrices for islet encapsulation, it is imperative to develop judicious system that would be islet-supporting, immunoisolatory and immunomodulatory. Recently, it has been reported that a non mulberry silk, Antheraea assama is enriched with intrinsic cell binding Arg-Gly-Asp (RGD) sequences (Gupta et al., 2015). Due to the presence of cell-binding RGD motif, this variety of silk supports better proliferation of cells from different tissues viz; bone, cartilage, blood vessels, skin, liver and cardiac tissues compared to its mulberry counterpart, B. mori (Gupta et al., 2016a; Gupta et al., 2016b; Konwarh et al., 2017). In this context, exploring non-mulberry silk-based matrix as a new biomaterial could provide a promising platform for successful islet encapsulation.
Despite the biological cues provided by 3D matrices, the islet transplantation process is associated with many challenges (Liu et al., 2016). Being an autoimmune disease, pancreatic beta cells are selectively destroyed by the patient’s own immune system. Also, surgery-associated tissue damages evoke inflammation and alter the immune environment of the implants (Piemonti et al., 2002; Weaver et al., 2015).
Under such adverse conditions, encapsulated islets undergo tremendous stress and secrete their own pro-inflammatory molecules amplifying the local inflammation and culminating in considerable islet loss and dysfunction (McDaniel et al., 1996; Weaver et al., 2015; Wilson and Chaikof, 2008). To prevent such drawbacks, the recipients require life-long administration of systemic immunosuppressive drugs which pose
systemic side-effects. In this circumstance, localized delivery of immunosuppressant at the implantation site has shown promising outcomes as an effective therapy to prevent immune targeting of islets and thereby eliminating the need for systemic immunosuppression (Weaver et al., 2015).
Herein, this thesis presents two possible strategies of silk biomaterial-based islet encapsulation for the local mitigation of inflammation in the islet microenvironment: 1) Encapsulation of islets to prevent infiltration of immune cells and, 2) local release of immunomodulatory molecules (dexamethasone and interleukin-4) from silk-based biomaterial systems that suppress local immunity. The aim of this work was to develop biomaterials with the capacity to reduce local inflammation, thereby improving long- term viability and functionality of the transplanted cells.
Based on this rationale, the initial attempt was to develop silk-based matrix that might display native islet-like niche and support islet culture and glucose responsive insulin secretion. Furthermore, the major emphases were on the development of matrices with immunoisolation and immunomodulation attributes and test their potential towards bio-artificial pancreas development. Moreover, with increasing number of type 1 diabetic patients and challenges encountered with several therapeutic approaches, this study strives to generate potent silk-based matrices addressing a serious health issue that demands attention. The following section provides a detailed literature review pertinent to the research area under investigation.