Full length articleSulfated alginate microspheres associate with factor H and dampen the inflammatory cytokine response
Graphical abstract
Introduction
Cell encapsulation therapy is a concept for transplantation of cells and sustainable delivery of therapeutic factors without immunosuppression. This concept could be particularly well suited in treatment of type I diabetes, currently extensively investigated with focus on device development and function. The concept encompasses the transplantation of immobilized cells in a matrix, providing a protective mechanical and semi-permeable barrier against immunological rejection. The most extensively explored encapsulation devices are microspheres of alginate hydrogels (diameter size of 0.5–1 mm). One of the major challenges is the host responses, resulting in fibrotic overgrowth depriving the free exchange of oxygen, nutrition and waste products required for a functional device [1]. New strategies to improve the alginate microsphere surface to avoid fibrotic overgrowth, and thus improve the biocompatibility, are critical toward constructing clinical suitable devices.
Alginates are a group of linear polysaccharides consisting of 1→4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G). Consecutive G units form binding sites for divalent cations, cross-linking the alginate and forming a hydrated gel network suitable for cell immobilization [2]. This hydrated gel-network formed as a microbead can be sufficient to provide protection and ensure cell viability upon transplantation in small animal models [3]. An outer polycation layer, such as poly-l-lysine or poly-l-ornithine, has commonly been employed to reduce the permeability of host proteins and to stabilize the microspheres. The cationic surface does, however, promote cell attachment and stimulates an inflammatory response [4], [5]. Cellular overgrowth and fibrosis are reduced by introducing a secondary alginate coat [6], which also provides a slight reduction in the inflammatory potential [4], [7]. While the alginate microbeads can be free of fibrotic overgrowth and show long-term function of the implant in small animal models, they have been prone to fibrotic overgrowth in primate models and humans. In clinical studies alginate microbeads were surrounded with fibrotic tissue, and the encapsulated pancreatic islets failed to restore the blood glucose [8], [9]. Although the clinical setting is complicated with the causative relationship involving oxygen deprivation and secretion of inflammatory and immunogenic components by the encapsulated cells, these findings still point to the requirement of device modifications. The severity of a fibrotic response to microspheres depends among other factors on the molecular composition and surface properties of the implanted material [1]. Other important variables are the choice of animal model, site of introduction and the implantation procedure [10]. There is an initial connection between the ability to trigger inflammation and the fibrotic responses [11], and the inflammatory potential can be studied by the use of a human whole blood model with the possibilities of comparing a set of variables during identical conditions [12], [13]. The human whole blood model is therefore valuable as a first-screening tool of the inflammatory properties caused by the biomaterial surface in order to define the most promising candidate device for transplantation.
We have previously shown a close connection between the potential of alginate microspheres to activate complement and the induction of inflammation [7]. A general strategy of increasing biomaterial biocompatibility is by inhibiting the coagulation and complement cascades by heparin coating of the surfaces [14], [15]. This strategy potentially also involves the binding of inflammatory cytokines [16]. In addition to directly associating with complement factors, heparin as well as other glycosaminoglycans has been shown to bind factor H [17]. Factor H possesses two important functions in reducing the complement activation, by hindering establishment of the C3 convertase (decay-accelerating activity) as well as assisting factor I-mediated cleavage of C3 convertase products C3b and iC3b [18], [19]. Due to its potent inhibiting properties, direct conjugation of factor H to biomaterials has been proposed for increasing complement compatibility [20]. Sulfated alginates can be viewed as structural analogs to heparin, and we have previously shown that sulfated alginates inhibit formation of the terminal complement complex in human plasma [21].
Heparin coating may efficiently improve the biocompatibility in blood contact devices for short-time exposure [22]. The biocompatibility has also been improved when used as a coating on alginate microbeads over a four week transplantation period in the peritoneal cavity of mice [23]. However, the heparin coating was gradually lost, demonstrating a challenge to the long-term stability. Alginate forms a relatively strong complex with polycations and is slowly degraded in vivo, making it suitable for long-term applications [24], [25]. The structural similarities of sulfated alginate to heparin, as well as their previously demonstrated anti-complement activity, motivated exploring its potential to reduce the inflammatory properties of alginate microspheres.
In the present work we designed alginate microspheres using sulfated alginate, incorporated as a secondary coat on PLL-alginate microcapsules or mixed with the gel core of a non-coated alginate microbead. A human whole blood model was used to evaluate the inflammatory properties of the microspheres [12], as well as the impact of the modifications on the complement activating protein C3 and the complement-inhibitory factor H. This is to our knowledge the first biocompatibility evaluation of sulfated alginates microspheres.
Section snippets
Materials
Ultra-pure (UP) grade LVG (FG = 0.67, Mw = 199 kDa, endotoxin < 43 EU/g) provided by FMC Biopolymer (Sandvika, Norway) was used as gelling alginate. The poly-M alginate (FM = 1.0) used for epimerization was ultra-pure (UP) grade (endotoxin < 100 EU/g) mannuronan produced by an AlgG- strain of Pseudomonas fluorescens [26]. Poly-l-lysine (Mw = 15,000–30,000 Da) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorosulfonic acid (99%) provided by Sigma-Aldrich and formamide provided by Merck (Whitehouse
Cytokine induction
The induction of selected inflammatory cytokines by the various alginate microspheres and controls is shown in Fig. 1. Most treatments resulted in a significantly elevated cytokine induction from the baseline value (T0, freshly drawn blood), with the exception of the different alginate microbeads inducing a slight but often non-significant elevation. The alginate microbeads showed cytokine induction at a similar or lower level compared to the saline control. Sulfated alginate microbeads (A/SMG)
Discussion
In this paper we introduced sulfated alginates as a strategy to modify alginate microspheres for improved biocompatibility, and assessed their inflammatory properties using a human whole blood model. Briefly, the sulfated polyalternating MG alginate, designated SMG, was mixed with alginate to form sulfated alginate microbeads (A/SMG) or incorporated as a secondary coat on alginate-poly-l-lysine (AP) microcapsules. Sulfated alginate microbeads induced the overall lowest cytokine responses of all
Conclusions
We present for the first time sulfated alginate microspheres with evaluation of their inflammatory properties. Inclusion of sulfated alginate in the gel core resulted in microbeads that were practically inert toward complement and cytokines in our model. We propose an inherent association of factor H with sulfated and non-sulfated alginate, contributing to its biocompatible nature. As a coating material, the sulfated alginate showed a complexity of responses dependent on the sulfation degree.
Disclosures
The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgements
Ø.A. and G.S.-B. were funded by the Norwegian Research Council (221576). A.M.R was funded by the Liaison Committee between the Central Norway Regional Health Authority (46056819). The authors would like to thank Liv Ryan for Multiplex analysis, and Berit Strand for discussions.
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