**5.2 Inflammation and the foreign body response**

*Alginates - Recent Uses of This Natural Polymer*

cell preservation and transplantation.

**5. Immune response**

**5.1 Alginate purity effect on immunogenicity**

**4.5 Conjugated alginate potential as drug-delivery system**

degree of cryoprotection during cryo processing [76, 77]. Transplantation research has particularly benefited from use of alginate technologies during cryopreservation. Recent studies in pancreatic islet transplantation have demonstrated that use of alginate microencapsulation helps maintain islet viability during preservation and improves islet secretory function during transplantation [67, 78, 79]. A 10 years study using encapsulated rat, pig, human islets showed that alginate encapsulated islets maintained significantly higher viability, secretory function, and yield compared to nonencapsulated islets after cryopreservation for 10 years [80]. Like pancreatic islets grafts, cryopreservation is also used for preservation of stem cells before transplantation. Alginate encapsulation has been shown repeatedly to improve both yield and function of multiple stem cell lines, including human stem cells [81, 82]. Recently, the use of alginate microcapsules improved viable recovery of human adipose-derived stem-cells after 72-h storage in hypothermic conditions [83]. The overall benefits of cryoprotection via alginate encapsulation will ensure relevance in future research of

Due to the relative feasibility of conjugation and semi-permeable wall structure of alginate microcapsules shows promising secretory capabilities for robust nanoparticle drug-delivery systems [8, 84–86]. Recently researchers have been able to conjugate alginate microcapsules with anti-HIV zidovudine nanoparticles. Results showed significant improvements in internalization of the nanoparticle into glioma cells during *in vitro* experiments marking for potential use as a targeted viral drug delivery system [87]. Another study demonstrated the conjugation of sodium alginate with graphene oxide which is known to create functional groups for synthesis. GO-conjugated alginate hydrogels readily loaded the anticancer drug oxorubicin hydrochloride (DOX·HCl) and caused high cytotoxicity when exposed to immortal HeLa cell lines [88]. When islet containing alginate microcapsules were conjugated with VEGF, increases in angiogenesis, islet viability, and islet function were observed [89]. More recently, similar benefits were observed through increases in bone formation and blood vessel growth after biomineral-conjugated alginate microcapsules containing MSCs were transplanted into sheep with ovine iliac crest bone defects [90]. The versatile conjugation behavior of alginate hydrogels has impressive potential for a variety of future medical applications in transplantation.

Crude unpurified alginate has been shown to provoke the immune system which

then causes fibrosis around the microcapsule [91]. This overgrowth of immunecells and fibroblasts interferes with the transfer of nutrients, hormones, and other proteins via the semi-permeable membrane of the capsule [92]. This is a difficult problem to avoid especially since the industrial processing used for extraction can leave contaminates in the alginate like polyphenols, endotoxins and algal proteins [92, 93]. Purification is a technique utilized to minimize the amount of contaminations in the alginate gels. Commercially available sodium alginate can be purchased in several different purified states including "ultra-purified" form from companies like Novamatrix® for use in hydrogel production [94–96]. Although this is highly purified there are still small concentrations of contaminants in the alginate that can prompt a severe foreign body response [91, 97]. If the alginate biomaterials

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The human immune system is broken into two parts, the innate and adaptive immune systems, which both work in tandem to defend and destroy external bacteria, viruses and various other microbes that may infiltrate a host's body [99]. The first aspect of the foreign body response (FBR) is the innate immune system which has no flexibility or memory to foreign antigens. This is followed by the adaptive immune system which generates a custom response to each specific pathogen while simultaneously building an immunological memory [92]. Alginate hydrogels used in implantations are highly purified so as to not present antigens on their surface and thus should not cause T cell activation due to antigen-presentation [100]. Despite lacking these immunoreactive properties, within nanoseconds of entering a host, biomaterials initiate an inflammatory FBR due to biomaterial surface interactions with tissue, proteins, and blood [100, 101]. The foreign body response results in fibrotic overgrowth surrounding the implanted alginate hydrogels which essentially isolates the biomaterial from host's body [102]. This fibrotic overgrowth is initiated by a sequence of cascading events including injury formation, recruitment of immune cells and fibroblasts, and cell adhesion which leads to fibrous tissue deposition [103]. Two stages of inflammation, namely acute followed by chronic, facilitate the foreign body response once alginate biomaterials are implanted [102].

It was determined that the immunogenic properties of alginate polymers were not directly responsible for the overgrowth around capsules but do contribute to is formation. In order for the immune cells to adhere to the cell surface, there must be a significant amount of protein adsorption or anchor sites for immune cell adhesion [92, 102]. Instead, the immune response occurs in the tissue immediately surrounding the alginate structure [92]. Studies have demonstrated that within a few days after alginate biomaterial implantation into animals, immune cells and differentially activated macrophages (i.e., stadia, granulocytes, and basophils) collect around, but not on, the alginate surface [92, 104]. This acute inflammatory period is characterized by the recruitment of macrophages and neutrophils in addition to the release of histamine and fibrinogen adsorption from mast cells to surrounding tissue [92, 102]. This is all done without significant immune cell adhesion to alginate biomaterials. A key player in immune cell recruitment are pattern recognizing receptors (PRRs) which act as sensors on cells of the innate immune system by responding to evolutionary conserved molecules with can contaminate alginate biomaterials [92]. Even after purification, lipoteichoic acid and LPS, the most commonly known endotoxin, can remain and are recognized by Toll-like Receptors (TLRs) [52]. It has been demonstrated that recognition of PAMPs by TLRs causes the activation of macrophages through the NF-κB pathway [105]. NF-κB results in the transcriptional upregulation of genes most associated with cytokine and chemokine production which serve to intensify humoral response [105]. Within a week, the initial acute inflammatory period transitions to a chronic inflammatory period.

The chronic inflammatory stage is characterized by the accumulation of lymphocytes and monocytes, among other mononuclear cells, in the tissue surrounding the alginate biomaterial [106, 107]. It was discovered *in vitro* that lymphocytes can adhere to alginate biomaterial surfaces, but when placed in co-cultures with macrophages, they appeared to attach predominantly to the macrophage rather than the alginate microcapsule surface [100]. Further research has suggested that macrophage adhesion and fusion to biomaterial surface is

assisted by T lymphocytes [100]. Both macrophages and lymphocytes secrete inflammatory mediators. Examples of these include cytokines IL-1β, IL-6, and TNFα and chemokines IL-8, MCP, MIP-1β, and ENA-78, all of which are known to activate and recruit inflammatory effector cells such as neutrophils, monocytes, T lymphocytes, and natural killer cells [100, 105]. Similarly, it was discovered that certain cytokine, chemokine levels, matrix metalloproteinases (MMPs) and tissue inhibitor MMPs (TIMP), fluctuated over time which suggests that T lymphocyte-macrophage interactions have the ability to facilitate the chronic inflammatory phase of the FBR [100, 108]. There have been *in vivo* studies that have shown active T cells in response to synthetic materials during an inflammatory response; however, *in vitro* studies have shown a lack of lymphocyte response to alginate biomaterials [100, 109]. A novel focus of study regarding mitogens, which are functional groups on the surface of biomaterials, led to the hypothesis that they can possibly trigger lymphocytes via cross-linking of glycoproteins, but these mitogenic properties have not been observed [100]. The chronic inflammatory period usually subsides 2 weeks after implantation and any longer periods of inflammation are usually the result of infection [100, 102]. A combination of granulation, fibroblast infiltration, and macrophage/fibroblast surface adhesion result in the final fibrotic encapsulation of alginate biomaterials.
