Imine Crosslinked, Injectable and Self-healing Fucoidan Based Hydrogels with Immunomodulatory Properties for Tissue Engineering

Introduction: Biomaterials based tissue-engineered grafts used for in-vivo applications often experience transplant rejection due to immune response mounted against them by the host immune system. To prevent this, immunomodulatory biomaterials could help in improving graft integration. Fucoidan is a natural sulfated polysaccharide that has innate immunomodulatory properties. Previously, fucoidan has been mostly reported to be delivered as a bioactive component loaded into other hydrogel systems, where the lack of chemical interactions between fucoidan and the main hydrogel matrix may lead to leaching of fucoidan, particularly in long term applications. In this study, we leveraged the immunomodulatory and anti-inflammatory properties of fucoidan to develop a novel biomaterial for tissue engineering applications. We report, for the time first time, a fucoidan based hydrogel by introducing aldehyde groups in fucoidan that formed imine crosslinks with amine containing polymers (e.g. gelatin and aminated PEG). The fucoidan-gelatin hydrogel was injectable and self-healable with tunable mechanical properties and showed remarkable stability in-vitro for a period of 1 month. Furthermore, this novel fucoidan-gelatin based hydrogel possessed immunomodulatory properties, showed biocompatibility, and allowed diverse cellular functions in in-vitro studies. Likewise, the in-vivo testing also validated the immunomodulatory nature of our hydrogels, as they interacted with the resident immune cells and reduced inflammatory responses by recruiting immune cells expressing anti-inflammatory markers. Lastly, we demonstrated that modified fucoidan can also form imine crosslinks with synthetic polymers such as 4 arm PEG amine while retaining its immunomodulatory properties both in-vitro and in-vivo.

Materials and

Methods: Fucoidan from Fucus vesiculosus source was oxidized using NaIO4 to introduce aldehyde groups (FuOx). The gelation of FuOx was tested by mixing it with unmodified gelatin in different ratios at physiological pH. The mechanical and rheological properties of oxidized fucoidan (FuOx) and gelatin (Gel) based hydrogels were quantified using a mechanical tester and a rheometer, respectively. The stability of these hydrogels was monitored over a period of 1 month and the rate of degradation was evaluated by incubating the hydrogels in collagenase solution and quantifying the protein release over time using BCA assay. Bioadhesion of 10% FuOx:Gel hydrogel was tested with mice brain. Moreover, the biocompatibility was analyzed by culturing PC-12 cells, a model neuronal cell line, on FuOx:Gel hydrogels with 5%, 10% and 15% polymer concentrations in 1:1 ratio. The number of cells adhered to the hydrogels after 6 hours of incubation were counted. Moreover, cell spreading, and differentiation was observed by staining differentiated PC-12 cells with β-III tubulin antibody after 7 days of cell culture. For in-vivo testing, 10% FuOx:Gel and 10% FuOx:PEG hydrogels were subcutaneously injected in the C57BL/6 mice along with an initial intraperitoneal dose of 10 µg/mL LPS to induce the inflammatory responses in the mice. PBS and LPS groups were used as controls. After 7 days, mice were euthanized and the tissue samples were collected for H&E staining and immunohistochemistry with CD206, CD86, Arginase-I and VEGFR2 primary antibodies. All the experiments were run in triplicates and the data is presented as mean ± SD.

Results, Conclusions, and Discussions: The reaction of native fucoidan with NaIO4 yielded an oxidized product (FuOx) with 33% degree of oxidation that formed an injectable and self-healable hydrogel at physiological pH by forming imine crosslinks with amine groups present on gelatin and 4-arm PEG amine. The FuOx:Gel and FuOx:PEG hydrogels had tunable mechanical properties that can be controlled by adjusting the concentration of the prepolymer solutions (Fig1. a,b). Furthermore, FuOx:Gel hydrogels showed self-healing property (Fig1. c) remained stable in PBS for a period of ~1 month (Fig1. d) and demonstrated collagenase mediated biodegradation as they dissolved completely in 8µg/mL and 20µg/mL collagenase solutions, while remained stable in PBS control (Fig1. e.i-iii). Moreover, FuOx:Gel hydrogels were bioadhesive likely due to the presence of aldehyde groups that can react to amines present in tissue (Fig1. f). The biocompatibility was monitored in-vitro by culturing PC-12 cells on 5%, 10% and 15% FuOx:Gel hydrogels. A significantly higher number of PC-12 cells adhered to 15% hydrogel compared to 10% and 5% that could be attributed to relatively greater abundance of RGD domains due to higher gelatin content in 15% hydrogels. (Fig1. g). Moreover, FuOx:Gel hydrogels allowed the differentiation of cultured PC-12 cells. The length of newly formed neurites, visualized through β-III tubulin antibody staining, was increased on stiffer gels compared to soft hydrogels (Fig1. h). For in-vivo biocompatibility and immunomodulatory analyses, the histological examination of 10% FuOx:Gel and FuOx:PEG hydrogels groups showed the recruitment of cells around these hydrogels. These infiltrating cells expressed CD206, Arginase-I (M2-type macrophage), CD86 (M1-type macrophage) and VEGFR2 (endothelial cell) markers (Fig1.i). Altogether, these results suggest the potential of FuOx containing hydrogels in interacting with the host immune system by recruiting cells expressing M2 and M1 macrophages and endothelial cell markers indicating their immunomodulatory properties. Our current studies include the quantification of phenotype of infiltrating cells.
To conclude, we have developed novel fucoidan based hydrogels via dynamic imine crosslinking that are injectable, self-healable, self-immunomodulatory and biocompatible, both in-vitro and in-vivo. Moreover, these findings demonstrate the versatility of our novel fucoidan biomaterial for various tissue engineering applications such as transplantation of cells and other therapeutics.

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