INTRODUCTION

Since ever scientists and engineers have been looking into Nature as inspiration for the development of new materials, architectures, design, functions, devices, etc. Marine environment in particular has been the source of a myriad of natural products with diverse chemistries and biological activities, which are being explored as pharmaceuticals, including some examples on the market. Besides, several marine biopolymers have been produced and are used in fields from food and biotechnology to cosmetics, pharma and medicine. In the latter case, marine biopolymers can be found as wound dressings, membranes, drug delivery devices and biomaterials¹. In this presentation, the authors will address the advances that are being made in our research group concerning the development of marine inspired biomaterials towards tissue regeneration, from biopolymer extraction and processing, to biomaterials development and applicability assessment, including nature-made scaffolds.

EXPERIMENTAL METHODS

Biopolymer extraction

Collagen has been extracted from fish skins with acid aqueous solutions, complemented by enzymatic treatment by using pepsin. In parallel, it has been also extracted from marine sponges with acid or basic aqueous solutions, including with chaotropic agents.

Chitin has been extracted from squid pens by NaHO treatment and further converted into chitosan with concentrated NaHO solution.

Sulfated polysaccharides have been extracted from algae with hot water and precipitated with organic solvents. Additional steps can be added for matters of purity improvement.

Development of biomaterials

Solutions of the extracted biopolymers with different concentrations (1 to 4%) have been prepared and further used to produce porous structures. Different processing techniques have been used, in particular, freeze-drying (chitosan and collagen), agglomeration of previously prepared particles (chitosan and composites with nanohydroxyapatite, coagulated in NaHO solutions) and rapid prototyping (carrageenans, following ionic gelation) and will be presented. Crosslinking with different chemical agents, namely genipin (chitosan and collagen), EDC/NHS (collagen) and HDMI (collagen), has also been explored to tune mechanical stability of the developed constructs. Their morphological properties were assessed by SEM and micro-computed tomography (μCT) and mechanical properties evaluated by compression tests.

Moreover, natural collagenous structures were produced from marine sponges after supercritical fluid processing to both decellularize and extract toxic compounds from the samples.

Applicability assessment

Biological performance of the developed constructs has been tested with chondrocyte-like cell line ATDC5 (chitosan and collagen scaffolds), osteoblast-like cell line SaOS2 (collagen structures) or adipose derived stem cells, ASCs (chitosan scaffolds).

RESULTS AND DISCUSSION

Marine collagen has been crosslinked with genipin for the production of hydrogels. Alternatively, freeze-drying of collagen solutions rendered membranes, which were successfully crosslinked with EDC/NHS. Crosslinking of freeze-dried collagen structures was also achieved with genipin, under dense CO₂, which supported the culture of ATDC5 cells.

The porous structure of marine sponges and its collagenous composition has inspired the development of nature made scaffolds by decellularization and removal of toxic compounds, in which osteoblast-like cells have been successfully cultured.

Carrageenan-based scaffolds were produced by 3D plotting and ionic gelation with K⁺ solutions, revealing to be non cytotoxic to a fibroblast cell line.

Chitosan scaffolds were produced by freeze-drying, with different pore sizes. ATDC5 cells adhered and proliferated to the interior of the scaffold when squid chitosan was used. Porous scaffolds made by agglomeration of squid chitosan particles or composites with nanohydroxyapatite supported the culture of ASCs for up to 7 days.

CONCLUSION

Several polymeric constructs have been produced with marine origin polymers and assessed as 3D support for culture of different cell types. The development of marine inspired biomaterials is being taken to the next level, envisaging the promotion of their use on tissue regeneration therapeutic approaches.

REFERENCES

1. TH Silva, …, RL Reis, Int Mater Rev 57: 276-306, 2012.

ACKNOWLEDGMENTS

Funding is acknowledged from projects IBEROMARE (POCTEP), MARMED (Atlantic Area), NOVOMAR (POCTEP), POLARIS (FP7) and SPECIAL (FP7).