Anabela Alves

Materials of marine origin: a review on polymers and ceramics of biomedical interest

Abstract Marine organisms are constituted by materials with a vast range of properties and characteristics that may justify their potential application within the biomedical field. Moreover, assuring the sustainable exploitation of natural marine resources, the valorisation of residues from marine origin, like those obtained from food processing, constitutes a highly interesting platform for development of novel biomaterials, with both economic and environmental benefits. In this perspective, an increasing number of different types of compounds are being isolated from aquatic organisms and transformed into profitable products for health applications, including controlled drug delivery and tissue engineering devices. This report reviews the work that is being developed on the isolation and characterisation of some polysaccharides, proteins, glycosaminoglycans and ceramics from marine raw materials. Emphasis is given to agar, alginates, carrageenans, chitin and chitosan, among other polysaccharides, collagen, glycosaminoglycans such as chondroitin sulphate, heparin and hyaluronic acid, calcium phosphorous compounds and biosilica. Finally, this report ends by reviewing the application of the previously mentioned materials on specific biomedical applications, in particular their participation on the development of controlled drug delivery systems and tissue engineering scaffolds.

A practical perspective on ulvan extracted from green algae

Researchers have many times turned their attention to nature and biological processes to develop novel technologies and materials. In a medical perspective, nature-based products are believed to be a strategic alternative approach to the use of fully synthetic materials, particularly in the design of medical devices. In the past decades, marine organisms have become the focus of considerable attention as potential sources of valuable materials. The sustainable exploitation and valorisation of natural marine resources constitutes a highly attractive and strategic platform for the development of novel biomaterials, with both economic and environmental benefits. In this context, algae are known to synthesise large quantities of polysaccharides and are well established sources of these particularly interesting molecules, many of which are known for their applicability in the design of biomaterials. Agar, carrageenan and alginates are some of the most known examples, and their uses can range from food to biomedical applications. However, few of the world’s available seaweed species are used commercially. Among the three main divisions of macroalgae (Chlorophyta, Phaeophyta and Rhodophyta), the green algae remain largely unexploited in this biomedical arena. While the demand for novel materials and technologies increases, so does the research of unexploited marine green algae including its unique polysaccharide ulvan.

Marine algae sulfated polysaccharides for tissue engineering and drug delivery approaches.

Biomedical field is constantly requesting for new biomaterials, with innovative properties. Natural polymers appear as materials of election for this goal due to their biocompatibility and biodegradability. In particular, materials found in marine environment are of great interest since the chemical and biological diversity found in this environment is almost uncountable and continuously growing with the research in deeper waters. Moreover, there is also a slower risk of these materials to pose illnesses to humans. In particular, sulfated polysaccharides can be found in marine environment, in different algae species. These polysaccharides don’t have equivalent in the terrestrial plants and resembles the chemical and biological properties of mammalian glycosaminoglycans. In this perspective, are receiving growing interest for application on health-related fields. On this review, we will focus on the biomedical applications of marine algae sulfated polymers, in particular on the development of innovative systems for tissue engineering and drug delivery approaches.

Extraction and physico-chemical characterization of a versatile biodegradable polysaccharide obtained from green algae.

During the last years, considerable attention has been given to different marine organisms, like algae, as potential sources of valuable materials. The continuous demand for novel materials and technologies is high and research on the underexploited marine green algae, including its polysaccharidic part-ulvan, has increased accordingly. In this research work, a novel method for extraction of ulvan from green algae is proposed and demonstrated successfully. Different characterization techniques were employed to characterize the isolated algal polysaccharide, namely, on what concerns its thermal trace and crystallinity. Upon heating, ulvan behaves as a non-meltable polysaccharide that is thermally stable before degradation at 220°C. Ulvan is semi-crystalline in nature and possesses high hygroscopic features, as revealed in this research work. Due to its properties, ulvan can be considered, pure or modified, as a versatile biodegradable polymer for different applications, including tissue engineering and regenerative medicine.

Comparative study about the effects of pollution on glass and yellow eels (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NW Portugal).

The health status of eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different types and levels of pollution was compared in relation to morphometric parameters, Fulton condition index (F index) and several biomarkers. Relatively to the reference population, glass eels from the Lima estuary had lower weight and length, cholinesterase (ChE) and lactate dehydrogenase (LDH) inhibition, and lower levels of some anti-oxidant parameters, while yellow eels also showed a decreased F index, and increased Na(+)/K(+)-ATPase and lipid peroxidation (LPO) levels. Relatively to the reference population, glass eels from the Douro estuary had increased Na(+)/K(+)-ATPase and glutathione-S-transferase activities and LDH inhibition, while yellow eels also had ChE inhibition and increased LPO. Overall, these results indicate that eels from polluted estuaries showed a poor health status than those from a reference estuary, and that adverse effects become more pronounced after spending several years in polluted estuaries.

Processing ulvan into 2D structures: Cross-linked ulvan membranes as new biomaterials for drug delivery applications

The polysaccharide ulvan, composed of sulphated rhamnose, glucoronic and iduronic acids was used to produce polymeric membranes by solvent casting. As ulvan is soluble in water, a cross-linking step was necessary to render the membrane insoluble in water and stable at physiological conditions. Cross-linked ulvan membranes were characterized by FTIR, SEM, swelling behaviour was investigated and the mechanical performance assessed by quasi-static tensile testing. Furthermore, the ability and mechanism of sustained release of a model drug from ulvan membranes was investigated. Produced membranes revealed remarkable ability to uptake water (up to ∼1800% of its initial dry weight) and increased mechanical performance (1.76 MPa) related with cross-linking. On the other hand, medicated ulvan dressings demonstrate the potential as drug delivery devices. Using a model drug we have observed an initial steady release of the drug - of nearly 49% - followed by slower and sustained release up to 14 days. The properties of ulvan membranes herein revealed suggest a great potential of this natural sulphated polysaccharide as a wound dressing.

Evaluation of anti-nociceptive and anti-inflammatory activity of low-level laser therapy on temporomandibular joint inflammation in rodents

The aim of this study was to investigate the analgesic and anti-inflammatory activity of low-level laser therapy (LLLT) on the nociceptive behavioral as well as histomorphological aspects induced by injection of formalin and carrageenan into the rat temporomandibular joint. The 2.5% formalin injection (FRG group) induced behavioral responses characterized by rubbing the orofacial region and flinching the head quickly, which were quantified for 45 min. The pretreatment with systemic administration of diclofenac sodium-DFN group (10 mg/kg i.p.) as well as the irradiation with LLLT infrared (LST group, 780 nm, 70 mW, 30 s, 2.1 J, 52.5 J/cm(2), GaAlAs) significantly reduced the formalin-induced nociceptive responses. The 1% carrageenan injection (CRG group) induced inflammatory responses over the time-course of the study (24 h, and 3 and 7 days) characterized by the presence of intense inflammatory infiltrate rich in neutrophils, scanty areas of liquefactive necrosis and intense interstitial edema, extensive hemorrhagic areas, and enlargement of the joint space on the region. The DFN and LST groups showed an intensity of inflammatory response that was significantly lower than in CRG group over the time-course of the study, especially in the LST group, which showed exuberant granulation tissue with intense vascularization, and deposition of newly formed collagen fibers (3 and 7 days). It was concluded that the LLLT presented an anti-nociceptive and anti-inflammatory response on the inflammation induced in the temporomandibular joint of rodents.

Carboxymethylation of ulvan and chitosan and their use as polymeric components of bone cements.

Ulvan, extracted from the green algae Ulva lactuca, and chitosan, extracted from Loligo forbesis squid-pen, were carboxymethylated, yielding polysaccharides with an average degree of substitution of ∼98% (carboxymethyl ulvan, CMU) and ∼87% (carboxymethyl chitosan, N,O-CMC). The carboxymethylation was confirmed by Fourier transform infrared spectroscopy and quantified by conductimetric titration and 1H nuclear magnetic resonance. The average molecular weight increased with the carboxymethylation (chitosan, Mn 145→296 kDa and Mw 227→416 kDa; ulvan, Mn 139→261 kDa and Mw 368→640 kDa), indicating successful chemical modifications. Mixtures of the modified polysaccharides were tested in the formulation of polyacrylic acid-free glass-ionomer bone cements. Mechanical and in vitro bioactivity tests indicate that the inclusion of CMU in the cement formulation, i.e. 0.50:0.50 N,O-CMC:CMU, enhances its mechanical performance (compressive strength 52.4±8.0 MPa and modulus 2.3±0.3 GPa), generates non-cytotoxic cements and induces the diffusion of Ca and/or P-based moieties from the surface to the bulk of the cements.

In Vitro Cytotoxicity Assessment of Ulvan, a Polysaccharide Extracted from Green Algae

Sustainable exploitation and valorization of natural marine resources represents a highly interesting platform for the development of novel biomaterials, with both economic and environmental benefits. In this context, toxicity data is regarded as a crucial and fundamental knowledge prior to any advances in the application development of natural derived polymers. In the present work, cytotoxicity of ulvan extracted from green algae Ulva lactuca was assessed by means of standard in vitro cytotoxicity assays. Fibroblast‐like cells were incubated in the presence of this green algaes polysaccharide, and cell viability was assayed through 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium test. In addition, double stranded DNA and total protein were quantified in order to assess cell number. In order to establish ulvans non‐cytotoxic behaviour, the effect of this polysaccharide on cellular metabolic activity and cell number was directly compared to hyaluronic acid (HA), used as a non‐cytotoxic control material. In this study, ulvan demonstrated promising results in terms of cytotoxicity, comparable to the currently used HA, which suggests that ulvan can be considered as non‐toxic in the range of concentrations studied. Copyright

Processing of degradable ulvan 3D porous structures for biomedical applications

The interest in ulvan within a biomedical framework increases as the knowledge of this polysaccharide evolves. Ulvan has been recently proposed as a potential biomaterial, and structures based on this polysaccharide are now being studied for different biomedical applications. In this work, a novel porous structure based on cross-linked ulvan was designed and characterized. Its mechanical performance, water-uptake ability and weight loss were assessed, morphology analyzed through scanning electron microscopy, and morphometric parameters quantified by microcomputed tomography. Cell viability and cell proliferation were evaluated in order to estimate the cytotoxicity of these structures and respective degradation products. Produced ulvan structures revealed remarkable ability to uptake water (up to ∼ 2000% of its initial dry weight) and are characterized by a highly porous and interconnected structure. Furthermore, these ulvan structures underwent nontoxic degradation, and cells remained viable through the time of culture. These results position ulvan structures as prospective blocks that can be further functionalized in order to acquire the desired stability and needed biological interactivity to be used as tissue-engineered structures.