The Characterization and Cytotoxic Evaluation of Chondrosia reniformis Collagen Isolated from Different Body Parts (Ectosome and Choanosome) Envisaging the Development of Biomaterials.

Chondrosia reniformis is a collagen-rich marine sponge that is considered a sustainable and viable option for producing an alternative to mammalian-origin collagens. However, there is a lack of knowledge regarding the properties of collagen isolated from different sponge parts, namely the outer region, or cortex, (ectosome) and the inner region (choanosome), and how it affects the development of biomaterials. In this study, a brief histological analysis focusing on C. reniformis collagen spatial distribution and a comprehensive comparative analysis between collagen isolated from ectosome and choanosome are presented. The isolated collagen characterization was based on isolation yield, Fourier-transformed infrared spectroscopy (FTIR), circular dichroism (CD), SDS-PAGE, dot blot, and amino acid composition, as well as their cytocompatibility envisaging the development of future biomedical applications. An isolation yield of approximately 20% was similar for both sponge parts, as well as the FTIR, CD, and SDS-PAGE profiles, which demonstrated that both isolated collagens presented a high purity degree and preserved their triple helix and fibrillar conformation. Ectosome collagen had a higher OHpro content and possessed collagen type I and IV, while the choanosome was predominately constituted by collagen type IV. In vitro cytotoxicity assays using the L929 fibroblast cell line displayed a significant cytotoxic effect of choanosome collagen at 2 mg/mL, while ectosome collagen enhanced cell metabolism and proliferation, thus indicating the latter as being more suitable for the development of biomaterials. This research represents a unique comparative study of C. reniformis body parts, serving as a support for further establishing this marine sponge as a promising alternative collagen source for the future development of biomedical applications.

Structure and Composition of the Cuticle of the Goose Barnacle Pollicipes pollicipes: A Flexible Composite Biomaterial.

Arthropods, the largest animal phylum, including insects, spiders and crustaceans, are characterized by their bodies being covered primarily in chitin. Besides being a source of this biopolymer, crustaceans have also attracted attention from biotechnology given their cuticles' remarkable and diverse mechanical properties. The goose barnacle, Pollicipes pollicipes, is a sessile crustacean characterized by their body parts covered with calcified plates and a peduncle attached to a substrate covered with a cuticle. In this work, the composition and structure of these plates and cuticle were characterized. The morphology of the tergum plate revealed a compact homogeneous structure of calcium carbonate, a typical composition among marine invertebrate hard structures. The cuticle consisted of an outer zone covered with scales and an inner homogenous zone, predominantly organic, composed of successive layers parallel to the surface. The scales are similar to the tergum plate and are arranged in parallel and oriented semi-vertically. Structural and biochemical characterization confirmed a bulk composition of ɑ-chitin and suggested the presence of elastin-based proteins and collagen. The mechanical properties of the cuticle showed that the stiffness values are within the range of values described in elastomers and soft crustacean cuticles resulting from molting. The removal of calcified components exposed round holes, detailed the structure of the lamina, and changed the protein properties, increasing the rigidity of the material. This flexible cuticle, predominantly inorganic, can provide bioinspiration for developing biocompatible and mechanically suitable biomaterials for diverse applications, including in tissue engineering approaches.

Potential of Atlantic Codfish (Gadus morhua) Skin Collagen for Skincare Biomaterials.

Collagen is the major structural protein in extracellular matrix present in connective tissues, including skin, being considered a promising material for skin regeneration. Marine organisms have been attracting interest amongst the industry as an alternative collagen source. In the present work, Atlantic codfish skin collagen was analyzed, to evaluate its potential for skincare. The collagen was extracted from two different skin batches (food industry by-product) using acetic acid (ASColl), confirming the method reproducibility since no significant yield differences were observed. The extracts characterization confirmed a profile compatible with type I collagen, without significant differences between batches or with bovine skin collagen (a reference material in biomedicine). Thermal analyses suggested ASColl's native structure loss at 25 °C, and an inferior thermal stability to bovine skin collagen. No cytotoxicity was found for ASColl up to 10 mg/mL in keratinocytes (HaCaT cells). ASColl was used to develop membranes, which revealed smooth surfaces without significative morphological or biodegradability differences between batches. Their water absorption capacity and water contact angle indicated a hydrophilic feature. The metabolic activity and proliferation of HaCaT were improved by the membranes. Hence, ASColl membranes exhibited attractive characteristics to be applied in the biomedical and cosmeceutical field envisaging skincare.

Insights on the properties of levofloxacin-adsorbed Sr- and Mg-doped calcium phosphate powders.

Several types of biodegradable materials have been investigated for the treatment of osteomyelitis. Calcium phosphate (CaP) ceramics are among the most performing materials due to their resemblance to human hard tissues in terms of mineralogical composition, and proven ability to adsorb and deliver a number of drugs. This research work was intended to study the suitability of modified CaP powders loaded with a fluoroquinolone as drug delivery systems for osteomyelitis treatment. Levofloxacin (LEV) was chosen due to the well-recognized anti-staphylococcal activity and adequate penetration into osteoarticular tissues. Substituted CaP powders (5 mol% Sr(2+) or 5 mol% Mg(2+)) were synthesised through aqueous precipitation. The obtained powders were characterised by X-ray diffraction, SEM and FTIR analysis. The X-ray diffraction patterns confirmed the presence of HA and ß-tricalcium phosphates (ß-TCP) phases in doped compositions, especially in the case of Mg-doped system. The fixation of LEV at the surface of the particles occurred only by physisorption. Both the in vitro microbiological susceptibility, against Staphylococcus spp, and biocompatibility of LEV-loaded CaP powders have not been compromised.

3D bioactive ionic liquid-based architectures: An anti-inflammatory approach for early-stage osteoarthritis.

3D bioprinting enables the fabrication of biomimetic cell-laden constructs for cartilage regeneration, offering exclusive strategies for precise pharmacological screenings in osteoarthritis (OA). Synovial inflammation plays a crucial role in OA's early stage and progression, characterized by the increased of the synovial pro-inflammatory mediators and cytokines and chondrocyte apoptosis. Therefore, there is an urgent need to develop solutions for effectively managing the primary events associated with OA. To address these issues, a phenolic-based biocompatible ionic liquid approach, combining alginate (ALG), acemannan (ACE), and cholinium caffeate (Ch[Caffeate]), was used to produce easily printable bioinks. Through the use of this strategy 3D constructs with good printing resolution and high structural integrity were obtained. The encapsulation of chondrocytes like ATDC5 cells provided structures with good cell distribution, viability, and growth, for up to 14 days. The co-culture of the constructs with THP-1 macrophages proved their ability to block pro-inflammatory cytokines (TNF-α and IL-6) and mediators (GM-CSF), released by the cultured cells. Moreover, incorporating the biocompatible ionic liquid into the system significantly improved its bioactive performance without compromising its physicochemical features. These findings demonstrate that ALG/ACE/Ch[Caffeate] bioinks have great potential for bioengineering cartilage tissue analogs. Besides, the developed ALG/ACE/Ch[Caffeate] bioinks protected encapsulated chondrocyte-like cells from the effect of the inflammation, assessed by a co-culture system with THP-1 macrophages. These results support the increasing use of Bio-ILs in the biomedical field, particularly for developing 3D bioprinting-based constructs to manage inflammatory-based changes in OA. STATEMENT OF SIGNIFICANCE: Combining natural resources with active biocompatible ionic liquids (Bio-IL) for 3D printing is herein presented as an approach for the development of tools to manage inflammatory osteoarthritis (OA). We propose combining alginate (ALG), acemannan (ACE), and cholinium caffeate (Ch[Caffeate]), a phenolic-based Bio-IL with anti-inflammatory and antioxidant features, to produce bioinks that allow to obtain 3D constructs with good printing resolution, structural integrity, and that provide encapsulated chondrocyte-like cells good viability. The establishment of a co-culture system using the printed constructs and THP-1-activated macrophages allowed us to study the encapsulated chondrocyte-like cells behaviour within an inflammatory scenario, a typical event in early-stage OA. The obtained outcomes support the beneficial use of Bio-ILs in the biomedical field, particularly for the development of 3D bioprinting-based models that allow the monitoring of inflammatory-based events in OA.

In vivo assessment of marine vs bovine origin collagen-based composite scaffolds promoting bone regeneration in a New Zealand rabbit model.

The ability of human tissues to self-repair is limited, which motivates the scientific community to explore new and better therapeutic approaches to tissue regeneration. The present manuscript provides a comparative study between a marine-based composite biomaterial, and another composed of well-established counterparts for bone tissue regeneration. Blue shark skin collagen was combined with bioapatite obtained from blue shark's teeth (mColl:BAp), while bovine collagen was combined with synthetic hydroxyapatite (bColl:Ap) to produce 3D composite scaffolds by freeze-drying. Collagens showed similar profiles, while apatite particles differed in their composition, being the marine bioapatite a fluoride-enriched ceramic. The marine-sourced biomaterials presented higher porosities, improved mechanical properties, and slower degradation rates when compared to synthetic apatite-reinforced bovine collagen. The in vivo performance regarding bone tissue regeneration was evaluated in defects created in femoral condyles in New Zealand rabbits twelve weeks post-surgery. Micro-CT results showed that mColl:BAp implanted condyles had a slower degradation and an higher tissue formation (17.9 ± 6.9 %) when compared with bColl:Ap implanted ones (12.9 ± 7.6 %). The histomorphometry analysis provided supporting evidence, confirming the observed trend by quantifying 13.1 ± 7.9 % of new tissue formation for mColl:BAp composites and 10.4 ± 3.2 % for bColl:Ap composites, suggesting the potential use of marine biomaterials for bone regeneration.

Marine Gelatin-Methacryloyl-Based Hydrogels as Cell Templates for Cartilage Tissue Engineering.

Marine-origin gelatin has been increasingly used as a safe alternative to bovine and porcine ones due to their structural similarity, avoiding the health-related problems and sociocultural concerns associated with using mammalian-origin materials. Another benefit of marine-origin gelatin is that it can be produced from fish processing-products enabling high production at low cost. Recent studies have demonstrated the excellent capacity of gelatin-methacryloyl (GelMA)-based hydrogels in a wide range of biomedical applications due to their suitable biological properties and tunable physical characteristics, such as tissue engineering applications, including the engineering of cartilage. In this study, fish gelatin was obtained from Greenland halibut skins by an acidic extraction method and further functionalized by methacrylation using methacrylic anhydride, developing a photosensitive gelatin-methacryloyl (GelMA) with a degree of functionalization of 58%. The produced marine GelMA allowed the fabrication of photo-crosslinked hydrogels by incorporating a photoinitiator and UV light exposure. To improve the biological performance, GelMA was combined with two glycosaminoglycans (GAGs): hyaluronic acid (HA) and chondroitin sulfate (CS). GAGs methacrylation reaction was necessary, rendering methacrylated HA (HAMA) and methacrylated CS (CSMA). Three different concentrations of GelMA were combined with CSMA and HAMA at different ratios to produce biomechanically stable hydrogels with tunable physicochemical features. The 20% (w/v) GelMA-based hydrogels produced in this work were tested as a matrix for chondrocyte culture for cartilage tissue engineering with formulations containing both HAMA and CSMA showing improved cell viability. The obtained results suggest these hybrid hydrogels be used as promising biomaterials for cartilage tissue engineering applications.

Mineralized collagen as a bioactive ink to support encapsulation of human adipose stem cells: A step towards the future of bone regeneration.

Bioprinting - printing with incorporated living cells - has earned special attention on tissue engineering approaches, aiming to closer reproduce the 3D microenvironment of the target tissue. However, it raises extra complexity related to the need to use cell-friendly printing conditions that still comply with material printing fidelity. Inspired by the composite nano structural organization of mineralized tissues, this work reports the efficiency of the chemical approach followed to in situ mineralize blue shark skin collagen, at a nano scale level, to ultimately produce stable inks. The influence of initial cellular density was evaluated by assessing three different concentrations (2.5, 5 and 7.5 × 106 cells·ml-1) of human adipose stem cells (hASC), with the higher density of encapsulated cells presenting improved viability in a long culture term. Immunodetection of osteogenic-related markers, like RUNX2 and osteopontin, 21 days after cell culture in basal conditions confirmed the potential of the ink to be applied for osteogenic purposes, which may be associated with the success of the cell-to-ink interaction and the Ca2+ ions released from the co-precipitated hydroxyapatite. A combination of mineralized shark collagen, alginate and hASC is thus proposed as a bioactive bioink with potential properties for regeneration of bone tissue.

Engineering 3D printed bioactive composite scaffolds based on the combination of aliphatic polyester and calcium phosphates for bone tissue regeneration.

In this study, polylactic acid (PLA) filled with hydroxyapatite (HA) or beta-tricalcium phosphate (TCP) in 5 wt% and 10 wt% of concentration were produced employing twin-screw extrusion followed by fused filament fabrication in two different architectures, varying the orientation of fibers of adjacent layers. The extruded 3D filaments presented suitable rheological and thermal properties to manufacture of 3D scaffolds envisaging bone tissue engineering. The produced scaffolds exhibited a high level of printing accuracy related to the 3D model; confirmed by micro-CT and electron microscopy analysis. The developed architectures presented mechanical properties compatible with human bone replacement. The addition of HA and TCP made the filaments bioactive, and the deposition of new calcium phosphates was observed upon 7 days of incubation in simulated body fluid, exemplifying a microenvironment suitable for cell attachment and proliferation. After 7 days of cell culture, the constructs with a higher percentage of HA and TCP demonstrated a significantly superior amount of DNA when compared to neat PLA, indicating that higher concentrations of HA and TCP could guide a good cellular response and increasing cell cytocompatibility. Differentiation tests were performed, and the biocomposites of PLA/HA and PLA/TCP exhibited earlier markers of cell differentiation as confirmed by alkaline phosphatase and alizarin red assays. The 3D printed composite scaffolds, manufactured with bioactive materials and adequate porous size, supported cell attachment, proliferation, and differentiation, which together with their scalability, promise a high potential for bone tissue engineering applications.

Cell-Laden Biomimetically Mineralized Shark-Skin-Collagen-Based 3D Printed Hydrogels for the Engineering of Hard Tissues.

Mineralization processes based on coprecipitation methods have been applied as a promising alternative to the most commonly used methods of polymer-ceramic combination, direct mixing, and incubation in simulated body fluid (SBF) or modified SBF. In the present study, for the first time, the in situ mineralization (ideally hydroxyapatite formation) of blue shark (Prionace glauca (PG)) collagen to fabricate 3D printable cell-laden hydrogels is proposed. In the first part, several parameters for collagen mineralization were tested until optimization. The hydroxyapatite formation was confirmed by FT-IR, XRD, and TEM techniques. In the second part, stable bioinks combining the biomimetically mineralized collagen with alginate (AG) (1:1, 1:2, 1:3, and AG) solution were used for 3D printing of hydrogels. The addition of Ca2+ ions into the system did present a synergistic effect: by one side, the in situ mineralization of the collagen occurred, and at same time, they were also useful to ionically cross-link the blends with alginate, avoiding the addition of any cytotoxic chemical cross-linking agent. Mouse fibroblast cell line survival during and after printing was favored by the presence of PG collagen as exhibited by the biological performance of the hydrogels. Inspired in a concept of marine byproduct valorization, 3D bioprinting of in situ mineralized blue shark collagen is thus proposed as a promising approach, envisioning the engineering of mineralized tissues.

Scaffolding Strategies for Tissue Engineering and Regenerative Medicine Applications.

During the past two decades, tissue engineering and the regenerative medicine field have invested in the regeneration and reconstruction of pathologically altered tissues, such as cartilage, bone, skin, heart valves, nerves and tendons, and many others. The 3D structured scaffolds and hydrogels alone or combined with bioactive molecules or genes and cells are able to guide the development of functional engineered tissues, and provide mechanical support during in vivo implantation. Naturally derived and synthetic polymers, bioresorbable inorganic materials, and respective hybrids, and decellularized tissue have been considered as scaffolding biomaterials, owing to their boosted structural, mechanical, and biological properties. A diversity of biomaterials, current treatment strategies, and emergent technologies used for 3D scaffolds and hydrogel processing, and the tissue-specific considerations for scaffolding for Tissue engineering (TE) purposes are herein highlighted and discussed in depth. The newest procedures focusing on the 3D behavior and multi-cellular interactions of native tissues for further use for in vitro model processing are also outlined. Completed and ongoing preclinical research trials for TE applications using scaffolds and hydrogels, challenges, and future prospects of research in the regenerative medicine field are also presented.

Novel sintering-free scaffolds obtained by additive manufacturing for concurrent bone regeneration and drug delivery: Proof of concept.

Advances on the fabrication of sintering-free biphasic calcium phosphate (BCP)/natural polymer composite scaffolds using robocasting as additive manufacturing technique are presented in the present work. Inks with high amounts of BCP powders (45 vol%) containing different HA/ß-TCP ratios, in presence of crosslinked polymer, were successfully fine-tuned for extrusion by robocasting. The non-existence of sintering step opened the possibility to obtain drug loaded scaffolds by adding levofloxacin to the extrudable inks. The drug presence induced slightly changes on the rheological behaviour of the inks, more emphasized for the BCP compositions with higher amounts of ß-TCP, and consequently, on the microstructure and on the mechanical properties of the final scaffolds. The strong interaction of ß-TCP with chitosan difficult the preparation of suitable rheological inks for printing. Drug delivery studies revealed a fast release of levofloxacin with a high burst of drug within the first 30 min. Levofloxacin loaded samples also presented bacteria growth inhibition ability, proving that antibiotic was not degraded during the fabrication process and its bactericidal efficacy was preserved. From the results obtained, the composite scaffolds containing higher amounts of HA (around 80% HA/20% ß-TCP) constitute a promising bi-functional synthetic bone substitute for simultaneous local bone regeneration and infection treatments.

Novel doped calcium phosphate-PMMA bone cement composites as levofloxacin delivery systems.

Antibiotic-loaded acrylic bone cements (ALABCs) are well-established and cost-effective materials to control the occurrence of bone and joint infections. However, the inexistence of alternative antibiotics other than those already commercially available and the poor ability to bind to bone tissue hampering its biological function are still major drawbacks of ALABCs clinical application. The concept of this research work is to develop a novel bone cement (BC) drug delivery system composed by Mg- and Sr-doped calcium phosphate (CaP) particles as drug carriers loaded into a lactose-modified acrylic BC, which, to the best of our knowledge, has never been reported. CaP particles are known to promote bone ingrowth and current research is focused on using these carriers as antibiotic delivery systems for the treatment of bone infections, like osteomyelitis. Levofloxacin is a fluoroquinolone with anti-staphylococcal activity and adequate penetration into osteoarticular tissues and increasingly being recommended to manage bone-related infections. Also, the lactose-modified BC matrix, with a more porous structure, has already proved to enhance antibiotic release from the BC inner matrix. This novel BC composite biomaterial has shown improved mechanical integrity, biocompatibility maintenance, and sustained release of levofloxacin, with concentrations over the minimum inhibitory concentration values after a 48h while maintaining antibacterial activity over an 8-week period against Staphyloccocus aureus and Staphyloccocus epidermidis, common pathogens associated with bone infections.