The expansion of fisheries and aquaculture in recent decades has led to a substantial increase in fish by-products. This study investigates the extraction and characterization of calcium phosphates from the by-products of representative species in these industries, aiming to identify potential sources for biotechnological and pharmaceutical applications. Clean bones obtained by enzyme hydrolysis from the heads, central skeletons, and/or tails of Atlantic horse mackerel, blue whiting, hake, mackerel, and farmed turbot were subjected to calcination to obtain calcium phosphates. The clean bone content in terms of nitrogen, lipids, organic matter, total protein, and amino acids was evaluated together with the chemical bonds, structures, and elemental composition of calcium phosphates. Results indicated a significantly higher yield of wet bone recovery (23%, p < 0.05) for the central skeleton of Atlantic horse mackerel and the highest mineral fraction for the heads of Atlantic horse mackerel (73.2%), followed by that of blue whiting (72.6%). Hake and turbot presented the lowest mineral fractions and, therefore, the highest protein content (27–31%, p < 0.05), with significant levels of collagen-related amino acids (p < 0.05). X-ray diffraction (XRD) and Fourier-transform Raman spectroscopy (FT-Raman) confirmed the biphasic calcium phosphate composition for most samples based on hydroxyapatite with contributions of whitlockite/β-tricalcium phosphate. The highest contribution to the non-apatite phase was made by the central skeletons of both mackerel and Atlantic horse mackerel.

A simple cascade process based on the hydrothermal fractionation of Ulva spp. biomass was proposed. Considering the overall extraction yields (50 %), ulvan recovery (23 %), and ulvan composition, structural, mechanical and cytotoxic properties, the selected optimal final heating temperature was 160 degrees C. Ethanol precipitation provided the highest ulvan recovery yields but choline chloride precipitated ulvans showed stronger mechanical properties, Gmoduli 1.5 & sdot;104 4 Pa and 3 & sdot;104 4 Pa for ethanol and for choline chloride, respectively. Both products were safe on NCTC 929 mouse fibroblasts and after a cooling stage, formed films without requiring any additives. From the ulvan-free liquid fraction, one product with 43 % (wt, d.b.) phenolics and moderate antiradical properties and a byproduct containing nutrients and minerals were separated. The methane potential of the corresponding residual solids was influenced by the hydrothermal heating temperature and was doubled compared to than for the untreated seaweed biomass (60 mL/g VS). This scheme could be also applied to the wet algal biomass, in a chemical free alternative to provide ready to use ulvan biopolymers, bioactives, nutrients, salts and biogas, conforming a biorefinery approach.

Hyperthermia is nowadays intensively investigated as a promising strategy to improve the therapeutic efficacy against different types of cancer and resistant infections. In particular, the remote generation of localized hyperthermia by magnetic field through iron-oxide nanoparticles (IONPs) offers good thermal conductivity in a controlled area. The incorporation of these IONPs in 3D-printed scaffolds designed for bone tissue regeneration has been scarcely addressed in the literature. This strategy would add the potential of magnetic-mediated hyperthermia against remnant cancer or resistant infections in the damaged tissue area to these personalized bone-related scaffolds. The present work proposes two methodologies to obtain 3D-printed bone-related scaffolds with magnetic properties: 1-Direct 3D printing with IONPs-embedded polylactic acid (PLA) and hydroxyapatite (HA), resulting in a uniform distribution of IONPs; and 2-Drop coating on 3D-printed PLA/HA scaffolds, resulting in the IONPs being concentrated on the scaffold surface. Physicochemical/mechanical characterizations were performed to confirm the IONPs' distributions and viability assays were carried out to validate the absence of cytotoxicity. Hyperthermia tests (314 kHz) were carried out, including the simulation/validation of the experimental equipment, to establish optimal distances from the planar coil. Temperature-time/distance curves were obtained and parametrized (R-2 > 0.96) for both methodologies in relation to the contribution of IONPs (0.20-1.00 mg), their distribution in the scaffold (uniform/concentrated), the electric-current intensity, and the distance. The results validated both methodologies to obtain personalized 3D-printed PLA/HA scaffolds with magnetic properties, reaching the required moderate/ablative hyperthermia levels.

This study investigates the effect of scaffold architecture on bone regeneration, focusing on 3D-printed polylactic acid–bioceramic calcium phosphate (PLA-bioCaP) composite scaffolds in rabbit femoral condyle critical defects. We explored two distinct scaffold designs to assess their influence on bone healing and scaffold performance. Structures with alternate (0°/90°) and helical (0°/45°/90°/135°/180°) laydown patterns were manufactured with a 3D printer using a fused deposition modeling technique. The scaffolds were meticulously characterized for pore size, strut thickness, porosity, pore accessibility, and mechanical properties. The in vivo efficacy of these scaffolds was evaluated using a femoral condyle critical defect model in eight skeletally mature New Zealand White rabbits. Then, the results were analyzed micro-tomographically, histologically, and histomorphometrically. Our findings indicate that both scaffold architectures are biocompatible and support bone formation. The helical scaffolds, characterized by larger pore sizes and higher porosity, demonstrated significantly greater bone regeneration than the alternate structures. However, their lower mechanical strength presented limitations for use in load-bearing sites.

The global tuna canning industry generates substantial volumes of by-products, comprising 50% to 70% of the total processed material. Traditionally, these by-products have been utilized in low-value products such as fish oils and fishmeal. However, there is significant potential to extract high-value compounds from these by-products, such as calcium phosphates (CaP), which can have pharmaceutical, agricultural and biotechnological applications. This work explores the potential of tuna canning by-products, particularly mineral-rich fractions (central skeleton, head and fish bones) as sources of calcium phosphates (CaP), offering a sustainable alternative to conventional synthetic derivatives within a circular bioeconomy framework. By-products from two of the most exploited species (yellowfin and skipjack) were subjected to enzymatic hydrolysis and chemical extraction, followed by controlled calcination to obtain CaP. The content of organic matter, nitrogen, total proteins, lipids and amino acids in the cleaned bones, as well as the main chemical bonds, structure and elemental composition (FT-Raman, XRD, XRF) were evaluated. Results indicated that the highest recovery yield of wet bones was achieved using the chemical method, particularly from the dorsal and caudal fins of yellowfin tuna. The proximal composition, with ash content ranging from 52% to 66% and protein content varying between 30% and 53%, highlights the potential of tuna skeleton substrates for plant growth formulations. Furthermore, variations in crystalline structures of the substrates revealed significant differences depending on the by-product source and species. XRD and Raman results confirmed a monophase calcium phosphate composition in most samples from both species, primarily based on hydroxyapatite (central skeleton, caudal and dorsal fin) or whitlockite/β-tricalcium phosphate (viscera), whereas the heads exhibited a biphasic composition. Comparing the species, yellowfin tuna (YF) exhibited a hydroxyapatite structure in the branchial arch and scales, while skipjack (SKJ) had a biphasic composition in these same regions.

Objective This study aimed to evaluate the in vivo biocompatibility, mechanical performance and osteoconductive potential of 3D-printed polylactic acid (PLA) scaffolds enriched with marine bioderived calcium phosphate (bioCaP) for bone tissue engineering. Materials and Methods PLA-bioCaP composite scaffolds were specifically designed for the rabbit cranial defect model by 3D printing, with a uniform distribution of open square-shaped pores and contributions in bioCaP. Physicochemical and mechanical characterization and the evaluation of biological response are presented. Results The scaffolds demonstrated mechanical properties comparable to human bones, integration with the host bone, and osteoconductive behavior promoting cell ingrowth from the defect edge. Strong mineralized tissue ingrowth through the scaffolds’ pores was observed, providing notable support to the host bone. In quantitative terms, micro-CT and histomorphometry analysis post-implantation revealed no significant differences in bone regeneration across all groups. Conclusion The 3D-printed scaffolds with perpendicular patterning, open porosity, and proposed composition displayed satisfactory mechanical properties, biocompatibility, and osteoconductive response. The scaffolds promoted bone regeneration at similar levels as the PLA. The highest contribution of bioCaP promoted a positive influence in certain histomorphometric parameters; however, it did not significantly improve their osteogenic capability. Further research is required to optimize scaffold composition and enhance their osteogenic potential. Clinical Relevance This study presents a significant advancement in bone tissue engineering through the development of personalized composite scaffolds for bone-related applications. The clinical implications of this research are profound, especially considering the increasing demand for functional bone regeneration technologies capable of producing cost-effective producing cost-effective customized scaffolds.

Hyperthermia-based therapies have shown great potential for clinical applications such as for the antitumor and antipathogenic activities. Within all strategies, the so-called photothermal therapy proposes to induce the hyperthermia by the remote laser radiation on a photothermal conversion agent, in contact with the target tissue. This paper reviews the most relevant in vitro and in vivo studies focused on NIR laser-induced hyperthermia due to photoexcitation of graphene oxide (GO) and reduced graphene oxide (rGO). Relevant parameters such as the amount of GO/rGO, the influence of the laser wavelength and power density are considered. Moreover, the required temperature and exposure time for each antitumor/antipathogenic case are collected and unified in a thermal dose parameter: the CEM43. The calculated CEM43 thermal doses revealed a great variability for the same type of tumor/strain. In order to detect potential tendencies, the values were classified into four ranges, varying from CEM43 < 60 min to CEM43 ≥ 1 year. Thus, a preference for moderate thermal doses of CEM43 < 1 year was detected in antitumor activity, with temperatures ≤ 50 °C and exposure time ≤ 15 min. In case of the antipathogenic studies, the most used thermal dose was higher, CEM43 ≥ 1 year, with ablative hyperthermia (> 60ºC). The ability of GO/rGO as effective photothermal conversion agents to promote a controlled hyperthermia is proven. The variability found for the CEM43 thermal doses on the reviewed studies reveals the potentiality to evaluate, for each application, the use of lower temperatures, by modulating time and/or repetitions in the doses.

The present work evaluates the thermal behavior of graphene oxide (GO) when deposited on 3D-printed polylactic acid (PLA), in order to develop a medical device for photothermal therapy applications. An experimental–numerical analysis was performed to assess the photothermal conversion capacity, based on the power emitted by a NIR (785 nm) laser, and the subsequent temperature distribution on the GO-PLA material. The influence of the deposited mass of GO and the PLA thickness was studied through 40 different scenarios. The results estimated a value of photothermal conversion efficiency of up to 32.6%, achieved for the lower laser power density that was tested (0.335 mW/mm²), and a high mass value of deposited GO (1.024 × 10−3 mg/mm²). In fact, an optimal mass of GO in the range of 1.024–2.048 × 10−3 mg/mm2 is proposed, in terms of absorption capacity, since a higher mass of GO would not increase the conversion efficiency. Moreover, the study allowed for an estimation of the thermal conductivity of this specific biomaterial (0.064 W/m·K), and proved that a proper combination of GO mass, PLA thickness, and laser power can induce ablative (>60 °C, in a concentrated area), moderate (50 °C), and mild (43 °C) hyperthermia on the bottom face of the biomaterial.

Graphene oxide (GO) has attracted increasing interest for biomedical applications owing to its outstanding properties such as high specific surface area, ability to bind functional molecules for therapeutic purposes and solubility, together with mechanical resistance and good thermal conductivity. The combination of GO with other biomaterials, such as calcium phosphate (CaP) and biodegradable polymers, presents a promising strategy for bone tissue engineering. Presently, the development of these advanced biomaterials benefits from the use of additive manufacturing techniques, such as 3D printing. In this study, we develop a 3D printed PLA:CaP:GO scaffold for bone tissue engineering. First, GO was characterised alone by XPS to determine its main bond contributions and C : O ratio. Secondly, we determined the GO dose which ensures the absence of toxicity, directly exposed in vitro (human osteoblast-like cells MG-63) and in vivo (zebrafish model). In addition, GO was microinjected in the zebrafish to evaluate its effect on immune cells, quantifying the genetic expression of the main markers. Results indicated that the GO tested (C : O of 2.14, 49.50% oxidised, main bonds: C–OH, C–O–C) in a dose ≤0.25 mg mL−1 promoted MG63 cells viability percentages above 70%, and in a dose ≤0.10 mg mL−1 resulted in the absence of toxicity in zebrafish embryos. The immune response evaluation reinforced this result. Finally, the optimised GO dose (0.10 mg mL−1) was combined with polylactic acid (PLA) and CaP to obtain a 3D printed PLA:CaP:GO scaffold. Physicochemical characterisation (SEM/EDS, XRD, FT-Raman, nano-indentation) was performed and in vivo tests confirmed its biocompatibility, enabling a novel approach for bone tissue-related applications.

The reconstruction or regeneration of damaged bone tissue is one of the challenges of orthopedic surgery and tissue engineering. Among all strategies investigated, additive manufacturing by fused deposition modeling (3D-FDM printing) opens the possibility to obtain patient-specific scaffolds with controlled architectures. The present work evaluates in depth 3D direct printing, avoiding the need for a pre-fabricated filament, to obtain bone-related scaffolds from direct mixtures of polylactic acid (PLA) and hydroxyapatite (HA). For it, a systematic physicochemical characterization (SEM-EDS, FT-Raman, XRD, micro-CT and nanoindentation) was performed, using different PLA/HA ratios and percentages of infill. Results prove the versatility of this methodology with an efficient HA incorporation in the 3D-printed scaffolds up to 13 wt.% of the total mass and a uniform distribution of the HA particles in the scaffold at the macro level, both longitudinal and cross sections. Moreover, an exponential distribution of the HA particles from the surface toward the interior of the biocomposite cord (micro level), within the first 80 µm (10% of the entire cord diameter), is also confirmed, providing the scaffold with surface roughness and higher bioavailability. In relation to the pores, they can range in size from 250 to 850 µm and can represent a percentage, in relation to the total volume of the scaffold, from 24% up to 76%. The mechanical properties indicate an increase in Young's modulus with the HA content of up to ~50%, compared to the scaffolds without HA. Finally, the in vitro evaluation confirms MG63 cell proliferation on the 3D-printed PLA/HA scaffolds after up to 21 days of incubation.

The regeneration of bone remains one of the main challenges in the biomedical field, with the need to provide more personalized and multifunctional solutions. The other persistent challenge is related to the local prevention of infections after implantation surgery. To fulfill the first one and provide customized scaffolds with complex geometries, 3D printing is being investigated, with polylactic acid (PLA) as the biomaterial mostly used, given its thermoplastic properties. The 3D printing of PLA in combination with hydroxyapatite (HA) is also under research, to mimic the native mechanical and biological properties, providing more functional scaffolds. Finally, to fulfill the second one, antibacterial drugs locally incorporated into biodegradable scaffolds are also under investigation. This work aims to develop vancomycin-loaded 3D-printed PLA–HA scaffolds offering a dual functionality: local prevention of infections and personalized biodegradable scaffolds with osseointegrative properties. For this, the antibacterial drug vancomycin was incorporated into 3D-printed PLA–HA scaffolds using three loading methodologies: (1) dip coating, (2) drop coating, and (3) direct incorporation in the 3D printing with PLA and HA. A systematic characterization was performed, including release kinetics, Staphylococcus aureus antibacterial/antibiofilm activities and cytocompatibility. The results demonstrated the feasibility of the vancomycin-loaded 3D-printed PLA–HA scaffolds as drug-releasing vehicles with significant antibacterial effects for the three methodologies. In relation to the drug release kinetics, the (1) dip- and (2) drop-coating methodologies achieved burst release (first 60 min) of around 80–90% of the loaded vancomycin, followed by a slower release of the remaining drug for up to 48 h, while the (3) 3D printing presented an extended release beyond 7 days as the polymer degraded. The cytocompatibility of the vancomycin-loaded scaffolds was also confirmed.

Graphene oxide (GO) has attracted increasing interest for biomedical applications owing to its outstanding properties such as high specific surface area, ability to bind functional molecules for therapeutic purposes and solubility, together with mechanical resistance and good thermal conductivity. The combination of GO with other biomaterials, such as calcium phosphate (CaP) and biodegradable polymers, presents a promising strategy for bone tissue engineering. Presently, the development of these advanced biomaterials benefits from the use of additive manufacturing techniques, such as 3D printing. In this study, we develop a 3D printed PLA:CaP:GO scaffold for bone tissue engineering. First, GO was characterised alone by XPS to determine its main bond contributions and C : O ratio. Secondly, we determined the GO dose which ensures the absence of toxicity, directly exposed in vitro (human osteoblast-like cells MG-63) and in vivo (zebrafish model). In addition, GO was microinjected in the zebrafish to evaluate its effect on immune cells, quantifying the genetic expression of the main markers. Results indicated that the GO tested (C : O of 2.14, 49.50% oxidised, main bonds: C-OH, C-O-C) in a dose <= 0.25 mg mL(-1) promoted MG63 cells viability percentages above 70%, and in a dose <= 0.10 mg mL(-1) resulted in the absence of toxicity in zebrafish embryos. The immune response evaluation reinforced this result. Finally, the optimised GO dose (0.10 mg mL(-1)) was combined with polylactic acid (PLA) and CaP to obtain a 3D printed PLA:CaP:GO scaffold. Physicochemical characterisation (SEM/EDS, XRD, FT-Raman, nano-indentation) was performed and in vivo tests confirmed its biocompatibility, enabling a novel approach for bone tissue-related applications.

Polylactic acid (PLA) has become one of the most commonly used polymers in medical devices given its biocompatible, biodegradable and bioabsorbable properties. In addition, due to PLA's thermoplastic behaviour, these medical devices are now obtained using 3D printing technologies. Once obtained, the 3D-printed PLA devices undergo different sterilisation procedures, which are essential to prevent infections. This work was an in-depth study of the physicochemical changes caused by novel and conventional sterilisation techniques on 3D-printed PLA and their impact on the biological response in terms of toxicity. The 3D-printed PLA physicochemical (XPS, FTIR, DSC, XRD) and mechanical properties as well as the hydrophilic degree were evaluated after sterilisation using saturated steam (SS), low temperature steam with formaldehyde (LTSF), gamma irradiation (GR), hydrogen peroxide gas plasma (HPGP) and CO2 under critical conditions (SCCO). The biological response was tested in vitro (fibroblasts NCTC-929) and in vivo (embryos and larvae wild-type zebrafish Danio rerio). The results indicated that after GR sterilisation, PLA preserved the O:C ratio and the semi-crystalline structure. Significant changes in the polymer surface were found after HPGP, LTSF and SS sterilisations, with a decrease in the O:C ratio. Moreover, the FTIR, DSC and XRD analysis revealed PLA crystallisation after SS sterilisation, with a 52.9% increase in the crystallinity index. This structural change was also reflected in the mechanical properties and wettability. An increase in crystallinity was also observed after SCCO and LTSF sterilisations, although to a lesser extent. Despite these changes, the biological evaluation revealed that none of the techniques were shown to promote the release of toxic compounds or PLA modifications with toxicity effects. GR sterilisation was concluded as the least reactive technique with good perspectives in the biological response, not only at the level of toxicity but at all levels, since the 3D-printed PLA remained almost unaltered.

3D printing allows the development of devices in a wide range of sectors, including the automotive industry, construction, aeronautics, food, textiles and also biomedical, among others. There are several technologies being the impression by molten deposition (FDM) one of the most widespread. In the particular case of biomedical engineering, three are the main application and research axes: (1) biomodels for the preparation of surgical interventions, (2) design and manufacture of customized biomedical devices and (3) porous scaffolds for regenerative medicine. The objective of the present study focuses on the development of a new filament for 3D-FDM printing composed of polylactic acid (PLA) and a biphasic crystalline bioceramic of marine origin (Biofast). Several filament formulations were obtained after extrusion of mixtures with variable proportions PLA / Biofast. The physical-chemical properties of both the filament and the printed pieces were studied by scanning electron microscopy, Raman and IR spectroscopy. The analyzes show that the bioceramic particles are evenly distributed in the polymer matrix and their incorporation can be controlled depending on the selected mixture. As for the crystal structure and links, manufacturing processes do not originate modifications that may affect the biocompatibility of the final device. Thus, these new filaments are shown as potential candidates for application in biomedical engineering.

Pulsed laser deposition (PLD) represents a promising bottom-up methodology for the synthesis and transference of nanoparticles to the surface of a biomedical device. Silver (Ag) nanoparticles directly incorporated on the metallic implant emerge as an alternative strategy for local action against prosthetic joint-associated infections. In the present research, a dual sequential PLD process is proposed to obtain a bilayer coating with (1) a bio-derived calcium phosphate (CaP) layer, to provide osteointegrative properties and (2) the controlled growth of the Ag nanoparticles over it, ranging the number of laser pulses from 100 to 500. The characterization by SEM, EDS, TEM, XPS and AFM revealed the uniform deposit of Ag rounded nanoparticles, with a narrow mean size distribution, in the original non-oxidized metallic state. Moreover, given the evidences from XPS and AFM techniques, the occurrence of a coalescence phenomenon from 400 pulses onwards was proposed together with the expected positive linear relation between the number of pulses and Ag contribution with a deposition rate of 0.05 at. % of Ag per pulse. Conversely, the decrease in roughness as the Ag content increased was also verified. Finally, the expected bacteriostatic activity for these PLD deposited metallic state Ag nanoparticles against the bacterial strainStaphylococcus aureuswas confirmed. Moreover, the evaluation of the osteoblast-like MG-63 cells viability on the Ag(100-500)-CaP coatings revealed a significant increased proliferation (p > 0.05) on the Ag100-CaP coating compared to the control (Ag0-CaP). When same coating was evaluated againstS. aureusthe effect was not significant. The possibility of modulating the amount of nanoparticles in the bilayer coating to obtain a greater or lesser effect in combination with CaP was revealed.

Autogenous cancellous bone graft is the current gold standard of treatment for the management of bone defects since it possesses the properties of osteoinduction, osteoconduction, and osteogenesis. Xenografts and synthetic grafts have been widely reported as available and low-cost alternatives, which retain good osteoconductive and mechanical properties. Given the rich biodiversity of ocean organisms, marine sources are of particular interest in the search for alternative bone grafts with enhanced functionalities. The purpose of this paper is to assess the biocompatibility of a marine-derived bone graft obtained from shark tooth, which is an environmentally sustainable and abundant raw material from fishing. This research presents the findings of a preclinical trial—following UNE-EN ISO 10993—that induced a critical-sized bone defect in a rabbit model and compared the results with a commercial bovine-derived bone graft. Evaluation by micro-computed tomography and histomorphometric analysis 12 weeks after implantation revealed good osseointegration, with no signs of inflammatory foreign body reactions, fibrosis, or necrosis in any of the cases. The shark tooth-derived bone graft yielded significantly higher new bone mineral density values (54 ± 6%) than the control (27 ± 8%). Moreover, the percentage of intersection values were much higher (86 ± 8%) than the bovine-derived bone graft (30 ± 1%) used as control. The area of occupancy by bone tissue in the test material (38 ± 5%) also gave higher values than the control (30 ± 6%). The role of physicochemical properties, biphasic structure, and composition on the stimulation of bone regeneration is also discussed.

In the present work, the potential of the Prionace glauca jaw as a source of both chondroitin sulfate and bioapatite is explored. The sandwich-type structure in cross section of the jaw based on alternate layers with prevalence in organic tissue or mineralized is shown and these bands respectively confirmed as CS or hydroxyapatite -enriched zones. As result of this, an optimized process in sequential steps for the recovery of both biomaterials and their purification process is proposed, by combining enzymatic proteolysis, chemical precipitation and separation using ultrafiltration membrane for CS production together with controlled thermal treatment for hydroxyapatite obtaining. The purified CS was characterized by Gel Permeation Chromatography, Nuclear Magnetic Resonance and Strong Anion Exchange Chromatography, revealing a polymeric material with a molecular weight of 67 kDa, and prevalent 6S-GalNAc sulfation (68%), followed by 4S-GalNAc (13%), a significant proportion of disulfated disaccharides (12%) and only 7% of non-sulfated units. In the case of the bioapatite a purified biphasic 60:40 porous calcium phosphate of hydroxyapatite: whitlockite/β-TCP was confirmed. Hydroxyapatite as major component (85%) was also obtained for jaws directly subjected to the thermal treatment. This proved the influence of the enzymatic hydrolysis and centrifugation on the composition of the mineral fraction.

Osteoarthritis is the most prevalent rheumatic disease. During disease progression, differences have been described in the prevalence of chondroitin sulfate (CS) isomers. Marine derived-CS present a higher proportion of the 6S isomer, offering therapeutic potential. Accordingly, we evaluated the effect of exogenous supplementation of CS, derived from the small spotted catshark (Scyliorhinus canicula), blue shark (Prionace glauca), thornback skate (Raja clavata) and bovine CS (reference), on the proliferation of osteochondral cell lines (MG-63 and T/C-28a2) and the chondrogenic differentiation of mesenchymal stromal cells (MSCs). MG-G3 proliferation was comparable between R. clavata (CS-6 intermediate ratio) and bovine CS (CS-4 enrichment), for concentrations below 0.5 mg/mL, defined as a toxicity threshold. T/C-28a2 proliferation was significantly improved by intermediate ratios of CS-6 and -4 isomers (S. canicula and R. clavata). A dose-dependent response was observed for S. canicula (200 µg/mL vs 50 and 10 µg/mL) and bovine CS (200 and 100 µg/mL vs 10 µg/mL). CS sulfation patterns discretely affected MSCs chondrogenesis; even though S. canicula and R. clavata CS up-regulated chondrogenic markers expression (aggrecan and collagen type II) these were not statistically significant. We demonstrate that intermediate values of CS-4 and -6 isomers improve cell proliferation and offer potential for chondrogenic promotion, although more studies are needed to elucidate its mechanism of action.

In this work, five biosurfactant extracts, obtained from different sources, all of them with demonstrated antimicrobial properties, were characterized and subjected to a cytotoxic study using mouse fibroblast cells (NCTC clone 929). Biosurfactant extracts obtained directly from corn steep water (CSW) showed similar surfactant characteristics to those of the extracellular biosurfactant extract produced by Bacillus isolated from CSW and grown in tryptic soy broth, observing that they are amphoteric, consisting of viscous and yellowish liquid with no foaming capacity. Contrarily, cell-bound biosurfactant extracts produced from Lactobacillus pentosus or produced by Bacillus sp isolated from CSW are nonionic, consisting of a white powder with foaming capacity. All the biosurfactants possess a similar fatty acid composition. The cytotoxic test revealed that the extracts under evaluation, at a concentration of 1 g/L, were not cytotoxic for fibroblasts (fibroblast growth > 90%). The biosurfactant extract obtained from CSW with ethyl acetate, at 1 g/L, showed the highest cytotoxic effect but above the cytotoxicity limit established by the UNE-EN-ISO10993-5. It is remarkable that the cell-bound biosurfactant produced by L. pentosus, at a concentration of 1 g/L, promoted the growth of the fibroblast up to 113%.