Flounder fish (Paralichthys sp.) collagen a new tissue regeneration: genotoxicity, cytotoxicity and physical-chemistry characterization.

Collagen dressings have been widely used as effective treatments for chronic wounds acting as barrier, protecting the area from infections and participating in the healing process. Collagen from fish skin is biocompatible, presents low immunogenicity and is able of stimulating wound healing. In this scenario, skin of flounder fish (Paralichthys sp.) may constitute a promising source for collagen. Then, our hypothesis is that fish collagen is able of increasing cell proliferation, with no cytotoxicity. In this context, the aim of the present study was to investigate the physicochemical and morphological properties of collagen using scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), mass loss and pH. Moreover, the cytotoxicity and genotoxicity of collagen were studied using in vitro studies (cell viability, comet assay and micronucleus assay). Fish collagen showed no variation of pH and mass weight, with characteristic peaks of collagen in FTIR. Furthermore, all the extracts presented cell viability at least over 50% and no cytotoxicity was observed. Regarding genotoxicity data, the results showed that only the extract of 100% showed higher values in comparison with negative control group for CHO-K1 cell line as depicted by comet and micronucleus assays. Based on the results, it is suggested that fish collagen is biocompatible and present non-cytotoxicity in the in vitro studies, being considered a suitable material for tissue engineering proposals.

3D Printed Scaffolds Manufactured with Biosilica from Marine Sponges for Bone Healing in a Cranial Defect in Rats.

The inorganic part of marine sponges, called Biosilica (BS), presents an osteogenic potential and the ability of consolidating fractures. Moreover, 3D printing technique is highly effective for manufacturing scaffolds for tissue engineering proposals. Thus, the aims of this study were to characterize the 3D rinted scaffolds, to evaluate the biological effects in vitro and to investigate the in vivo response using an experimental model of cranial defects in rats. The physicochemical characteristics of 3D printed BS scaffolds were analyzed by FTIR, EDS, calcium assay, evaluation of mass loss and pH measurement. For in vitro analysis, the MC3T3-E1 and L929 cells viability was evaluated. For the in vivo evaluation, histopathology, morphometrical and immunohistochemistry analyses were performed in a cranial defect in rats. After the incubation, the 3D printed BS scaffolds presented lower values in pH and mass loss over time. Furthermore, the calcium assay showed an increased Ca uptake. The FTIR analysis indicated the characteristic peaks for materials with silica and the EDS analysis demonstrated the main presence of silica. Moreover, 3D printed BS demonstrated an increase in MC3T3-E1 and L929 cell viability in all periods analyzed. In addition, the histological analysis demonstrated no inflammation in days 15 and 45 post-surgery, and regions of newly formed bone were also observed. The immunohistochemistry analysis demonstrated increased Runx-2 and OPG immunostaining. Those findings support that 3D printed BS scaffolds may improve the process of bone repair in a critical bone defect as a result of stimulation of the newly formed bone.

Effects of ZnO/TiO2 nanoparticle and TiO2 nanotube additions to dense polycrystalline hydroxyapatite bioceramic from bovine bones.

OBJECTIVES: A bovine dense hydroxyapatite ceramic (HA) was produced as new biomaterial, however, the production of a material with consistently high flexural strength remains challenging. The objective of this study was to evaluate the effects of ZnO nanoparticles, TiO2 nanoparticles, and TiO2 nanotubes (1%, 2%, and 5% by weight) on the microstructure and flexural strength of a bovine dense hydroxyapatite ceramic (HA). METHODS: Discs (Ø=12.5mm; thickness=1.3mm) were prepared and subjected to X-ray diffraction (XRD), and observation with a field emission scanning electron microscope (FE-SEM), biaxial flexural strength (BFS) testing, and Vickers hardness (VH) testing. The BFS and VH data were subjected to ANOVA and Tukey post-hoc tests (α=0.05) and Weibull analysis. RESULTS: The XRD showed that the addition of nanomaterials caused the formation of a secondary phase when 5% of the ZnO nanoparticles was used, or when all percentages of the TiO2 nanoparticles/nanotubes were used, and the HA crystallographic planes were maintained. Differences were not observed between the higher BFS values obtained with pure HA and those obtained with the 5% addition of TiO2 nanoparticles. However, the results were different compared with the other groups (α=0.05). The results obtained by Weibull analysis revealed that the 1%, 2%, and 5% addition of TiO2 nanotubes, and the 1% and 2% addition of TiO2 nanoparticles decreased the HA characteristic strength (σ0), while the Weibull modulus (m) increased when 5% of TiO2 nanoparticles, 1% and 2% of ZnO nanoparticles, and 2% of TiO2 nanoparticles were added, but with no statistical difference from the pure HA. The 5% addition of ZnO2 nanoparticles decreased the σ0 without changing m. Moreover, the 5% addition of TiO2 nanoparticles resulted in an m closest to that of pure HA. Regarding the VH results, the blend of HA with 1% and 2% addition of TiO2 nanoparticles exhibited the higher values, which were similar between the different addition ratios (p=0.102). Moreover, the addition of 5% TiO2 nanoparticles resulted in higher value compared with pure HA. SIGNIFICANCE: This study demonstrated that the HA blend with 5% of TiO2 nanoparticles has the greatest potential as a bovine HA dense bioceramic reinforcement.