Use of Zebrafish (Danio rerio) for Biosafety Evaluation of Strontium Nanostructured Hydroxyapatite.

Despite the numerous studies on biocompatibility with nano-biomaterials, the biological effects of strontium-substituted HA nanoparticles (nSrHA) need to be better understood. So, we conducted an embryotoxicity test using zebrafish (Danio rerio) according to the OECD 236 guideline, a model that represents a viable alternative that bridges the gap between in vitro and mammalian models. Zebrafish embryos were exposed for 120 h to microspheres containing nSrHA nanoparticles with low and high crystallinity, synthesized at temperatures of 5°C (nSrHA5) and 90°C (nSrHA90). We evaluated lethality, developmental parameters, and reactive oxygen species (ROS) production. The larval behavior was assessed at 168 hpf to determine if the biomaterials affected motor responses and anxiety-like behavior. The results showed that the survival rate decreased significantly for the nSrHA5 group (low crystalline particles), and an increase in ROS was also observed in this group. However, none of the biomaterials caused morphological changes indicative of toxicity during larval development. Additionally, the behavioral tests did not reveal any alterations in all experimental groups, indicating the absence of neurotoxic effects from exposure to the tested biomaterials. These findings provide valuable insights into the biosafety of modified HA-based nanostructured biomaterials, making them a promising strategy for bone tissue repair. As the use of hydroxyapatite-based biomaterials continues to grow, it is crucial to ensure rigorous control over the quality, reliability, and traceability of these materials.

Embryotoxicity and visual-motor response of functionalized nanostructured hydroxyapatite-based biomaterials in zebrafish (Danio rerio).

Hydroxyapatite (HA) is a biomaterial widely used in biomedical applications. Many studies have shown that ionic substituents can be incorporated into HA to produce a mineral composition more similar to natural bone tissue with more favorable biological characteristics for application in bone regeneration. However, its potentially toxic effects need to be evaluated before full approval for human use. For this purpose, an embryotoxicity test was performed on zebrafish according to OECD guideline 236. Zebrafish embryos were exposed to 1 or 3 microspheres of alginate containing nanoparticles of HA and carbonate (CHA), strontium (SrHA), and zinc-substituted HA (ZnHA) from 4 to 120 h post-fertilization (hpf). Lethality and developmental endpoints were evaluated. In addition, larval behavior at 168 hpf was also analyzed to observe whether biomaterials adversely affect optomotor and avoidance responses (neurotoxicity), as well as the oxidative stress pattern through qPCR. After 120 h exposure to all microspheres with different patterns of crystallinity, porosity, nanoparticle size, surface area, and degradation behavior, there was no mortality rate greater than 20%, indicating the non-embryotoxic character of these biomaterials. All experimental groups showed positive optomotor and avoidance responses, which means that embryo exposure to the tested biomaterials had no neurotoxic effects. Furthermore, larvae exposed to one SrHA microsphere showed a better optomotor response than the control. Furthermore, the biomaterials did not change the pattern of mRNA levels of genes related to oxidative stress even after 120 hpf. The growing number of new HA-based biomaterials produced should be accompanied by increased studies to understand the biosafety of these compounds, especially in alternative models, such as zebrafish embryos. These results reinforce our hypothesis that ion-substituted HA biomaterials do not impose toxicological effects, cause development and neuromotor impairment, or increase oxidative stress in zebrafish embryos being useful for medical devices and in the process of bone regeneration.

In Vitro Degradation of Electrospun Poly(Lactic-Co-Glycolic Acid) (PLGA) for Oral Mucosa Regeneration.

Poly(lactic-co-glycolic acid) (PLGA) has been used in the field of tissue engineering as a scaffold due to its good biocompatibility, biodegradability and mechanical strength. With the aim to explore the degradability of PLGA electrospun nonwoven structures for oral mucosa tissue engineering applications, non-irradiated and gamma irradiated nonwovens were immersed in three different solutions, in which simulated body fluid (SBF) and artificial saliva are important for future oral mucosa tissue engineering. The nonwovens were immersed for 7, 15 and 30 days in SBF, culture media (DMEM) and artificial saliva at 37 °C. Before immersion in the solutions, the dosage of 15 kGy was applied for sterilization in one assay and compared with non-irradiated samples at the same timepoints. Samples were characterized using different techniques such as scanning electron microscopy (SEM), differential scanning calorimetric (DSC) and gel permeation chromatography (GPC) to evaluate the nonwoven degradation and Fourier-transform infrared spectroscopy (FTIR) to evaluate the chain scissions. Our results showed that PLGA nonwovens were constituted by semicrystalline fibers with moderate degradation properties up to thirty days. The non-irradiated samples exhibited slower kinetics of degradation than irradiated nonwovens. For immersion times longer than 7 days in the three different solutions, the mean diameter of irradiated fibers stayed in the same range, but significantly different from the control sample. On non-irradiated samples, the degradation kinetics was slower and the plateau in the diameter value was only attained after 30 days of immersion in the fluids. Plasticization (fluid absorption into the fiber structure) occurred in the bulk material, as confirmed by a decrease in Tg observed by DSC analyses of non-irradiated and irradiated nonwovens, in comparison with the respective controls. In addition, artificial saliva showed a higher capacity of influencing PLGA crystallization than SBF and DMEM. FTIR analyses showed typical PLGA chemical functional groups changes. These results will be important for future application of those PLGA electrospun nonwovens for oral mucosa regeneration.

Doxycycline containing hydroxyapatite ceramic microspheres as a bone-targeting drug delivery system.

Drug delivery technology is a promising way to enhance the therapeutic efficacy of drugs. The purpose of this study is to evaluate the physical and chemical properties of hydroxyapatite ceramic microspheres loaded with doxycycline (HADOX), their effects on in vitro osteoblast viability, and their antimicrobial activity, and to determine the effects of DOX on the healing of rat sockets after tooth extraction. The internal microsphere porosity was sensitive to the treatment used to adsorb DOX onto microsphere surface; HA microspheres without DOX presented 26% of pores, whereas HADOX0.15 microspheres presented 52.0%. An initial drug release of 49.15 µg/ml was observed in the first 24 hr. The minimal inhibitory concentration (MIC) tested against Enterococcus faecalis demonstrated that bacterial growth was inhibited for up to 7 days. Results of cell viability and cell proliferation did not indicate statistical differences in the metabolic activity of HADOX samples relative to HA without DOX microspheres (p > .05). After 1 week, a discreet inflammation reaction was observed in the control group, and after 6 weeks, newly-formed bone was observed in the HADOX0.15 (p < .05). The HADOX did not interfere in the bone repair and controlled the early inflammatory response. HADOX could be a promising biomaterial to promote bone repair in infected sites.