Titanium (Ti) and its alloys are widely used in dental implants and hip-prostheses due to their excellent biocompatibility. Growing evidence support that surface degradation due to corrosion and wear processes, contribute to implant failure, since the release of metallic ions and wear particles generate local tissue reactions (peri-implant inflammatory reactions). The generated ions and wear debris (particles at the micron and nanoscale) stay, in a first moment, at the interface implant-bone. However, depending on their size, they can enter blood circulation possibly contributing to systemic reactions and toxicities. Most of the nanotoxicological studies with titanium dioxide nanoparticles (TiO 2 NPs) use conventional two-dimensional cell culture monolayers to explore macrophage and monocyte activation, where limited information regarding bone cells is available. Recently three-dimensional models have been gaining prominence since they present a greater anatomical and physiological relevance. Taking this into consideration, in this work we developed a human osteoblast-like spheroid model, which closely mimics bone cell-cell interactions, providing a more realistic scenario for nanotoxicological studies. The treatment of spheroids with different concentrations of TiO 2 NPs during 72 h did not change their viability significantly. Though, higher concentrations of TiO 2 NPs influenced osteoblast cell cycle without interfering in their ability to differentiate and mineralize. For higher concentration of TiO 2 NPs, collagen deposition and pro-inflammatory cytokine, chemokine and growth factor secretion (involved in osteolysis and bone homeostasis) increased. These results raise the possible use of this model in nanotoxicological studies of osseointegrated devices and demonstrate a possible therapeutic potential of this TiO 2 NPs to prevent or reverse bone resorption.

The progressively increasing use of nanomaterials (NMs) has awakened issues related to nanosafety and its potential toxic effects on human health. Emerging studies suggest that NMs alter cell communication by reshaping and altering the secretion of extracellular vesicles (EVs), leading to dysfunction in recipient cells. However, there is limited understanding of how the physicochemical characteristics of NMs alter the EV content and their consequent physiological functions. Therefore, this review explored the relevance of EVs in the nanotoxicology field. The current state of the art on how EVs are modulated by NM exposure and the possible regulation and modulation of signaling pathways and physiological responses were assessed in detail. This review followed the manual for reviewers produced by The Joanna Brigs Institute for Scoping Reviews and the PRISMA extension for Scoping Reviews (PRISMA-ScR): checklist and explanation. The research question, “Do NMs modulate cellular responses mediated by EVs?” was analyzed following the PECO model (P (Population) = EVs, E (Exposure) = NMs, C (Comparator) = EVs without exposure to NMs, O (Outcome) = Cellular responses/change in EVs) to help methodologically assess the association between exposure and outcome. For each theme in the PECO acronym, keywords were defined, organized, and researched in PubMed, Science Direct, Scopus, Web of Science, EMBASE, and Cochrane databases, up to 30 September 2021. In vitro, in vivo, ex vivo, and clinical studies that analyzed the effect of NMs on EV biogenesis, cargo, and cellular responses were included in the analysis. The methodological quality assessment was conducted using the ToxRTool, ARRIVE guideline, Newcastle Ottawa and the EV-TRACK platform. The search in the referred databases identified 2944 articles. After applying the eligibility criteria and two-step screening, 18 articles were included in the final review. We observed that depending on the concentration and physicochemical characteristics, specific NMs promote a significant increase in EV secretion as well as changes in their cargo, especially regarding the expression of proteins and miRNAs, which, in turn, were involved in biological processes that included cell communication, angiogenesis, and activation of the immune response, etc. Although further studies are necessary, this work suggests that molecular investigations on EVs induced by NM exposure may become a potential tool for toxicological studies since they are widely accessible biomarkers that may form a bridge between NM exposure and the cellular response and pathological outcome.

TiO2 NPs’ nano–bio-interactions mediate a distinct intracellular trafficking and destiny in human skin cells.

Titanium implants undergo tribocorrosion processes releasing particles that interact with several cells at the implant–bone interface. Osteoblasts-derived exosomal proteins reduce osteogenic differentiation of HMSCs contributing to joint failure.

BackgroundThe inflammatory response to titanium implant-derived wear particles is considered as the hallmark of periprosthetic osteolysis, an event that cause pain, reduce patient motility, ultimately leading to the need of a revision surgery. Although macrophages are major cell players, other cell types such as bone cells can indirectly contribute to periprosthetic osteolysis, however the mechanisms are not fully understood. Exosomes (Exos) has been related with several bone pathologies, with growing body of literature recognizing them as actively shuttle molecules through the body, with their cargo being completely dependent of external stimuli (e.g. chemicals and metals ions and particles). Till the moment, the role of wear debris on osteoblasts exosomes biogenesis is absent and the possible contribution of Exos to osteoimmune communication and periprosthetic osteolysis is still in its infancy. Taking that in consideration, in this work we investigate the effect of wear debris on Exo biogenesis, where two bone cell models were exposed to titanium dioxide nanoparticles (TiO2 NPs) similar in size and composition to wear debris associated with prosthetic implants. The contribution of Exos to periprosthetic osteolysis was evaluated performing functional tests stimulating primary human macrophages with bone-derived Exos.ResultsFor the first time, we report that TiO2 NPs enter in multivesicular bodies, the nascent of Exos and altered osteoblasts derived exosomes secretion and cargo. No significant differences were observed in Exos morphology and size, however mass spectrometry analysis identified urokinase-type plasminogen activator (uPA), specifically enriched in Exos derived from bone cells pre-incubated with TiO2 NPs. Functional tests confirmed the activation of human macrophages towards a mixed phenotype with consequent secretion of pro and anti-inflammatory cytokines. ConclusionsThe external stimuli of osteoblasts to TiO2 NPs induced a dose dependent secretion of Exos, suggesting alterations in their biogenesis as well as in their cargo. Functional tests reveal that enriched uPA exosomal cargo is stimulating macrophages towards a mixed M1 and M2 phenotype inducing the release of pro-and anti-inflammatory signals that are characteristic of periprosthetic osteolysis. Interestingly, uPA may be proposed, in the future, as a possible candidate biomarker to early diagnose particle induced periprosthetic osteolysis, since uPA was also detected in the pseudocapsular interface around implants of patients with loosening of total hip prosthesis and joint replacement surgery, suggesting their active role in disease progression.

Although several studies assess the biological effects of micro and titanium dioxide nanoparticles (TiO2 NPs), the literature shows controversial results regarding their effect on bone cell behavior. Studies on the effects of nanoparticles on mammalian cells on two-dimensional (2D) cell cultures display several disadvantages, such as changes in cell morphology, function, and metabolism and fewer cell–cell contacts. This highlights the need to explore the effects of TiO2 NPs in more complex 3D environments, to better mimic the bone microenvironment. This study aims to compare the differentiation and mineralized matrix production of human osteoblasts SAOS-2 in a monolayer or 3D models after exposure to different concentrations of TiO2 NPs. Nanoparticles were characterized, and their internalization and effects on the SAOS-2 monolayer and 3D spheroid cells were evaluated with morphological analysis. The mineralization of human osteoblasts upon exposure to TiO2 NPs was evaluated by alizarin red staining, demonstrating a dose-dependent increase in mineralized matrix in human primary osteoblasts and SAOS-2 both in the monolayer and 3D models. Furthermore, our results reveal that, after high exposure to TiO2 NPs, the dose-dependent increase in the bone mineralized matrix in the 3D cells model is higher than in the 2D culture, showing a promising model to test the effect on bone osteointegration.