Nanoplastic impact on bone microenvironment: A snapshot from murine bone cells

. This study aimed to assess if NPs influence skeleton remodeling through OS. Murine bone cell cultures (MC3T3-E1 preosteoblasts, MLOY-4 osteocyte-like cells, and RAW264.7 pre-osteoclasts) were used to test the NPs detrimental effects on bone cells. NPs affect cell viability and induce ROS production and apoptosis (by caspase 3/7 activation) in pre-osteoblasts, osteocytes, and pre-osteoclasts. NPs impair the migration capability of pre-osteoblasts and potentiate the osteoclastogenesis of preosteoclasts. NPs affected the expression of genes related to inflammatory and osteoblastogenic pathways in pre-osteoblasts and osteocytes, related to the osteoclastogenic commitment of pre-osteoclasts. A better understanding of the impact

Our world is made of plastic.Plastic waste deeply affects our health entering the food chain.The degradation and/or fragmentation of plastics due to weathering processes result in the generation of nanoplastics (NPs).Only a few studies tested NPs effects on human health.NPs toxic actions are, in part, mediated by oxidative stress (OS) that, among its effects, affects bone remodeling.This study aimed to assess if NPs influence skeleton remodeling through OS.Murine bone cell cultures (MC3T3-E1 preosteoblasts, MLOY-4 osteocyte-like cells, and RAW264.7 pre-osteoclasts) were used to test the NPs detrimental effects on bone cells.NPs affect cell viability and induce ROS production and apoptosis (by caspase 3/7 activation) in pre-osteoblasts, osteocytes, and pre-osteoclasts.NPs impair the migration capability of pre-osteoblasts and potentiate the osteoclastogenesis of preosteoclasts.NPs affected the expression of genes related to inflammatory and osteoblastogenic pathways in pre-osteoblasts and osteocytes, related to the osteoclastogenic commitment of pre-osteoclasts.A better understanding of the impact

Introduction
Plastics are synthetic or semisynthetic polymers with thermoplastic or thermostable properties.The production of plastics is increasing year by year, starting either from fossil or renewable sources.Plastic items are an active part of our everyday life, and for this reason, the production of these materials increased by 660% between 1976 and 2016, reaching 300 million tons (PlasticsEurope, 2022).The massive demand, production, and use of plastics, coupled with their inappropriate end-life management and disposal, have resulted in the contamination of both aquatic and terrestrial ecosystems worldwide [1].For instance, in 2019, 25.9 Mt of plastic waste was generated In European countries (Plas-ticsEurope, 2022), and most of them accumulated in the environment [2].
The degradation and/or fragmentation of plastics due to weathering processes result in the generation of microplastics (MPs) and nanoplastics (NPs) [3].Because of their size, MPs and NPs enter the food chain, potentially threatening human health (European Food Safety Authority (2017).Moreover, some recent studies have demonstrated that inhalation represents a crucial exposure pathway for MPs and NPs in humans [4].
A plastic particle can be considered "nano" if at least two of its dimensions are less than 100 nm [5], but a shared definition of NPs is still lacking [6].Existing wastewater treatment processes fail to remove NPs [7].The breakdown of larger plastic items, such as the fragmentation of synthetic fibers during clothes washing [8], is an additional source of MPs and NPs [9].NPs may be carriers of chemical and biological contaminants, thus increasing their transport and uptake by organisms and further amplifying their toxicity [10].
MPs and NPs from synthetic textile fibers can persist up to five years in the air and can be transported by the wind from clothes drying in the open or numerous other sources; hence, inhalation is an important entry route for MPs and NPs which are potentially resuspended in the atmosphere and easily transported in the wind due to their low density and small size [4,11].In addition, the current pandemic associated with COVID-19 and the wearing of single-use face masks is leading to the release of large quantities of polypropylene MPs with consequences for marine and terrestrial ecosystems and potential adverse effects on human health [12].
A recent study demonstrated the presence of NPs composed of polyethylene terephthalate, polystyrene, or polyethylene in the blood of human donors with a mean concentration of 1.6 µg total plastic particles/ml blood sample [13] and, also, for MPs, in human placenta samples [14].Once NPs enter the human body, they are uptaken by cells through different routes (i.e.phagocytosis, endocytosis) [15].NPs also penetrate bone marrow [16,17], disrupting hemopoietic activity and possibly the bone microenvironment.In vitro and in vivo studies indicated that NPs could severely impact the human body, including physical stress and damage, cell death, inflammation, and immune responses [18].The environmental impact of NPs is an increasingly worrying problem for human health due to the large-scale growth of plastic production and its resistance to degradation.
Studies focused on the toxicity of NPs showed that they induce reactive oxygen species (ROS) overproduction and oxidative stress (OS) in experimental animal models [2,19,20], and human cell lines [21].
Among the negative impacts of ROS overproduction, the disruption of skeleton integrity is a major effect, particularly during aging [22].In physiological conditions (naive), ROS affects the balance of critical processes in bone remodelling (resorption and deposition) [23].Indeed, previous studies showed that OS resulting from excessive levels of ROS or dysfunction of antioxidant defence is involved in osteoporosis [24].In mice, it has been reported that increased cellular OS induces low-turnover osteopenia [25] and that bone mass progressively declines in association with decreased antioxidant enzyme levels [26].
In a recent paper from our laboratory, ROS increase has been identified as a common mechanism of sex steroid deficiency and aging in bone homeostasis [27].Moreover, sex steroid deficiency may accelerate the effects of aging on skeletal involution, partially mediated by epigenetic mechanism [27].As NPs are considered potential inductors of oxidative stress, exposure to NPs could affect bone cell activities, thus disrupting bone accrual during childhood and increasing bone loss during aging.
A better understanding of the impact of NPs on bone cell activities could give the necessary information on the possible consequence of NPs-mediated toxicity on bone turnover and subsequent human health problems, including bone disease.
Although several invivo and invitro studies confirmed that the internalization of NPs results in the onset of an oxidative stress condition, coupled with the induction of apoptosis and the modulation of inflammatory markers, to the best of our knowledge, no studies have investigated the role of NPs in the bone microenvironment, which remains an unexplored research field.Thus, the present study aimed at investigating if NPs can induce an impact on healthy bone cells.In detail, we explored whether NPs may influence skeleton remodeling through OS, testing our hypothesis on bone cells (including cell cultures representing preosteoblasts, osteocyte-like cells, and pre-osteoclasts).Cell viability, apoptosis level, cellular differentiation, and transcriptomic profile were investigated after NPs exposure.Considering the consistency in the outcomes of previous experiments investigating the effects of NPs exposure on different cell types, we expect that bone cell can internalize NPs and experience an oxidative stress condition.

NPs treatment
Fluorescent, spherical polystyrene NPs (diameter 50 nm, Excitation/ emission peaks of 468/508 nm (Green), 542/612 nm (Red), and 365/ 447 nm, aqueous solution 1% solids) were purchased by ThermoFisher Scientific.NPs were used following the manufacturer instruction.The range of treatments was from 1 µg/ml to 200 µg/ml, and the time of the cell exposure was from 4 h to 96 h, depending on the experimental plan.NPs were used to treat MC3T3-E1, MLOY-4, RAW264.7.For uptake experiments NPs green were used at 1-200 µg/ml for 48 h.For immunofluorescence experiments NPs red were used at 100 µg/ml, for 48 h.For cell viability, Nitrite assay, NPs green were used at 1-200 µg/ml for 48 h.For Caspase activity and ROS production NPs red ere used at 1-200 µg/ml for 48 h.For the scratch assay NPs green were used at 10-100 µg/ml for 48 h.For the osteoclastogenic assay NPs green were used at 10 µg/ml for 48 h.For the transcriptomic analysis NPs green were used at 100 µg/ml for 48 h.

Cas3-7 enzymatic activity (MC3T3-E1, MLOY-4, RAW264.7 -NPs 1-200 µg/ml, 48 h).
The Caspase 3-7 activation, measured by using the Casp3/Casp7 Apoptosis Kit (Abnova kit), was evaluated as it is considered a reliable indicator of cell apoptosis.We followed the instruction of protocol manufacturer.Briefly, we seeded 5000 cells for well in 96 multiwell.To test the Caspase activity, the half of the medium of each well was replaced with 100 µl of caspase 3/7 substrate mixed with Assay buffer.The plate was incubated at 37 • C for 1 h in the dark.The plate was read with a fluorescence microplate reader at Ex/Em = 490/525 nm (ENSIGHT, Perkin Elmer).

Nitrite analysis (MC3T3-E1, MLOY-4, RAW264.7 -NPs 1-200 µg/ml, 48 h).
Nitrite production was measured as a marker of nitrosative stress to investigate the contribution of both the radical species inducing oxidative stress.Nitrite assay was performed following manufacturer's protocol.Briefly, a standard curve was prepared starting from the stock solution of sodium nitrite 0,1 M. Cells were seeded in 96well plates and cultured for 24 h 100 µl of supernatants were used for nitrite dosage.We added the Griess reagent previously mixed as reported by the manufacturer protocol (Panreac).Reaction was incubated at 37 • C for 15 min and samples were analyzed in a microplate reader at the absorbance of 550 nm.

Bone activity assay
2.3.3.1.Scratch wound healing assay (MC3T3-E1, NPs 10-100 µg/ml, h).A linear scratch on confluent MC3T3-E1 cells was performed by using a sterile tip.We removed the cellular debris by washing with PBS, and then we incubated the cells in DMEM 1% FBS alone or in the presence of NPs for 48 h (see [32] for reference).Images were acquired immediately after the scratch (t0) and at various times after treatment (t24, and t48) using an Olympus U-CMAD3 phase-contrast microscope equipped with a Zeiss Axiocam ICc1 camera at a 4x magnification.As previously reported, the images were analyzed by using ImageJ software (NIH, USA) [33].The covered area percentage was calculated for each experimental group by measuring the wound size at different times from treatment compared with the initial (t0) wound size considered as 100%.

Osteoclastogenic differentiation assay (RAW264.7, NPs 10 µg/ml, 48 h).
As shown in our previous paper [34], RAW264.7 cells were seeded in a 24-well plate in 250 µl of DMEM supplemented with 10% FBS at a density of 2,5 × 104 cells/ well with or without 30 ng/ml RANKL and with or without 10 µg/ml NPs.The exposure with NPs lasted 48 h.After 7 days, RAW264.7 cells were fixed on the culture plates with citrate-acetone solution and stained for tartrate resistant acid phosphatase (TRAP kit, Sigma-Aldrich).Osteoclasts were identified and counted under light microscopy considering TRAP positive cells the ones with ≥ 3 nuclei.Representative images of TRAP positive osteoclasts were acquired with Olympus U-CMAD3 phase-contrast microscope equipped with a Zeiss Axiocam ICc1 camera at 4x magnification.
For the PCR Prime Arrays we used the PCR arrays commercially available from SAB (Qiagen) and from Biorad.Following the instruction, we extracted the total mRNA using Biorad Mini kit.The quality of RNA was evaluated with electrophoresis.The samples were read at the spectrophotometer.Total RNA (1 μg) was retrotranscribed using iScript Advanced cDNA kit from Biorad.For the PRIME PCR we analyzed 10 μg of cDNA for well.We used both negative and positive control for PCR, as requested by the assay.The reaction was carried on QuantStudio Fast (Thermofisher Scientific) using 20 μl of the total volume for each well.
Q-PCR was run following the manufacturer protocol (Biorad).

Statistical analysis
We used GraphPad Prism8 software (GraphPad Software, USA) for all statistical analyses.The results are expressed as the mean ± SEM of at least 3 independent experiments (6 replicates for each experiment).Differences between groups were evaluated by one-way or two-way ANOVA followed by Bonferroni post-hoc test.Significance was set up at p-value lower than 0.05.

Results
To assess the effect of NPs on biological functions of bone cells, we used murine bone cell cultures (i.e., MC3T3-E1 preosteoblasts, osteocyte-like MLOY-4 cells, and RAW264.7 pre-osteoclasts) as experimental models.These cell lines were treated in a time course from 24 to 96 h with increasing concentrations of fluorescent, spherical polystyrene NPs (diameter 50 nm, concentration range 0.1-200 µg/ml).We tested cell viability, ROS level, and apoptosis by MTT, DCF, and fluorometric assay, respectively.Cellular morphology, as well as NPs localization and uptake, were also assessed.To investigate the effect of NPs on the bone remodeling process, we analyzed changes in the migration capability of MC3T3-E1 cells and the osteoclastogenic potential of RAW264.7 cells.Lastly, the transcriptomic profile of MC3T3-E1, MLOY-4, and RAW264.7 cells was evaluated.

Nanoplastics Localization and Uptake
The three cell lines were exposed to increasing NPs concentrations (1-200 µg/ml) according to the concentration range already used in in vitro studies [35].The cellular uptake of NPs was evaluated by exposing for 4 h the cell lines to increasing concentrations of polystyrene NPs emitting fluorescence at 508 nm when excited at 468 nm.The internalization was analyzed at 37 • and at 4 • C to verify the inhibition of uptake by flow cytometry in the FITC channel.For each cell line, we show the MFI (mean fluorescence intensity) in the panels A (Fig. 1) and the SSC (side scatter channel) parameters in the panels B (Fig. 1).The MFI indicates the shift of fluorescence of the cells positive for NPs, and SSC is an index of the complexity degree and granularity of the cell surface.In Fig. 1, we observed that in MC3T3-E1 cells, the MFI increases in a dose-dependent manner, indicating that the higher the concentration of NPs, the higher uptake of NPs in the cells.Additionally, the significant decrease of MFI at 4 • C compared to 37 • C suggests that even though a passive uptake is present at high NPs concentrations, NPs route of access in these cells is at least partly controlled by the cell membrane.Finally, upon cell uptake, a slight change in cell complexity was observed by analyzing the SSC.In the osteocyte-like MLOY-4 cells, NPs uptake occurred with a similar trend, although no changes in SSC were associated with NPs exposure.Differently, the monocyte cell line RAW264.7 cells, displayed an increased control of NPs uptake with a more linear uptake of NPs at 37 • C and 4 • C. Also, the SSC parameter indicated a more marked change in cell complexity.
NPs localization within the cellular compartment was confirmed by microscope immunofluorescence after 48 h-exposure to 100 μg /ml polystyrene.In Fig. 2, results for MC3T3-E1, MLOY-4, and RAW264.7 cells are reported.The fluorescent signal associated with NPs was localized in the cytoplasm.

Nanoplastic effect on cell viability, apoptosis, and ROS production
To assess NP effect on cytotoxicity, we evaluated the cell viability by MTT assay, the caspase-3 activity related to the induction of the apoptotic process, and the ROS and nitrite production.The three cell lines were exposed to increasing NPs concentrations (from 1 to 200 µg/ ml) according to concentration range already used in in-vitro studies [35].The most effective time of exposure, 48 h, was set up with a time course (data not shown).As shown in Fig. 3A, the outcome on cell viability is different for the three cell lines.In particular, NPs caused a similar effect on mesenchymal cells (preosteoblasts and osteocytes), while it had a different action on macrophage-derived cells (preosteoclasts).NPs at the concentration of 100 µg/ml significantly reduced cell viability of MC3T3-E1 cells (around 20%), and caused a similar trend in MLOY-4 cells, even if more intense at the concentration of NPs of 200 µg/ml which induced the reduction of MLOY-4 cell viability around 35% compared to controls.RAW264.7 cells resulted to be more sensitive to NPs, indeed a statistically significant reduction of the cellular viability was observed at 10 µg/ml and an approximate decrease of 50% at the concentration of 200 µg/ml NPs.To assess if the effect of NPs on cell viability could be ascribed to an increase in apoptosis, we evaluated the apoptotic process through the enzymatic activity of caspase 3/7 after 48 h of NPs exposure.The results reported in Fig. 3B indicate that NPs induce the caspase 3/7 enzymatic activity with both concentrations, 100 and 200 µg/ml.
The assessment of radical oxygen production by DCFHDA assay and nitrite levels by Griess assay after 48 h of NPs exposure is reported in Fig. 4A.The two mesenchymal cell types (MC3T3-E1 and MLOY-4 cells) displayed similar ROS production, with a slight, but significant, increase (around 10%− 15) at the higher NPs concentrations.On the contrary, as reported in Fig. 4B, in MC3T3-E1 cells, NPs induced nitrite production at a value around 4 µM, while MLOY-4 cells did not produce nitrites.The effects of NPs on RAW264.7 cells are more significant than those in MC3T3-E1 cells.RAW264.7 cells exhibit an increase of ROS production of 40% at NPs concentration of 200 µg/ml and an enhancement of nitrite release of ca. 15 µM, which is more than double in comparison to the control group.

Nanoplastics effect on bone cell biology
Osteoblast migration represents a key process in the physiological control of bone metabolism, including responses to mechanical loading [32].As reported in our previous paper, impairment of the migratory potential of osteoblastic cells could also play a role in bone disease onset [27].For this reason, we analysed the NPs effect on the migratory capability of MC3T3-E1 cells by using the scratch assay.As reported in Fig. 5, a pre-treatment of 48 h with NPs reduced the wound healing capability of MC3T3-E1 cells, after 24 h from scratch.This negative effect was observed only at the highest concentration of NPs (100 µg/ml).
As osteoclastogenesis is a fundamental prerequisite for bone remodelling, we investigated the role of NPs on osteoclast differentiation.For this purpose, tartrate-resistant acid phosphatase (TRAP) analysis was performed after a RANK-L-induced differentiation and exposure to 50 µg/ml of NPs.After 7 days of differentiation, TRAP + cells with  more than 3 nuclei were counted.As shown in Fig. 6, in the presence of a differentiation factor such as RANKL, NPs displayed an osteoclastogenic activity, increasing the number of osteoclasts as well as their size.

Transcriptomic Profile
To complete the landscape of the effect of NPs on bone cells, we performed a transcriptomic analysis on MC3T3-E1, MLOY-4, and RAW264.7 cells exposed to NPs (100 µg/ml) for 48 h by RT-PCR focusing on the profile of 88 genes involved in bone remodelling using a commercial PCR array for 88 genes putative involved in osteoporosis pathway.
Fig. 7 shows the profile of the genes affected after exposure to NPs in MC3T3-E1 cells.In detail, NPs significantly reduced the mRNA expression of the following genes HSD11b1, TNFRSF11b, and IGF1.Moreover, a decrease of mRNA expression of IGFBP2 coupled with an increase of IL-15 was observed, although not statistically significant.HSD11b1 gene codes for hydroxysteroid 11-β dehydrogenase-1 enzyme, which is involved in converting cortisol into the inactive metabolite cortisone (Fig. 7A).TNFSR11B, also known as osteoprotegerin, is involved in inhibiting osteoclastogenesis (Fig. 7B).IGF1 encodes for the insulin-like growth factor 1, a critical mediator of bone growth (Fig. 7C).IGFBP2 encodes for Insulin-like growth factor binding protein 2, crucial for acquiring correct bone mass in mice (Fig. 7D).IL-15 codes for a proinflammatory cytokine involved in osteoclastogenesis (panel E).The observed modulation of IGF1, TNFSR11B1, IGFBP2, and IL-15 is consistent with the decrease of bone deposition and an increase in bone resorption.

Discussion
Our results indicate that NPs affect cell viability, induce ROS production (oxygen and nitrogen reactive species), and caspase 3/7 mediated apoptosis in all the cell lines tested.Moreover, NPs hampered the preosteoblast migration capability, which is directly involved in bone remodeling, and affected the gene expression of osteogenic pathways and related to inflammatory processes in the cell lines we used as biological models.In our opinion, the most relevant effect of NPs emerging from this study is the osteoclastogenic effects on the precursors of preosteoclasts, RAW264.7 cells.Indeed, NPs induced an increase of ROS levels and affected cell viability.Most importantly, NPs induced osteoclastogenesis, stimulating a transcriptomic profile consistent with osteoclastogenic differentiation.
Plastics represent one of our time's most important environmental pollutants.They are used in many activities and objects and strongly influence our daily lives in various ways.At the same time, these materials are also a significant source of environmental pollution to which human organism is exposed, unknowingly [36].However, only a few studies analyze their true accumulation process and the mechanism of their toxicity at the cellular level [37].A common mechanism involved in NPs cellular toxicity is the generation of free radical species.Several studies have highlighted NPs-induced molecular events such as free radical generation, activation of oxidative stress metabolism, lipid peroxidation, DNA damage, and signaling pathway activation.These mechanisms precede branched molecular changes that can lead to irreparable oxidative damage and inflammatory processes [38].ROS are chemically active molecules involved in many biological processes.
When produced in excessive amounts, ROS are known to cause potentially toxic, mutagenic, or carcinogenic oxidative damage.Several  studies have shown that free radicals and ROS can affect the growth and function of osteoblasts and osteocytes [22][23][24][25][26].The balance between the removal of old bone by osteoclasts and the synthesis of new bone by osteoblasts maintains the structural integrity of bone.If this mechanism is disrupted, it leads to various pathologies, such as osteoporosis or diseases that alter the bone environment.
NPs can cross biological membranes and interact with many intracellular sites [39].The literature reports that NPs exposure reduce cell viability, leading to cell membrane instability, deterioration of cell organelles, ROS and nitrite generation, mitochondrial damage, and apoptotic and necrotic processes [40].Our results clearly show that cells from the bone microenvironment can uptake NPs and cystosolic localization of the NPs in cellular compartment and is consistent with a previous study on the bone marrow demonstrating the accumulation of polystyrene MPs [16,17].
Besides cellular uptake, we show that NPs alter bone cells, often with a cell-specific effect.The alteration of the cellular morphology observed in SSC parameters linked to cell granularity, particularly for RAW264.7 cells and not for the others, could be likely related to the different derivation and the role of these cells.RAW264.7 cells are monocyte/ macrophage-like cells originating from Abelson leukaemia virus transformed cell line derived from BALB/c mice.These cells are capable to perform pinocytosis and phagocytosis.Upon LPS stimulation, RAW264.7 cells increase nitric oxide (NO) production and enhance phagocytosis.NPs, inducing inflammatory/OS cues, could increase phagocytosis of NPs themselves, changing the granularity of the cells.Furthermore, the SSC parameter is related to a greater activation and maturation of human mononuclear phagocytes [41].
The reduction of cell viability was directly related to ROS production in the cells of bone component.Our previous studies indicate that osteocytes and osteoblasts are sensitive to ROS production [42].Also, RAW264.7 cells are sensitive to OS [38]: our data clearly indicate a reduction of cell viability in line with the decrease of ROS production observed in RAW264.7 cells.The investigation of the role of NPs in the apoptosis pathway, indicated an increase of the Cas3/7 enzymatic activity in the all the three studied cell lines (MC3T3-E1, RAW264.7,MLOY-4 cells), although the sensitivity of the response is quite different among them.The apoptosis activation appears to be directly related to ROS production since apoptosis represents the main stress-induced cell death [43], so the differences found among the different cellular models is likely related to the difference of stimuli caused by the different ROS production induced by NPs in the three cell lines.
NPs induced an increase in osteoclastogenesis and a reduction of the level of genes involved in bone deposition.In detail, NPs increased differentiation of RAW264.7 cells toward osteoclast cells at the morphological and molecular levels.These could be related to the OS action, which acts toward osteoclastogenesis directions [29,44].
As highlighted in our previous paper [32], the analysis of osteoblast migratory activity could be considered as a new parameter for testing innovative therapeutical strateies for bone disease.We have already put in relation the effect of steroids and nutraceutical compounds in promoting migratory capability of preosteoblast MC3T3-E1 cells by Wnt/beta catenin pathway [27,32].This pathway, crucial for the osteogenesis [45], is disrupted by OS [46].NPs inducing OS affect also migratory capability of MC3T3-E1 cells.
Importantly, we observed an alteration in the transcriptomic profile, related to an induction towards a differentiated osteoclast phenotype in RAW264.7 cells, together to a disruption of the genes related to osteoblastogenesis in MC3T3-E1 cells and on inflammatory condition in the osteocyte-like MLOY-4 cells.The transcriptomic profile analyzed in MC3T3-E1 cells indicates an inhibition of the genes involved in osteoblastogenesis.NPs can affect the crosstalk between osteoblast and osteoclast by reducing OPG level (TNFg1), which is fundamental in controlling bone remodelling [47].NPs are involved in reducing the gene expression of IGF1 and IGF2B, which are critical mediators for bone growth and deposition, respectively [48,49].From the literature, it is known the role of oxidative stress in counteracting osteoblastogenesis [22].We found induction of the gene expression also of IL15, which is a widely expressed pro-inflammatory cytokine.Elevated IL15 receptor alpha (IL15RA) levels are found in the synovial fluid of patients affected by rheumatoid arthritis and other chronic inflammatory diseases that are associated with bone loss [50,51].Moreover, IL15 was shown to activate the osteoclasts mediated by NK cells [52].
The transcriptomic analysis of MLOY-4 cells after NPs exposure show an interesting profile related to the induction of inflammation and related to unhealthy aging.We observed induction of Cnr2 and Cd40, which are both involved in the inflammatory process in the osteocytes [36,37].
In detail, CD40 encodes for a transmembrane glycoprotein of around 48 kDa, belonging to TNSFR superfamily.CD40 activates a pathway involved in the transcription of pro-inflammatory cytokines [36], whereas Cnr2 is involved in the modulation of general inflammatory status [37].In the transcriptomic profile of MLOY-4 cells, it was also found an alteration of Nog gene in common with RAW264.7 cells.Noggin protein can inhibit BMP2 and BMP-4 expression and can counteract bone mass deposition [53].Studies from Sandri's group have highlighted that Noggin is involved in developing unhealthy aging and cachexia, and it is also derived from a reduction of bone deposition [54].
The NPs exposure in RAW264.7 cells shows a transcriptomic profile coherent with activation for these cells in the osteoclast direction.The genes induced by NPs are Acp5, Alox15, Itgb3, Mab21l2, Nog, Npy, Twist1, and Ccln7, all involved in osteoclastogenic pathways.As known, TRAP (tartrate-resistant acid phosphatase) is a key differentiation marker of osteoclastogenesis [55].Alox15 is a lipoxygenase identified as a negative modulator of bone mineral density peak [56].Itgb3 is the subunit b induced in osteoclasts during differentiation [57].Mab21l2 was identified as a novel repressor of BMP-induced transcription [58].As discussed above, Nog is involved in BMP inhibition [54].The neuropeptide Npy is a multifunctional neuropeptide that participates in various physiological and pathological processes and is present in both the nerve system and bone tissue: of note, Npy negatively direct the osteogenesis [59,60].Twist is a transcription factor that educates the metastatic niche in the bone [61] and its induction in osteoclasts could be related to a wider change involving the bone microenvironment.Clcn7 is a chloride channel expressed in the ruffled board of the osteoclasts, and it is a key target gene of bone resorption [62].The fact that all these genes are induced by NPs exposure, indicates that NPs can induce an osteoclastogenic profile in RAW264.7 cells.All data derived from the three cell lines that resemble the bone components indicate an unbalance between bone deposition and resorption caused by NPs exposure in the bone microenvironment.

Conclusions
Our data first showed that NPs can be internalized into different types of murine bone cells, efficiently passing through membranes.Once in the cells, NPs can induce diverse detrimental effects on bone cells by modifying (increasing or inhibiting) specific functions.Specifically, NPs affected cell viability, induced ROS production and triggered the apoptotic process in pre-osteoblasts, osteocytes, and pre-osteoclasts, confirming that the mechanism of toxic action of NPs in cells is mediated by the onset of an oxidative stress condition.Moreover, NPs impaired the migration capability of pre-osteoblasts and potentiated the osteoclastogenesis of preosteoclasts.Finally, NPs exposure affected the gene expression profile of murine bone cell lines, modulating the expression of genes related to inflammatory process and of genes related to the osteoclastogenic commitment of pre-osteoclasts.Despite our findings, further studies are necessary to describe the complex landscape of NP effects on the bone microenvironment.Although our experiments showed that NPs exposure can affect the health status of different murine bone cells treated individually, considering their intimate and reciprocal interactions, assessing the effects of NPs in co-cultured bone cells should be a priority.This approach allows to shed light on the effects on individual cell types subjected to paracrine interactions.Lastly, our experimental design should be replicated in normal human cells having pattern of proliferation/differentiation in culture more representative of the in vivo condition, to understand the link between NPs exposure, bone health and disease.

•
Nanoplastic influence bone remodelling through oxidative stress.• Nanoplastics enter in bone cells affecting their viability.• Nanoplastics potentiate the osteoclastogenesis and impair the osteoblastogenesis • Nanoplastics affect the inflammatory and osteoblastogenic pathways in bone cells A R T I C L E I N F O Editor: Karina S. B. Miglioranza

D
.Giannandrea et al.

Fig. 1 .
Fig. 1.Murine bone cells can uptake nanoplastics in a dose-dependent manner.MC3T3-E1, MLOY-4 and RAW264.7 cell lines were treated for 4 h at 37 • C and 4 • C with increasing concentrations of NPs (1-200 µg/ml) and the uptake was evaluated by flow cytometry analysing the median fluorescence intensity (MFI) in the FITC channel at FACS verse (panel A).For each cell line, changes in cell complexity assessed by the side scatter (SSC) channel parameter analysis were also analysed (panel B).The graphs report the mean ( ± SEM) of at least 3 independent experiments (6 replicates for each experiment).Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test.37 • C vs 4 • C: $ =p < 0.05; $$=p < 0.01.

Fig. 3 .
Fig. 3. Effect of NPs on the cell viability and apoptosis.MC3T3-E1, MLOY-4 and RAW264.7 cell lines were treated for 48 h with increasing concentrations of NPs (1-200 µg/ml).Cell viability was assessed by MTT assay (panel A) and apoptotic activity was performed by the enzymatic activity of caspase 3/7 by using a commercial kit from AbNova (panel B).The graphs report the mean ( ± SEM) of at least 3 independent experiments (6 replicates for each experiment) in which data are reported as the percentage vs control.Statistical analysis was performed by one way ANOVA followed by Bonferroni post-hoc test.Conditions vs control: * =p < 0.05; ** =p < 0.01; *** =p < 0.001.

Fig. 4 .
Fig. 4. NPs affect the ROS production in murine bone cell lines.MC3T3-E1, MLOY-4 and RAW264.7 cells were exposed for 48 h to increasing concentrations of NPs (1-200 µg/ml) and the radical oxygen production was assessed by DCFHDA assay (panel A), while nitrite levels by Griess assay (panel B).The graphs report the mean ( ± SEM) of at least 3 independent experiments (6 replicates for each experiment) showing the data as the percentage vs control.Statistical analysis was performed by ANOVA followed by Bonferroni post-hoc test.Conditions vs control: * =p < 0.05; ** =p < 0.01; *** =p < 0.001.

Fig. 5 .
Fig. 5. NPs affect the migratory ability of MC3T3-E1 cells.The effect of NPs on the migratory ability of MC3T3-E1 cells, treated for 48 h with 10 and 100 µg/ml NPs, was evaluated by a wound healing assay assessed 24 h and 48 hoursafter the scratch (panel A).The graph (panel B) reports the mean ( ± SEM) of at least 3 independent experiments (6 replicates for each experiment) considering the data as the percentage vs control.Statistics analysis was performed by one way ANOVA followed by Bonferroni post-hoc test.Conditions vs control: ** =p < 0.01.Representative images reported above for each condition were acquired with Olympus U-CMAD3 phase-contrast microscope equipped with a Zeiss Axiocam ICc1 camera at a 4x magnification.

Fig. 8 Fig. 6 .
Fig.6.NPs affect the osteoclastogenic ability of RAW264.7 cells.RAW264.7 cells were treated for 7 days with or without 50 µg/ml NPs after a RANK-L-induced differentiation.After 7 days of differentiation, TRAP + cells with ≥ 3 nuclei were identified and counted under light microscopy as TRAP positive cells.Graph shows the mean ( ± SEM) of at least 3 independent experiments (6 replicates for each experiment).Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test.Conditions vs control: ** =p < 0.01; NPs+RANKL vs RANKL: $$=p < 0.01.Representative pictures of TRAP positive osteoclasts were acquired with Olympus U-CMAD3 phase-contrast microscope equipped with a Zeiss Axiocam ICc1 camera at 4x magnification.
pattern of several genes related to osteoclastogenesis (panels A-G), indicating that these cells are susceptible to NPs.In detail, NPs induced the gene expression of ACP5/TRAP, encoding for the enzyme involved in bone matrix degradation and, thereby, a biomarker of active osteoclasts (panel A).As shown in Panel B, NPs induced the gene expression of Alox15, a negative regulator of bone mass acquisition.NPs also increased the gene expression of IgtB3 encoding for the Integrin Subunit Beta 3, a key mediator of osteoclast-bone interaction (panel C).NPs exposure also induced the expression of Mab21l2, encoding for Mab21-like-2 gene, a novel repressor of BMP-induced transcription (panel D).In Panel E, it is possible to observe that Nog, similarly to MLOY-4 cells, is induced by NPs.Panel F shows that NPs induced Npy mRNA, encoding for Neuropeptide Y, a negative osteogenesis regulator.In Fig. 9, panel G shows the gene expression of the Twist1 gene, a gene coding for a transcription factor involved in preparing metastatic niches in tumors, especially in bone districts.NPs induce the mRNA expression of Twist1.Clcn7, a gene coding for chloride voltage-gated channel 7, usually expressed in osteoclast ruffled board and osteopetrosis, is highly induced
• C or 4 • C. The NPs incorporation was evaluated in the FITC channel at the FACS Verse.