Bisphenol A (BPA) is a chemical compound that has been used in commercial plastics and epoxy resins since the 1950s. The widespread use of BPA in manufacturing has led to its ubiquitous presence in industrialized environments (13). The primary method of exposure in humans is through the diet, mainly through drinking water in industrialized regions, as well as from food and drink storage or use containers (14). Specifically, BPA can leach into foods from the plastics or resins used during the manufacturing of such containers (e.g., water bottles, food cans, etc.). Up to 90% of the US population has detectable blood levels of BPA, which has been a growing public health concern (15). Thus far, chronic exposure has been linked to cancer, metabolic disorders, and a range of reproductive and cardiovascular diseases (16, 17).

Bisphenol A functions as an endocrine disruptor through binding to ERα and ERβ with roughly 1/1,000th the affinity of estradiol (18, 19). Because estradiol can attenuate osteoclastogenesis and induce osteoblast differentiation (20, 21), Hwang et al. recently examined the effects of BPA on osteoclast formation and osteoblast differentiation in vitro (22). The authors found that BPA directly inhibited both osteoclastogenesis and osteoblastogenesis, and increased apoptosis in both types of progenitors. BPA suppressed RANK expression in differentiating osteoclasts and RunX2 and Osterix expression in preosteoblasts. Wnt/β-catenin signaling and bone nodule formation was also reduced. These authors demonstrate that BPA promotes apoptosis of osteoclasts and osteoblasts through MAPK cascades and death receptor pathways in a dose-dependent manner (22).

Despite the effects of BPA on osteoblasts and osteoclasts, our search criteria yielded no studies examining BPA-induced chemokine expression changes in osteogenic models. However, given its endocrine-disrupting status, several studies have examined its impact on chemokines in reproductive tissues such as mouse mammary, breast cancer, and ovarian cancer cells (5–8). Significant decreases in CXCL2, CXCL4, CXCL14, and CCL20 have been observed in these various tissues with results that suggest BPA may act through pathways independent of estrogen. Additionally, the related compound, Bisphenol AF (BPAF), has demonstrated upregulation of CXCL12 in T47D ERα-positive cancer cell lines (23). Considering the critical role of the CXCL12/CXCR4 axis on bone regeneration, the known effects of BPA and BPAF on tissues other than bone make clear the importance of evaluating their effects in the context of bone healing.

Phthalate esters comprise another group of EDCs that are used to increase the flexibility of polymers, such as polyvinyl chloride (PVC)-based products (e.g., toys, food wraps, medical devices, and flooring). Similar to BPA, humans are exposed to phthalates through ingestion, inhalation, and physical contact on a daily basis (24, 25). Two particular phthalates, di-(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DINP), have been examined in the context of the chemokine expression and bone formation (10, 11).

DEHP is cytotoxic to neonatal rat calvarial osteoblasts at high doses (1,000 µM), while inducing proliferation at low doses (10 µM) (26). In that system, osteogenic differentiation was inhibited at high doses of DEHP, with reduced RunX2 and ALP expression and decreased collagen and mineralization staining (26).

Inflammation is an important early step in the bone healing cascade. Chemokines play a critical role in recruitment of macrophages and progenitor cells to the site of injury, where they remove necrotic tissue and initiate the regenerative process. However, this process is tightly regulated, and chronic inflammatory diseases are associated with systemic bone loss (27). Even subclinical inflammation has been shown to increase fracture risk and alter bone remodeling (28). Nishioka et al. examined the effects of DEHP on the production of inflammatory cytokines in activated macrophage-like THP-1 cells. A 3-h exposure to 200 µM DEHP significantly induced CXCL1, CXCL2, CXCL3, CXCL6, and CCL3 transcripts (10). PTH induces CXCL1 expression in osteoblasts, which then attracts osteoclasts via CXCR2 (29). Interestingly, CXCL1 acts as a chemoattractant for osteoclast precursors without promoting osteoclastic differentiation, acting as a “primer” for additional factors (29). That said, it is reasonable to speculate that increased CXCL1 expression by macrophages recruited during bone repair could also prove chemotactic for osteoclasts. Further research should examine whether DEHP influences CXCL1 secretion in osteoblasts in a similar manner.

Human osteoblasts express CCL2, which is an important chemoattractant for monocytes and macrophages (30). Additionally, CCL2 is critical for osteoclastogenesis, and its absence results in inhibition of osteoclast formation (31). In studies examining the hypothalami of DEHP- and DINP-treated male mice, Win-Shwe et al. observed decreases in CCL2 and TNF-α expression in the DINP-exposed groups (11). Considering the important role that CCL2 plays on osteoclastogenesis, further investigation to the effects of DINP in bone models should be explored.

Perfluoroalkyl substances have been widely used as protective coatings in water- and stain-resistant clothing, furnishings, and non-stick home goods for over 60 years (32). These chemicals have been classified as EDCs based on their hormonal and metabolic actions (33, 34). PFASs are ubiquitous in the environment, and detectable amounts are found in humans worldwide (35). Examination of the U.S. National Health and Nutritional Examination Survey (NHANES) from 1999 to 2008 showed that four PFASs were found in 95% of the U.S. population: perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), and perfluorononanoic acid (PFNA) (36, 37). No research has been conducted to examine the effects of PFASs on chemokine production. However, there is evidence justifying further investigation into the mechanisms of PFAS action in osteogenic models. In a representative sample of the U.S. population collected from the NHANES 2009–2010, serum PFAS levels were inversely correlated with bone mineral density (BMD) of the femur and lumbar spine (32). Most of the associations were limited to women, despite men having higher serum PFAS levels. In general, postmenopausal women had stronger associations with lower BMD than their premenopausal counterparts. Osteoporosis was associated with exposure to PFOA, PFNA, and PFHxS in women (32).

Organotins are environmental contaminants considered to be obesogens because they activate peroxisome proliferator-activated receptor γ (PPARγ), the primary regulator of adipogenesis (38, 39). These compounds are broadly used as pesticides, catalytic agents, plastic stabilizers, and antifouling agents (40). The actions of organotins on skeletal development have been examined in animal models (41, 42). Tsukamoto et al. demonstrated that mouse fetuses exposed to tributyltin chloride (TBT) showed reduced calcification of the supraoccipital bone (41). In vitro, TBT dose-dependently inhibited differentiation of primary rat calvarial osteoblasts, with reduced ALP activity, mineral deposition rate, and osteocalcin expression levels.

Despite these data, few studies have been published examining the effects of organotins on chemokine expression. Schutte et al. examined chemokine expression after implanting an organotin-stabilized PVC cage implant in Sprague-Dawley rats (43). They demonstrated a long-term (56 days postimplantation) 10-fold increase of CCL3, a pro-inflammatory chemokine responsible for increasing osteoclast motility (44). CCL2 demonstrated a significant increase on postoperative day 1, but then returned to normal levels. Given the endocrine activity associated with these compounds and their known effects on skeletal development, the mechanisms by which this class of compounds affects osteogenesis should be clarified.

Benzene is a colorless, flammable organic liquid that can volatize to vapors at room temperatures. Used as an industrial chemical in the manufacturing of other compounds, it is also a component of crude oil, gasoline, and cigarette smoke (57, 58). Benzene was among the first compounds identified as a Group I known human carcinogen by the International Agency for Research on Cancer (59). The role of benzene in the development of acute myeloid leukemia and other myelodysplastic syndromes is well established (60–63). Human exposure to benzene occurs through inhalation and dermal absorption as well as ingestion of contaminated food and water (64). It is widely agreed that the toxicity of benzene results from its metabolism to reactive intermediates. In the liver, benzene is metabolized by CYP2E1 to phenol, which undergoes hydroxylation to hydroquinone, catechol, and 1,2,4-benzenetriol (65). Catechol and hydroquinone persist in bone marrow, where they are oxidized to 1,2-benzoquinone and 1,4-benzoquinone by myeloperoxidase (54, 66). The mechanism(s) by which these metabolites influence carcinogenesis have not been fully clarified (67).

While the effects of benzene and its metabolites on the immune and hematopoietic systems are well established, the influence of these compounds on bone development and skeletal remodeling is less fully understood. Zolghadr et al. examined the effects of benzene, hydroquinone, and 1,4-benzoquinone on human-derived mesenchymal stem cells (MSCs) (53). They examined cell viability, apoptosis, and expression of genes relevant to hematopoiesis and skeletal remodeling. At the lowest concentrations tested, all three compounds increased cell division rate after only 24 h, and the effects were attributed to cell cycle control defects (68). Interestingly, RunX2 expression was significantly upregulated (up to eightfold) by all three chemicals, as was Wnt5a, which is a non-canonical Wnt ligand. Simultaneously, Dkk1 expression was induced, which inhibits canonical Wnt signaling by interacting with the Wnt co-receptors, LRP-5/6. The authors hypothesized that the increase in Dkk expression was a response to induction of RunX2 and canonical Wnt signaling.

CXCL12, along with its receptor, CXCR4, plays a critical role in mesenchymal and hematopoietic stem cell recruitment, as well as BMP-mediated osteoblastic differentiation (69, 70). A major regulator of BMSC growth, CXCL12 expression is thought to decrease acutely after osteoblastic lineage commitment (71, 72), suggesting that its role is critical in the early stages of osteogenesis (69). In human MSC, CXCL12 expression was upregulated after short-term (1 h) exposure to 1,4-benzoquinone, but was downregulated after 24 h exposure to hydroquinone. The dysregulation of the Wnt and CXCL12 signaling pathways could have pronounced implications on bone regeneration. Further research needs to be conducted to define the upstream mechanisms regulating the effects of benzene metabolites on the CXCL12/CXCR4 axis.

Chlorobenzene is one of the most widely used chlorinated benzenes (55). It functions as a solvent for many substances, such as paints, adhesives, waxes, and polishes, and it is also commonly used in the dry-cleaning industry (73). Chlorobenzene inhalation can lead to irritation of the eyes and respiratory tract, drowsiness, loss of coordination, CNS depression, and loss of consciousness (73, 74). Additionally, epidemiological studies suggest that exposure to chlorobenzene is associated with allergic sensitizations and Th2-primed T cell immunity (75).

Chlorobenzene has been shown to induce CCL2, a chemokine critical for osteoclast formation, production at indoor-relevant concentrations in TNF-α-primed alveolar A549 cells (55, 56). Additional research has shown that the mechanism through which chlorobenzene induces CCL2 is through the NF-kB and MAPK pathways (76). Despite the CCL2 connection, however, no studies have attempted to correlate exposure with impaired osteoclastic differentiation, osteogenesis, or bone healing.

Similar to chlorobenzene, m-Xylene and styrene have been shown to induce CCL2 production, albeit in A549 cells. Still, dose-dependent alterations in osteoclast precursor recruitment and differentiation are worth investigating, which could lead to impaired bone remodeling and healing.

Fischader et al. additionally examined the effects of several aliphatic compounds (n-non-ane, n-decane, n-undecan, n-dodecane, n-tridecane, and methylcyclopentane) on A549 cells (56). Neither the single aliphatic compounds nor a mixture of the group demonstrated any effect on cytokine/chemokine release.

The most toxic and well-studied dioxin and AhR ligand is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD was first shown to downregulate CXCL12 and CXCR4 expression in breast and ovarian cancer cells (23). A study conducted by Casado et al. demonstrated diminished CXCL12 migration of LSK cells exposed to TCDD with increased cell surface expression of CXCR4 (80). However, levels of CXCR4 mRNA were not induced, suggesting that AhR activation resulted in either upregulation of posttranslation modification or downregulation of degradation of the receptor.

Uncleaved osteopontin (OPN) is associated with cell adhesion, facilitating osteoclast attachment to bone matrix (105). On the other hand, cleaved OPN is chemotactic for neutrophils and leukocytes. OPN knockout bone marrow cells showed reduced migratory capacity in vivo (80), and antibody blockade of the binding domain reduced cell migration as well as PTH-induced osteoclast bone resorption (106). TCDD exposure reproduced the effects of antibody blockade, with a similar inhibition of OPN-induced cell migration (80). Our group has shown that TCDD inhibits BMP-2-mediated bone regeneration and spine fusion in the rat, with only a partial recovery of fusion capacity after cessation of exposure for a period of four half-lives (T_1/2 for TCDD is 19 days in the rat) (89).

Vogel et al. demonstrated upregulation of CCL2 in the liver, thymus, kidney, adipose tissue, and heart of C57/BL6 mice injected with TCDD (107). Other in vitro studies in cell lines of various sources also suggest a direct correlation between CCL2 and TCDD exposure. Upregulation of CCL1, CXCL1, CXCL13, and CXCL8 (IL-8) has been observed as well (91, 108–110), while CCL5 was downregulated in mouse CD4+ T-cells ex vivo (111). The induction of IL-8 has been proposed to be as a result of NF-kB cross-talk through RelB activation (90, 91, 112). The upregulation of IL-8 and CCL2 by TCDD corresponds with findings of decreased expression in AhR-KO mice spleen (96). Notably, there was a 15-fold increase in CCR2 (the receptor for CCL2) expression in AhR-KO mice. These mice also demonstrated a >2-fold increase in CXCR5 and CCL20 (96). This supports the notion that the hyperactivated AhR may act as a negative regulator of BMSC proliferation and trafficking.

Dioxin-like compounds include other polycyclic aromatic hydrocarbons, such as Benzo[a]pyrene (BaP), polychlorinated dibenzofurans, and non-ortho-substituted or mono-ortho-substituted pentachlorobiphenyls (PCBs). Scant evidence can be found on the effects of these dioxin-like compounds on bone healing or any associated chemokine alterations. PCB-118 (2,3′,4,4′,5-pentachlorobiphenyl) exposure correlates with lower BMD (84) and induction of osteoclastic activity in vivo (113). No studies have found any associations with chemokine expression changes and PCB-118. Three additional environmental dioxin-like toxicants, PCB-126 (3,3′,4,4′,5-pentachlorobiphenyl), PCB-77 (3,3′,4,4′-tetrachlorobiphenyl), and BaP, have been shown to upregulate expression of CCL2 and CXCL13 in both in vitro and in vivo models, the latter of which contributes to osteoblast development (114–119). BaP has also been shown to reduce CCL5/RANTES mRNA expression during osteoclast formation in human keratinocytes (118). Considering that CCL5 promotes osteoclast development and chemotaxis (44), further work is needed to identify any direct effects of BaP on CCL5 expression in developing osteoclasts. Table 3 reviews the changes in bone repair chemokines by TCDD and the other dioxin-like compounds found in current literature.

We previously found that TCDD exposure inhibits spine fusion in rats (89). Subsequent work has shown that similar to breast and ovarian cancer cells, TCDD downregulates CXCL12 and CXCR4 expression in differentiating primary rat BMSC (our own unpublished data and Figures 1A,B). Concordant with this, BMSC chemotaxis toward CXCL12 was also reduced after TCDD treatment (unpublished data and Figure 1C). Since no such studies have investigated the effects of dioxin-like compounds on the CXCR4/CXCL12 axis, we performed similar work to examine the effects of these compounds on CXCL12 and CXCR4 expression in primary rat BMSC grown in both standard and osteogenic conditions.

A select group of dioxins and dioxin-like compounds were chosen based on representative classification (dioxin, furan, or PCB) as well as the compound’s toxic equivalence within its class according to the World Health Organization (120). Northwestern University Institutional Animal Care and Use Committee (IACUC) approval was obtained prior to animal procedures and bone marrow stromal cell harvest. All experimental procedures were conducted in accordance with the recommendations of the IACUC. Rat BMSC were treated under standard or osteogenic conditions with various concentrations determined to activate the Ahr without inducing cell death (Table S1 in Supplementary Material). Total RNA was isolated using TRIzol (Invitrogen) and mRNA expression of CXCL12 and CXCR4 were analyzed using qPCR (Figures 1A,B). For chemotaxis assays, cells were counted by three blinded, independent reviewers (Figure 1C). Groups were compared using ANOVA and unpaired t-tests post hoc, with significance of p < 0.05.

CXCR4 expression was significantly decreased in primary rat BMSC grown under both standard and osteogenic conditions for all but three of the treatment groups (PCB-118, -126, and -156). PCB-126 showed a significant decrease of CXCR4 expression only under standard conditions. PCB-118 and -156 showed no changes in expression in either conditions. CXCL12 was significantly reduced for all treatment groups in both conditions. Additionally, we evaluated the capacity of BMSC to migrate toward a CXCL12 gradient after treatment with these compounds. Chemotaxis was significantly reduced in cells pretreated with all but PCB-118, which was likely a result of a lack of CXCR4 downregulation by PCB-118. Interestingly however, PCB-156 had no significant impact on CXCR4 expression, yet CXCL12-induced chemotaxis was significantly decreased.

There is a growing appreciation for the deleterious effects of dioxin-like compounds on bone. As the physiologic role of the AhR pathway becomes better understood, so too will AhR-mediated changes in chemotactic signaling. Further research is prudent to better understand the mechanisms of dioxin-induced inhibition of bone healing.

Environmental metal pollution has long been a topic of interest to toxicologists, given the growth of industry and technology, which has led to supraphysiologic metal exposure in humans. Of particular interest are metals that are commonly encountered in developed or developing nations through food, drinking water, air, and soil. Lead, cadmium, strontium, and lithium have all been shown to affect levels of chemokines related to bone healing. Tungsten, arsenic, iron, boron, and mercury have been shown to impact bone homeostasis and healing potential, albeit with lacking evidence of alterations in chemokine expression levels (Table 4).

Aseptic periprosthetic osteolysis is one of the most common causes of long-term prosthetic joint failure. Reports have cited failure rates as high as 56% at 6.5 years postoperative (180–182). Constant friction through the joint surface of metal-on-metal (MoM) and polyethylene-on-metal prostheses generates non-biodegradable particulate debris. In addition to polyethylene particles, metal ions such as titanium (Ti), titanium-6%/aluminum-4%/vanadium (Ti6Al4V) alloy, zirconium (Zr) oxide, Zr alloy, cobalt, cobalt chrome alloy, cobalt nickel chrome alloy, and cobalt chrome molybdenum (CoCrMo) alloy are produced from wear. Elevated concentrations of these metals have been measured in periprosthetic tissue, serum, urine, and in distant organs (liver, lymph nodes, spleen) (183–186).

Metal-on-metal implants became very popular in the late 1980s, with over one million implanted in the US to date (187). Because they are thought to deteriorate more slowly, MoM implants are used increasingly in young patients. However, elevated serum levels of metal ions, adverse reactions to metal debris, aseptic lymphocyte-dominated vasculitis, pseudotumors, and metal hypersensitivity are thought to lead to early loosening (188–199).

Metal-on-metal implants were initially thought to cause an immune reaction solely through the macrophage response; however, recent studies suggest that systemic metal ions, organometallic protein complexes, and particulate debris can also be immunoreactive (200). The mechanisms leading to pathologic bone resorption surrounding MoM implants are poorly understood. Numerous inflammatory cytokines and chemokines have been shown to be upregulated in peri-implant tissues of these patients, leading to a state of chronic inflammation and osteoclast activation/proliferation (201). Drynda et al. sought to clarify how circulating metal ions affect the CXCL12/CXCR4 axis and determine the impact this may have in BMSC homing/differentiation. Cultures of MG-63 (early-stage) and SaOs-2 (late-stage) osteoblast-like cells were exposed to Cobalt (Co), CoNiCrMo alloy (Nickel containing), or CoCrMo alloy (Nickel free). CXCL12/CXCR4 protein expression was dose-dependently activated in both cell lines with all metal exposures (202). Upregulation of CXCL12 in preosteoclasts has been associated with increased osteoclastic activity in several bone diseases, which may participate in eventual periprosthetic osteolysis (203–205). When AMD3100 was administered to block the interaction of CXCL12 with CXCR4, a partial reduction in TNF-α expression was observed. The same group also recovered periprosthetic tissue from patients undergoing revision for MoM aseptic loosening and found CXCR4 to be upregulated. Others have also reported similar increases in CXCR4 and TNF-α in vivo after exposure to Ti ions (206). Although no direct correlation can be drawn from either of these studies, it is possible that TNF-α may act as a trigger for CXCR4 upregulation.

Ti has been shown to increase CCL17 (TARC), CCL22 (MDC), and CCR4 expression (207). Cadosch et al. found that secretion of pro-inflammatory TNF-α, IL-6, and IL-1a/β was increased after Ti exposure, thereby leading to either direct osteoclast precursor activation or indirect activation through RANKL/M-CSF secretion by osteoblasts. RANKL activation also increased osteoclastic expression of CCL22 (208, 209), which lead to increased recruitment of CCR4⁺ osteoclast precursors. Correspondingly, there was an increase in CCL17 expression in hFOB 1.19 fetal osteoblastic cells and human osteoclasts. CCL17 functions in chemotactic recruitment of osteoclast precursors, likely through NF-kB activation (207).

Recent studies suggest that CCR4 is expressed in both Th2 and Th17 cells, and microscopic analysis of periprosthetic tissue reveals an observed increase in Th17 cell number (210). These periprosthetic Th17 cells may have been recruited and deposited through CCL22+ CCL17/CCR4-mediated chemotaxis and arrest (210). Furthermore, these deposited Th17 cells promote osteolysis through an IL-17-dependent increase in RANKL. Collectively, this is suggestive of a Ti-induced vicious cycle of osteoclastogenesis and inflammation (207).

Interestingly, the majority of Ti particle-induced osteolysis is associated with CXCR2, which is the receptor for IL-8 (211, 212). This receptor can be found on macrophages, osteoclasts, osteoblasts, and neutrophils (213), where ligand binding promotes neutrophil attraction, osteoclastic differentiation, and bone resorption (214). Silencing CXCR2 mRNA reduced Ti-induced bone resorption rates in a mouse calvarial defect model by suppressing osteoclastogenesis indirectly through osteoblastic downregulation of RANKL (215). Several other groups have observed Ti-induced upregulation of CXCL8 (IL-8), CCL5 (RANTES), CCL3 (MIP-1α), and CCL2 (MCP-1) as a function of time-dependent NF-kB activation (216–220). Of these, IL-8 and CCL2 are strong chemoattractants for monocytes/macrophages/osteoclasts and neutrophils (221), which recruit bone-resorbing cells to induce periprosthetic osteolysis. Similar results were found with cobalt exposure in cultured human osteoblasts (222–224). Dalal et al. found that a CoCrMo alloy produced the greatest inflammatory response in comparison to Ti, Zr oxide, or Zr alloy, with a 100-fold increase (>2,000 pg/mL) of IL-8, a 30-fold increase of IL-6, and a 15-fold increase of TNF-α levels (224).

Metal particle-induced osteolysis is largely due to excessive osteoclast/inflammatory cell recruitment, osteoclastic activation, and inhibition of osteoblast activity. This occurs locally through phagocytosis and/or by the metal ions triggering toll-like receptors (214). These processes (is this true?) are mediated primarily by chemokines (225). Thus far, CXCL12/CXCR4, CCL17/CCL22/CCR4, IL-8/CXCR2, CCL5, CCL3, and CCL2 have all been implicated as mediators of prosthetic metal-induced bone destruction (Table 5). More research is needed to identify the chemokines responsible for recruitment of osteoclasts and Th17 cells, which in turn contribute to osteolysis and aseptic loosening. This understanding would provide for targeted approach to early diagnosis and treatment of prosthetic-induced loosening.

Fluoride is found naturally and as an additive in tap water. It is well known for its ability to reduce the rate of dental caries. In low doses, fluoride increases osteoblast proliferation (226) and inhibits osteoclastic bone resorption (226, 227). Some clinical evidence suggests that fluoride may improve BMD (228), although other studies suggest that this more dense bone may in fact be more brittle and prone to fracture (226).

With high dose or prolonged exposure time, skeletal fluorosis and other toxic side effects may ensue. Skeletal fluorosis occurs secondarily to an increase in bone density, through a supraphysiologic increase of the growth of osteophytes. This results in symptoms and disability similar to what is observed in osteoarthritic patients. A safe level of fluoride in drinking water has proven difficult to establish. Many factors, including climate, air temperature, patient age, duration of exposure, and dietary calcium intake may play a role in the variability of doses reported in the literature, as each of these can alter fluoride bioavailability (229, 230). In an attempt to maintain safe doses of fluoride in drinking water, the EPA established and enforces a maximum allowable fluoride concentration of 4 mg/L; however, given recent experimental findings, the EPA now suggests maintaining fluoride levels at less than 2 mg/L to prevent possible deleterious effects.

Rat osteoblasts undergo apoptosis upon exposure to sodium fluoride (NaF) at doses of 500 µM and higher (231). At lower doses however, fluoride has positive effects on Wnt signaling, which leads to upregulation of osteoblastic differentiation makers, including ALP, COL1A1, osteonectin, and RunX2 in rat primary osteoblasts (232). Fluoride was also found to increase phosphorylation and inhibition of GSK-3β, resulting in prolonged activation of the Wnt pathway. Other groups have found COL1A1 and COL1A2 mRNA levels to be induced in rat osteoblasts after 24-h NaF exposure, although by 72 h, expression decreases in a dose-dependent fashion (231).

In addition to its direct effects on osteoblasts, fluoride has also been linked to anti-osteoclastic activity, with reduced cathepsin K, IL-1B, MMP-9, and TRAP activity upon RANKL- and M-CSF-induced osteoclastogenesis (233). In IL-1B-induced gingival inflammation, NaF was strongly anti-inflammatory, downregulating IL-1B, IL-8, and TNF-α expression at doses that did not induce apoptosis (234); however, the effects of fluoride on inflammation appear to depend on route of administration and are tissue specific (235–237). The inflammatory effects for fluoride in the context of bone have not been studied.

Fluoride may play a more direct role in calcium metabolism and bone turnover. In MC3T3E1 preosteoblasts, low-dose fluoride increased free calcium ion in the cell culture supernatant and inhibited PTH and PTHrP expression, with opposite effects on Ca++ and PTHrP at high NaF doses. In vivo, the same group found that NaF reduced serum calcium levels, and the effect of fluoride on PTHrP depended on whether the rats received standard or low calcium diet (238). These results suggest that high fluoride ingestion causes hypocalcemia by upregulating calcitonin through PTH and PTHrP secretion (238). Fluoride treatment also increased RANKL and OPG expression, hypothetically promoting osteoclast activation. Further research regarding chemokine signaling after fluoride exposure may explain the dose-dependent effects of fluoride on skeletal health.

Fungi and molds are common naturally occurring environmental contaminants. Toxic effects related to mold exposure are most commonly caused by mycotoxins and the various VOCs they produce. A study by Hokeness et al. evaluated the effects of two common VOCs ((E)-2-octenal and oct-1-en-3-ol) on BMSC viability (239). Both VOCs were cytotoxic, and the effect was determined to be secondary to alterations to membrane fluidity. The authors postulated that this could lead to breakdown of the immune system, but did not comment on the potential impact this could have on bone. However, membrane fluidity is known to affect osteoblastic differentiation of MSC (240).

The active compound from the mushroom Cordyceps militaris, cordycepin, is used medicinally for anti-inflammatory and chemotherapeutic purposes. Both C. militaris extract (CME) and isolated cordycepin have been shown to reduce RANKL-mediated osteoclastogenesis in vitro. In an in vivo murine model, cordycepin reduced LPS-induced inflammatory bone loss. Further studies will need to be conducted to determine whether cordycepin treatment is suitable in humans for the prevention of bone loss.

Peripheral blood mononuclear cells from mold-exposed workers expressed higher levels of eotaxin, INF-α, IL-1α, IL-12 p40, IL-12 p70, IP-10, PDGF-AA, TNF-β, and VEGF when exposed to a panel of common mold toxins ex vivo relative to cells from control patients (241). Asthmatic patients had a significant difference in chemokine expression, regardless of mold exposure. When exposed to aflatoxin B1 in vitro, polymorphonuclear leukocytes harvested from peripheral blood demonstrated decreased IL-8, CXCL1, and CXCL2 (242). While these studies demonstrate a link between exposure to fungal toxins and chemokines, it is unclear whether this has an impact on bone health. Subsequent research will be necessary to determine if fungal extracts and toxins are related to bone regeneration through chemokine mediation.

Asbestos has a well-established role in inflammation and disease of respiratory tissues (243). Asbestos-derived ROS have been shown to activate TGF-β in lung epithelial tissue (244). CXCR3 levels have been reported to be decreased in CD4+ T-cells, inhibiting chemotaxis and potentially antitumor immunity in patients with asbestos-related lung disease (245). Other systemic markers of inflammation including IgE, IgA, IL-6, IL-8, and ICAM-1 have also been found to be elevated in asbestos-exposed persons (246). Upregulation of gremlin plays a role in asbestos-induced pulmonary fibrosis, which has been linked to decreased BMP and increased TGF-β signaling locally (247). No literature connecting asbestos to BMP, IL-8, or other chemokine levels in the context of bone were found. Further studies will be needed to determine if asbestos affects growth factor signaling in the bone, or has any impact on bone-related chemokines.

While chlorinated compounds have been studied, no research examining the relationship between chlorine itself and bone-related chemokines has been conducted. Chlorinated and fluorinated hydroxyapatite-based biomaterials are currently under development. The addition of chlorine and fluorine to these scaffolds creates an acidic environment that simulates the osteoclastic conditions necessary for bone resorption and remodeling while simultaneously increasing apatite formation in vitro (248). Further studies will be necessary to determine if chlorine impacts bone healing through chemokine modulation, which may enhance or inhibit the osseointegration of chlorinated biomaterials.