Effect of phosphodiester composition in polyphosphoesters on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells

Abstract Osteoporosis is a common bone disorder characterized by reduced bone density and increased risk of fractures. The modulation of bone cell functions, particularly the inhibition of osteoclastic differentiation, plays a crucial role in osteoporosis treatment. Polyphosphoesters (PPEs) have shown the potential in reducing the function of osteoclast cells, but the effect of their chemical structure on osteoclastic differentiation remains largely unexplored. In this study, we evaluated the effect of PPE’s chemical structure on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells (BMNCs). PPEs containing phosphotriester and phosphodiester units at varying compositions were synthesized. Cytotoxicity testing confirmed the biocompatibility of the copolymers at concentrations below 0.5 mg/mL. Isolated from long bones, BMNCs were cultured in a differentiation medium supplemented with different PPE concentrations. Osteoclast formation was assessed through tartrate-resistant acid phosphatase and phalloidin staining. A significant decrease in the size of osteoclast cells formed upon BMNC contact with PPEs was observed, with a more pronounced effect observed at higher PPE concentrations. In addition, an increased composition of phosphodiester units in the PPEs yielded a decreased density of differentiated osteoclasts. Furthermore, real-time PCR analysis of major osteoclastic markers provided gene expression data that correlated with microscopic observations, confirming the effect of phosphodiester units in suppressing osteoclast differentiation of BMNCs from the early stages. These findings highlight the potential of PPEs as polymers are capable of modulating bone cell functions through their chemical structures.


Introduction
Osteoporosis, a prevalent disease in an aging society, is characterized by reduced bone density and increased fracture risk.As the population ages, the incidence and impact of osteoporosis continue to grow, making it a significant health concern.Recent studies have focused on developing reliable and safe drugs for osteoporosis treatment.Although several approved drugs possessing supplemental, antiresorptive, and anabolic activities for treating patients with osteoporosis disease abound [1], some treatment limitations and adverse effects remain a serious concern [2,3], considering the fact that osteoporosis is considered a complication of several diseases such as breast cancer metastasis [4] and diabetes [5].Therefore, developing alternative strategies for designing bone-targeting drugs with multiple efficacies and minimal side effects is now required [6,7].Macromolecular drugs offer the potential for prolonged circulation within the bloodstream while also enabling active targeting of specific tissues through ligand conjugation.In addition, they can accommodate multiple-component loadings.Kopezeck and coworkers successfully delivered anticancer drugs to bone tissue using N-(2-hydroxypropyl methacrylamide) (HPMA) copolymers conjugated with bisphosphonates (BPs) or prostaglandins [8].Drugs and imaging probes can be easily incorporated into this polymer through simple chemical ligations [9].The utilization of an HPMA polymer bearing FITC and TNP-470 resulted in reduced growth and vascularity of primary tumors and metastases in various human tumor xenograft models [10].The authors targeted both tumor and endothelial compartments of bone metastases and calcified neoplasms with a single administration.The HPMA polymer substantially inhibited osteosarcoma growth.Notably, immobilizing BPs onto polymers remains a primary strategy for obtaining bone-targeting macromolecular vehicles [11,12].However, the biological functions of the polymers themselves, beyond their bone affinity, are rarely discussed.
We previously investigated polyphosphoesters (PPEs) as alternative biodegradable and biocompatible polymers, primarily due to their structural similarity to the nucleic acid backbone.Penczek and coworkers extensively studied the synthesis and fundamental properties of PPEs during 1970-1980 [13].Among the various established synthetic routes for PPEs, the ring-opening polymerization of cyclic phosphoesters is one of the most reliable processes for obtaining PPEs [14].Biomedical research on PPEs has been conducted since the 1990s, and early studies have elucidated the usefulness of PPEs as gene carriers and tissue engineering substrates [15,16].PPEs outperform conventional degradable polymers such as aliphatic polyesters in the control of solubility and alterations in molecular structures [17,18].Furthermore, the environmental pH values and chemical structure of the side chains of PPEs can effectively regulate PPE degradation [19].Notably, the incorporation of ribonucleic acid (RNA)-inspired phosphoester linkages into polymers can accelerate their degradation [20,21].
We have previously clarified that PPEs comprising a phosphodiester backbone showed high affinity for hydroxyapatite (HAp) and bone minerals.Altering the composition of phosphodiester units in PPEs could also control mineral affinity strength [22].The selective accumulation of poly(sodium ethylene phosphate) (PEP•Na) in bone tissue was observed when PEP•Na comprising entirely a phosphodiester unit was injected into the tail vein [23].In addition to PPE bone affinity, our recent interests have been directed toward determining the effect of PPEs on the functions of bone cells.The addition of PEP•Na to the differentiation medium for osteoblast cultivation resulted in the upregulation of the expression of osteoblastic differentiation marker genes and mineralization of osteoblasts [24].Polyphosphodiesters (PPDEs) may influence the influx and support the nucleation of calcium ions to form calcium phosphate crystals in osteoblastic cells [25].We also reported that PEP•Na reduced the resorptive function of osteoclast differentiation [26].However, the mechanistic understanding of the effect of PPE's chemical structures on osteoclastic differentiation remains limited.Therefore, we hypothesized that synthesizing various copolymers comprising phosphotriester and phosphodiester units with varying molar ratios and determining the effect of their composition on the differentiation of murine bone marrow mononuclear cells (BMNCs) into osteoclasts would reveal the optimal chemical structure of PPEs to regulate osteoclastic differentiation.Tartrate-resistant acid phosphatase (TRAP) and phalloidin staining assays were used to determine the differentiation of BMNCs.Furthermore, the gene expression of major osteoclastic markers from BMNCs cultured with PPEs was also investigated.This study provides insight into the effect of PPE's chemical structures on osteoclast differentiation, revealing promising information for creating new polymeric drugs for osteoporosis treatment.Louis, MO, USA) and used without purification.TRAP staining kit was purchased from Sigma-Aldrich Co. (St.Louis, MO, USA) and used following the manufacturer's protocol.Cytoskeleton Inc. (Denver, CO, USA) supplied fluorescence-labeled phalloidin (Acti-Stain TM 535).Recombinant macrophage colony-stimulating factor (M-CSF) and recombinant receptor activator of nuclear factor-kB ligand (RANKL) were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA).Prostaglandin E 2 (PGE 2 ) was obtained from Cayman Chemical (Ann Arbor, MI, USA).Real-time PCR reagents and primers were obtained from Takara Bio (Otsu, Japan) and Life Technologies Japan Ltd. (Tokyo, Japan).Table S1 lists the primers used in this study.Other chemicals were purchased from Fujifilm Wako Pure Chemical Co.(Osaka, Japan) as extra-pure grades and were used without further purification.Water was purified using a Merk Millipore Simplicity UV system, which involves UV irradiation, ion exchange, activated carbon adsorption, and filtration (18.2 MΩ cm).
PPE copolymers of phosphotriester (T) and phosphodiester (D) units (PT x D y, x: T (mol%) in feed, y: D (mol%) in feed) were synthesized using a modification method previously reported by our group [28].The synthetic route is shown in Scheme 1.Typically, the synthetic procedure for PT 80 D 20 is described.MMP (28.8 mmol) and BzMP (7.2 mmol) were placed into a thoroughly dried 50-mL round-bottom flask equipped with a three-way stopcock.After 30-min drying of the mixture under reduced pressure, 1-propanol (0.48 mmol) was added to the flask and stirred under an argon atmosphere while cooling with ice.Next, 5.55 g of a 5 wt% solution of TBD in super-dehydrated dimethyl sulfoxide (equivalent to a final amount of 2.0 mmol of TBD) was added, and the ring-opening polymerization was allowed to proceed overnight.The polymerization was quenched by adding a small amount of acetic acid, and the resulting polymer was purified through reprecipitation using diethyl ether.Deprotection of the benzyl group in the copolymer was performed by Pd/C-catalyzed hydrogenation.The copolymer (1.0 g) was dissolved in 50 mL methanol, and Pd/C (1.28 g) was added to the solution.The reaction flask was purged with hydrogen gas, followed by a 3-h rigorous stirring of the solution, which was then filtered with celite to remove Pd/C.The filtrate was dialyzed using a dialysis membrane (MWCO 3.5 kD) with distilled water for 3 days.To form the sodium salt of PPEs, the polymer was dissolved in an aqueous solution whose pH was adjusted to 7.0 by adding 0.1 N and 0.01 N NaOH.The solution was dialyzed with distilled water for 1 day.Freeze drying of the aqueous solution yielded PT 80 D 20 .Table 1 contains the synthetic results of the PPEs in this study.

Cytotoxicity test
The animal experiment protocol was approved by the animal experimentation committee of Kansai University (Permit Number: 2208).Mouse bone marrow (BM) cells were isolated from the long bones (humerus, femur, and tibia) of two donor mice by modifying a previously described method [29].The BM cell suspension was prepared with alpha-MEM containing 10% fetal calf serum (FCS) and 100 U/mL Scheme 1. synthetic scheme of polyphosphoesters (PPes).Pt x d y represents the composition of PPes.x: mol% of phosphotriester unit (t), y: mol% of phosphodiester unit (d).penicillin-streptomycin.After removing bigger particles by sedimentation for a few minutes, the BM cell suspension was transferred to a new tube and centrifuged.The BM cells were suspended in a fresh medium, and the suspension was seeded into a cell culture dish.After 2 h, nonadherent cells were collected, and BMNCs were separated using Ficoll 1.084.M-CSF and PGE 2 were added to the suspension of BMNCs (1.67 × 10 5 cells/mL, 10 mL) to achieve a final concentration of 10 ng/mL and 10 −7 M, respectively.The cell suspension (600 µL) was added to a 48-well cell culture plate and incubated at 37 °C, 5% CO 2 for 3 days.After 300 µL of medium was removed from each well, 240 µL of fresh medium containing M-CSF, PGE 2 , and RANKL, and 60 µL polymer/PBS were added to the well.The final concentrations of M-CSF, PGE 2 , and RANKL were adjusted to 10 ng/mL, 0.1 µM, and 10 ng/mL, respectively.In contrast, the final concentration of polymers varied from 0 to 0.5 mg/mL.After 1-day cultivation, the viability of cells in contact with polymers was investigated using a WST-8 Cell Counting Kit (Dojindo, Kumamoto, Japan).

Rhodamine phalloidin and TRAP staining
After BMNCs were cultured in a 48-well cell culture plate with M-CSF, PGE 2 , RANKL, and polymers as mentioned above for 4 days, cells were washed twice with PBS and fixed with 4% paraformaldehyde overnight.The cells were then rinsed with PBS twice and treated with 0.1% Triton X-100 in PBS for 5 min.After rinsing the cells with PBS twice again, fluorescence-labeled phalloidin solution (Acti-stain 535) was in contact with the cells according to the instructions.After Hoechst staining, the cells were rinsed with PBS.Subsequently, the fluorescence-stained adherent cells were stained with the TRAP staining kit.The morphology and fluorescent signals of the cells were observed using an all-in-one fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan).The density of TRAP-positive cells with three or more nuclei was counted on each surface.

Real-time PCR analysis
The total messenger RNA (mRNA) from cultured osteoclast cells in contact with each polymer was extracted using NucleoSpin RNA (MACHEREY-NAGEL GmbH, Düren, Germany), and complementary DNA (cDNA) was synthesized using 5× Primer Script RT MasterMix (Takara Bio) following the manufacturers' instructions.Then, the expression levels of representative differentiation markers and transcription factors in differentiation were evaluated by real-time PCR using TaKaRa PCR Thermal Cycler Dice (Takara Bio) and TB Green Primer Ex Taq II (Takara Bio) following the manufacturer's instructions.Table S1 lists the primers used in this study.

Statistical analysis
All data were expressed as mean ± standard deviation (SD).Statistical analysis was performed using Student's t test.

Polymer synthesis
Several processes for synthesizing phosphodiester polymers have been reported [30].To control the compositions of the phosphodiester and phosphotriester units, Pd/C-catalyzed hydrogenation was used in the current study.In our previous investigations, 2-benzyloxy-2-oxo-1,3,2-dioxaphospholane (BzP) was used to synthesize PPEs with phosphodiester backbones through hydrogenation [28].Because we could not obtain the BzP homopolymer, BzMP was employed as a monomer in the present study.Table 1 contains the synthetic results of the PPEs.The molar fractions in the copolymers closely matched the feed.The polymerization degree of the copolymers was 80-100.A trend of decreasing conversion with an increasing BzMP fraction was observed.The bulky benzyloxy pendant groups may reduce the polymerization ability of cyclic phosphoesters.The deprotection of the benzyl groups was performed by Pd/C-catalyzed hydrogenation.Supplementary Figure S1 shows 1 H NMR spectra of P(MMP/BzMP) 80/20 and PT 80 D 20 before and after hydrogenation.The signals of benzyl groups in the 1 H NMR spectrum disappeared after hydrogenation.The number-averaged polymerization degree of the PPEs was determined through 1 H NMR end-group analysis, and no change was observed before and after hydrogenation; no degradation of the PPE backbone or methoxy side chain was observed due to hydrogenation.This observation suggests that the process of transitioning from phosphotriesters to phosphodiesters is reliable and specific.Although PT 100 D 0 (i.e.P(MMP)) was obtained as a colorless high-viscosity liquid, other PT x D y were obtained as a white solid.Moreover, every PT x D y was well soluble in aqueous media.

Cytotoxicity test
In the current study, the viability of BMNCs in contact with polymers for only 1 day was investigated to determine acute cytotoxicity of the polymers on predifferentiated BMNCs.Figure 1 shows the cell viability of BMNCs in contact with different concentrations of polymers for a day.The data were standardized with the control condition as 100% using PBS instead of polymer solutions.No significant difference was observed in cell viability when the polymer concentration of the culture medium was less than 0.5 mg/mL (p > .01).In our previous study, the cytocompatibility of PPEs was investigated using cell lines.The 50% inhibition concentration (IC 50 ) for PEP•Na was about 20 mg/mL [23], and an increasing trend in IC 50 was observed with the composition of the phosphotriester unit.The cytocompatibility of PPEs for BMNCs could be confirmed, and subsequent studies were conducted under conditions that did not affect the viability of BMNCs.

Osteoclast differentiation
To clarify the effect of PPEs on osteoclastic differentiation of BMNCs, BMNCs were cultured in a differentiation medium with PPEs for 4 days.Figures 2 and 3 show the micrographs of cultured cells treated with TRAP and Rhodamine phalloidin staining, respectively.To demonstrate the distribution of the experiment results, additional images are shown in Supplementary Figures S2 and S3.When BMNCs were cultured with 60 µL of PBS containing no PPEs, TRAP-positive large multinuclear cells with a remarkable actin ring were well observed (see a micrograph at the top left of Figures  2 and 3).Therefore, the differentiation medium used in this study worked well for the differentiation of BMNCs into osteoclastic cells.When the polymer concentration in media was 5 × 10 −3 mg/mL, the osteoclastic differentiation of BMNCs proceeded in the same manner as the control condition.In contrast, increased PPE concentrations tended to decrease both the size and density of the multinuclear cells.Decreased osteoclastic differentiation of BMNCs became remarkable upon increased composition of the phosphodiester unit in PPEs when the PPE concentration in the media was 5 × 10 −1 mg/mL.In particular, almost all the observed cells were mononuclear when BMNCs were cultured under media containing 5 × 10 −1 mg/mL of PT 20 D 80 and PT 0 D 100 .Figures 4 and 5 show the quantitative data for the size and density of TRAP-positive cells with three or more nuclei.Figure 4(a) summarizes the average sizes of multinuclear cells as determined by microscopic observations.When BMNCs were cultured with 5 × 10 −1 mg/mL of PPEs, the size of osteoclasts formed was significantly smaller than that of osteoclasts formed in the medium with no PPEs.Figure 4(b) shows a magnified graph for the size of cells cultured with 5 × 10 −1 mg/mL of PPEs.The effect of polymer composition on the size of osteoclast cells was observed.
Because the size and nuclei number of osteoclasts are related to their resorption activity [31], the regulation of osteoclastic activity could be performed with PPEs.
Figure 5 shows the density of osteoclast cells formed after 4-day cultivation with PPEs.The number of fused cells with three or more nuclei was significantly less when PT 20 D 80 and PT 0 D 100 were added to the cultivation medium, suggesting that phosphodiester units have a reducing effect on osteoclast formation from BMNCs.To clarify the effect of polymers on osteoclastic differentiation, the gene expression of several osteoclastic markers was determined using real-time PCR. Figure 6 shows the relative amount of each maker gene against the housekeeping β-actin gene.Overall, the trend of gene expression corresponded well with the microscopic observation results.Receptor activator of nuclear factor κB (RANK), c-Fos, and nuclear factor of activated T-cells c1 (NFATc1) are early osteoclastic marker proteins secreted by osteoclast precursor cells [32].The expression levels of Rank, c-fos, and Nfatc1 moderately  decreased with increased compositions of phosphodiester in PPEs.M-CSF in the differentiation medium induces RANK in early-stage precursors [33].While the specific mechanism by which PPEs influence the interaction between M-CSF and its receptor remains unclear, the real-time PCR analysis results indicate that PPEs with a high phosphodiester content have a reducing effect on osteoclastic differentiation from the early stage.A similar effect of poly-γ-glutamic acid, a natural polyanion derived from Bacillus subtilis, was reported by Lee and coworkers.Poly-γ-glutamic acid downregulated the expression of the cell surface and total cellular M-CSF receptor protein, resulting in suppressed osteoclastogenesis [34].Osteoclast-associated receptor (OSCAR) [35] and dendritic cell-specific transmembrane protein (DC-Stamp) [36] are essential molecules that are upregulated by NFATc1 and important for osteoclast cell-cell fusion.The expression pattern of these genes is similar to that of Nfatc1.Cathepsin K (CtsK) is responsible for type I collagen degradation in osteoclast-mediated bone resorption [37].The suppression pattern of Ctsk also corresponded with others.The decreased expression of these major osteoclastic marker genes upon treatment with PPEs containing high phosphodiester units was significant.In particular, PT 20 D 80 and PT 0 D 100 reduced the gene expression of osteoclastic markers.Due to their hydrophobic side chains, PPEs can induce cellular internalization [38].Therefore, PT 20 D 80 may show a strong reducing effect for osteoclastic differentiation due to its unique structure for interacting with cells.

Conclusion
This study investigated the effect of phosphodiester unit composition in PPEs on osteoclastic differentiation.All synthesized PPEs exhibited no adverse effects on the viability of BNMCs at concentrations below 0.5 mg/mL.Increased PPE concentration in the studied medium yielded a reduced size and density of osteoclasts.In particular, PPEs with high phosphodiester units effectively reduced osteoclastic formation.This trend was also confirmed by real-time PCR analysis for the gene expression of several osteoclastic markers.Notably, our prior studies revealed the promoting effect of phosphodiester polymers on osteoblastic differentiation [24,25].Hence, the treatment of PPEs holds the potential for anabolic function in moderating bone formation.Furthermore, PPEs containing entirely phosphodiester units have a high affinity for bone tissue in vivo [23,25].Overall, PPEs are interesting polymers that can effectively modulate bone cell function through their chemical structure variations.These polymers hold promise as platforms for developing polymeric drugs aimed at treating osteoporosis.