Decalcification of calcified tissues induced by inorganic polyphosphate in chondrogenic ATDC5 cells in the presence of insulin

Abstract

Purpose: Inorganic polyphosphate (PolyP), a polymer of orthophosphate, strongly promotes mineralized tissue formation. This study explored the conditions necessary for PolyP to induce calcified deposits in cartilage and assessed the role of insulin in modulating PolyP-induced tissue calcification.

Methods: Murine chondrogenic ATDC5 cells were cultured under growth, mineralization, or PolyP-induced calcification conditions, with or without insulin. Calcified nodules were stained with Alizarin Red S, and conditioned media were analyzed for pH and lactate concentration using a pH meter and a lactate assay kit-WST.

Results: PolyP treatment of ATDC5 cells led to calcified deposits by day 5, both with and without insulin. However, in the presence of insulin, these deposits were nearly fully decalcified by day 14. Conditioned media with insulin had a lower pH and a higher lactate concentration compared to those without insulin, with lactate levels sufficient to demineralize the PolyP-induced calcified deposits.

Conclusion: These data suggest that treatment of ATDC5 chondrogenic cells with PolyP accelerates the formation of mineralized tissue. However, PolyP-induced calcified nodules undergo demineralization owing to lactate production by cells in the presence of insulin.

Introduction

In clinical practice, bone grafts are commonly used to repair defects. Grafting with vascularized bone tissue is necessary for the reconstruction of large bone defects, such as those resulting from ameloblastoma excision [1,2], as nutrients and oxygen must be delivered to the center of the graft [3,4]. Tissue engineering approaches focus on inducing mesenchymal stem cells to differentiate into osteoblasts, thereby promoting bone formation through intramembranous ossification, which directly produces the bone extracellular matrix [4]. However, ensuring adequate blood supply within the newly formed bone tissue remains a significant challenge. Consequently, there has been a growing interest in the transplantation of cartilage into bone defects and the use of endochondral ossification for bone regeneration [4]. This approach is advantageous due to the avascular nature of cartilage, which enables chondrocytes to tolerate relatively low oxygen levels, and facilitates the repair of larger bone defects. Additionally, cartilage contains factors that promote both angiogenesis and osteogenesis, such as basic fibroblast growth factor, bone morphogenetic proteins-2, and vascular endothelial growth factor [4,5,6,7,8]. However, studies on the application of cartilage tissue in the repair of bone defects are limited.

Inorganic polyphosphates (PolyPs) are linear polymers composed of orthophosphate residues linked via high-energy phosphoanhydride bonds [9,10]. These molecules are found across a wide range of organisms, from bacteria to humans, and participate in various biological processes [9]. In mammalian cells, PolyP is involved in tissue mineralization [11], blood coagulation [12], immune response [13], angiogenesis [14], energy metabolism [15], and tumor metastasis [14]. Mikami et al. demonstrated that PolyP treatment induces mineralized tissue formation in both osteoblastic and chondrogenic ATDC5 cells [16]. Therefore, the application of PolyP for bone formation via endochondral ossification may accelerate the development of calcified tissues. In addition, because insulin promotes the proliferation and differentiation of chondrocytes [17], it is likely that insulin plays a crucial role in bone tissue formation through endochondral ossification. Accordingly, the effects of insulin on calcified tissue formation in chondrocytes influenced by PolyP were investigated.

Materials and Methods

Cell culture

The murine ATDC5 chondrogenic cell line was acquired from RIKEN BioResource Research Center (#RCB0565, Tsukuba, Japan). Cells were maintained at 37°C in a 5% CO₂ atmosphere using a growth medium composed of Dulbecco’s Modified Eagle Medium/F-12 (DMEM/F-12; Fujifilm Wako, Osaka, Japan), supplemented with 5% fetal bovine serum (FBS; Cytiva, Marlborough, MA, USA) and penicillin/streptomycin (Fujifilm Wako). For mineralization experiments, growth medium containing 10 mM disodium β-glycerophosphate pentahydrate (Fujifilm Wako) and 50 µg/mL L-ascorbic acid magnesium salt n-hydrate (Fujifilm Wako) was used as a mineralization medium.

Mineralization experiments

The experimental schedule is shown in Fig. 1A. ATDC5 cells (2 × 10⁵ cells/well) were seeded in type I collagen-coated 24-well plate (AGC techno glass, Yoshida, Japan) and cultured in growth medium (control), mineralization medium, or PolyP medium (mineralization medium containing 1 mM exclusive polyphosphate medium-chain [60-mer average chain length; RegeneTiss, Okaya, Japan]) in the presence or absence of 10 µg/mL recombinant human insulin, 0.5 µg/mL human transferrin, and 6.7 ng/mL sodium selenite (ITS, R&D systems, Minneapolis, MN, USA). The medium was changed every three days. Cells were treated for 5, 7, and 14 days and stained with alizarin red S (Muto Pure Chemicals, Tokyo, Japan). The stained plates were scanned using a GT-X830 scanner (Seiko Epson, Suwa, Japan), and mean red intensities of each well were measured using Fiji/ImageJ software (https://imagej.net/software/fiji/). High-magnification images were photographed using Keyence BZ-X810 microscope (Keyence, Osaka, Japan).

Fig. 1 Polyphosphate (PolyP)-induced mineralized tissue was demineralized in the presence of insulin

(A) Experimental timeline (B) ATDC5 cells were treated for the indicated durations with growth (control), mineralization, or PolyP media in the absence or presence of insulin. On days 0, 5, 7, and 14, mineralized tissues were stained with alizarin red S. (C) Red intensities were measured using Fiji/ImageJ software. C, control growth medium; M, mineralization medium; P, PolyP medium. (D) High magnification image of the cells treated for 14 days with PolyP in the presence of insulin. Bar scale, 100 µm

pH and lactate measurement in conditioned medium

ATDC5 cells (2 × 10⁵ cells/well) were cultured in type I collagen-coated 24-well plates in growth medium (control), mineralization medium, or PolyP medium, in the presence or absence of ITS media supplement. Conditioned media were collected on days 9 and 12 and stored at −80°C. The pH of each conditioned medium sample was measured using a LAQUA D-210 pH meter equipped with a 9618S-10D electrode (Horiba, Kyoto, Japan). Lactate content in the conditioned media was measured using the Lactate Assay Kit-WST (Dojindo, Mashiki, Japan) according to the instructions provided by the manufacturer. These experiments were conducted in 6 replicates.

Examination of chondrogenic markers

ATDC5 cells (4 × 10⁵ cells/well) were cultured in type I collagen-coated 12-well plates for 0, 5, 7, and 14 days in growth medium (control), mineralization medium, or PolyP medium, with or without the addition of the ITS media supplement. Cells were lysed with lysis buffer (50 mM Tris-HCl [pH7.5], 150 mM NaCl, and 1% triton-X100) and total protein concentrations were measured using BCA protein assay kit (Takara, Kusatsu, Japan). Two micrograms each of total protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Fujifilm Wako). The membrane was applied to western blot. An anti-type II collagen (SouthernBiotech, Birmingham, AL, USA), an anti-type X (Merck, Darmstadt, Germany), and HRP-conjugated anti-β-tubulin (Proteintech, Rosemont, IL, USA) antibodies were used for primary antibodies. WestVision Peroxidase Polymer, anti-rabbit and mouse IgGs (Vector Laboratories, Newark, CA, USA) and a biotin-conjugated anti-goat IgG (Zymed, South San Francisco, CA, USA) were used as secondary antibodies. When the biotin-conjugated IgG was used, a Streptavidin-Peroxidase (KPL, Gaithersburg, MD, USA) was also used. Band images were developed using a Clarity Western ECL reagent (Bio-Rad Laboratories, Hercules, CA, USA) and photographed using a ChemiDoc XRS system (Bio-Rad Laboratories).

Alcian blue staining

ATDC5 cells (2 × 10⁵ cells/well) were cultured for 0, 5, 7, and 10 days in type I collagen-coated 24-well plates with growth medium (control), mineralization medium, or PolyP medium, in the presence or absence of ITS media supplement. Cells were fixed for 20 min with 20% formaldehyde and washed three times with deionized water. They were then treated for 5 min with 3% acetate, and subsequently for 1 h with alcian blue staining solution (pH2.5; Muto Pure Chemicals, Tokyo, Japan), followed by washing with 5-times 3% acetate and 3 times deionized water. Plates were scanned using a GT-X830 (Seiko Epson, Suwa, Japan) and mean blue intensities of each well were measured using Fiji/ImageJ software.

Statistical analysis

Statistical analysis was conducted using the EZR program. Normality was assessed using the Shapiro-Wilk test. Due to the violation of normality assumptions, the Kruskal-Wallis test, followed by the Steel-Dwass test, were utilized to evaluate the differences. Results yielding a P-value of less than 0.05 were interpreted as statistically significant.

Results

Previous studies demonstrated that PolyP induces mineralized nodule formation in chondrogenic ATDC5 cells [16]. Additionally, insulin stimulates chondrocyte differentiation and induces mineralized tissue formation [17,18,19]. Therefore, the effects of insulin on PolyP-induced mineralization in ATDC5 cells were examined. In the absence of insulin, treatment of ATDC5 cells with mineralization medium slightly induced the formation of mineralized nodules, whereas PolyP treatment strongly induced nodule formation after day 5 (Fig. 1B, C). In the presence of insulin, mineralization medium treatment induced nodule formation in a time-dependent manner after day 7 (Fig. 1B, C). In contrast, PolyP treatment induced the formation of mineralized nodules after day 5; however, interestingly, the nodules underwent demineralization by day 14 (Fig. 1B, C). A highly magnified image of cells treated with PolyP in the presence of insulin revealed that the cells remained attached to the bottom of the well (Fig. 1D).

To clarify why PolyP-induced mineralized nodules were demineralized in the presence of insulin, conditioned media were collected on days 9 and 12, and their pH were measured (Fig. 2A). Given the significant reduction in mineralization observed between days 7 and 14 (Fig. 1B, C), days 9 and 12 were selected for further analyses. On day 9, the pH of each conditioned medium in the presence of insulin was significantly lower than that in its absence (P < 0.05, Fig. 2B). The pH values of conditioned control, mineralization, and PolyP media were similar in the presence of insulin (Fig. 2B). In the PolyP medium, the pH of the conditioned medium in the absence and presence of insulin was 7.98 ± 0.02 and 7.01 ± 0.03, respectively (Fig. 2B). On day 12, the pH of all conditioned media was significantly lower in the presence of insulin than that in its absence (P < 0.05, Fig. 2B). However, the pH of conditioned medium treated with the mineralization medium was lower than that of the control (growth medium), even in the absence of insulin (P < 0.05, Fig. 2B). In the PolyP medium, the pH of conditioned medium in the absence and presence of insulin was 7.75 ± 0.03 and 6.97 ± 0.01, respectively (Fig. 2B).

As acid production lowers pH, lactate production was measured (Fig. 3A). All conditioned media in the presence of insulin contained higher concentrations of lactate (33.0-36.7 mM) than those in its absence (12.2-29.6 mM, P < 0.05), with nearly the same lactate levels on days 9 and 12 (Fig. 3B). PolyP-conditioned medium contained 22.5-22.8 and 33.7-33.8 mM lactate in the absence and presence of insulin, respectively (Fig. 3B).

To examine whether similar lactate concentrations induced demineralization of PolyP-induced nodule, ATDC5 cells were treated for 5 days to form PolyP-induced mineralized nodules, after which the medium was replaced with mineralization medium containing varying doses of lactate (Fig. 3C). Five days of PolyP treatment induced mineralized nodule formation, and lactate subsequently demineralized them in a dose-dependent manner (Fig. 3D, E). A strong demineralization effect of lactate was observed at concentrations > 30 mM (Fig. 3D, E).

On day 14, the mineralization medium-treated culture displayed mineralized nodules, even in the presence of insulin (Fig. 1B, C); however, this condition produced a high concentration of lactate that decalcified the PolyP-induced nodules (Fig. 3B). To investigate whether calcification mechanisms differ between mineralization and PolyP-containing medium, mineralization medium conditions on day 14 and the PolyP medium conditions on day 7, both exhibiting comparable red intensities in alizarin red S staining, were selected. High-magnification images were compared (Fig. 4). In the PolyP medium, the staining was uniformly distributed across the entire area, whereas the mineralization medium treatment demonstrated notable variations in staining intensity (Fig. 4).

ATDC5 cells are prechondrocytes capable of differentiating into mature chondrocytes [20,21,22]. To investigate the effects of mineralization medium and PolyP medium on ATDC5 cells, collagen II and collagen X, representing early- and late-phase differentiation markers, respectively, were analyzed via western blot (Fig. 5A). Under the control medium condition, the expression levels of collagen II and collagen X were upregulated after day 5, both in the absence and presence of insulin (Fig. 5A). Notably, the induction levels seemed relatively higher in the presence of insulin compared to its absence (Fig. 5A). In the mineralization medium, collagen II and collagen X levels gradually increased over time, both in the absence and presence of insulin (Fig. 5A). Collagen X induction occurred earlier in the presence of insulin compared to its absence, with a pronounced increase observed by day 5 (Fig. 5A). Treatment with PolyP medium resulted in a marked induction of collagen II expression in ATDC5 cells by day 5, followed by a time-dependent increase observed both in the presence and absence of insulin (Fig. 5A). Collagen X expression remained at basal levels during PolyP medium treatment without insulin; however, it was upregulated in the presence of insulin under the same conditions (Fig. 5A).

Alcian blue staining was performed as an alternative method to assess changes in aggrecan levels (Fig. 5B, C). The Alcian blue staining intensity under mineralization medium conditions was nearly identical at both 5 and 7 days, regardless of the presence or absence of insulin. Notably, a significant increase in staining intensity was observed at 14 days. Treatment with PolyP medium slightly enhanced staining intensity by day 7 in the absence of insulin. Interestingly, in the presence of insulin, staining intensity was upregulated by day 7 but strongly decreased by day 14.

Fig. 2 pH of conditioned media decreased in the presence of insulin

(A) Experimental timeline (B) ATDC5 cells were treated with growth medium (control), mineralization medium, or mineralization medium containing 1 mM polyphosphate (PolyP) in the absence or presence of insulin. On days 9 and 12, conditioned media were collected, and their pH values were measured (n = 6; *P < 0.05). C, control growth medium; M, mineralization medium; P, PolyP medium

Fig. 3 Insulin-induced lactate production demineralizes PolyP-induced mineralized tissue

(A) Timeline for lactate measurement in conditioned media. (B) ATDC5 cells were treated with growth medium (control), mineralization medium, or mineralization medium containing 1 mM polyphosphate (PolyP) in the absence or presence of insulin. On days 9 and 12, conditioned media were collected, and lactate concentrations in them were measured (n = 6; *P < 0.05). C, control medium; M, mineralization medium; P, PolyP medium. (C) Experimental protocol to investigate the effect of lactate on PolyP-induced mineralized tissue. (D) ATDC5 cells were treated for 5 days with 1 mM PolyP in mineralization medium in the absence of insulin. The medium was replaced with mineralization medium containing the specified concentrations of lactate until day 14, after which alizarin red S staining was conducted. (E) Red intensities were measured using Fiji/ImageJ software.

Fig. 4 PolyP-induced calcification may differ from that induced by mineralization medium

The two conditions that yielded nearly identical staining intensities to alizarin red S (Fig. 1B, C) were selected for comparison, focusing on high-magnification images (×40) of alizarin red S-stained samples between these conditions. (A) Cells were treated for 14 days with mineralization buffer in the presence of insulin. (B) Cells were treated for 7 days with PolyP medium in the presence of insulin. Bar scale, 100 µm

Fig. 5 Changes in cartilage differentiation markers in response to culture conditions

(A) ATDC5 cells were cultured for the indicated durations with growth (control), mineralization, or PolyP media in the absence or presence of insulin. The expression of chondrogenic markers, such as type II collagen (an early-stage marker) and type X collagen (a late-stage marker), was analyzed by western blot. β-tubulin served as a loading control. Arrowheads indicate the type X collagen bands. (B) On days 0, 5, 7, and 14, acidic proteoglycans such as aggrecan were stained with alcian blue, which targets sulfate and carboxyl groups. (C) Blue intensities were measured using Fiji/ImageJ software. C, control growth medium; M, mineralization medium; P, PolyP medium

Discussion

This study demonstrated that PolyP treatment induced mineralization in chondrogenic ATDC5 cells after day 5, both in the presence and absence of insulin (Fig. 1B, C). Although mineralization medium treatment did not form mineralized tissue in the absence of insulin, even on day 14, the number of mineralized nodules increased in a time-dependent manner (after day 7) in the presence of insulin (Fig. 1B, C). These results suggest that PolyP treatment effectively induced calcified tissue formation (within 5 days). However, in the presence of insulin, the red intensity of alizarin red S was strongly decreased in PolyP medium treated cells by day 14 (Fig. 1B, C). Additionally, cells of the condition did not detach from the bottom of the well (Fig. 1D). These data suggest that the mineralized nodules formed by PolyP treatment by day 5 demineralized by day 14. The pH of the PolyP-conditioned medium containing insulin was lower than that without insulin both on days 9 and 12 (Fig. 2B). Given that lactate concentrations in PolyP-conditioned medium on days 9 and 12 were 33.8 ± 0.49 and 33.7 ± 0.51 mM, respectively (Fig. 3B), and that these lactate concentrations effectively decalcified PolyP-induced mineralized tissue (Fig. 3D, E), the demineralization effect of PolyP-conditioned medium in the presence of insulin may be attributed to insulin-induced lactate production.

The pH of the culture supernatant presented in this study remained within the neutral range (pH6.97-8.21), exceeding the critical pH of hydroxyapatite (pH5.5), even under conditions where PolyP-induced calcified nodules were demineralized. However, the PolyP-induced calcified tissues underwent demineralization. This observation could be attributed to the following. The medium used in this study (DMEM/F-12) is buffered bicarbonate. This buffering property likely contributed to maintaining the pH in the neutral range. Additionally, after collecting the culture supernatants, they were stored at −80°C, and subsequently measured its lactate concentration and pH after thawing. During the freezing and thawing process, a portion of the bicarbonate converts to CO₂, which is released into the gas layer, resulting in an increase in pH. These may be reasons for the neutral range pH of the conditioned media in the presence of insulin.

Cells adhered to the bottom of the wells and produced lactate in situ in the presence of insulin. The PolyP-induced calcified tissues were localized to areas containing cells or their surroundings. These observations suggest that much higher concentrations of lactate than measured directly interacted with the calcified nodules. Thus, the localized environment where calcified tissues are present likely had a lower pH sufficient to facilitate demineralization. Moreover, the calcified tissues induced by PolyP treatment may consist of calcium phosphate crystals which can be easily demineralized compared to hydroxyapatite, as discussed in next paragraph.

Although PolyP medium treatment in the presence of insulin decalcified the PolyP-induced mineralized tissue, mineralization medium treatment in the presence of insulin increased the number of mineralized nodules by day 14 (Fig. 1B, C). However, the pH and lactate concentrations in conditioned media derived from ATDC5 cultures treated with mineralization and PolyP media in the presence of insulin were almost the same (Fig. 2B, 3B). High-magnification images were compared to explore the reasons for this discrepancy. Alizarin red S-stained images of the culture treated with the PolyP medium differed from those treated with mineralization medium. In PolyP medium-treated cultures, staining was evenly distributed throughout the area. In contrast, mineralization observed in cultures treated with mineralization medium showed substantial variability in staining intensity (Fig. 4). This discrepancy may reflect distinct mineralization mechanisms. The images of the culture treated with the mineralization medium closely resembled those observed during matrix vesicle-mediated mineralization of mesenchymal stem cells [23]. In contrast, cultures treated with the PolyP medium exhibited more uniform calcification (Fig. 4), implying possible mineralization mechanisms other than the matrix vesicle theory. Although treatment of osteoblastic cells with PolyP is believed to produce hydroxyapatite crystals, represented by the chemical formula Ca₁₀(PO₄)₆(OH)₂, the mechanisms of PolyP-induced mineralization remain unclear. Therefore, other types of calcium phosphates, such as tricalcium phosphate and tetracalcium phosphate, may also form. Klein et al. compared the solubilities of hydroxyapatite, tricalcium phosphate, and tetracalcium phosphate particles in lactate buffer at pH6.2 or 7.2 [24]. The solubility order was tetracalcium phosphate > tricalcium phosphate > hydroxyapatite. Therefore, calcium phosphate (other than hydroxyapatite) induced by PolyP treatment may be more readily demineralized than the hydroxyapatite generated through mineralization medium treatment.

The expression of collagen X in ATDC5 cells cultured in PolyP medium without insulin supplementation remained unchanged, maintaining basal levels (Fig. 5A). Furthermore, cells cultivated in PolyP medium with insulin exhibited a marked induction of collagen II expression by day 5, followed by a substantial decrease by day 14 (Fig. 5A). These expression patterns were quite different from those treated with mineralization medium. These characteristics might be related to the difference of mineralization mechanisms between PolyP and mineralization media treated ATDC5 cells. Therefore, investigation of the mechanisms of PolyP-induced mineralization is the next issue for PolyP mineralization research.

Insulin binding to the insulin receptor tyrosine kinase initiates its autophosphorylation, which subsequently facilitates the phosphorylation and recruitment of adaptor proteins, such as the insulin receptor substrate (IRS) family [25,26]. The phosphorylated tyrosine residues on IRSs serve as docking sites for various signaling molecules. Of these pathways, phosphatidylinositol-3 kinase (PI3K) plays a pivotal role in insulin signaling, primarily by activating the protein kinase Cζ and Akt signaling cascades. These activations facilitate glucose uptake by promoting the translocation of glucose transporter 4 vesicles to the plasma membrane [27]. Furthermore, Akt activation stimulates mechanistic target of Rapamycin complex1 (mTORC1), which enhances the transcription, translation, and stabilization of hypoxia-inducible factor (HIF)-1α [28,29,30]. HIF-1α is reported to promote the expression of lactate dehydrogenase (LDH)A [31,32], suggesting that the insulin-induced increase in lactate production observed in this experiment may be attributed to the activation of the IRS/PI3K/Akt/mTORC/HIF-1α pathway, triggered by insulin binding to its receptor, leading to enhanced LDHA expression on ATDC chondrogenic cells. Elucidation of the mechanism will be the next challenge.

The data shown in this study demonstrate that treatment of ATDC5 chondrogenic cells with PolyP strongly promotes tissue mineralization within 5 days (Fig. 1B, C). In addition, the absence of insulin resulted in a significantly stronger mineralization effect than its presence (Fig. 1B, C). Therefore, treatment with PolyP in the absence of insulin appears to be the most effective method for obtaining chondrocyte-derived calcified tissue. However, the nature of calcified structures induced by PolyP treatment in chondrocytes remains unclear. Further studies are required to support the potential clinical application of this method. In addition, the effects of insulin on the decalcification of mineralized tissues formed through PolyP treatment should also be considered. The clinical application of bone formation accompanied by endochondral ossification necessitates investigation into whether the mineralized deposits formed by PolyP result from endochondral ossification. Otherwise, inducing mineralization using a calcification medium may be more appropriate for the intended purpose. However, this topic requires further extensive research.

These data suggest that treating ATDC5 chondrogenic cells with polyphosphate accelerates the formation of mineralized tissue. However, calcified nodules induced by polyphosphate treatment undergo demineralization owing to lactate production by cells in the presence of insulin.

Abbreviations

DMEM: Dulbecco’s modified eagle’s medium; FBS: fetal bovine serum; ITS: mixture of insulin, transferrin and sodium selenite; PolyP: inorganic polyphosphate

Ethical Statements

Not applicable

Conflicts of Interest

The authors declare no potential conflicts of interest with respect to the research, authorship, and publication of the article.

Funding

This work was supported by JSPS KAKENHI (23K09222), the Sato Funds (SATO-2023-4, SATO-2024-24) Nihon University School of Dentistry, and a grant from the Dental Research Center Nihon University School of Dentistry (DRC(B)-2024-4).

Author Contributions

TF: investigation, formal analysis, validation, visualization, writing – original draft; HS: investigation, writing – review and editing; YM: conceptualization, writing-review and editing; TT: writing – review and editing; YY: writing – review and editing; HT: conceptualization, funding acquisition, methodology, project administration, supervision, writing-original draft, writing – review and editing. All authors read and approved the final version of the manuscript.

ORCID iD

^1,2)TF: [e-mail], https://orcid.org/0009-0003-6200-0348

²⁾HS: [e-mail], https://orcid.org/0000-0002-5379-0603

³⁾YM: [e-mail], https://orcid.org/0000-0001-9263-3937

⁴⁾TT: [e-mail], https://orcid.org/0009-0004-0275-425X

²⁾YY: [e-mail], https://orcid.org/0000-0002-3626-9023

^5,6)HT*: [e-mail], https://orcid.org/0000-0002-2047-2262

Acknowledgments

The authors would like to thank Editage (www.editage.com) for English language editing.

Data Availability Statements

Data generated during the current study are available from the corresponding author on reasonable request.

References

• 1.  Watari S, Ikuta Y, Adachi N, Murase M, Tsuge K (1978) Vascular pedicle fibular transplantation as treatment for bone tumor. Clin Orthop Relat Res 133, 158-164.
• 2.  Ihara K, Doi K, Yamamoto M, Kawai S (1998) Free vascularized fibular grafts for large bone defects in the extremities after tumor excision. J Reconstr Microsurg 14, 371-376. doi: 10.1055/s-2007-1000193
• 3.  Gupte MJ, Ma PX (2012) Nanofibrous scaffolds for dental and craniofacial applications. J Dent Res 91, 227-234. doi: 10.1177/0022034511417441
• 4.  Bardsley K, Kwarciak A, Freeman C, Brook I, Hatton P, Crawford A (2017) Repair of bone defects in vivo using tissue engineered hypertrophic cartilage grafts produced from nasal chondrocytes. Biomaterials 112, 313-323. doi: 10.1016/j.biomaterials.2016.10.014
• 5.  Luan Y, Praul CA, Gay CV, Leach RM (1996) Basic fibroblast growth factor: an autocrine growth factor for epiphyseal growth plate chondrocytes. J Cell Biochem 62, 372-382. doi: 10.1002/(sici)1097-4644(199609)62:3<372::aid-jcb7>3.0.co;2-o
• 6.  Bluteau G, Julien M, Magne D, Mallein-Gerin F, Weiss P, Daculsi G et al. (2007) VEGF and VEGF receptors are differentially expressed in chondrocytes. Bone 40, 568-576. doi: 10.1016/j.bone.2006.09.024
• 7.  Schmal H, Zwingmann J, Fehrenbach M, Finkenzeller G, Stark GB, Südkamp NP et al. (2007) bFGF influences human articular chondrocyte differentiation. Cytotherapy 9, 184-193. doi: 10.1080/14653240601182846
• 8.  Shu B, Zhang M, Xie R, Wang M, Jin H, Hou W et al. (2011) BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development. J Cell Sci 124, 3428-3440. doi: 10.1242/jcs.083659
• 9.  Kornberg A, Rao NN, Ault-Riché D (1999) Inorganic polyphosphate: a molecule of many functions. Annu Rev Biochem 68, 89-125. doi: 10.1146/annurev.biochem.68.1.89
• 10.  Müller WEG, Schröder HC, Wang X (2019) Inorganic polyphosphates as storage for and generator of metabolic energy in the extracellular matrix. Chem Rev 119, 12337-12374. doi: 10.1021/acs.chemrev.9b00460
• 11.  Kawazoe Y, Shiba T, Nakamura R, Mizuno A, Tsutsumi K, Uematsu T et al. (2004) Induction of calcification in MC3T3-E1 cells by inorganic polyphosphate. J Dent Res 83, 613-618. doi: 10.1177/154405910408300806
• 12.  Ruiz FA, Lea CR, Oldfield E, Docampo R (2004) Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem 279, 44250-44257. doi: 10.1074/jbc.M406261200
• 13.  Hassanian SM, Avan A, Ardeshirylajimi A (2017) Inorganic polyphosphate: a key modulator of inflammation. J Thromb Haemost 15, 213-218. doi: 10.1111/jth.13580
• 14.  Han KY, Hong BS, Yoon YJ, Yoon CM, Kim YK, Kwon YG et al. (2007) Polyphosphate blocks tumour metastasis via anti-angiogenic activity. Biochem J 406, 49-55. doi: 10.1042/BJ20061542
• 15.  Pavlov E, Aschar-Sobbi R, Campanella M, Turner RJ, Gómez-García MR, Abramov AY (2010) Inorganic polyphosphate and energy metabolism in mammalian cells. J Biol Chem 285, 9420-9428. doi: 10.1074/jbc.M109.013011
• 16.  Mikami Y, Tsuda H, Akiyama Y, Honda M, Shimizu N, Suzuki N et al. (2016) Alkaline phosphatase determines polyphosphate-induced mineralization in a cell-type independent manner. J Bone Miner Metab 34, 627-637. doi: 10.1007/s00774-015-0719-6
• 17.  Yao Y, Zhai Z, Wang Y (2014) Evaluation of insulin medium or chondrogenic. medium on proliferation and chondrogenesis of ATDC5 cells. Biomed Res Int 2014, 569241. doi: 10.1155/2014/569241
• 18.  Phornphutkul C, Wu KY, Gruppuso PA (2006) The role of insulin in chondrogenesis. Mol Cell Endocrinol 249, 107-115. doi: 10.1016/j.mce.2006.02.002
• 19.  Okita K, Hikiji H, Koga A, Nagai‐Yoshioka Y, Yamasaki R, Mitsugi S et al. (2023) Ascorbic acid enhances chondrocyte differentiation of ATDC5 by accelerating insulin receptor signaling. Cell Biology International 47, 1737-1748. doi: 10.1002/cbin.12067
• 20.  Atsumi T, Miwa Y, Kimata K, Ikawa Y (1990) A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells. Cell Differ Dev 30, 109-116. doi: 10.1016/0922-3371(90)90079-c
• 21.  Shukunami C, Shigeno C, Atsumi T, Ishizeki K, Suzuki F, Hiraki Y (1996) Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor. J Cell Biol 133, 457-468. doi: 10.1083/jcb.133.2.457
• 22.  Shukunami C, Ishizeki K, Atsumi T, Ohta Y, Suzuki F, Hiraki Y (1997) Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro. J Bone Miner Res 12, 1174-1188. doi: 10.1359/jbmr.1997.12.8.1174
• 23.  Li P, Yang YM, Sanchez S, Cui DC, Dang RJ, Wang XY et al. (2016) Deubiquitinase MYSM1 is essential for normal bone formation and mesenchymal stem cell differentiation. Sci Rep 6, 22211. doi: 10.1038/srep22211
• 24.  Klein CP, de Blieck-Hogervorst JM, Wolke JG, de Groot K (1990) Studies of the solubility of different calcium phosphate ceramic particles in vitro. Biomaterials 11, 509-512. doi: 10.1016/0142-9612(90)90067-z
• 25.  Cheng Z, Tseng Y, White MF (2010) Insulin signaling meets mitochondria in metabolism. Trends Endocrinol Metab 21, 589-598. doi: 10.1016/j.tem.2010.06.005
• 26.  Guo S (2014) Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms. J Endocrinol 220, T1-23. doi: 10.1530/JOE-13-0327
• 27.  Rowland AF, Fazakerley DJ, James DE (2011) Mapping insulin/GLUT4 circuitry. Traffic 12, 672-681. doi: 10.1111/j.1600-0854.2011.01178.x
• 28.  Dunlop EA, Tee AR (2009) Mammalian target of rapamycin complex 1: signaling inputs, substrates and feedback mechanisms. Cell Signal 21, 827-835. doi: 10.1016/j.cellsig.2009.01.012
• 29.  Hoeffer CA, Klann E (2010) mTOR signaling: at the crossroads of plasticity, memory and disease. Trends Neurosci 33, 67-75. doi: 10.1016/j.tins.2009.11.003
• 30.  Laplante M, Sabatini DM (2013) Regulation of mTORC1 and its impact on gene expression at a glance. J Cell Sci 126, 1713-1719. doi: 10.1242/jcs.125773
• 31.  Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet JP, Maire P et al. (1996) Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol Chem 271, 32529-32537. doi: 10.1074/jbc.271.51.32529
• 32.  Cui XG, Han ZT, He SH, Wu X, Chen TR, Shao CH et al. (2017) HIF1/2α mediates hypoxia-induced LDHA expression in human pancreatic cancer cells. Oncotarget 8, 24840-24852. doi: 10.18632/oncotarget.15266