Apoptotic bodies from endplate chondrocytes enhance the oxidative stress‐induced mineralization by regulating PPi metabolism

Abstract This study aimed to investigate the role of apoptotic bodies (Abs) from the oxidative stressed endplate chondrocytes in regulating mineralization and potential mechanisms. Endplate chondrocytes were isolated from rats and treated with H2O2 to induce oxidative stress. The calcium deposition for matrix mineralization in the cells was examined by histological staining. The expression levels of calcification‐related genes in individual groups of cells were determined by quantitative real time‐PCR (qRT‐PCR). Subsequently, extracellular vesicles (EVs) were purified and characterized. The effect of treatment with H2O2 and/or Abs on the mineralization, extracellular PPi metabolism and related gene expression were determined. Oxidative stress significantly increased the mineralization and promoted the generation of main Abs from endplate chondrocytes. Abs were effectively endocytosed by endplate chondrocytes and co‐localized with collagen (COL)‐II in the cytoplasm, which enhanced the mineralization, alkaline phosphatase (ALP), osteocalcin (OCN), Runt‐related transcription factor 2 (RUNX2) and COL‐I expression in endplate chondrocytes. Furthermore, treatment either H2O2 or Abs significantly decreased PPi, but increased Pi production and treatment with both further enhancing the changes in endplate chondrocytes. Similarly, treatment either H2O2 or Abs significantly decreased the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and ankylosis protein (ANK) expression and ENPP1 promoter activity, but increased the tissue‐nonspecific alkaline phosphatase (TNAP) expression and TNAP promoter activity in endplate chondrocytes. Oxidative stress promoted the generation of Abs, which might enhance the oxidative stress‐mediated mineralization in endplate chondrocytes by regulating the PPi metabolism.


| INTRODUC TI ON
Low back pain is a major cause of work-related disabilities worldwide and causes significanthealthcare-related costs. 1 A leading cause of low back pain is degeneration of the intervertebral disc (IVD). 2 Although many factors are associated with the occurrence of IVD degeneration nutritional dysregulation is crucial for the IVD degeneration as the IVD is the largest avascular organ in the body. 3 Foundational solutes diffusing through the cartilage endplate (CEP) is the primary route to supply nutrients to the IVD. 4 The CEP is a thin layer of hyaline cartilage, which lies between the vertebral body and the intervertebral disc. 5 Previous studies have shown that CEP calcification promotes the degradation of IVD because it prevents the diffusion of nutrients into the disc. 6 Hence, understanding the mechanisms underlying CEP calcification will be of great significance in developing new therapies for prevention and intervention of IVD degeneration and low back pain.
Increased oxidative stress can cause inflammation and promote the IVD degeneration. 7 Oxidative stress can produce excessive reactive oxygen species (ROS) and cause inflammation, which can promote catabolism and premature senescence, affect the matrix homoeostasis and induce apoptosis of IVD cells, leading to IVD degeneration. 8 Actually, high levels of oxidative stress occur during the process of CEP degeneration, participating in the degeneration of CEP. 9 A recent study has shown that increased oxidative stress induces apoptosis and promotes calcification in CEP cells. 10 However, how apoptosis participates in oxidative stress-induced calcification of CEP cells has not been clarified. Extracellular vesicles, including exosomes (Exo), microvesicles (MVs) and apoptotic bodies (Abs) can regulate the process of calcification in the arteries and heart valves. 11 These membrane-bound vesicles contain the necessary calcium-binding proteins and phosphatases for nucleation of hydroxyapatite. Actually, extracellular vesicle-like structures have also been found in mineralizing cartilage. 12,13 These extracellular vesicles participate in cellular communication between cells in joint tissues and regulate the turnover of the extracellular matrix. 14 It is well known that autophagy and apoptosis can increase the generation of extracellular vesicles in cells. A recent study indicates that Abs from chondrocytes may contribute to the pathogenic process of cartilage calcification in aging patients with osteoarthritis. 15 However, it is unclear whether oxidative stress can increase the production of Abs and whether Abs can regulate the expression of calcification-related genes, such as ALP, osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2) and collagen II (COL-II) remain unclear. In addition, it is well known that the levels of extracellular PPi and Pi are critical for mineralization in cartilage. 15 However, there is no information on whether Abs can regulate the levels of PPi and Pi and the expression of PPi-related genes, such as ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and ankylosis protein (ANK) and tissue-nonspecific alkaline phosphatase (TNAP) in primarily cultured endplate chondrocytes.
In the present study, we isolated endplate chondrocytes of the IVD of rats and explored whether increased oxidative stress could increase the production of extracellular vesicles. Subsequently, we examined the effect of Abs on mineralization, PPi metabolism and related gene expression in primarily cultured endplate chondrocytes ex vivo. Our findings indicated that increased oxidative stress promoted the generation of Abs, which enhanced the oxidative stress-induced mineralization by modulating the PPi metabolism in endplate chondrocytes.

| Ethics statement
The experimental protocol was approved by the Institutional Animal Committee of Nantong University (Nantong, China, Permit Number: ntdx2017-0082). All animals were cared, in accordance with the 'Guidelines for the Care and Use of Laboratory Animals' as stated in the Helsinki Declaration. At the end of the experiment, the animals were killed using pentobarbital sodium.

| Culture and treatment of endplate chondrocytes
Endplate chondrocytes were isolated from the endplate cartilage of 4-week-old Sprague-Dawley (SD) rats, as described previously. 13 Endplate chondrocytes were cultured overnight in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% foetal bovine serum (FBS; Gibco), and exposed to fresh medium every 3 days. The cells were treated with, or without, different concentrations  For von Kossa staining, the fixed cells in each well were stained with 5% silver nitrate solution (Sigma, USA) and exposed to UV radiation for 1.5 hour. After being washed, individual wells were added with 250 μL of sodium thiosulfate and incubated for 5 minutes to remove nonspecific staining. The percentages of von Kossa positive staining were quantified in each experimental group.

| Abs Purification by centrifugation
Abs from the supernatants of culturedendplate chondrocytes were isolated by a sequential centrifugation approach. 16 Following treatment with H 2 O 2 [1 mmol/L]) for 12 hours to induce endplate chondrocyte apoptosis, the supernatants were subjected to multiple steps of centrifugations, first at 300 g for 10 minutes and 3000 g for 20 minutes to pellet Abs. Subsequently, the pellets were washed, resuspended and passed through a 0.8-mm pore filter, followed by centrifuging at 16 000 g for 40 minutes. Finally, the resulting supernatants were passed through a 0.2-mm pore filter, and centrifuged at 100 000 g for 1 hour to pellet exosomes.
The concentrations of proteins in individual Abs were determined by Bradford protein assay.

| Electron microscopy
After being washed, the H2O2-treated endplate chondrocytes were fixed immediately in 2.5% glutaraldehyde, post-fixed in 2% osmium tetroxide in 0.15 mol/L cacodylate buffer for 1 hour, washed in distilled water, dehydrated in a graded series of acetone series (30%-100%) and embedded in Spurr resin, followed by ultrathin sectioning at 70 nm. The sections were stained with uranyl acetate and lead citrate and analysed by transmission electron microscopy (JEM-1230; Jeol).
For scanning electron microscope, the samples were further fixed in 1% osmium tetroxide aqueous solution for 10 minutes and then dehydrated in a graded ethanol series (60%-100%). The slides were covered in hexamethyldisilazane for 30 seconds, left to dry in a dessicator, sputter-coated in gold and viewed in a scanning electron microscope (JSM-840; Jeol).

| Inorganic pyrophosphate and inorganic phosphate assay
The concentrations of Inorganic pyrophosphate (PPi) and inorganic phosphate (Pi) in the supernatants of cultured cells were measured by enzyme-linked immunosorbent assay (ELISA) using the Pyrophosphate Assay Kit and Phosphate Assay Kit (Molecular Probes, USA), respectively, according to the manufacturer's instructions. The reaction mixtures were incubated in triplicate for 10 minutes at room temperature, and measured for absorbance at 360 nm.  Table 1.

| Western blot analysis
The different groups of cells were harvested and lyzed in lysis

| Statistical analysis
Data are expressed as mean ± standard error of mean (SEM). The difference between two groups was analysed by Student's t test and the difference among groups was analysed by one-way analysis of variance (ANOVA) and post hoc least significant difference (LSD)

| Oxidative stress induces mineralization in primary endplate chondrocytes from rat IVDs
To explore the effects of oxidative stress on mineralization of endplate chondrocytes from IVDs, endplate chondrocytes were isolated from SD rats and treated with different concentrations of H 2 O 2 , followed by Alizarin Red and von Kossa staining. As shown in Figure 1A

| Oxidative stress increases the generation of apoptotic bodies (Abs) by endplate chondrocytes from rat IVDs
Increased oxidative stress can induce chondrocyte apoptosis. 18,19 To Exo, respectively, Figure 2E) in the purified EVs, consistent with the character of Abs. 20,21 Hence, oxidative stress increased the production of Abs in the primary endplate chondrocytes from rat IVDs, which may be associated with mineral deposition in endplate chondrocytes.

| Abs from the oxidative stressed endplate chondrocytes promote mineralization
To

| Abs from the oxidative stressed cells alter the levels of extracellular PPi in the supernatants of cultured endplate chondrocytes
It is wellknown that the levels of extracellular PPi and Pi are critical for mineralization in cartilage. 22,23 Accordingly, we tested the lev- These results support the notion that oxidative stress modulates calcification and promotes chondrocyte mineralization by increasing the contents of Pi.

| Abs form the oxidative stressed cells alter the expression of PPi metabolism-related genes in endplate chondrocytes
To understand the molecular mechanisms underlying the action of Abs in regulating the metabolism of PPi, we examined the relative levels of ENPP1, TNAP and ANK mRNA transcripts, which are primarily involved in PPi metabolism. 25,26 In comparison with that in

| D ISCUSS I ON
In this study, our data indicated that oxidative stress significantly promoted mineralization in primarily cultured endplate chondrocytes from IVDs of rats, associated with increased levels of ALP, RUNX2, OCN and COL-I expression in the endplate chondrocytes.
Because oxidative stress can modulate the generation of extracellular vesicles, including Abs, we found that treatment with H 2 O 2 significantly increased the production of Abs in endplate chondrocytes, probably contributing to mineral deposition. Actually, we found that Abs were effectively endocytosed and co-localized with COL-II in endplate chondrocytes in a short period after treatment with Abs.  increased Pi to promote the process of calcification in endplate chondrocytes. It is well known that the EVs contain many components, including mRNAs, long non-coding RNAs, rRNA, miRNAs or their fragments. 26 We are interested in further investigating how oxidative stress regulates the expression of those regulators and PPi metabolism and which components in Abs can enhance the H2O2-induced mineralization in chondrocytes.
In conclusion, our data indicated that oxidative stress induced the generation of Abs that promoted the mineralization in primarily cultured endplate chondrocytes. Oxidative stress and its related Abs enhanced PPi metabolism by modulating ENPP1, ANK and TNAP expression, contributing to the mineralization in endplate chondrocytes ( Figure 6). Our data suggest that both oxidative stress and its related Abs may be new therapeutic targets for intervention of CEP degeneration. Our findings may uncover new insights in the pathogenesis of CEP degeneration.

CO N FLI C T O F I NTE R E S T
The auths declare that there are no conflicts of interest associated with this study.