A higher expression of P2X7 receptor at the site of heterotopic ossication promotes osteogenesis

Heterotopic ossication (HO) refers to a painful and complex disease. Adenosine triphosphate (ATP), as a key modulator of inammation, is veried to promote the development of HO. However, the mechanism remains to be illustrated. The ionotropic P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed in the majority of stem cells. Here, this paper hypothesizes that P2X7R may be activated by extracellular ATP and promote osteogenesis of stem cells under inammatory condition, ending up in the formation of ectopic bone.


Methods
The tenotomy puncture and burn injury-induced HO model was constructed. The expression of P2X7R was found increasing at the site of injured Achilles tendon where HO occurs. Mesenchymal stem cells (MSCs) were cultivated under an in ammatory condition plus Bz-ATP treatment which mimicked a microenvironment of HO site. An induction in P2X7R expression was also observed along with an enhancement of osteogenesis. In addition, an inhibition of P2X7R expression by its speci c antagonist successfully reversed the above process.

Results
P2X7R expression of the Achilles tendon and osteogenic capability of SCs is higher in HOG than in other two groups. Bz-ATP promoted osteogenesis under in ammation condition. BBG impeded the heterotopic bone formation in animal model.

Conclusions
P2X7R is a crucial mediator in ATP-signaling promotion of HO, blocking which may represent a potential therapeutic target for HO.

Background
Heterotopic ossi cation (HO) refers to a complex, reactive, musculoskeletal condition characterized by ectopic bone formation in muscles, tendons, or other soft tissues [1] . HO tends to be accompanied by trauma, burns, and orthopedic surgeries etc. There are many theories explaining why it develops. However, the mechanism remains largely unknown. Therefore, there are no completely effective treatment methods that can be taken to prevent this clinically devastating pathological condition.
Tissues vulnerable to HO act out a dysregulated in ammatory response to injury. Dwelling in a prolonged or abnormally heightened in ammatory microenvironment [2][3] , stem cells including mesenchymal stem cells (MSCs) that attempt to repair tissue differentiate into osteogenic cells instead of its original fate. Moreover, such osteogenic cells have witnessed the intensifying formation of bone which has been recognized as a fundamental process for HO. Adenosine triphosphate (ATP) is a key modulator of in ammation [4] . Prior research has illustrated that extracellular ATP is of importance to in ammationinduced HO, as it interacts with bone morphogenetic protein-mediated canonical SMAD signaling [5] .
Moreover, with the application of apyrase, an enzyme that hydrolyzes ATP to adenosine monophosphate (AMP), sees a rise in intracellular adenylate cyclase activity and cyclic AMP (cAMP). The reduction of bone formation was achieved at the site of HO [5] .
The ionotropic P2X7R is an ATP-gated ion channel expressed in most stem cells. Activated by extracellular ATP, P2X7R plays a wide range of physiological and pathological roles in in ammation and in ammation-related condition [6] . It is noteworthy that P2X7R expression on bone marrow MSCs has been found to regulate the formation of osteoblasts at different stages to orchestrate bone metabolism [7] . Also, recent researches have con rmed the crucial role for P2X7R in periodontal ligament stem cells (PDLSC) osteogenesis under in ammatory condition [8] . Nonetheless, the expression pattern of P2X7R on MSCs at the HO site and whether it is essential to the mediation of ATP signaling-related bone formation remain unknown.
Here, this paper applied a tendon puncture and burn injury-induced HO model. The increasing expression of P2X7R emerged at the site of injured Achilles tendon where HO occurs. Subsequently, in vitro model mimicking an in ammatory microenvironment with ATP intervention around MSCs was establishment.
An induction in P2X7R expression was also observed along with an enhancement of osteogenesis. In addition, an inhibition of P2X7R expression by its speci c antagonist successfully reversed the above process, which may suggest that it could be a potential therapeutic target for HO.

Animal model
The animal experiment was grouped into three cohorts, sham group(Sham), Achilles tendon puncture group (Tendon puncture group, TPG), 30% total body surface area (TBSA) partial thickness burn injury combined Achilles tendon puncture group (HOG), respectively. Partial thickness scald injury was performed as Peterson JR et al. described [9] . In brief, 8 weeks, male, C57BL/6 mice in sham group, the Achilles tendons were exposed alone. In TPG, after Achilles tendon exposed, a 27G needle was punctured into the Achilles tendon body perpendicularly at different parts from proximal to distal and this process was repeated three times. In HOG, apart from the Achilles tendon punctured, mice also received about 30% TBSA burn injury with exposure to 60℃ for 18 s (Fig. 1a) [10] .
Cell harvest and in vitro culture assays Achilles tendon stem cells were harvested from the different groups. Stem cells (SCs) from Achilles tendon were isolated based on the research Xu et al. reported [8] , and modi ed slightly. In short, freshly harvested tendon was washed with sterile phosphate-buffered saline (PBS; Gibco, New York, USA), and then tendon tissue was placed into 30mm dish. The tissue was cut into fragments and digested in 3 mg/ml type I collagenase (Sigma-Aldrich, St. Louis, USA) for 45 minutes. Then, the digested fragments were cultivated in 35 mm dish (Corning, New York, USA) in α-minimum essential medium (α-MEM, Gibco) containing 100 U/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco), and 10% fetal bovine serum (FBS; Gibco, Australian). In in vitro experiment, mesenchymal stem cells (MSCs) were purchased from the Peking Union Medical College Hospital cell bank. The complete culture medium was changed every 3 days. Cell passage was performed until the cell fusion rate reached about 80 ~ 90%. MSCs at passage 2-5(P2-P5) were used for further experiment. For osteogenic differentiation, complete osteo-inductive medium was replaced every 2 days when the percentage of cell fusion dipped to 60-70%. Osteoinductive medium contained α-MEM and 10% FBS supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM β-glycerophosphate, 50 µg/mL L-ascorbic-2-phosphate and 10 nM dexamethasone (all from Cyagen). TNF-α(10 ng/ml) and IL-1β 5 ng/ml was added to the medium of MSCs to simulate in ammation environment in vitro [11] .

Micro CT
Micro CT imaging of HO of Achilles tendon was obtained from all animals by means of the highresolution Micro CT equipment (Siemens, Munich, Germany). CT scan settings: 80 kV, 500µA, and 1100ms exposure. The bone mineral in the soft tissues was quanti ed for HO bone volume using standard protocols as Peterson et al. described [12] .

Immuno uorescent Staining
MSCs were seeded into a 96-well culture plate designed for confocal microscopy and cultivated in α-MEM containing 10% FBS. When the con uence of cells reached 80 ~ 90% (day 0), different condition was added respectively for intervention. After 7d culture or 14d osteogenesis inducing, adherent cells were xed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 15 min and blocked with 10% goat serum for 40 min at room temperature. The cells were then incubated at 4°C with an antibody against P2X7R (1:200, Abcam, ab48871) overnight. The samples were washed with PBST, and then incubated in the dark place at 25℃ with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:500; Abcam, ab150077) for 2 h. Finally, the cells were stained in a dark room at 25℃ with DAPI for 30 min.
ALP staining ALP activity in MSCs was analyzed with ALP staining. In brief, cells were seeded at 1⊆10 4 cells/well in 96-well plates. ALP staining was performed after 14 days of osteo-inductive culture using an ALP staining kit (Beyotime Biotechnology, China) according to the instructions. The quantitative analysis of ALP staining was performed with the help of an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, China).

ARS staining
According to the manual instructions, Alizarin red staining (ARS, Cyagen,Suzhou, China) was completed which was used to evaluate the osteogenic differentiation of cells. To quantify the staining for statistical analysis, the mineralized nodes were dissolved in 2% cetylpyridinium chloride for 20 min, and then OD values were measured at 560 nm wavelength [13] .

Protein assays
The protein expression pro les of cells or tissue (prepared for analysis) were measured by ProteinSimple Jess Capillary electrophoresis instrument. In brief, total proteins were extracted using RIPA lysis (Proteintech, PR20001). After cell lysis of 30 minutes, the supernatant was collected by centrifugation with 13,000g. Protein concentration was determined by BCA kit (Keygen BioTECH, Jiangsu, China). Supernatant were run on a ProteinSimple Jess Capillary electrophoresis instrument using instrument default settings and manufacturer's standard protocol [14] . Approximately 3µg (1µg/µl) total protein were loaded per capillary. Quanti cation of the nal images was carried out using compass for SW software (Version 5.0.1).

qRT-PCR assays
Total RNA was extracted using RNAeasy Kit (Vazyme, RC112-01) and 1 µg of total RNA was used to generate cDNA using HiScript II Q RT SuperMix (Vazyme, R223-01) for qPCR. Quantitative Real time PCR (qRT-PCR) was performed on cDNA samples diluted twentyfold in double-distilled water using SYBR Green Master Mix (Vazyme, Q711). The CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was used. To amplify them under the following conditions: denaturation at 95°C for 5 min; 40 cycles at 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s, with melt curve analysis from 65 to 95°C in increments of 0.5°C. The gene-speci c qRT-PCR primers were speci c for mouse. Relative mRNA gene expression levels were measured by qRT-PCR. Relative quanti cation for qRT-PCR was calculated using the 2 − ΔΔCT method. The sequences of primers used in the present study are shown in supplementary Table. 1.

Statistical Analysis
All results are calculated by the mean and standard deviation (mean ± S.D.). There are more than three samples. Experiments for each cell were performed independently for three times. GraphPad Prism 9 software was utilized for statistical tests. The Student's t-test was used for analysis of two unpaired groups, and one-way ANOVA followed by Tukey's posttest for analysis of multiple groups. Statistical signi cance was established at p < 0.05.

Ethics statement
The ethics committee of the general hospital of People Liberate Army of China and Harbin Medical University approved this experiment.

Results
P2X7R expression of the Achilles tendon and osteogenic capability of SCs is higher in HOG than in other two groups In this study, TPG and HOG all signi cantly had given rise to the HO. The largest size of heterotopic ossi cation formation was in HOG, and the no HO was observed in the sham group (Fig. 1b). Then, this paper compared the bone volume of HO in different groups. These disparities were of statistical signi cance. Bone volume (BV) of HO in HOG and TPG outstripped that of Sham, p values were less than 0.0001 and 0.0001, respectively. There was a signi cantly higher BV in the HOG than in the TPG (p< 0.01) Fig. 1b). Burn injury enhanced bone formation as previous reports [15][16] .
After the establishment of the model, at 7 days, the Achilles tendon was resected for further study. Then, the Achilles tendon tissues of different groups received pathological analysis. Among three groups (HOG, TPG and Sham), the uorescence intensity of P2X7R in Achilles tendon was considerably different from each other (p<0.05) (Fig. 1c). In HOG and TPG, the immuno uorescence (IF) intensity of P2X7R was signi cantly higher compared with sham group (p < 0.001) (Fig. 1d). The expression of P2X7R protein also differed in three groups. HOG and TPG had higher expression level compared to sham group. However, there was no signi cant difference between HOG and TPG (p>0.05) (Fig. 1e).
Then, the osteogenic capabilities of SCs extracted from Achilles tendon in different groups were examined. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining. After 14 days of the osteogenic induction, osteogenesis differentiation of the SCs was assessed by ALP (Fig. 1f). The results of the ALP positive cells suggested the difference among the different groups. In HOG, SCs had better osteogenesis compared with the other groups. The software of Image J was employed to quantify ALP staining. The ability of osteogenesis of SCs from HOG was higher than that of the TPG and sham on account of a higher cell ALP staining positive area rate (Fig. 1g) (p<0.0001,0.001, respectively).
The result indicates that the higher expression of P2X7R, the higher ability of osteogenesis of SCs. Therefore, the ATP and in ammation condition plays an important role in the formation of HO.

P2X7R agonist promoted the expression of P2X7R of MSCs under in ammation condition
During the HO formation, in ammation environment is an essential part [10] . To exclude other purinergic receptors' interference, Bz-ATP, a P2X7R speci c agonist was used. This investigated the effect of Bz-ATP (100μM) under in ammation condition on P2X7R of MSCs. MSCs grown in normal growth medium with the Bz-ATP added served as the control. MSCs grown in in ammation condition medium with the Bz-ATP served as the experimental group. IF analysis illustrated that under in ammation condition, Bz-ATP promoted the expression of P2X7R (Fig.2a). Relative uorescence intensity of P2X7R between two groups had signi cant difference. In the Bz-ATP combined in ammation group was higher than the other group signi cantly (p<0.001) (Fig.2b). Similarly, the results of protein assays and qRT-PCR analysis supported the outcome of IF (Fig.2c, d). These results con rm that under in ammation condition, Bz-ATP stimulates the overexpression of P2X7R.

Bz-ATP promoted osteogenesis under in ammation condition
To further investigate the role of increased expression of P2X7R on osteogenesis under in ammatory condition, Bz-ATP was added in medium. To test the osteogenic differentiation variances among the different groups ALP staining and Alizarin Red staining(ARS) were performed, and then ALP activity and ARS solution absorbance was measured. Under in ammation condition, Bz-ATP intervention brought about a remarkable surge of ALP activity and the mineralized nodes formation, compared with no in ammation condition intervention. ALP activity and staining revealed higher ALP activity in in ammation condition intervention group than in the no in ammation condition intervention group (p<0.05). The results of ARS staining was also similar to ALP (p<0.05) (Fig.3a). According to the qRT-PCR analysis, under the same Bz-ATP intervention, in the group of in ammation condition intervention, mRNA expression levels of osteogenic-associated genes such as ALP, RUNX2, OPN were signi cantly higher compared with the other group (p<0.001, p<0.05, p<0.0001, respectively) (Fig.3b). Similarly, protein analysis maintained that under the same Bz-ATP intervention, the expression of ALP, RUNX2 and OPN protein were signi cantly higher in the in ammation condition intervention than that of without the in ammation condition (p<0.001, p<0.001, p<0.001, respectively) (Fig.3c). Based on the above data, when Bz-ATP was added into osteo-inductive medium, the expression of P2X7R and the ability of osteogenesis of MSCs were enhanced under in ammation condition.

Brilliant Blue G (BBG) decreased the osteogenetic ability of MSCs promoted by Bz-ATP under in ammation condition
To diminish the effect between overexpression of P2X7R and the enhancement of osteogenesis of MSCs under Bz-ATP and in ammatory condition, the P2X7R antagonist BBG was employed. When BBG (10 μM) was added into osteo-inductive medium under Bz-ATP and in ammation condition, it signi cantly crippled the expression of P2X7R proved by the IF analyse (Fig4. a). Relative uorescence intensity of P2X7R between two groups had signi cant difference (Fig4. b). Similarly, qRT-PCR analysis showed that the inhibition of P2X7R with BBG in MSCs signi cantly curtailed P2X7R mRNA expression (Fig4. c). Then, the ALP and ARS staining were conducted to explore the intervention of BBG on MSCs osteogenesis under in ammation and Bz-ATP condition. The results of ALP activity and staining revealed that ALP activity in no BBG intervention group was higher than that of BBG intervention (Fig. 4d) (p<0.05). The results of ARS staining also had the similar result as ALP (Fig. 4d) (p<0.01).
To further investigate the mechanism of BBG on osteogenesis of MSCs under Bz-ATP and in ammation condition, the methods of qRT-PCR and protein assays were used. According to the qRT-PCR, with the BBG intervention, the mRNA expression of osteogenic-related genes, such as ALP, RUNX2, OPN, deceased signi cantly (p<0.0001, p<0.0001, p<0.01, respectively) (Fig. 4e). Similarly, the results of protein assays con rmed that BBG also decreased the expression of ALP, RUNX2, OPN of MSCs in comparison to the no BBG intervention (p<0.01, p<0.05, p<0.01, respectively). Taken together, under Bz-ATP and in ammation condition, the intervention of BBG could control the osteogenesis of MSCs. Cytological results of BBG intervention may provide a novel treatment for HO.

Intervention of BBG reduced heterotopic bone formation in animal model
To further con rm whether the therapeutic effects of intervention of BBG in animal model, animal experiments were carried out. HO animal model of the 30% TBSA partial thickness burn injury combined Achilles tendon puncture was used. 6 hours after the modeling, the intervention of BBG (50 mg/kg) by intraperitoneal administration was employed. The BBG administered once daily for 14 consecutive days, and the mice were watched for 12 weeks. Saline solution was injected as the control. Micro CT analysis was conducted to evaluate heterotopic bone formation at 1st, 6th and 12week (Fig. 5a). At the 1st week, there was no heterotopic bone formation between two groups. At the 6th week, there was heterotopic bone formation in two groups. The difference of bone volume (BV) of heterotopic bone between two groups was statistically signi cant (p < 0.05). At the 12th week, the difference of BV was more signi cant between the two groups (p < 0.0001) (Fig. 5b). Based on the result of animal experiment, treatment of BBG reduced heterotopic bone formation in HO model.

Discussion
In ammation has been con rmed to be a key driver of HO [17] . A commonality across many of the conditions that predispose to HO formation such as autoimmune diseases, trauma, burn etc. is that they are more or less related to in ammation [15] . Clinical application of NSAIDs for HO prophylaxis rests on the theory that reducing postoperative in ammation will likewise reduce HO formation albeit that no robust evidence has been provided [1] . Prior review by Benjamin Levi et al. summarized the crucial role of in ammation in development of HO [18] . Among which, processes involved in innate immunity, adaptive immunity, secretion of in ammatory cytokines had all been demonstrated to be of importance underlying the mechanism of HO [18] .
As a key in ammatory mediator, ATP acts as not only an energy supply molecule but also a signal transducer released upon stimulation or due to passive leakage of damaged or dying cells. The role of extracellular ATP in in ammation is supported by the direct in vivo demonstration that in ammatory sites contain high (hundred micromolar) extracellular ATP concentration [4] . In HO model, prior research has shown that extracellular ATP acted as a signal transducer, which interacts with bone morphogenetic protein mediated canonical SMAD signaling to promote HO development [5] . Moreover, with application of apyrase, a rise occurs in intracellular adenylate cyclase activity and cAMP and a reduced bone formation was achieved at the site of HO [5] . In this study, when MSCs were cultivated under Bz-ATP and in ammation condition, the result showed a signi cant enhancement of osteogenesis compared with those without in ammation condition, which also con rmed the crucial role of in ammation condition in HO formation.
Almost all living organisms, from protozoa to higher mammals, have evolved a sophisticated receptor set for extracellular nucleotides. In higher mammals, the nucleotide receptor family (P2 receptors, P2Rs) is comprised of two subfamilies: G protein-coupled metabotropic P2Y (P2YR) and ligand (ATP)-gated ionotropic P2X receptors (P2XR) [19] . Among these family, P2X7R is the most studied and outstanding as the single member of the P2XR family with a rmly established role in multiple in ammatory and immune responses [6] . Prior studies have shown that P2X7R plays a crucial role of bone homeostasis with controversial conclusions [19][20] . The P2X7R has previously been shown that it plays a crucial role in the bone anabolic under the mechanical loading [21] . P2X7R involves bone formation through Wnt/βcatenin signaling [22] . But another study points out that at low concentrations of Bz-ATP (from 0.5 to 5.0 mM), Bz-ATP not only decreases the mRNA expression of Wnt3a, but also reduces mineralization without effect on proliferation [23] . In this study, in vivo model showed an intensi ed expression of P2X7R in the injured Achilles tendons together with a formation of exotic bone. Using in vitro model mimicking an in ammatory microenvironment with BZ-ATP, an induction in P2X7R expression was also observed. As exposed to an osteogenesis condition medium, an enhancement in osteogenesis was achieved in MSCs and they expressed higher P2X7R. Moreover, this enhancement was attenuated when BBG was added into the medium. These results suggested that P2X7R plays a crucial role in the mechanism underlying in ammation-induced HO.
BBG is a P2X7R antagonist of low toxicity and high selectivity. Previous studies have reported that BBG treatment reduces tissue injury and promotes motor function recovery after spinal cord injury (SCI) [24][25] . There are some limitations in this research. Although the treatment of BBG reduces progression of heterotopic ossi cation in animal model, the role of BBG in the initiation occurrence of heterotopic ossi cation is uncertain. In clinical research, progression of heterotopic ossi cation in tendon leads to restricted joint mobility and pain [26] . Nevertheless, there are differences in current HO prophylaxis. Taken together, the treatment of reducing the rate of surgery is a key for HO prevention and management. Because BBG inhibits osteogenesis of MSCs under in ammation and ATP condition, it may provide a new therapeutic target for HO in the future.

Conclusion
The expression of P2X7R was found increasing in tendon puncture and burn injury-induced HO. A combination of in ammatory condition and ATP may be the trigger of this up-expression of P2X7R. An induction in P2X7R expression led to an enhancement of osteogenesis while an inhibition of P2X7R attenuated it, which may suggest that P2X7R is a crucial mediator underlying the mechanism of HO. This study may bring forward a novel therapy for the HO.  Figure 1 In HOG, apart from the Achilles tendon punctured, mice also received about 30% TBSA burn injury with exposure to 60℃ for 18 s (Fig. 1a) .The largest size of heterotopic ossi cation formation was in HOG, and the no HO was observed in the sham group (Fig. 1b). Then, this paper compared the bone volume of HO in different groups. These disparities were of statistical signi cance. Bone volume (BV) of HO in HOG and TPG outstripped that of Sham, p values were less than 0.0001 and 0.0001, respectively. There was a signi cantly higher BV in the HOG than in the TPG (p< 0.01) Fig. 1b). Burn injury enhanced bone formation as previous reports [15][16]. After the establishment of the model, at 7 days, the Achilles tendon was resected for further study. Then, the Achilles tendon tissues of different groups received pathological analysis. Among three groups (HOG, TPG and Sham), the uorescence intensity of P2X7R in Achilles tendon was considerably different from each other (p<0.05) (Fig. 1c). In HOG and TPG, the immuno uorescence (IF) intensity of P2X7R was signi cantly higher compared with sham group (p < 0.001) (Fig. 1d). The expression of P2X7R protein also differed in three groups. HOG and TPG had higher expression level compared to sham group. However, there was no signi cant difference between HOG and TPG (p>0.05) (Fig. 1e).

Figure 2
IF analysis illustrated that under in ammation condition, Bz-ATP promoted the expression of P2X7R (Fig.2a). Relative uorescence intensity of P2X7R between two groups had signi cant difference. In the Bz-ATP combined in ammation group was higher than the other group signi cantly (p<0.001) (Fig.2b).
Similarly, the results of protein assays and qRT-PCR analysis supported the outcome of IF (Fig.2c, d).
These results con rm that under in ammation condition, Bz-ATP stimulates the overexpression of P2X7R.

Figure 3
ALP activity and staining revealed higher ALP activity in in ammation condition intervention group than in the no in ammation condition intervention group (p<0.05). The results of ARS staining was also similar to ALP (p<0.05) (Fig.3a). The results of ARS staining was also similar to ALP (p<0.05) (Fig.3a). According to the qRT-PCR analysis, under the same Bz-ATP intervention, in the group of in ammation condition intervention, mRNA expression levels of osteogenic-associated genes such as ALP, RUNX2, OPN were signi cantly higher compared with the other group (p<0.001, p<0.05, p<0.0001, respectively) (Fig.3b). Similarly, protein analysis maintained that under the same Bz-ATP intervention, the expression of ALP, RUNX2 and OPN protein were signi cantly higher in the in ammation condition intervention than that of without the in ammation condition (p<0.001, p<0.001, p<0.001, respectively) (Fig.3c).

Figure 5
Saline solution was injected as the control. Micro CT analysis was conducted to evaluate heterotopic bone formation at 1st, 6th and 12week (Fig. 5a). At the 1st week, there was no heterotopic bone formation between two groups. At the 6th week, there was heterotopic bone formation in two groups. The difference of bone volume (BV) of heterotopic bone between two groups was statistically signi cant (p < 0.05). At the 12th week, the difference of BV was more signi cant between the two groups (p < 0.0001) (Fig. 5b).