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 significantly had given rise to the HO. The largest size of heterotopic ossification 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 significance. 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 significantly 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 fluorescence intensity of P2X7R in Achilles tendon was considerably different from each other (p<0.05) (Fig. 1c). In HOG and TPG, the immunofluorescence (IF) intensity of P2X7R was significantly 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 significant 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 inflammation condition plays an important role in the formation of HO.
P2X7R agonist promoted the expression of P2X7R of MSCs under inflammation condition
During the HO formation, inflammation environment is an essential part. To exclude other purinergic receptors’ interference, Bz-ATP, a P2X7R specific agonist was used. This investigated the effect of Bz-ATP (100μM) under inflammation condition on P2X7R of MSCs. MSCs grown in normal growth medium with the Bz-ATP added served as the control. MSCs grown in inflammation condition medium with the Bz-ATP served as the experimental group. IF analysis illustrated that under inflammation condition, Bz-ATP promoted the expression of P2X7R (Fig.2a). Relative fluorescence intensity of P2X7R between two groups had significant difference. In the Bz-ATP combined inflammation group was higher than the other group significantly (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 confirm that under inflammation condition, Bz-ATP stimulates the overexpression of P2X7R.
Bz-ATP promoted osteogenesis under inflammation condition
To further investigate the role of increased expression of P2X7R on osteogenesis under inflammatory 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 inflammation condition, Bz-ATP intervention brought about a remarkable surge of ALP activity and the mineralized nodes formation, compared with no inflammation condition intervention. ALP activity and staining revealed higher ALP activity in inflammation condition intervention group than in the no inflammation 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 inflammation condition intervention, mRNA expression levels of osteogenic-associated genes such as ALP, RUNX2, OPN were significantly 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 significantly higher in the inflammation condition intervention than that of without the inflammation 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 inflammation condition.
Brilliant Blue G (BBG) decreased the osteogenetic ability of MSCs promoted by Bz-ATP under inflammation condition
To diminish the effect between overexpression of P2X7R and the enhancement of osteogenesis of MSCs under Bz-ATP and inflammatory condition, the P2X7R antagonist BBG was employed. When BBG (10 μM) was added into osteo-inductive medium under Bz-ATP and inflammation condition, it significantly crippled the expression of P2X7R proved by the IF analyse (Fig4. a). Relative fluorescence intensity of P2X7R between two groups had significant difference (Fig4. b). Similarly, qRT-PCR analysis showed that the inhibition of P2X7R with BBG in MSCs significantly curtailed P2X7R mRNA expression (Fig4. c). Then, the ALP and ARS staining were conducted to explore the intervention of BBG on MSCs osteogenesis under inflammation 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 inflammation 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 significantly (p<0.0001, p<0.0001, p<0.01, respectively) (Fig. 4e). Similarly, the results of protein assays confirmed 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 inflammation 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 confirm 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 significant (p < 0.05). At the 12th week, the difference of BV was more significant 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.