ObjectiveTo explore the value of matrix metalloproteinase 9 (MMP-9) in predicting the occurrence of heterotopic ossification by observing the expression of MMP-9 in heterotopic ossification of the early trauma rat model. MethodsA total of 132 male Sprague Dawley rats, aged 4-5 weeks, weighing (135.0±6.5) g, were randomly divided into experimental group and control group (n=66). In experimental group, the Achilles tendon was cut off and clamped to prepare heterotopic ossification model; in control group, only Achilles tendon was exposed by making a incision. The general condition of the rats was observed after operation; at 2, 3, 4, 5, 6, 7, and 8 days after operation, the Achilles tendon tissue was harvested for gross observation, histological observation, and immunohistochemical staining observation; the serum and Achilles tendon tissue were harvested to detect the expressions of MMP-9 protein and mRNA by ELISA and RT-PCR. The X-ray films at 5 and 10 weeks and histological examination at 10 weeks after operation were used to observe heterotopic ossification. ResultsAll rats survived to the end of the experiment. The Achilles tendon had no significant change in control group at each time point, showing normal tendon structure. In experimental group, the hardness of Achilles tendon tissue gradually increased with the time; there were a large number of irregular connective tissue and cartilage cells; and immunohistochemical staining for MMP-9 was positive results. The MMP-9 protein and mRNA expression levels of experimental group were significantly higher than those of the control group at each time point (P < 0.05). MMP-9 protein and mRNA expression levels of experimental group showed an increasing tendency (P < 0.05). According to the results of X-ray films and histological observation, heterotopic ossification occurred at 10 weeks after operation in experimental group, but no heterotopic ossification was observed in control group. ConclusionIn early heterotopic ossification of rat Achilles tendon, the expression of MMP-9 increases significantly, indicating that it has reference significance in predicting heterotopic ossification.
ObjectiveTo compare the efficacy of selective cyclooxygenase 2 (COX2) inhibitor and non-selective COX2 inhibitor drugs in prevention of heterotopic ossification in rats model so as to provide reference for clinical drugs selection of heterotopic ossification prevention. MethodsFifty male Sprague Dawley rats, 6 to 8 weeks old, weight (190.0±8.5) g, were selected; the right Achilles tendon was cut off to induce ectopic bone formation. The rats were randomly divided into 5 groups (n=10):on the 1st day after modeling, celecoxib was given in groups A[2 mg/(kg·d)] and B[10 mg/(kg·d)], indomethacin in groups C[(2 mg/(kg·d) and D[10 mg/(kg·d)], and 2 mL of saline in group E for 10 weeks. The general condition of rats was observed after operation. At 5 and 10 weeks after operation, X-ray films of the right lower limb were taken to observe new bone formation. At 10 weeks after operation, the right Achilles tendon tissue was harvested for histological observation. Based on X-ray and histological results, heterotopic ossification was assessed. Immunohistochemical staining was used to evaluate COX2 and bone morphogenetic protein 2 (BMP-2) expression levels in local Achilles tendon. ResultsDuring the experiment, 5 rats died (2 in group B, 1 in group C, and 2 in group D), the other rats survived to the end of the experiment. General observation of Achilles tendon tissue showed that the tendon tissue volume of group B was the smallest, with soft texture and no cartilage-like tissue; the tendon tissue volume of group E was the biggest, with hard texture and cartilage-like tissue. The incidence of heterotopic ossification was 80.0% (8/10), 25.0% (2/8), 88.9% (8/9), 50.0% (4/8), and 100% (10/10) in groups A-E respectively at 10 weeks after operation; significant differences were found between groups B, D and group E (P=0.002,P=0.023) and between groups B and C (P=0.015), but no significant difference was found among the other groups (P>0.05). COX2 expression level in groups B and D was significantly lower than that in group E (P<0.05), but no significant difference was found among the other groups (P>0.05); BMP-2 expression level in group B was significantly lower than that in groups A, C, and E (P<0.05), but no significant difference was found among the other groups (P>0.05). ConclusionCelecoxib at a dose of 10 mg/(kg·d) can effectively reduce the incidence of heterotopic ossification in rats.
ObjectiveTo explore the role and significance of hypoxia inducible factor lα (HIF-lα) and hypoxia microenvironment in the pathogenesis of post-traumatic heterotopic ossification by detecting the expression of HIF-lα in rat model of heterotopic ossification after Achilles tenotomy. MethodsA total of 140 male Sprague Dawley rats, aged 8-10 weeks, and weighing (210.1±10.6) g, were randomly divided into experimental group (n=70) and control group (n=70). In experimental group, the Achilles tendon was cut off and clamped to prepare post-traumatic heterotopic ossification model; in control group, only Achilles tendon was exposed. The general condition of rats was observed after operation, and at 2, 3, 4, 5, 6, 7, 8, 10, 12, and 14 days after operation, the Achilles tendon tissue was harvested from 6 rats for gross observation, histological observation, and immunohistochemical staining observation, and real-time fluorescence quantitative PCR and Western blot were used to detect the expressions of HIF-lα gene and protein at different time points in 2 groups. The X-ray films were taken and histological examination was done at 10 weeks after operation to evaluate the formation of heterotopic ossification. ResultsDuring the experiment, 1 rat died in experimental group at 3 days after operation, and the other rats survived to the end of the experiment. Gross and histological staining showed that the Achilles tendon had no obvious change, with normal tendon structure in control group at each time point. In experimental group, atrophy and necrosis of Achilles tendon stump were observed, with infiltration of inflammatory cells; and the hardness of Achilles tendon tissue gradually increased with the time; there were a large number of irregular connective tissue and cartilage cells. When compared with control group, the HIF-lα mRNA and protein expressions were significantly increased in experimental group at each time point (P < 0.05). Immunohistochemical staining showed that HIF-lα was positive in experimental group. According to the results of X-ray films and histological examination at 10 weeks after operation, heterotopic ossification was found in experimental group, but no heterotopic ossification in control group. ConclusionThe expression of HIF-lα significantly increases at early stage of post-traumatic heterotopic ossification after Achilles tenotomy, suggesting that the local hypoxia microenvironment plays an important role in the pathogenesis of heterotopic ossification.