Effect of COX-2 inhibitors and non-steroidal anti-inflammatory drugs on a mouse fracture model

A randomised, blinded, prospective animal study with 296 male C57BL/6N mice was performed to evaluate the biomechanical, biomolecular, biochemical, and histological impact of anti-inflammatory medications on fracture healing. A reproducible closed tibia fracture was created and stabilised with an intramedullary pin. Animals were randomised to placebo, ketorolac, ibuprofen, celecoxib, or rofecoxib treatment groups with biomechanical and biochemical testing at 4, 8, and 12 weeks. A second arm of the study was conducted in which animals were randomised to indomethacin or placebo treatment with biomechanical testing at 12 weeks. Histological and biomolecular studies were performed at 2 weeks on all groups in the first arm of the study. Biomechanical testing consisted of three-point bending evaluating maximum load, energy absorbed to maximum load, and stiffness. Safranin O-Fast Green stain was performed for histology. Biochemical quantifications of chondroitin and dermatan sulphate, hydroxyproline, total protein, and DNA content were performed. Osteocalcin and collagen types II and X were evaluated by in situ hybridisation. Some mechanical differences were seen between ketorolac and placebo at 4 weeks with respect to energy absorbed, but there were no differences in maximum load or stiffness seen between any treatment group and placebo at any time point. Indomethacin, celecoxib, rofecoxib, ibuprofen, and ketorolac did not significantly affect fracture healing in this young murine model.     

非甾体抗炎药临床应用的研究进展

非甾体抗炎药（non-steroidal anti-inflammatory drugs，NSAIDs）应用广泛，不仅可用于抗炎、镇痛，在防治肿瘤、阿尔茨海默病及脑保护等方面也发挥一定疗效。过去认为其药理作用与抑制环氧合酶-2（COX-2）有关，而其副作用与抑制COX-1有关。近期的研究则发现，COX-1和COX-2在炎症反应部位和胃肠道、肾脏等器官均有表达，NSAIDs的药理作用和不良反应均与两者密切相关；同时发现特异性COX-2抑制剂因打破体内COX-1与COX-2的平衡而具有潜在心血管危险性。因而传统所谓“COX-2抑制作用越特异，副作用越小”的观点可能是错误的。     

Age related effects of selective and non-selective COX-2 inhibitors on bone healing

IntroductionFractures are increasing worldwide and with an aging population, are frequent in the elderly. The healing of fractures progresses through various phases including the inflammatory stage. Aging is associated with slower healing and the use of non steroidal anti-inflammatory drugs (NSAIDs) may interrupt bone healing processes. We designed a study to compare the effect of diclofenac and celecoxib on fracture callus histomorphometry in a rat model of different age groups.MethodsUsing 5 and 15 month old rats, fractures were induced on the left tibia and the animals allocated to receive one of the drugs. Animals were sacrificed at day 21 and 42 and the fracture callus harvested for processing and histological evaluation. Tissue proportions and histological grades were determined and compared across the groups.ResultsAcross all groups, the histological grade increased with time and animals in the young diclofenac group had the highest grade at day 42 (p = 0.004). The proportion of bone increased in all groups and was highest in the young diclofenac group at day 21 and day 42 (p = 0.003). Post hoc analysis showed that the young celecoxib and old celecoxib groups had the least proportion of bone (p = 0.032 and p = 0.003). The proportion of cartilage reduced in all groups at both time points.ConclusionCelecoxib was associated with lower histological grade and lower proportion of bone in older animals. We urge for caution regarding the use of celecoxib in older people for the management of pain associated with fractures. Diclofenac may be a better option in this group.     

Effect of non-selective, non-steroidal anti-inflammatory drugs and cyclo-oxygenase-2 selective inhibitors on the PFA-100 closure time

The place of cyclo-oxygenase (COX)-2 selective non-steroidal anti-inflammatory drugs (NSAIDs) in the peri-operative period remains under discussion. Due to the absence of COX-2 in platelets, the risk of bleeding in patients who use selective NSAIDs is thought to be decreased. We studied the influence of aspirin, diclofenac, lornoxicam and rofecoxib on the in vitro bleeding time using the platelet function analyser (PFA-100). The PFA-100 simulates the process of platelet adhesion and aggregation after vascular injury in vitro. Measurements in 43 volunteers were performed at three time points: before, 3 h, and 12 h after oral ingestion of one of the randomly assigned study medications. Aspirin, diclofenac and lornoxicam had a significant effect on the in vitro closure time, while rofecoxib did not show this effect. This supports the use of COX-2 selective drugs in the peri-operative period to minimise the risk of bleeding.     

非甾体类消炎药对骨折愈合影响的研究进展

骨折后或骨折术后为了缓解疼痛临床上常用非甾体类消炎药（non—steroidalanti—inflammatorydrug,NSAID）镇痛。近年来研究发现NSAID在镇痛的同时对骨折愈合会产生不良影响。这一发现越来越引起各国学者的注意,笔者就此研究进展综述如下。     

Influence of diclofenac (group of nonsteroidal anti-inflammatory drugs) on fracture healing

Introduction Nonsteroidal antirheumatics (NSAR; NSAID) are often used in patients with fractured bones for analgetic reasons. This animal experiment was performed to determine the influence of NSAR on the process of fracture healing. As an alternative, tramadol, the centrally acting analgetic without peripheral effects, was included in this experiment.Materials and methods Wistar rats were operated on by a transverse osteotomy of the proximal tibia of the left leg. The fracture was stabilized by intramedullary nailing (healing period 21 days). All drugs were applied orally twice a day. The animals were divided into four groups with 10 rats each: Group 1 was treated with placebo (P), group 2 with tramadol (T; 20 mg/kg body weight/day), group 3 with diclofenac sodium (DS; 5 mg/kg bw/day) for 7 days followed by 14 days of placebo, group 4 with diclofenac sodium (DL; 5 mg/kgbw/day) over 21 days. On day 21 the rats were killed, and each leg was examined by X-ray, then the tibia was examined by CT scan, three-point bending, and histology.Results The results of CT and three-point bending showed that rats treated by diclofenac presented with delayed fracture healing compared with those treated by placebo or tramadol. Bone density in CT was highest in group 1 (mean 611.4±50.1 mg/ml), followed by group 2 (mean 542.5±29.5 mg/ml). Groups 3 (mean 411±34.0 mg/ml; p=0.006) and 4 (mean 395.2±15.4 mg/ml; p=0.009) were significantly lower. The stability of the bones, as measured by the breaking force (F_max), was highest in group 1 (mean 45.8±19.0 N), followed by group 2 (mean 39.0±7.9 N; NS); group 3 (mean 20.6±7.8 N; p=0.01) was significantly lower than the placebo animals, followed by group 4 (mean 26.5±8.3 N; p=0.03). Similar results were shown for bending stiffness: group 1 (mean 1404.6±611.4 Nmm/mm), group 2 (mean 1033.2±232.1 Nmm/mm; NS), group 3 (mean 564.2±457 Nmm/mm; p=0.045), and group 4 (mean 494.8±340.2 Nmm/mm; p=0.028). There were no significant differences between groups 1 and 2 and between groups 3 and 4, respectively. Diclofenac serum levels on day 21 in rats with long-term diclofenac application (mean 301.4±83.3 ng/ml) were comparable to those in humans.Conclusion Oral application of diclofenac significantly delayed fracture healing in rats. This effect might be comparable to other NSAR and fracture healing in humans.     

Paracetamol and selective and non-selective non-steroidal anti-inflammatory drugs for the reduction in morphine-related side-effects after major surgery: a systematic review

Non-opioid analgesics, paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs), or cyclo-oxygenase 2 (COX-2) inhibitors are often given along with morphine as part of multimodal analgesia after major surgery. We have undertaken a systematic review and a mixed treatment comparison (MTC) analysis in order to determine explicitly which class of non-opioid analgesic, paracetamol, NSAIDs, or COX-2 inhibitors is the most effective in reducing morphine consumption and morphine-related adverse effects. Sixty relevant studies were identified. The MTC found that when paracetamol, NSAIDs, or COX-2 inhibitors were added to patient-controlled analgesia (PCA) morphine, there was a statistically significant reduction in morphine consumption: paracetamol [mean difference (MD) -6.34 mg; 95% credibility interval (CrI) -9.02, -3.65], NSAIDs (MD -10.18; 95% CrI -11.65, -8.72), and COX-2 inhibitors (MD -10.92; 95% CrI -12.77, -9.08). There was a significant reduction in nausea and postoperative nausea and vomiting with NSAIDs compared with placebo (odds ratio 0.70; 95% CrI 0.53, 0.88) but not for paracetamol or COX-2 inhibitors, nor for NSAIDs compared with paracetamol or COX-2 inhibitors. There was no statistically significant difference in sedation between any intervention and comparator. On the basis of six trials (n=695), 2.4% of participants receiving an NSAID experienced surgical-related bleeding compared with 0.4% with placebo. The MTC found that there is a decrease in 24 h morphine consumption when paracetamol, NSAID, or COX-2 inhibitors are given in addition to PCA morphine after surgery, with no clear difference between them. Similarly, the benefits in terms of reduction in morphine-related adverse effects do not strongly favour one of the three non-opioid analgesics.     

雷帕霉素调节自噬抑制凋亡促进骨折愈合的机制研究

目的探讨雷帕霉素调节细胞自噬抑制凋亡促进骨折愈合的机制。 方法建立SD大鼠右侧股骨干骨折模型,随机分为两组：空白对照组和雷帕霉素组（1 mg/kg/d）。空白对照组无特殊处理,雷帕霉素组在术后12 h、24 h,3 d和7 d腹腔注射1 mg/kg的雷帕霉素。分别在造模后2 w,4 w和6 w应用X线和Micro CT评估骨组织愈合情况,免疫荧光检测LC3-Ⅱ,免疫组化检测Beclin-1的表达水平,western blot检测Bax、VEGF及p-mTOR,HE染色计数骨折后骨组织中成骨细胞数量来评估成骨活性。 结果与对照组相比,雷帕霉素组骨折端骨组织中p-mTOR及p-mTOR/mTOR比值降低[2 w（t=6.703,P=0.004;t=16.346,P=0.000）、4 w（t=16.407,P=0.000;t=16.738,P=0.000）、6 w（t=6.101,P=0.001;t=6.104,P=0.001）];自噬蛋白LC3-Ⅱ和Beclin-1表达明显增加[2 w（t=9.509,P=0.000;t=9.962,P=0.000）、4 w（t=3.868,P=0.008;t=24.490,P=0.000）、6 w（t=5.071,P=0.002;t=12.454,P=0.001）];抗凋亡蛋白Bcl-2表达增加[2 w（t=-9.099,P=0.000）、4 w（t=-8.128,P=0.000）、6 w（t=-9.497,P=0.000）];促凋亡蛋白Bax表达降低[2 w（t=-4.263,P=0.005）、4 w（t=-2.936,P=0.026）和6 w（t=-8.343,P=0.000）];VEGF表达增加[2 w（t=-5.754,P=0.001）、4 w（t=-5.077,P=0.002）和6 w（t=-12.200,P=0.000）],差异均具有统计学意义。X线结果显示雷帕霉素组术后2 w、4 w和6 w Garrett评分高于对照组（t=4.371,P=0.005;t=5.166,P=0.002;t=4.243,P=0.005）,micro-CT结果显示雷帕霉素组术后2 w、4 w和6 w骨密度评分高于对照组（t=8.765,P=0.000;t=25.649,P=0.000;t=11.199,P=0.000）,差异具有统计学意义。 结论雷帕霉素通过诱导细胞自噬,抑制细胞凋亡促进骨折愈合。     

Postfracture irradiation effects on the biomechanical and histologic parameters of fracture healing

The effects of single-dose local irradiation on the biomechanical properties of closed femoral fractures were studied in 75 mature Sprague-Dawley rats. Ten days after fracture, the rats were irradiated with 900 rads at 250 kV to the entire fractured femur. At 2, 3, 4, 8, and 16 weeks after fracture, both fractured and contralateral intact femurs were recovered and evaluated biomechanically by testing to failure in torsion. Results were compared with those from a similar study involving fractures irradiated 3 days after fracture as well as nonirradiated control fractures. Fracture healing progressed faster when irradiation was delayed 10 days than when delayed 3 days, and control fractures healed more rapidly than after either delay. In the 10-day delay group, fractures showed greater strength than did those in the 3-day delay group at 8 weeks, but the strength of irradiated fractures in both groups was similarly depressed at 16 weeks, with a maximum torque well below that of control fractures. These results suggest that delaying radiation exposure of a fracture may mitigate short-term deleterious effects on fracture repair, but that long-term results may be similar to those associated with expeditious irradiation.     

Shear movement at the fracture site delays healing in a diaphyseal fracture model.

This study tested the hypothesis that interfragmentary axial movement of transverse diaphyseal osteotomies would result in improved fracture healing compared to interfragmentary shear movement. Ten skeletally mature merino sheep underwent a middiaphyseal osteotomy of the right tibia, stabilized by external fixation with an interfragmentary gap of 3 mm. A custom made external fixator allowed either pure axial (n=5) or pure shear movement (n=5) of 1.5 mm amplitude during locomotion by the animals. The movement of the osteotomy gap was monitored weekly in two sheep by an extensometer temporarily attached to the fixator. After 8 weeks the sheep were killed, and healing of the osteotomies was evaluated by radiography, biomechanical testing, and undecalcified histology. Shear movement considerably delayed the healing of diaphyseal osteotomies. Bridging of the osteotomy fragments occurred in all osteotomies in the axial group (100%), while in the shear group only three osteotomies (60%) were partially bridged. Peripheral callus formation in the shear group was reduced by 36% compared to the axial group (p<0.05). In the axial group bone formation was considerably larger at the peripheral callus and in between the osteotomy gaps but not in the intramedullary area. The larger peripheral callus and excess in bone tissue at the level of the gap resulted in a more than three times larger mechanical rigidity for the axial than for the shear group (p<0.05). In summary, fixation that allows excessive shear movement significantly delayed the healing of diaphyseal osteotomies compared to healing under axial movement of the same magnitude.     

Gene expression of periostin in the early stage of fracture healing detected by cDNA microarray analysis.

To comprehensively evaluate gene expression in the early stage of fracture healing, we used a cDNA microarray with 2304 cDNA clones derived from an oligo-capped mouse embryo library. Closed mid-diaphyseal fractures were created in mouse tibiae and expression profiles were analyzed 3 days after fracture. Six genes were up-regulated in comparison to those in unfractured bones and these included three genes previously identified but never shown to be present in fractures, periostin, calumenin, and FHL-1. Cloning of these genes has been completed but their expression pattern and function during fracture healing and bone formation remain to be elucidated. Up-regulation of the six genes was reconfirmed by semi-quantitative RT-PCR analysis. Spatial and temporal expression of one of the newly identified fracture-induced genes, periostin, was analyzed using in situ hybridization, because it displayed the highest up-regulation ratio. A signal for periostin was detected in undifferentiated mesenchymal cells and immature preosteoblastic cells in the periosteal tissues between days 3 and 14 after fracture. Northern analysis showed that periostin gene expression rapidly increased by day 3, reached a peak on day 7, and declined by day 14. These findings suggest that periostin is a specific marker for preosteoblasts and may play an important role in periosteal callus formation during the early stage of fracture healing.     

The non-steroidal anti-inflammatory drug diclofenac reduces appearance of osteoblasts in bone defect healing in rats

Introduction

Non-steroidal anti-inflammatory drug (NSAID) is well known to significantly delay fracture healing. Results from in vitro studies implicate an impairment of osteoblast proliferation due to NSAIDs during the initial stages of healing. We studied whether diclofenac, a non-selective NSAID, also impairs appearance of osteoblasts in vivo during the early phase of healing (at 10 days).

Materials and methods

Two defects (Ø 1.1 mm) were drilled within distal femurs of 20 male Wistar rats. Ten rats received diclofenac continuously; the other obtained a placebo until sacrificing at 10 days. Osteoblast proliferation was assessed by cell counting using light microscopy, and bone mineral density (BMD) was measured using pQCT.

Results

Osteoblast counts from the centre of bone defect were significantly reduced in the diclofenac group (median 73.5 ± 8.4 cells/grid) compared to animals fed with placebo (median 171.5 ± 13.9 cells/grid). BMD within the defect showed a significant reduction after diclofenac administration (median 111.5 ± 9.3 mg/cm³) compared to the placebo group (median 177 ± 45.4 mg/cm³).

Conclusion

The reduced appearance of osteoblasts in vivo implicates an inhibiting effect of diclofenac on osteoblasts at a very early level of bone healing. The inhibition of proliferation and migration of osteoblasts, or differentiation from progenitor cells, is implicated in the delay of fracture healing after NSAID application.

     

Effects of a selective cyclooxygenase-2 inhibitor (celecoxib) on fracture healing in rats

Background:

Several studies suggested that celecoxib interferes with bone healing while others contradict these findings. This study was conducted to investigate the effects of celecoxib on bone healing in rats femur mold with a dose based on body surface area conversion.

Materials and Methods:

72 adult female Sprague Dawley rats were randomly divided into three groups after the internal fixation operation of nondisplaced transverse mid diaphyseal fractures of the right femurs. Each group was treated with 1% methylcellulose, celecoxib (21 mg/kg/d) for 1 week, or celecoxib (21 mg/kg/d) for 4 weeks after surgeries respectively. Bone healing scores and callus formation were evaluated by radiographs at 3, 4, 6 weeks after surgeries. Half of these rats were sacrificed for histological analysis at 4 weeks after surgery. The remaining fractured femurs were evaluated by biomechanical tests at 6 weeks after surgery.

Results:

The mean radiographic scores for fracture healing of both short and long term groups were lower than that of the control group and the differences among the three groups were statistically significant (P < 0.05) at 3, 4, 6 weeks after surgery. The mean bone trabecula density of both groups was smaller than that of the control group and the differences were also statistically significant (P < 0.05) at 4 week. The maximum load, total energy and stiffness in both the short term and long term groups were significantly decreased compared with those in the control group (P < 0.05) at 6 week.

Conclusion:

Both short term and long term sustained use of celecoxib in rat models has significantly inhibitory effects on rat fracture healing.     

Calcitonin and fracture healing. An experimental study on rats

The effects of systemically administered calcitonin (CT) on fracture healing were analyzed in an experimental study on rats. The healing of a fracture was followed from 3 days up to 9 weeks postoperatively. Half of the rats in each age group were given daily CT 10 MRC-U/kg body wt s.c. Mechanical properties of the healing tibial fractures (tension strength) as well as various connective tissue components of the callus tissue were analyzed. No difference in the radiological or microscopical appearance of the fractures was detectable between the animals receiving CT and the controls. In the biochemical analysis matrix production as assessed from the concentrations of nitrogen, hexosamines, and hydroxyproline within the callus followed the usual lines of undistributed fracture union without any difference between the groups with and without CT. No differences could be detected in the mineralization of the callus between the specimens from animals receiving CT and those without. The tensile strength values of the fractures increased almost linearly up to 9 weeks. At 1 week the tensile strength values for fractures union in the animals without CT were approximately 50% higher, but later on no differences could be detected between the groups. These results indicate that although in the early phases of long-term CT therapy collagen synthesis may be impaired, there will be no effect on the net content of collagen or calcifying tissue in the callus or on the mechanical strength of healing fractures.     

围手术期应用选择性环氧化酶-2抑制剂对胫腓骨中段骨折愈合的影响

目的 探讨围手术期短期应用选择性环氧化酶-2(COX-2)抑制剂进行镇痛对胫腓骨中段骨折愈合的影响. 方法 采用前瞻性对照研究方法,将2008年10月至2009年10月收治的胫腓骨中段闭合性骨折患者分为对照组和研究组.对照组55例,男35例,女20例;平均年龄为(35.5±2.4)岁.研究组52例,男30例,女22例;平均年龄为(34.6±3.2)岁.研究组患者在围手术期短期(7～10 d)联合应用选择性COX-2抑制剂(塞来昔布胶囊和帕瑞昔布)进行镇痛,对照组未应用选择性COX-2抑制剂.观察两组患者术后1、2、3、4、6、8、12、18个月骨折端出现明显骨痂及骨折线消失的病例数,比较两组患者骨痂出现时间、骨折线消失时间及骨折愈合时间等的差异. 结果 107例患者术后获18 ～ 30个月(平均22个月)随访.对照组术后1、2、3、4个月骨折端可见明确骨痂生成的病例数分别为7、15、52、55例,研究组分别为6、13、49、52例;对照组术后6、8、12、18个月骨折端骨折线消失的病例数分别为3、42、53、55例,研究组分别为2、38、49、52例,以上项目两组间比较差异均无统计学意义(P＞0.05).对照组和研究组的骨折愈合时间平均分别为(10.0±2.3)、(10.2±2.5)周,差异无统计学意义(t=0.689,P=0.425).两组患者均无骨不愈合、感染、成角畸形等并发症发生.结论 围手术期短期应用选择性COX-2抑制剂进行镇痛对胫腓骨中段骨折愈合无明显影响.