骨折愈合应力机制研究进展

骨折愈合是个复杂的过程.研究表明应力是影响骨折愈合的主要因素之一,应力使骨细胞间连接的树状突起增加,骨细胞骨架变形,骨细胞形态改变,细胞基质分泌增加.应力导致骨小管流体运动异常时,骨细胞可将这种信号传递到成骨细胞和破骨细胞,诱导骨细胞凋亡、成骨细胞和骨膜细胞增殖.应力影响骨代谢激素的作用,使成骨作用和破骨作用均增强,但以成骨作用增强为主.应力使多种细胞因子的合成增加,多种骨细胞特异性信号传导通路打开.此外,随着对应力遮蔽作用认识的深入,有限元分析已越来越多地应用到骨科学研究和治疗中.应力和骨骼微损伤对骨折愈合的作用也直接关系到骨折的预后情况,现也受到关注.     

骨折愈合的应力适应性研究

目的 探讨骨折端受力、肌肉动力、骨痂密度与骨折愈合的关系。方法 通过传感器电测技术与X线灰度分析的方法，从三个方面对骨折愈合的应力适应性进行了研究：①分别对14只1岁龄山羊进行了断端受力与骨折愈合的关系的研究；②对10只健康成年家兔进行了肌肉动力与骨折愈合的关系的研究；③对56只健康成年家兔进行了骨痂密度与骨折愈合的关系的研究。结果与结论 ①理想的骨折愈合与最佳的应力状态相适应；②肌肉动力是应力适应的反馈调节因素；③骨痂密度是应力适应的反馈结果。     

Early muscle-periosteal lesion inhibits fracture healing in rats

We assessed the effects of muscular detachment from the periosteum on fracture healing, focusing on a muscle-periosteal lesion in the initial healing process. In 30 male Wistar rats we produced a partial osteotomy in the mid-diaphysis of the left femur which was then manually broken. All fractures were reamed and stabilized with a 1.6 mm steel pin. The animals were randomly assigned to 3 groups. In group 1, an extraperiosteal detachment between muscle and periosteum was created in the middle third of the diaphysis. In group 2, an extraperiosteal detachment was created with application of an ePTFE sheath (Gore-Tex expanded polytetrafluoroethylene) around the shaft between muscle and periosteum during the first 2 weeks following fracture. In group 3, the dissection was identical, while the ePTFE sheath was installed after 2 weeks. The rats were killed after 4 weeks, and their bones were evaluated radiographically and mechanically by the three-point bending test. The fractures healed by production of external callus, and radiographs revealed various degrees of periosteal callus with a radiolucent fracture line, most evident after early muscle-periosteal isolation. The callus area was significantly smaller after early muscle isolation, compared to extraperiosteal dissection alone and later tissue isolation. Bending moment and stiffness were also less in this group than in groups 1 and 3, while fracture energy was less than in group 1. No differences in mechanical properties were detected between extraperiosteal dissection alone and late-tissue isolation. This animal study underlines the importance of early muscle-periosteal apposition for fast periosteal healing of diaphyseal fractures.     

Early period of fracture healing in ovariectomized rats

Objective. To evaluate the effect of osteoporosis on fracture healing through observing the hlstomorphological changes, bone mineral density of callus and expression and distribution of transforming growth factor beta 1 (TGF-β1 ), basic fibroblast growth factor (bFGF)and bone morphogenetic protein.2 (BMP-2) in ovariectomized rats. Methods. Sixty female Sprague-Dawley rats ( aged 12 weeks and weighing 235 g on average ) were randomly divided into an ovariectomized (OVX) group (n =30) anda sham-operated (SO) group ( n = 30). Ovariectomy was performed in the OVX rats and same incision was made in the SO rats. Three months later, fracture of femoral shaft was made on all the rats. Then they were killed at different time points. Callus formation was observed with histological and imethods. Results: A reduction in callus and bone mineral density in the healing femur and a decrease of osteoblasts expressing TGF-β1 near the bone trabecula were observed in the OVX rats 3-4 weeks after fracture.Histomorphological analysis revealed a higher content of soft callus in the OVX rats than that in the SO rats.Immunohistochemistry results showed that no remarkable difference in expression and distribution of BMP-2 and bFGF between the OVX and SO groups was found. Conclusions: Osteoporosis influences the quantity and quality of callus during the early period of fracture healing. The effect of osteoporosis on fracture healing has no relationship with the expression of BMP-2 or bFGF. The decreased expression of TGF-I31 in osteoblasts may cause a decrease in quality of facture healing after osteoporosis.     

Early muscle-periosteal lesion inhibits fracture healing in rats

We assessed the effects of muscular detachment from the periosteum on fracture healing, focusing on a muscle-periosteal lesion in the initial healing process. In 30 male Wistar rats we produced a partial osteotomy in the mid-diaphysis of the left femur which was then manually broken. All fractures were reamed and stabilized with a 1.6 mm steel pin. The animals were randomly assigned to 3 groups. In group 1, an extraperiosteal detachment between muscle and periosteum was created in the middle third of the diaphysis. In group 2, an extraperiosteal detachment was created with application of an e-PTFE sheath (Gore-Tex® expanded polytetrafluoro-ethylene) around the shaft between muscle and periosteum during the first 2 weeks following fracture. In group 3, the dissection was identical, while the e-PTFE sheath was installed after 2 weeks. The rats were killed after 4 weeks, and their bones were evaluated radiographically and mechanically by the three-point bending test. The fractures healed by production of external callus, and radiographs revealed various degrees of periosteal callus with a ra-diolucent fracture line, most evident after early muscle-periosteal isolation. The callus area was significantly smaller after early muscle isolation, compared to extraperiosteal dissection alone and later tissue isolation. Bending moment and stiffness were also less in this group than in groups 1 and 3, while fracture energy was less than in group 1. No differences in mechanical properties were detected between extraperiosteal dissection alone and late-tissue isolation. This animal study underlines the importance of early muscle-periosteal apposition for fast periosteal healing of diaphyseal fractures.     

Poor muscle coverage delays fracture healing in rats

We undertook this study in rats to ascertain the influence of muscle coverage on tibial fracture healing. 30 rats were randomly assigned to three intervention groups. Following a mid-diaphyseal osteotomy in the left tibia, reamed nailing was performed in all animals. In one group (A), the antero-lateral muscles were detached from the fractured bone, while the anterolateral compartment was excised in another group (B). In the third group (C), the muscle compartment was resected, and the superficial gluteal muscle was mobilized and transposed over the fractured area. Muscle intervention, like that in group A and C, had no effect on the blood flow. The fibular nerve was resected in all the rats. At 4 weeks, we studied the healing bones in each group clinically, radiologically and mechanically. At 4 weeks, radiographs in two planes revealed a clearly visible fracture line in the three experimental groups. Mechanical testing of the healing fractures showed significantly lower bending moment and bending rigidity in group B than in groups A and C. No difference in mechanical characteristics was detected between the healing bones in groups A and C. This animal study indicates that in tibial fractures, an extensive muscle tissue defect may have negative effects on early bone healing.     

Oxidant status increased during fracture healing in rats

We evaluated oxidant status during bone healing in 50 rats. In 40 rats, the right tibia was fractured and fixed intramedullarlyy (study leg) and the left tibia was pinned but not fractured (control leg). Rats were killed on days 1, 3, 7, 14, 28 and malondialdehyde (MDA) levels were determined in tibial bone tissue. The MDA levels of study and control legs were compared with basal MDA levels in bone in 10 rats. There was no apparent difference between the basal level and control legs, but the study legs showed a statistically significant increase in MDA levels on days 7 and 14. We conclude that no oxidative stress injury occurs during the ischemic period of fracture healing, but it may be significant during inflammation and the formation of callus.     

Cyclooxygenase-2 inhibitor delays fracture healing in rats

Background Cyclooxigenase-2 (COX-2) inhibitors have been reported to delay fracture healing. To investigate the major inhibitory period of COX-2 inhibitors in fracture healing, we administrated etodolac, a COX-2-specific inhibitor, to a rat fracture model by altering the period of administration from early to late.

Method After closed fractures had been created at the middle of the femoral shafts in 12-week-old Wister rats, a standardized dose of etodolac was administrated in three ways: group I received it for 3 weeks, group II for just the first week after operation, and group III for just the third (final) week. Group IV was the vehicle control group. Bone maturation was estimated by radiographic scoring system, and mechanically by a three-point bending test.

Results and interpretation In both the radiographic and mechanical studies, groups I and II showed lower scores than group IV, indicating that even a short period of administration of a COX-2-specific inhibitor in the early phase of fracture healing creates a risk of delayed healing. ▪     

Osteoformin accelerates fresh fracture healing in rats.

PURPOSE: Negatively charged resins have been shown to stimulate bone repair. In previous studies, the negatively charged polypeptide polyaspartate, which has been named Osteoformin, has been shown to stimulate osteoblast differentiation in vitro. The objective of the current study was to investigate the potential effect of Osteoformin on experimental femoral fracture healing in vivo. MATERIALS AND METHODS: Thirty-six male Sprague-Dawley rats were used. The femurs of anesthetized rats were stabilized with intramedullary pins and subjected to closed midshaft transverse facture by bending to failure. In experimental rats, fracture sites were injected with 100 microg of Osteoformin dissolved in 0.1 mL phosphate buffered saline (PBS) at day 1 and day 7 after surgery; in controls groups, fracture sites received 0.1 mL PBS at the intervals indicated above. Between 2 and 4 weeks after fracture, the animals were sacrificed and the healing femurs were removed for radiographic and histologic analysis. RESULTS: Osteoformin decreased the healing time of fresh fractures in rats, as indicated by histologic and radiographic assessments. CONCLUSION: The results of this study show that Osteoformin improves femoral fracture healing in rats.     

Oxidant status increased during fracture healing in rats

We evaluated oxidant status during bone healing in 50 rats. In 40 rats, the right tibia was fractured and fixed intramedullarly (study leg) and the left tibia was pinned but not fractured (control leg). Rats were killed on days 1, 3, 7, 14, 28 and malondialdehyde (MDA) levels were determined in tibial bone tissue. The MDA levels of study and control legs were compared with basal MDA levels in bone in 10 rats. There was no apparent difference between the basal level and control legs, but the study legs showed a statistically significant increase in MDA levels on days 7 and 14. We conclude that no oxidative stress injury occurs during the ischemic period of fracture healing, but it may be significant during inflammation and the formation of callus.     

骨折愈合的应力适应性研究

设计了生物适应性很强的力学控制装置──滑动机械加载控制器,对骨折断端没有应力遮挡,把肌肉动力与肢体负重力作为执行力学加载的源动力,通过压力传感器、多导传感放大器动态记录生理活动状态下断端受力情况及愈合过程中断端的应力状态。结果表明,同一时期内骨折断端压力随肌肉收缩及步态发生变化,不同愈合时间断端压力均值随时间的增加而逐渐增加,而滑动机械加载控制器上承载由术后当日平均2.4kg逐渐变小至术后五周平均0.78kg.同时,通过解剖显微镜进行断端骨痂显微观察,显示断端完全由外骨痂包绕,从而认为骨折断端压力变化是肢体功能恢复,断端骨痂承受载荷的表现,提示临床骨折固定后应适时地进行功能锻炼,肌肉收缩、患肢负重可为断端提供最好的力学环境──生理应力状态。     

骨折愈合的应力适应性研究──功能活动时兔胫骨断端应力状态的动态观察

设计了生物适应性很强的力学控制装置──滑动机械加载控制器，对骨折断端没有应力遮挡，把肌肉动力与肢体负重力作为执行力学加载的源动力，通过压力传感器、多导传感放大器动态记录生理活动状态下断端受力情况及愈合过程中断端的应力状态。结果表明，同一时期内骨折断端压力随肌肉收缩及步态发生变化，不同愈合时间断端压力均值随时间的增加而逐渐增加，而滑动机械加载控制器上承载由术后当日平均２．４ｋｇ逐渐变小至术后五周平均０．７８ｋｇ。同时，通过解剖显微镜进行断端骨痂显微观察，显示断端完全由外骨痂包绕，从而认为骨折断端压力变化是肢体功能恢复，断端骨痂承受载荷的表现，提示临床骨折固定后应适时地进行功能锻炼，肌肉收缩、患肢负重可为断端提供最好的力学环境──生理应力状态。     

Experimental blunt chest trauma impairs fracture healing in rats

In poly‐traumatic patients a blunt chest trauma is an important trigger of the posttraumatic systemic inflammatory response. There is clinical evidence that fracture healing is delayed in such patients, however, experimental data are lacking. Therefore, we investigated the influence of a thoracic trauma on fracture healing in a rat model. Male Wistar rats received either a blunt chest trauma combined with a femur osteotomy or an isolated osteotomy. A more rigid or a more flexible external fixator was used for fracture stabilization to analyze whether the thoracic trauma influences regular healing and mechanically induced delayed bone healing differently. The blunt chest trauma induced a significant increase of IL‐6 serum levels after 6 and 24 h, suggesting the induction of a systemic inflammation, whereas the isolated fracture had no effect. Under a more rigid fixation the thoracic trauma considerably impaired fracture healing after 35 days, reflected by a significantly reduced flexural rigidity (three‐point‐bending test), as well as a significantly diminished callus volume, moment of inertia, and relative bone surface (µCT analysis). In confirming the clinical evidence, this study reports for the first time that a blunt chest trauma considerably impaired bone healing, possibly via the interaction of the induced systemic inflammation with local inflammatory processes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:734–739, 2011     

Cyclo-oxygenase 2 function is essential for bone fracture healing.

Despite the molecular and histological similarities between fetal bone development and fracture healing, inflammation is an early phase of fracture healing that does not occur during development. Cyclo-oxygenase 2 (COX-2) is induced at inflammation sites and produces proinflammatory prostaglandins. To determine if COX-2 functions in fracture healing, rats were treated with COX-2-selective nonsteroidal anti-inflammatory drugs (NSAIDs) to stop COX-2-dependent prostaglandin production. Radiographic, histological, and mechanical testing determined that fracture healing failed in rats treated with COX-2-selective NSAIDs (celecoxib and rofecoxib). Normal fracture healing also failed in mice homozygous for a null mutation in the COX-2 gene. This shows that COX-2 activity is necessary for normal fracture healing and confirms that the effects of COX-2-selective NSAIDs on fracture healing is caused by inhibition of COX-2 activity and not from a drug side effect. Histological observations suggest that COX-2 is required for normal endochondral ossification during fracture healing. Because mice lacking Cox2 form normal skeletons, our observations indicate that fetal bone development and fracture healing are different and that COX-2 function is specifically essential for fracture healing.     

An experimental study on fracture healing in paraplegic rats

Fracture healing in denervated limbs was studied using paraplegic rats of Wistar stain. Femoral fractures were made at the same time as spinal cord injury or at regular intervals after spinal cord injury, for roentgenological and histological observation. In the former, proliferation and differentiation of osteogenic cells derived from the periosteum was almost the same as controls, with earlier bone union than controls. In the latter, with longer intervals between spinal cord injury and fracture, osteogenic cells were less proliferated and differentiated resulting in scant callus or delayed union. The environment of paralytic limbs was evidently altered substantially from 2 to 3 weeks after spinal cord injury, because thereafter fracture healing seemed to become poor. Circulatory disturbance plays a major role in fracture healing in paralytic limbs. Although healing is accelerated by increased circulatory volume at the acute phase of spinal cord injury, this potentiality is gradually decreased because of the regressive degeneration of long-term vasomotor nerve insufficiency.     

Effect of clodronate on fracture healing in denervated rats

The effect of clodronate on healing of the fracture of osteopenic bone was studied in rats. A total of 165 female rats (14 ± 1 weeks, 216 ± 2 g) were divided into five fracture groups (n = 30), and a neurectomized group (n = 15). Osteopenia (op) was induced by right sciatic neurectomy 4 weeks before the fracture. Nonosteopenic (nop) rats were not operated. A closed prepinned diaphyseal fracture of the right femur was done by three-point bending method both to op and nop rats, and the left femur served as an unoperated control. All the fracture groups were divided into treatment (clodronate 10 mg/kg/day sc) and control (saline sc) groups, and the administration was continued throughout the study. The op rats were killed 2, 4, 8, and 12 weeks and nop rats 8 weeks after the fracture. Fracture healing was examined by x-ray and bone-bending strength. Neurectomy reduced bone strength (p < 0.01) at 4 weeks. Clodronate did not affect the bending strength of healing callus of op rats at 2, 4, 8, or 12 weeks after fracture, but reduced the strength of healing callus in nop rats (p < 0.05) at 8 weeks. Radiologic callus width increased in clodronate-treated groups both in op (8 and 12 weeks, p < 0.001) and nop rats (8 weeks, p < 0.05) when compared with saline-treated groups. Clodronate did not affect normal bone strength.

In conclusion, clodronate did not affect the bending strength of op fracture nor the strength of the control bones. The remodeling of the fracture was delayed with clodronate.     

The role of hematoma and periosteal sealing for fracture healing in rats

Bilateral closed femoral fractures were produced in two groups of rats. Reaming was made from the trochanteric area before fracture, and the fractures were stabilized by intramedullary pinning. In one (hematoma) group, both femurs were exposed subperiosteally at the midshaft prior to fracture. At one side, the hematoma was evacuated 30 min following fracture, at the other side the hematoma was left undisturbed. In the other (periosteal) group, the femur was exposed subperiosteally at one side, while the periosteum was left intact at the other side. The healing of the fractures was evaluated at 4 weeks. In the hematoma group, no differences were found in callus production, while bending moment and bending rigidity were greater at the side where the hematoma was not removed. No differences were found in fracture energy. In the periosteal group, marginal differences were found in the callus area. Bending moment, bending rigidity and fracture energy were greater at the side where the periosteum was left intact. A comparison of all four limbs, showed that the periosteal sealing was of particular importance for rapid healing.     

可控性应力与微动对骨折愈合影响的组织学研究

目的 探讨骨折端在不同轴向应力作用下,不同骨折愈合时期所需轴向应力的适宜力值. 方法 32只青山羊均行股骨干中段横行截骨制作骨折模型,按骨折端施加实验动物自身体质量的0倍(对照组)、1/6(A组)、1/3(B组)、1/2(C组)应力分为4组,每组8只.术后4、8周分批处死,每次每组处死4只,行大体观察和组织学观察,测量骨折端骨外膜骨痂面积. 结果 对照组有1只动物骨折端发生成角畸形,实验A、B、C组分别有1、2、4只动物骨折端发生成角畸形.术后4周,对照组、A、B、C组骨折端骨外膜骨痂面积平均值分别为(1.15±0.34)、(1.86±0.28)、(2.18±0.36)、(1.99±0.33)cm~2,A、B、C组分别与对照组比较,骨折端骨痂生成多,骨外膜骨痂面积差异有统计学意义(P<0.05).术后8周,沿轴向排列骨外膜骨痂中骨性骨痂多、致密,皮质骨松化明显;对照组、A、B、C组骨折端骨外膜骨痂面积平均值分别为(1.38±0.31)、(2.09±0.23)、(2.69±0.28)、(2.71±0.31)cm~2,A、B、C组分别与对照组比较,B、C组分别与A组比较,差异均有统计学意义(P<0.05). 结论 骨折端施加轴向应力时能促进骨折端骨痂生长,较大的应力强度能更好地促进骨折端骨痂生长,但同时会造成骨折愈合成角畸形发生率增高.骨折端施加自身体质量的1/3应力时骨折端成角畸形发生率较低,最适宜促进骨折端骨痂生长.     

降钙素对卵巢切除大鼠股骨骨折愈合的影响

目的观察降钙素对卵巢切除大鼠股骨骨折愈合的作用，为临床上治疗骨质疏松性骨折提供实验依据。方法50只雌性、14周龄SD大鼠共分成5组，每组10只。分成假手术组（Sham，G1），双侧卵巢切除术组（OVX，G2），假手术＋骨折组（Sham＋F，G3），卵巢切除术＋骨折组（OVX＋F，G4），卵巢切除＋骨折＋降钙素药物组（OVX＋F＋CT，G5），骨折组大鼠均采用右股骨中段横行骨折，髓内针固定；降钙素采用皮下给药，隔日1次（16IU·kg^-1）。所有大鼠于术后4周杀死，取右侧股骨标本。然后，分别进行CR摄片、组织形态学观察，并应用双能X线骨密度仪（DEXA）测量右股骨整体、远段和中段骨密度以及BMP-2免疫组化观察，并应用病理图像分析仪对BMP-2免疫组化进行光密度测量。结果（1）OVX组与Sham组比较，BMD显著下降。（2）OVX＋F＋CT组与OVX＋F组比较：骨痂mBMD显著增高；BMP-2的表达无显著性差异。结论降钙素对OVX大鼠股骨骨折具有明显促进骨折愈合的作用，加速编织骨向板层骨的演变过程。     

Bone morphogenetic protein-7 accelerates fracture healing in osteoporotic rats

Background:

Osteoporosis is characterized by low bone mass, bone fragility and increased susceptibility to fracture. Fracture healing in osteoporosis is delayed and rates of implant failure are high with few biological treatment options available. This study aimed to determine whether a single dose of bone morphogenetic protein-7 (BMP-7) in a collagen/carboxy-methyl cellulose (CMC) composite enhanced fracture healing in an osteoporotic rat model.

Materials and Methods:

An open femoral midshaft osteotomy was performed in female rats 3 months post-ovarectomy. Rats were randomized to receive either BMP-7 composite (n = 30) or composite alone (n = 30) at the fracture site during surgery. Thereafter calluses were collected on days 12, 20 and 31. Callus cross-sectional area, bone mineral density, biomechanical stiffness and maximum torque, radiographic bony union and histological callus maturity were evaluated at each time point.

Results:

There were statistically significant increases in bone mineral density and callus cross-section area at all time points in the BMP-7 group as compared to controls and biomechanical readings showed stronger bones at day 31 in the BMP-7 group. Histological and radiographic evaluation indicated significant acceleration of bony union in the BMP-7 group as compared to controls.

Conclusion:

This study demonstrated that BMP-7 accelerates fracture healing in an oestrogen-deficient environment in a rat femoral fracture healing model to scientific relevance level I. The use of BMP-7 composite could offer orthopedic surgeons an advantage over oestrogen therapy, enhancing osteoporotic fracture healing with a single, locally applied dose at the time of surgery, potentially overcoming delays in healing caused by the osteoporotic state.