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.     

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.     

Effects of lesion between bone, periosteum and muscle on fracture healing in rats

We assessed the effects of periosteal detachment from bone and musculature on the healing of diaphyseal fracture. In 30 male Wistar rats we produced a partial osteotomy, which was manually broken in the middiaphysis of the left femur. All fractures were reamed and stabilized with an 1.6 mm steel pin. The animals were randomly assigned to 3 groups. In group 1, a subperiosteal detachment between cortex and periost was created in the middle third of the diaphysis. An extraperiosteal detachment between periost and the surrounding musculature was performed in group 2. In group 3, the periosteum was isolated from the musculature by an extraperiosteal detachment and application of an e-PTFE sheath (Gore-Tex ® expanded polytetrafluoroethylene) around the shaft between the periost and the surrounding muscles. The rats were killed after 4 weeks and callus formation and mechanical characteristics were measured. All fractures healed by production of external callus. The callus area was significantly less in the group where periost was mechanically isolated from the surrounding muscles compared to the other groups. Bending moment, bending rigidity and fracture energy were less in this group than in groups 1 and 2. No differences were detected between the sub- and extraperiosteal groups, either in callus formation or in mechanical measurements. Our findings underline the importance of the muscle-periosteal connection for periosteal healing of diaphyseal fractures.     

骨折愈合早期磁共振成像的实验研究

目的 探讨骨折愈合过程中磁共振成像(MRI)的表现及变化规律.方法 6只新西兰白兔右侧桡骨中段制造垂直长度为10 mm、断端间隙为1 mm的长斜形骨折愈合模型.左侧桡骨中段制造10 mm骨质缺损模型.于术后1、2、4、6、8、12周对动物模型行X线及MRI扫描,于术后4 周及12周行螺旋CT扫描,观察骨折愈合过程中T1序列、T2序列、T2压脂(FS-T2)序列、质子(PD)序列及质子压脂(FS-PD)序列影像学表现.结果 骨折后早期周围软组织在T2序列、FS-T2序列、PD序列及FS-PD序列显现为弥漫高信号,随着时间推移,信号强度逐渐下降,至骨折后6周恢复中等信号.骨髓腔在骨折后早期T1序列、T2序列及PD序列由正常高信号变为中等信号,随着髓腔的修复,逐渐恢复为高信号;而FS-T2序列及FS-PD序列髓腔早期显现为中等信号内混杂高信号,随着骨折愈合,混杂高信号逐渐消散.骨折后2周,在FS-T2序列及FS-PD序列发现的中等信号颗粒与之后骨痂的形成位置一致.骨质缺损模型无上述表现.结论 MRI可以比X线更早发现骨折愈合的变化,FS-T2序列及FS-PD序列对骨折愈合早期能更好地加以显像.骨折愈合早期MRI成像的变化有待于病理学的进一步验证.     

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.     

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.     

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. ▪     

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     

Angiogenesis is required for stress fracture healing in rats

Although angiogenesis and osteogenesis are critically linked, the importance of angiogenesis for stress fracture healing is unknown. In this study, mechanical loading was used to create a non-displaced stress fracture in the adult rat forelimb. Fumagillin, an anti-angiogenic agent, was used as the water soluble analogue TNP-470 (25 mg/kg) as well as incorporated into lipid-encapsulated α_vβ₃ integrin targeted nanoparticles (0.25 mg/kg). In the first experiment, TNP-470 was administered daily for 5 days following mechanical loading, and changes in gene expression, vascularity, and woven bone formation were quantified. Although no changes in vascularity were detected 3 days after loading, treatment-related downregulation of angiogenic (Pecam1) and osteogenic (Bsp, Osx) genes was observed at this early time point. On day 7, microCT imaging of loaded limbs revealed diminished woven bone formation in treated limbs compared to vehicle treated limbs. In the second experiment, α_vβ₃ integrin targeted fumagillin nanoparticles were administered as before, albeit with a 100-fold lower dose, and changes in vascularity and woven bone formation were determined. There were no treatment-related changes in vessel count or volume 3 days after loading, although fewer angiogenic (CD105 positive) blood vessels were present in treated limbs compared to vehicle treated limbs. This result manifested on day 7 as a reduction in total vascularity, as measured by histology (vessel count) and microCT (vessel volume). Similar to the first experiment, treated limbs had diminished woven bone formation on day 7 compared to vehicle treated limbs. These results indicate that angiogenesis is required for stress fracture healing, and may have implications for inducing rapid repair of stress fractures.     

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.     

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.     

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.     

Locally delivered lovastatin nanoparticles enhance fracture healing in rats.

Statins stimulate bone formation in vitro and in vivo and, when given in large doses or by prolonged infusions, stimulate biomechanical strength of murine long bones with healing fractures. However, administration of statins by large oral doses or prolonged infusions to a fracture site is not a feasible therapeutic approach to hasten healing of human fractures. We administered lovastatin in biodegradable polymer nanobeads of poly(lactic-co-glycolide acid) to determine if lovastatin delivered in low doses in nanoparticles of a therapeutically acceptable scaffold could increase rates of healing in a standard preclinical model of femoral fracture. We found that these nanobeads: (1) stimulated bone formation in vitro at 5 ng/mL, (2) increased rates of healing in femoral fractures when administered as a single injection into the fracture site, and (3) decreased cortical fracture gap at 4 weeks as assessed by microcomputed tomography. These preclinical results suggest that lovastatin administered in a nanobead preparation may be therapeutically useful in hastening repair of human fractures.     

Lipopolysaccharide impairs fracture healing: an experimental study in rats

Background It has been shown that trauma causes translocation of lipopolysaccharide (LPS) endotoxins from the gut. LPS has been identified as a major bacterial bone resorbing factor. The effects of LPS on bone healing are therefore of clinical interest, as trauma involving fractures followed by sepsis is a clinical scenario. We investigated the effects of systemic and local administration of LPS on the healing of femoral fractures in rats.

Animals and methods In 3 groups, each consisting of 9 rats, a mid-diaphyseal osteotomy/fracture of the femoral bone was performed and then nailed. In one group of animals, LPS was applied intraperitoneally (systemically), and in another group, LPS was applied locally at the fracture site. The third group served as a control. The animals were killed after 6 weeks, and the mechanical characteristics of the healing osteotomies were evaluated.

Results We found that LPS induced a hypertrophic and immature callus, as evaluated by bone mineral content and density. In the rats given LPS intraperitoneally, the mechanical strength characteristics were reduced, as evaluated by bending moment, rigidity, and energy absorption.

Interpretation The rats given LPS intraperitoneally reflect a clinical situation with fracture trauma and endotoxinemia. Our findings indicate that endotoxinemia may impair the fracture healing processes.

        ▪     

Calcium-dependent neutral proteinase (calpain) in fracture healing in rats

Calpain refers to Ca²⁺-dependent neutral cysteine proteinase, which originally was thought to be an intracellular proteinase but recently has been shown to function extracellularly as well. This report describes the immunohistochemical demonstration of calpain and biochemical changes in the amount of calpain during fracture healing in rats. The tibiae of 6-week-old Wistar rats were fractured, and calluses were obtained 5–28 days after fracture. A frozen section of the fracture callus was stained by the immunoperoxidase method with use of polyclonal antibodies of calpains I and II. Positive staining was noted with the anti-calpain II antibody in the perivascular areas, chondrocytes, and cartilage matrix in calluses at 5, 7, and 10 days. Less intense staining was seen in older calluses. The caseinolytic activity of calpain II reached its maximum on the 5th day, was high on the 7th and 10th days, and decreased rapidly thereafter. The quantity of calpain II was dependent on the process of fracture healing. It was concluded that calpain was working as one of the matrix proteinases in fracture callus.