A Fracture Pain Model in the Rat: Adaptation of a Closed Femur Fracture Model to Study Skeletal Pain

Background: Because of the relative lack of understanding of the mechanisms that drive skeletal pain, the purpose of this study was to adapt a previously validated closed femur fracture model to quantitatively evaluate skeletal pain in female and male rats.

Methods: Three-month-old female and male Sprague-Dawley rats were anesthetized, and a stainless steel pin was inserted into the intramedullary space of the left femur. Three weeks later, the rats were reanesthetized, and left femoral diaphyses were fractured using a standardized impactor device. At 1-21 days after fracture, skeletal pain was measured by quantitatively assessing spontaneous guarding, spontaneous flinching, and weight bearing of the fractured hind limb.

Results: Females and males showed highly robust pain behaviors that were maximal at day 1 after fracture and returned gradually to normal nonfractured levels at days 14-21 after fracture. The magnitude of fracture pain was not significantly different at most time points between female and male rats. In both females and males, the pain-related behaviors were attenuated by subcutaneous morphine in a dose-dependent manner.     

Effect of administration of antibodies against nerve growth factor in a rat model of muscle injury

IntroductionAlthough muscle injury is a common source of pain, the mechanism causing such pain is not completely known. We have previously reported nerve growth factor (NGF) as a proinflammatory mediator involved in acute pain, and clinical trials have shown the effectiveness of anti-NGF antibodies for management of low back pain. Here, we aim to examine the effects of anti-NGF antibodies on muscle-derived pain by studying their effects on sensory innervation in a rat muscle injury model.MethodsA nervous system tracer, Fluoro-Gold, was applied to both gastrocnemius muscles of 24 male Sprague Dawley rats to stain the sensory nerves. Then, the drop-mass method was used to damage the right gastrocnemius muscle of the posterior limb. Anti-NGF antibodies (50 μL) were injected into the injured muscles in 12 rats. Tissues were evaluated 1, 3, and 7 days post-injury by performing haematoxylin-and-eosin (HE) staining. The percentage of the total number of FG-positive cells that were also positive for a pain-related neuropeptide, calcitonin gene-related peptide (CGRP), was determined for the bilateral dorsal root ganglia from L1 to L6 7 days post-injury.ResultsHE staining showed active inflammation, indicated by increased basophil and eosinophil accumulation, at the injury site 1 and 3 days post-injury, as well as scar tissue formation 7 days post-injury. Injection of anti-NGF reduced muscle necrosis 1 and 3 days post-injury, and resulted in replacement of granulation tissue and muscle fibre regeneration 7 days post-injury. Anti-NGF also significantly inhibited CGRP among FG-positive cells (treatment group 38.2%, control group 49.6%; P < 0.05).DiscussionThis study found active inflammation induced by NGF, which may contribute to pain after muscle injury. Anti-NGF antibodies successfully suppressed the pain mediator NGF and inhibited inflammation, suggesting NGF as a target for control in pain management.     

Delayed Fracture Healing in Aged Senescence-Accelerated P6 Mice

ABSTRACT

Background: Osteoporosis is characterized by poor bone quality. However, it is still controversially discussed whether osteoporosis compromises fracture healing. Herein, we studied whether the course of healing of a femur fracture is affected by osteoporosis or age. Methods: Using the senescence-accelerated osteoporotic mouse, strain P6 (SAMP6), and a closed femur fracture model, we studied the process of fracture healing in 5- and 10-month-old animals, including biomechanical, histomorphometric, and protein biochemical analysis. Results: In five-month-old osteoporotic SAMP6 mice, bending stiffness, callus size, and callus tissue distribution as well as the concentrations of the bone formation marker osteocalcin and the bone resorption markers tartrate-resistant acid phosphatase form 5b (TRAP) and deoxypyridinoline (DPD) did not differ from that of non-osteoporotic, senescence-resistant, strain 1 (SAMR1) controls. In contrast, femur fractures in 10-month-old SAMP6 mice showed a significantly reduced bending stiffness and an increased callus size compared to fractures in age-matched SAMR1 controls. This indicates a delayed fracture healing in advanced age SAMP6 mice. The delay of fracture healing was associated with higher concentrations of TRAP and DPD. Significant differences in osteocalcin concentrations were not found between SAMP6 animals and SAMR1 controls. Conclusion: In conclusion, the present study indicates that fracture healing in osteoporotic SAMP6 mice is not affected in five-month-old animals, but delayed in animals with an age of 10 months. This is most probably due to the increased osteoclast activity in advanced age SAMP6 animals.     

Comparison of healing process in open osteotomy model and open fracture model: Delayed healing of osteotomies after intramedullary screw fixation

Murine osteotomy and fracture models have become the standard to study molecular mechanisms of bone healing. Because there is little information whether the healing of osteotomies differs from that of fractures, we herein studied in mice the healing of femur osteotomies compared to femur fractures. Twenty CD‐1 mice underwent a standardized open femur osteotomy. Another 20 mice received a standardized open femur fracture. Stabilization was performed by an intramedullary screw. Bone healing was studied by micro‐CT, biomechanical, histomorphometric and protein expression analyses. Osteotomies revealed a significantly lower biomechanical stiffness compared to fractures. Micro‐CT showed a reduced bone/tissue volume within the callus of the osteotomies. Histomorphometric analyses demonstrated also a significantly lower amount of osseous tissue in the callus of osteotomies (26% and 88% after 2 and 5 weeks) compared to fractures (50% and 100%). This was associated with a delayed remodeling. Western blot analyses demonstrated comparable BMP‐2 and BMP‐4 expression, but higher levels of collagen‐2, CYR61 and VEGF after osteotomy. Therefore, we conclude that open femur osteotomies in mice show a markedly delayed healing when stabilized less rigidly with an intramedullary screw. This should be considered when choosing a model for studying the mechanisms of bone healing in mice. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:971–978, 2015.     

Sildenafil accelerates fracture healing in mice

Sildenafil, a cyclic guanosine monophosphate (cGMP)‐dependent phospodiesterase‐5 inhibitor, has been shown to be a potent stimulator of angiogenesis through upregulation of pro‐angiogenic factors and control of cGMP concentration. Herein, we determined whether sildenafil also influences angiogenic growth factor expression and bone formation during the process of fracture healing. Bone healing was studied in a murine closed femur fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analysis at 2 and 5 weeks after fracture. Thirty mice received 5 mg/kg body weight sildenafil p.o. daily. Controls (n = 30) received equivalent amounts of vehicle. After 2 weeks of fracture healing sildenafil significantly increased osseous fracture bridging, as determined radiologically and histologically. This resulted in an increased biomechanical stiffness compared to controls. A smaller callus area with a slightly reduced amount of cartilaginous tissue indicated an accelerated healing process. After 5 weeks the differences were found blunted, demonstrating successful healing in both groups. Western blot analysis showed a significantly higher expression of the pro‐angiogenic and osteogenic cysteine‐rich protein (CYR) 61, confirming the increase of bone formation. We show for the first time that sildenafil treatment accelerates fracture healing by enhancing bone formation, most probably by a CYR61‐associated pathway. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:867–873     

Comparison of Fracture Healing Among Different Inbred Mouse Strains

Quantitative trait locus analysis can be used to identify genes critically involved in biological processes. No such analysis has been applied to identifying genes that control bone fracture healing. To determine the feasibility of such an approach, healing of femur fractures was measured between C57BL/6, DBA/2, and C3H inbred strains of mice. Healing was assessed by radiography and histology and measured by histomorphometry and biomechanical testing. In all strains, radiographic bridging of the fracture was apparent after 3 weeks of healing. Histology showed that healing occurred through endochondral ossification in all strains. Histomorphometric measurements found more bone in the C57BL/6 fracture calluses 7 and 10 days after fracture. In contrast, more cartilage was present after 7 days in the C3H callus, which rapidly declined to levels less than those of C57BL/6 or DBA/2 mice by 14 days after fracture. An endochondral ossification index was calculated by multiplying the callus percent cartilage and bone areas as a measure of endochondral ossification. At 7 and 10 days after fracture, this value was higher in C57BL/6 mice. Using torsional mechanical testing, normalized structural and material properties of the C57BL/6 healing femurs were higher than values from the DBA/2 or C3H mice 4 weeks after fracture. The data indicate that fracture healing proceeds more rapidly in C57BL/6 mice and demonstrate that genetic variability significantly contributes to the process of bone regeneration. Large enough differences exist between C57BL/6 and DBA/2 or C3H mice to permit a quantitative trait locus analysis to identify genes controlling bone regeneration.     

Inducible expression of Wnt7b promotes bone formation in aged mice and enhances fracture healing

There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as a promising target pathway for developing novel bone anabolic drugs. Although neutralizing antibodies against the Wnt antagonist sclerostin have been tested,Wnt ligands themselves have not been fully explored as a potential therapy. Previous work has demonstrated Wnt7b as an endogenous ligand upregulated during osteoblast differentiation, and that Wnt7b overexpression potently stimulates bone accrual in the mouse. The earlier studies however did not address whether Wnt7b could promote bone formation when specifically applied to aged or fractured bones. Here we have developed a doxycycline-inducible strategy where Wnt7b is temporally induced in the bones of aged mice or during fracture healing. We report that forced expression of Wnt7b for 1 month starting at 15 months of age greatly stimulated trabecular and endosteal bone formation, resulting in a marked increase in bone mass. We further tested the effect of Wnt7b on bone healing in a murine closed femur fracture model. Induced expression of Wnt7b at the onset of fracture did not affect the initial cartilage formation but promoted mineralization of the subsequent bone callus. Thus, targeted delivery of Wnt7b to aged bones or fracture sites may be explored as a potential therapy.     

Internal remodeling of periosteal new bone during fracture healing

A closed fracture model of the rat tibia was employed to study internal remodeling of periosteal new bone during fracture repair. Static histomorphometric parameters of osteoid surface (or perimeter) and eroded surface (resorption surface) were used as indicators of appositional bone formation and resorption of bone trabeculae, respectively. Intracortical remodeling at the fracture site was evaluated using quantitative tetracycline histology and microradiography. The extents of osteoid and eroded bone surfaces did not differ significantly in the periosteal woven new bone in the early phases of fracture healing. Later on, the periosteal new bone had significantly more osteoid surface than eroded surface (p less than 0.001). The number of osteoclasts also decreased significantly over time during fracture healing (p = 0.028). Cortical bone showed a continuous increase of porosity (p less than 0.01) between 1 and 6 weeks after fracture. These results suggest that there is a time-related change in the balance of periosteal bone formation and resorption during the progress of fracture repair. We hypothesize that this change was related to the restoration of bony continuity. Further studies are, however, needed to indicate the histomorphometric features of periosteal new bone in fracture nonunions.     

Elcatonin injections suppress systemic bone resorption without affecting cortical bone regeneration after drill‐hole injuries in mice

It is assumed that there are systemic changes in mineral metabolism during fracture healing that may cause a predisposition to sequential fractures in osteoporotic patients who suffered from previous fractures. Initial therapies for patients with osteoporotic fractures are important to prevent disabilities in daily life consequent to bone and muscle atrophies, and sequential fractures, although systemic and local bone metabolism during fracture healing have not been well understood. We evaluated the effects of bone injury and elcatonin injection as an initial therapy on systemic and local bone turnover and bone wound healing. Two drill holes were made in the diaphysis of the left femur and tibia of 12‐week‐old male C57BL/6J mice. They were treated with three doses of elcatonin or a vehicle thrice a week until the end of the 28‐day experiment. Urinary crosslinked C‐telopeptide of type I collagen (CTX) increased and the bone mineral densities (BMDs) in the lumbar vertebrae decreased in the vehicle‐treated mice. Elcatonin injection prevented increases in urinary CTX and reduction of the BMDs. In the noninjured femoral metaphysis, osteoclast surface increased until day 28, whereas elcatonin suppressed it. In the fracture site, elcatonin facilitated osteoblast proliferation and did not delay the healing of the bone defect. Bone injuries accelerated bone turnover systemically and locally, and the elcatonin injections suppressed the systemic acceleration of bone resorption without a delay of filling regenerated cortical bone in the bone defect. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1652–1658, 2009     

Characterization of a closed femur fracture model in mice

OBJECTIVES: The goal of this study was to develop and characterize a closed femur fracture model for mice that can be used for the molecular and genetic analysis of fracture healing. STUDY DESIGN: Longitudinal time study of species-specific fracture healing. METHODS: A protocol was developed for creating reproducible, closed femur fractures in mice. Impending fractures were stabilized by retrograde insertion of a 0.01-inch-diameter, stainless steel wire into the intramedullary canal. The intramedullary wire was held in place with a wedge made from the first 2 mm of a 30-gauge needle. Fractures were produced by 3-point bending. Fracture healing was assessed by radiography, histology, and torsional mechanical testing. RESULTS: The mouse femur fracture technique produced good results with minimal loss of animals. Of the 246 mice used in the study, 22 mice were excluded due to poor fracture quality (8), loss of fracture stabilization (6), or to anesthesia death (8). Radiography showed a consistent pattern of fracture healing between mice with peak fracture callus volume evident at 10 (15 mice) to 14 days (18 mice) after fracture. Fracture bridging was apparent in all 3-week postfracture radiographs (35 mice). Histologic examination of 117 specimens at 9 time points showed chondrocyte differentiation within the fracture callus by 7 days after fracture, endochondral ossification occurring by 10 days after fracture, and bone remodeling evident as early as 3 weeks after fracture. Despite radiologic and histologic evidence of fracture bridging after 3 weeks, torsional mechanical testing of 68 mice at 3, 4, 6, and 12 weeks after fracture (group size of 15 to 18 mice at each time point) indicated that significant increases in structural or material strength did not occur until 6 to 12 weeks after fracture. CONCLUSIONS: Femur fracture healing in mice follows a typical endochondral ossification pathway with fracture bridging occurring approximately 1 week faster in mice than rats. This fracture model is amenable to the molecular and genetic analysis of fracture healing using different inbred, transgenic, and knockout strains of mice.     

A novel mouse model to study fracture healing of the proximal femur

The majority of fractures, especially in elderly and osteoporotic patients, occurs in metaphyseal bone. However, only a few experimental models exist to study metaphyseal bone healing in mice. Currently used mouse models of metaphyseal fracture healing are either based on drill hole defects, lacking adequate biomechanical stimulation at the site of fracture and therefore endochondral ossification in the fracture callus, or are introduced into the distal part of the mouse femur stabilized by a locking plate, which is challenging due to the small specimen size. Therefore, the aim of the current study was to develop a new mouse model to study metaphyseal fracture healing of the proximal femur. We chose a combination between an open osteotomy and a closed intramedullary stabilization. A 24 G needle was inserted into the femur in a closed manner, then an osteotomy was made with a 0.4-mm Gigli wire saw between the third and the lesser trochanter of the femur using an open approach. Fractured femurs were analyzed using microcomputed tomography and histology at days 14 and 21 after surgery. No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first three days after fracture. We found robust endochondral ossification during the fracture healing process with high expression of late chondrocyte and early osteogenic markers at day 14 (d14). By day 21 (d21), all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume significantly decreased from d14 to d21, whereas high numbers of osteoclasts appeared at the fracture callus until d21, indicating that callus remodeling had already started at d21. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur. This model might be useful for future studies using transgenic animals to unravel molecular mechanisms of osteoporotic metaphyseal fracture healing.     

Strain-dependent variations in the response of cancellous bone to ovariectomy in mice.

The goal of this study was to characterize the skeletal response to ovariectomy in mice (129P3, C57BL/6, and B6129PF2) commonly used in gene manipulation studies to evaluate their potential as preclinical models of postmenopausal osteoporosis. The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to ovariectomy varied with mouse type and skeletal site, but in general, were less pronounced and less consistent than in Sprague-Dawley rats, the established preclinical model for postmenopausal bone loss. INTRODUCTION: The ovariectomized (OVX) rat is the most widely used preclinical rodent model for postmenopausal osteoporosis. However, the underlying mechanisms of bone disorders, including osteoporosis, have been explored predominantly in the mouse. The purpose of this study was to evaluate mice (129P3 and C57BL/6 inbred strains and their F2 hybrid offspring, B6129PF2), commonly used for gene knockout and overexpression studies, for their potential as preclinical models of postmenopausal bone loss. MATERIALS AND METHODS: The mice were OVX or sham-operated at 4 months of age and killed at 1 or 3 months after surgery. Lumbar vertebrae and distal femora were subjected to histomorphometric assessment. RESULTS: Mice in the two strains and the F2 hybrids (will be referred to as strain for the remainder of the abstract) lost vertebral cancellous bone after OVX; bone volume (BV/TV) was 20% and 27% lower at 1 and 3 months after surgery, respectively. The decreased cancellous BV/TV was associated with an increase in osteoclast surface at 1 month after OVX in the 129P3 strain only. Osteoblast surface was increased by 20% with OVX at both 1 and 3 months after surgery, irrespective of mouse strain. However, bone formation rate was not altered by OVX in any of the mouse strains. In contrast to the lumbar vertebrae, cancellous bone loss in response to OVX differed in the distal femur among the three mouse strains. OVX had no significant effect on distal femur BV/TV in the B6129PF2 mouse strain. In the C57BL/6 strain, cancellous BV/TV was reduced by OVX at 1 month after surgery but not at 3 months after surgery, whereas distal femur BV/TV in 129P3 mice was reduced at 3 months after surgery. Osteoclast surface was not affected by OVX at either time-point in the C57BL/6 strain, but was increased by 116% at 1 month after surgery in the 129P3 strain. Osteoblast surface was increased with OVX at 1 month after surgery, irrespective of strain, whereas bone formation rate was not altered by OVX at either time-point in any of the strains. CONCLUSIONS: The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to OVX varied with mouse strain and skeletal site, but in general, were less pronounced and less consistent than in the Sprague-Dawley rat. Although mouse models will continue to provide insights into genetic influences on bone mass and turnover, caution should be exercised when using 129P3 and C57BL/6 mice, and their F2 hybrids, as models for postmenopausal bone loss and preclinical testing of potential therapies for osteoporosis.     

Biological effects of extracorporeal shockwave in bone healing: a study in rabbits

INTRODUCTION: This study is an investigation of the biological effects of extracorporeal shockwave treatment (ESWT) on bone healing in a rabbit model. MATERIALS AND METHODS: Sixteen 12-month-old New Zealand white rabbits with body weight ranging from 2.5 to 3.5 kg were used in the study. An intra-medullary pin was inserted retrograde into the femur canal. A closed fracture of the femur was created with a three-point bend method. The animals were randomly divided into the study group and the control group with eight rabbits in each group. The study group received shockwave treatment, whereas the control group did not. The animals were killed at 12 weeks, and a 5-cm long femur bone including the callus was harvested. The specimens were subjected to biomechanical study, histomorphological examination, and immunohistochemical analysis. RESULTS: The shockwave group showed significantly better bone strength in biomechanical study, more cortical bone formation in histomorphological examination and higher number of neo-vessels and angiogenic and osteogenic growth markers including VEGF, eNOS, PCNA, and BMP-2 on immunohistochemical stains than the control group. CONCLUSION: ESWT significantly improved bone healing after fracture of the femur in rabbit. ESWT promoted the formation of cortical bone what might have been associated with increased biomechanical results. ESWT-promoted bone healing was associated with increased neovascularization and up-regulation of angiogenic and osteogenic growth factors.     

Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14 days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair.     

Long-term effect of incadronate disodium (YM-175) on fracture healing of femoral shaft in growing rats.

The aim of this study was to investigate the long-term effect of incadronate on fracture healing of the femoral shaft in rats. Female Sprague-Dawley 8-week-old rats were injected subcutaneously (sc) with either vehicle (V group) or two doses of incadronate (10 microg/kg and 100 microg/kg) three times a week for 2 weeks. Right femoral diaphysis was then fractured and fixed with intramedullary stainless wire. Just after fracture, incadronate treatment was stopped in pretreatment groups (P groups: P-10 and P-100) or continued in continuous treatment groups (C groups: C-10 and C-100). All rats were killed at 25 weeks or 49 weeks after surgery. Fractured femur was evaluated radiologically and mechanically and then stained in Villanueva bone stain and embedded in methyl methacrylate. Undecalcified cross-sections from the fracture area were evaluated microradiologically and histomorphometrically. Radiographic observation showed that the fracture line disappeared in all groups. Cross-sectional area in the C-100 group was the biggest among all groups and in the C-10 group was larger than that in the V group at 25 weeks. Histological and histomorphometric observations showed that the process of fracture healing was delayed under continuous treatment with incadronate as evidenced by the delay of both lamellar cortical shell formation and resolution of original cortex in C groups. Percent linear labeling perimeter, mineral apposition rate (MAR), and bone formation rate (BFR) in C groups significantly decreased compared with the other groups, indicating that the callus remodeling was suppressed under continuous treatment, especially with a high dose. Mechanical study showed that the stiffness and ultimate load of the fractured femur in the C 100 group were the highest among all groups at both 25 weeks and 49 weeks. In conclusion, this study showed that long-term continuous treatment with incadronate delayed the process of fracture healing of femur in rats, especially under high dose but it did not impair the recovery of mechanical integrity of the fracture.     

Recurrent Proximal Femur Fractures in a Teenager With Osteogenesis Imperfecta on Continuous Bisphosphonate Therapy: Are We Overtreating?

Long‐term bisphosphonate (BP) therapy in adults with osteoporosis is associated with atypical femoral fractures, caused by increased material bone density and prolonged suppression of bone remodeling which may reduce fracture toughness. In children with osteogenesis imperfecta (OI), long‐term intravenous BP therapy improves bone structure and mass without further increasing the already hypermineralized bone matrix, and is generally regarded as safe. Here we report a teenage girl with OI type IV, who was started on cyclical intravenous pamidronate therapy at age 6 years because of recurrent fractures. Transiliac bone biopsy revealed classical structural features of OI but unusually low bone resorption surfaces. She made substantial improvements in functional ability, bone mass, and fracture rate. However, after 5 years of pamidronate therapy she started to develop recurrent, bilateral, nontraumatic, and proximal femur fractures, which satisfied the case definition for atypical femur fractures. Some fractures were preceded by periosteal reactions and prodromal pain. Pamidronate was discontinued after 7 years of therapy, following which she sustained two further nontraumatic femur fractures, and continued to show delayed tibial osteotomy healing. Despite rodding surgery, and very much in contrast to her affected, untreated, and normally mobile mother, she remains wheelchair‐dependent. The case of this girl raises questions about the long‐term safety of BP therapy in some children, in particular about the risk of oversuppressed bone remodeling with the potential for microcrack accumulation, delayed healing, and increased stiffness. The principal concern is whether there is point at which benefit from BP therapy could turn into harm, where fracture risk increases again. This case should stimulate debate whether current adult atypical femoral fracture guidance should apply to children, and whether low‐frequency, low‐dose cyclical, intermittent, or oral treatment maintenance regimens should be considered on a case‐by‐case basis. © 2016 American Society for Bone and Mineral Research.     

成骨活性肽对骨折愈合生物力学性能的影响

目的观察自行设计合成的具有成骨活性的小分子多肽对小鼠股骨骨折愈合生物力学性能的影响,评价其促进骨折愈合的能力。方法选择8周龄雌性C57BL/6J小鼠40只,随机分为对照组和多肽组两组,每组各20只。所有动物制备左侧股骨骨折模型,分组植入复合PBS或多肽的胶原材料。术后第3周行三点弯曲实验,观察各标本最大载荷、弹性载荷、最大挠度、弹性挠度、最大应力、弹性模量等生物力学指标的变化情况。结果多肽组各项生物力学指标均显著高于对照组(P<0.01)。结论合成的成骨活性多肽能明显增强小鼠股骨骨折愈合的生物力学性能。     

Effect of Stabilization on the Healing Process of Femur Fractures in Aged Mice

Background: The influence of mechanical stability on fracture healing has previously been studied in adult mice, but is poorly understood in aged animals. Therefore, we herein studied the effect of stabilization on the healing process of femur fractures in aged mice. Methods: Twenty-four 18-month-old CD-1 mice were stabilized after midshaft fracture of the femur with an intramedullary screw. In another 24 18-month-old mice, the femur fractures were left unstabilized. Bone healing was studied by radiological, biomechanical, histomorphometric, and protein expression analyses. Results: After 2 and 5 weeks of healing, the callus of nonstabilized fractures compared to stabilized fractures was significantly larger, containing a significantly smaller amount of osseous tissue and a higher amount of cartilaginous tissue. This was associated with a significantly lower biomechanical stiffness during the early phase of healing. However, during the late phase of fracture healing both nonstabilized and stabilized fractures showed a biomechanical stiffness of ～40%. Of interest, Western blot analyses of callus tissue demonstrated that the expression of proteins related to angiogenesis, bone formation and remodeling, i.e. VEGF, CYR61, BMP-2, BMP-4, Col-2, Col-10, RANKL, OPG, did not differ between nonstabilized and stabilized fractures. Conclusion: Nonstabilized fractures in aged mice show delayed healing and remodeling. This is not caused by an altered protein expression in the callus but rather by the excessive interfragmentary movements.     

Osteoclast depletion with clodronate liposomes delays fracture healing in mice

Osteoclasts are abundant within the fracture callus and also localize at the chondro‐osseous junction. However, osteoclast functions during fracture healing are not well defined. Inhibition of osteoclast formation or resorptive activity impairs callus remodeling but does not prevent callus formation. Interestingly, though anti‐osteoclast therapies differentially affect resolution of callus cartilage into bone. Treatments that inhibit osteoclast formation or viability tend to impair callus cartilage resolution, while treatments that target inhibition of bone resorption generally do not affect callus cartilage resolution. Here, we tested whether depletion of osteoclasts by systemic treatment with clodronate liposomes would similarly impair callus cartilage resolution. ICR mice were treated by intraperitoneal injections of clodronate‐laden liposomes or control liposomes and subjected to closed femur fracture. Femurs were resected at multiple times after fracture and analyzed by radiography, histology, and mechanical testing to determine effects on healing. Clodronate liposome treatment did not prevent callus formation. However, radiographic scoring indicated that clodronate liposome treatment impaired healing. Clodronate liposome treatment significantly reduced callus osteoclast populations and delayed resolution of callus cartilage. Consistent with continued presence of callus cartilage, torsional mechanical testing found significant decreases in callus material properties after 28 days of healing. The results support a role for osteoclasts in the resolution of callus cartilage into bone. Whether the cartilage resolution role for osteoclasts is limited to simply resorbing cartilage at the chondro‐osseous junction or in promoting bone formation at the chondro‐osseous junction through another mechanism, perhaps similar to the reversal process in bone remodeling, will require further experimentation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1699–1706, 2017.