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.     

Stimulation of angiogenesis by cilostazol accelerates fracture healing in mice

Cilostazol, a selective phosphodiesterase‐3 inhibitor, is known to control cyclic adenosine monophosphate (c‐AMP) and to stimulate angiogenesis through upregulation of pro‐angiogenic factors. There is no information, however, whether cilostazol affects fracture healing. We, therefore, studied the effect of cilostazol on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine femur fracture stabilized with an intramedullary screw. Radiological, biomechanical, histomorphometric, histochemical, and protein biochemical analyses were performed at 2 and 5 weeks after fracture. Twenty‐five mice received 30 mg/kg body weight cilostazol p.o. daily. Controls (n = 24) received equivalent amounts of vehicle. In cilostazol‐treated animals radiological analysis at 2 weeks showed an improved healing with an accelerated osseous bridging compared to controls. This was associated with a significantly higher amount of bony tissue and a smaller amount of cartilage tissue within the callus. Western blot analysis showed a higher expression of cysteine‐rich protein 61 (CYR61), bone morphogenetic protein (BMP)‐4, and receptor activator of NF‐kappaB ligand (RANKL). At 5 weeks, improved fracture healing after cilostazol treatment was indicated by biomechanical analyses, demonstrating a significant higher bending stiffness compared to controls. Thus, cilostazol improves fracture healing by accelerating both bone formation and callus remodeling. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1880–1887, 2015.     

An internal locking plate to study intramembranous bone healing in a mouse femur fracture model

In most murine fracture models, the femur is stabilized by an intramedullary implant and heals predominantly through endochondral ossification. The aim of the present study was to establish a mouse model in which fractures heal intramembranously. Femur fractures of 16 SKH‐mice were stabilized by an internal locking plate. Femur fractures of another 16 animals were stabilized by an intramedullary screw. Bone repair was analyzed by radiographic, biomechanical, and histological methods. At 2 weeks, histological analysis showed a significantly smaller callus diameter and callus area after locking plate fixation. Cartilage formation within the callus could only be observed after screw fixation, but not after fracture stabilization with the locking plate. Radiological and biomechanical analysis after 2 and 5 weeks showed a significantly improved healing and a higher bending stiffness of fractures stabilized by the locking plate. Fractures stabilized by the locking plate healed exclusively by intramembranous ossification, which is most probably a result of the anatomical reduction and stable fixation. The fractures that healed by intramembranous ossification showed an increased stiffness compared to fractures that healed by endochondral ossification. This model may be used to study molecular mechanisms of intramembranous bone healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:397–402, 2010     

Bone morphogenetic protein-2 coating of titanium implants increases biomechanical strength and accelerates bone remodeling in fracture treatment: a biomechanical and histological study in rats

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor (TGF)-beta superfamily, is known to be a very potent osteoinductive growth factor. The purpose of this study was to investigate the effect of BMP-2 (5% [w/w], 50 microg on each nail), locally released from poly(D,L-lactide) (PDLLA)-coated intramedullary implants, on fracture healing. A closed fracture of the right tibia of 5-month-old Sprague-Dawley rats (n = 64) was intramedullary stabilized with uncoated vs. BMP-2-coated titanium Kirschner wires. X-ray examinations (posteroanterior and lateral) were performed throughout the experiment. At 28 and 42 days after fracture, the animals were killed and both tibiae were dissected for biomechanical torsional testing. For histological and histomorphometric evaluation, 5 microm sections were obtained, stained with Safranin-O/light green and von Kossa, and examined using an image analysis system. The radiological results demonstrated progressed callus consolidation in the BMP-2-treated groups compared with the uncoated groups at both timepoints. Histomorphometric evaluation showed progressed callus remodeling with significantly increased mineralization and less cartilage of the periosteal callus. Due to the BMP-2 treatment, increased mineralization of the cortices was detected at 28 and 42 days after fracture. Biomechanical testing revealed significantly elevated maximum load and torsional stiffness in the BMP-2-treated groups compared with controls at both timepoints. The results clearly demonstrate that local application of BMP-2 from PDLLA-coated implants is feasible and significantly accelerates fracture healing. Local administration of growth factors from coated implants could reduce clinical problems in fracture treatment without opening of the fracture, implantation of further devices, or injection with the risk of infection or side effects caused by other carriers.     

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.     

Simvastatin locally applied from a biodegradable coating of osteosynthetic implants improves fracture healing comparable to BMP-2 application

Many clinical and experimental investigations address the influence of statins on bone formation and fracture healing. Simvastatin was shown to increase the expression of Bone morphogenetic protein (BMP-2), which is one of the most potent growth factors targeting bone formation. In this study, the effect of simvastatin locally applied from a bioactive polymer coating of implants on fracture healing was investigated. A closed fracture of the right tibia of 5-month-old Sprague–Dawley rats was performed. Intramedullary stabilization was achieved with uncoated vs. polymer-only coated vs. polymer plus drug coated titanium Kirschner wires. Test substances (either simvastatin low- or high dosed or BMP-2) were incorporated into a biodegradable layer of poly(d,l-lactide). Tibiae were harvested after 28 or 42 days, respectively and underwent biomechanical testing (torsional stiffness and maximum load) and histomorphometric analysis. Radiographic results demonstrated progressed callus consolidation in the BMP-2- and simvastatin-treated groups compared to the uncoated group at both timepoints. The simvastatin-high-dosed group revealed an increased torsional stiffness and significantly elevated maximum load (d 28) compared to control group as well as a significant increase in both parameters at d 42. BMP-2-treated animals showed significantly elevated maximum load and stiffness at the early timepoint and elevated torsional stiffness after d 42.The histomorphometric analysis revealed a significantly decreased cartilage area for BMP-2 treated animals at d 28. Even though an increase of mineralized areas among periosteal callus was found at d 42 for simvastatin-high as well as BMP-2 treated animals, no significant difference could be detected at both timepoints compared to the uncoated group. However, simvastatin-high treated animals revealed significantly reduced cartilage areas within the periosteal callus at d 42. The present study revealed a dose-dependent effect and improved fracture healing under local application of simvastatin. Biomechanical, radiographic and histomorphometric properties showed comparable results to BMP-2- treated animals in this study.     

BMP-14 deficiency inhibits long bone fracture healing: a biochemical, histologic, and radiographic assessment

OBJECTIVES: Bone morphogenetic proteins (BMPs) represent a distinct subset of the transforming growth factor-beta family best known for their role in joint formation and bone growth, and several recent clinical trials have begun to look at their efficacy in the augmentation of fracture healing. The goal of this research is to examine the effect of BMP-14, also known as growth differentiation factor-5 and cartilage- derived morphogenetic protein-1 (GDF-5, CDMP-1) on fracture healing by studying the long bone repair process in mice with a deficiency in this signaling peptide. METHODS: The animal model used for these studies was the BMP-14 (-/-) brachypodism (bp) mouse. Phenotypically normal heterozygous (+/-) littermates were used as controls. Closed mid shaft femur fractures were created and stabilized with intramedullary fixation in 8-week-old female mice. Forty-eight mice per genotype group were examined. On postoperative days 4, 7, 10, 14, 21, 28, 35, and 42, the mice were killed and the femurs and repair tissue were harvested for analysis. At each time point, the fracture sites were analyzed radiographically, histologically, and biochemically. For all quantitative analyses, the data were normalized and analyzed statistically using a 2-factor ANOVA test. RESULTS: Biochemically, peak values of normalized proteoglycan content were approximately 3 times less in the mutant fractures early in the time course of healing compared with the controls (P < 0.05). Histologically, BMP-14-deficient fractures exhibited a delay in peak area cell density, callus organization, and bone formation compared with controls. Radiographic analysis demonstrated that the peak callus was 2 weeks delayed and approximately 2 times less in the mutants compared with controls (P < 0.05). Radiographic grading of callus also demonstrated a significant difference after day 14. CONCLUSIONS: Based on histologic, radiographic, and biochemical analysis, BMP-14-deficient mice display a short-term delay in healing of approximately 1 to 2 weeks. The observed abnormalities seem to be the result of a delay in cellular recruitment and chondrocyte differentiation in the early stages fracture repair in the absence of BMP-14. These results support the hypothesis that BMP-14 deficiency leads to a delay in fracture healing. Further studies are warranted to more closely examine the role of BMP-14 in normal fracture healing and the mechanism by which it works.     

Recombinant human BMP-7 effectively prevents non-union in both young and old rats.

The purpose of this study was to evaluate the influence of age on the effectiveness of rhBMP-7 treatment in a fracture with severe periosteal damage that is known to result in non-union formation. Closed stabilized femur fractures were produced in 3-month-old and 18-month-old rats. The fracture site was exposed and 2 mm of the periosteum cauterized circumferentially to impair normal fracture healing. The cauterized fracture site was immediately treated with either 100 microg rhBMP-7 (BMP-7 group), or with 25 microL of vehicle alone (control group). Fracture healing was evaluated with radiographs taken at 3 and 6 weeks. Animals were sacrificed at 3 and 6 weeks and specimens subjected to biomechanical and histological evaluation. In both age groups, none of the control animals healed throughout the 6 weeks experimental duration. All of the rhBMP-7-treated 3-month-old animals were radiographically healed at 3 weeks. In comparison, only 56% (9/16) of the rhBMP-7-treated 18-month-old animals were radiographically healed at 3 weeks. At 6 weeks, however, all of the 18-month-old rhBMP-7-treated animals had healed. Histology revealed slower healing in the 18-month-old animals. Treatment with rhBMP-7 significantly increased all of the biomechanical properties in both age groups. In the 3-month-old animals the mechanical strength approached that of the intact femur at 3 weeks, while in the 18-month-old animals this did not occur until 6 weeks. In conclusion, rhBMP-7 can effectively stimulate fracture repair in both young (3-month-old) and old (18-month-old) rats. However, the effect of rhBMP-7 on the rate of fracture healing is greater in young rats compared to old rats.     

Pantoprazole, a Proton Pump Inhibitor, Delays Fracture Healing in Mice

Proton pump inhibitors (PPIs), which are widely used in the treatment of dyspeptic problems, have been shown to reduce osteoclast activity. There is no information, however, on whether PPIs affect fracture healing. We therefore studied the effect of the PPI pantoprazole on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analyses at 2 and 5 weeks after fracture. Twenty-one mice received 100 mg/kg body weight pantoprazole i.p. daily. Controls (n = 21) received equivalent amounts of vehicle. In pantoprazole-treated animals biomechanical analysis revealed a significantly reduced bending stiffness at 5 weeks after fracture compared to controls. This was associated with a significantly lower amount of bony tissue within the callus and higher amounts of cartilaginous and fibrous tissue. Western blot analysis showed reduced expression of the bone formation markers bone morphogenetic protein (BMP)-2, BMP-4, and cysteine-rich protein (CYR61). In addition, significantly lower expression of proliferating cell nuclear antigen indicated reduced cell proliferation after pantoprazole treatment. Of interest, the reduced expression of bone formation markers was associated with a significantly diminished expression of RANKL, indicating osteoclast inhibition. Pantoprazole delays fracture healing by affecting both bone formation and bone remodeling.     

The influence of compression on the healing of experimental tibial fractures

Purpose

Experimental studies of the effects of various mechanical conditions and stimuli on bone healing have disclosed an improvement potential in bone fracture mineralization and biomechanical properties. We therefore evaluated the effect of a clinically practicable application of a mechanical compressive interfragmentary stimulus on the healing of experimental tibial diaphyseal fractures.

Methods

Sixty Male rats received a standardized tibial shaft osteotomy stabilized with a unilateral external fixator with a zero interfragmentary distance, and then randomly assigned to the compression (N = 20), control (N = 20) or distraction (N = 20) group. From days 4 to day 14, the external fixator was either tightened (compression group) or loosened (distraction group) once daily to gradually induce a total axial displacement of the external fixator pin clamps of 1.25 mm. Evaluation at 30 and 60 days post-osteotomy included radiography, dual-energy X-ray absorptiometry (DXA), quantitative CT and mechanical testing.

Results

All fractures healed radiographically with sparse callus. At 60 days, the compression and control groups exhibited significantly less amount of mineralized callus in terms of DXA measured callus area and bone mineral content (BMC) compared to the distraction group. These groups also demonstrated a smaller volume of low-mineralized bone tissue (callus) and a larger volume of highly mineralized bone tissue (cortical bone) measured by QCT than in the distraction group. Both mechanical strength and stiffness was significantly higher in the compression and control groups than in the distraction group at 60 days.

Discussion

Compression did not enhance fracture healing in terms of mineralization, bending strength, or stiffness at the time of union, compared with the control condition. The compression and control groups exhibited improved healing in terms of mechanical strength and stiffness and a more mature callus mineralization compared with the distraction group.     

Systemic leptin administration alters callus VEGF levels and enhances bone fracture healing in wildtype and ob/ob mice

IntroductionLeptinʼs role in bone formation has been reported, however, its mechanism of affecting bone metabolism is remaining unclear. In this study, we aimed to test whether leptin has a positive effect on fracture healing through the possible mechanism of increasing vascular endothelial growth factor (VEGF) expression in callus tissue.MethodsStandardized femur fractures were created in leptin-deficient ob/ob and wildtype C57BL/6J mice, and recombinant mouse leptin or its vehicle (physiological saline) was administered intraperitoneally during the study. Body weight, radiological, histologic and immunoblotting analyses were performed at different stages of fracture healing.Key findingsThe results showed that leptin treatment led to lower rate of body weight change in both mice genotypes. Radiological and histological analyses showed that the experimental groups had better fracture healing at 14, 21 and 28 days compared to the control groups. Leptin-treated groups had significantly higher VEGF expression in callus compared with the control groups at 2 and 3 weeks post-fracture except normal mice at 2 weeks, and leptin-deficient mice had higher VEGF levels in calluses than normal mice at the same timepoint.ConclusionLow-dose systemically-administered leptin has a positive effect on promoting fracture healing during the latter stages in a clinically-relevant mouse bone fracture model, and increase callus VEGF levels.     

Osteoporosis influences the early period of fracture healing in a rat osteoporotic model

Osteoporotic fractures commonly occur in the elderly. Although current therapies are aimed at the prevention and treatment of osteoporotic fractures, studies examing the fracture healing process in osteoporotic bone are limited. We produced an osteoporotic rat model by ovariectomy (ovx) and maintained a low calcium diet (LCD) in order to evaluate the influence of osteoporosis on fracture healing. Callus formation and strength was monitored over a 3 week period by histological and biomechanical assessment. Data collected simultaneously on a group of rats undergoing sham surgery (sx) were used for comparison. A 40% reduction in fracture callus cross-sectional area and a 23% reduction in bone mineral density in the healing femur of the ovx rats was observed on day 21 following fracture as compared with the sx group (p < 0.01). Biomechanical data from the healing femur of the ovx rats revealed a fivefold decrease in the energy required to break the fracture callus, a threefold decrease in peak failure load, a twofold decrease in stiffness and a threefold decrease in stress as compared with the sx group (p < 0.01, respectively). Histomorphological analysis revealed a delay in fracture callus healing with poor development of mature bone in the ovx rats. This study provides physical evidence of altered fracture healing in osteoporotic bone, which may have important implications in evaluating the effects of new treatments for osteoporosis on fracture healing.     

Effect of osteoporosis medications on fracture healing

Antiosteoporotic medications are often used to concurrently treat a patient’s fragility fractures and underlying osteoporosis. This review evaluates the existing literature from animal and clinical models to determine these drugs’ effects on fracture healing. The data suggest that these medications may enhance bone healing, yet more thorough prospective studies are warranted. Pharmacologic agents that influence bone remodeling are an essential component of osteoporosis management. Because many patients are first diagnosed with osteoporosis when presenting with a fragility fracture, it is critical to understand how osteoporotic medications influence fracture healing. Vitamin D and its analogs are essential for the mineralization of the callus and may also play a role in callus formation and remodeling that enhances biomechanical strength. In animal models, antiresorptive medications, including bisphosphonates, denosumab, calcitonin, estrogen, and raloxifene, do not impede endochondral fracture healing but may delay repair due to impaired remodeling. Although bisphosphonates and denosumab delay callus remodeling, they increase callus volume and result in unaltered biomechanical properties. Calcitonin increases cartilage formation and callus maturation, resulting in improved biomechanical properties. Parathyroid hormone, an anabolic agent, has demonstrated promise in animal models, resulting in accelerated healing with increased callus volume and density, more rapid remodeling to mature bone, and improved biomechanical properties. Clinical data with parathyroid hormone have demonstrated enhanced healing in distal radius and pelvic fractures as well as postoperatively following spine surgery. Strontium ranelate, which may have both antiresorptive and anabolic properties, affects fracture healing differently in normal and osteoporotic bone. While there is no effect in normal bone, in osteoporotic bone, strontium ranelate increases callus bone formation, maturity, and mineralization; forms greater and denser trabeculae; and improves biomechanical properties. Further clinical studies with these medications are needed to fully understand their effects on fracture healing in order to simultaneously treat fragility fractures and underlying osteoporosis.     

Recombinant human bone morphogenetic protein‐7 enhances fracture healing in an ischemic environment

Ischemia predisposes orthopedic trauma patients to delayed fracture healing or nonunion. The goal of this study was to test the ability of bone morphogenetic protein 7 (BMP7) to stimulate fracture repair in an ischemic environment. Ischemic fractures were generated in male adult mice by resecting the femoral artery prior to the creation of a nonstabilized tibia fracture. Recombinant human BMP7 (rhBMP7, 50 µg) was injected into the fracture site immediately after surgery. At 7 days after injury, more tissue vascularization was observed in rhBMP7 treated fractures. Histomorphometric analyses revealed that rhBMP7 induced more cartilage at day 7, more callus and bone at days 14 and 28, and more adipose tissue and fibrous tissue at days 7, 14, and 28 compared to controls (n = 5/group/time). At day 28, all fractures treated with rhBMP7 (50 µg, n = 5) healed, whereas only three of five control fractures exhibited slight bony bridging. In addition, we found that rhBMP7 (both 10 and 50 µg) significantly increased the amount of cartilage compared to controls in stabilized fractures, confirming its chondrogenic effect. Lastly, using bone marrow transplantation, we determined that no donor‐derived osteocytes or chondrocytes were present in rhBMP7‐treated fractures, suggesting rhBMP7 did not recruit mesenchymal stem cells from the bone marrow to the fracture site. In conclusion, our results indicate that rhBMP7 is a promising treatment for fractures with severely disrupted blood supply. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:687–696, 2010     

Fracture healing in protease‐activated receptor‐2 deficient mice

Protease‐activated receptor‐2 (PAR‐2) provides an important link between extracellular proteases and the cellular initiation of inflammatory responses. The effect of PAR‐2 on fracture healing is unknown. This study investigates the in vivo effect of PAR‐2 deletion on fracture healing by assessing differences between wild‐type (PAR‐2^+/+) and knock‐out (PAR‐2^?/?) mice. Unilateral mid‐shaft femur fractures were created in 34 PAR‐2^+/+ and 28 PAR‐2^?/? mice after intramedullary fixation. Histologic assessments were made at 1, 2, and 4 weeks post‐fracture (wpf), and radiographic (plain radiographs, micro‐computed tomography (µCT)) and biomechanical (torsion testing) assessments were made at 7 and 10 wpf. Both the fractured and un‐fractured contralateral femur specimens were evaluated. Polar moment of inertia (pMOI), tissue mineral density (TMD), bone volume fraction (BV/TV) were determined from µCT images, and callus diameter was determined from plain radiographs. Statistically significant differences in callus morphology as assessed by µCT were found between PAR‐2^?/? and PAR‐2^+/+ mice at both 7 and 10 wpf. However, no significant histologic, plain radiographic, or biomechanical differences were found between the genotypes. The loss of PAR‐2 was found to alter callus morphology as assessed by µCT but was not found to otherwise effect fracture healing in young mice. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1271–1276, 2012     

Osteoprotegerin Treatment Impairs Remodeling and Apparent Material Properties of Callus Tissue without Influencing Structural Fracture Strength

The influence of osteoprotegerin (OPG) treatment on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. OPG was given intravenously (10 mg/kg twice weekly) during the entire observation period, and control animals with fractures received vehicle only. When compared with control fractures after 3 weeks of healing, OPG treatment reduced the number of osteoclasts in the callus tissue (93%, P < 0.001) and hampered resorption of genuine cortical bone in the fracture line; OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or callus tissue apparent material properties. When compared with control fractures after 8 weeks of healing; OPG treatment reduced the number of osteoclasts in callus tissue (92%, P < 0.001), augmented callus dimensions (anteriorposterior diameter: 12%, P = 0.034, mediolateral diameter: 13%, P = 0.013), and increased callus BMC (50%, P = 0.007); OPG treatment hampered deposition of new woven bone at the fracture line of the genuine cortical bone (new woven bone present in all vehicle animals, but only in 13% of the OPG-treated animals (P < 0.001)); OPG treatment did not influence structural strength of the fractures, but decreased apparent material properties of the callus tissue (ultimate stress: 51%, P < 0.001; elastic modulus: 42%, P = 0.033). The experiment demonstrates that OPG treatment does not influence the early callus expansion and fracture strength. However, during the subsequent period of remodelling, OPG treatment impairs the normal remodeling and consolidation processes.     

Reduced COX‐2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX‐2), the inducible regulator of prostaglandin E₂ (PGE₂) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX‐2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX‐2 is expressed primarily in early cartilage precursors that co‐express col‐2. COX‐2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX‐2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX‐2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging.     

Quantification of fracture healing with three-dimensional computed tomography

Quantitative methods are necessary for an objective evaluation of fracture healing. Three-dimensional computed tomography (CT) for the measurement of callus volume and density could be such a method and was investigated in an animal model. In 23 goats a closed tibial fracture was created and stabilized with a cast. The animals were killed at 2, 4 and 6 weeks for radiographical, CT and biomechanical analysis. From the CT scans a three-dimensional reconstruction of the callus was made to measure its volume and mean density. At 2 weeks the callus volume had already reached its maximum. In contrast, callus density, torsional strength and torsional stiffness increased over time (P < 0.0001, analysis of variance, ANOVA). Multiple regression analysis showed that the callus volume was not related to the torsional properties. However, callus density was a significant explanatory variable for both torsional strength (R ² = 0.72, P < 0.0001) and torsional stiffness (R ² = 0.82, P < 0.0001). Therefore, callus density as measured by three-dimensional CT is a predictor of the extent of fracture consolidation. CT with three-dimensional reconstruction of the callus seems a valid technique for the quantification of fracture healing. Received: 11 August 1997