In vivo gait analysis in a mouse femur fracture model

Although the mouse has become a preferred species for molecular studies on fracture healing, gait analysis after fracture fixation and during bone healing has not yet been performed in mice. Herein, we introduce a novel technique for gait analysis in mice and report the change of motion pattern after fracture and fixation. A standardized femur fracture was stabilized by a common pin. The non-fractured tibia was additionally marked with a pin, allowing continuous analysis of the tibio-femoral angle by digital video-radiography. Dynamic gait analysis was performed at day fourteen after surgery in a radio-opaque running wheel. Fracture fixation resulted in a significantly reduced range and maximum of the tibio-femoral angle compared to non-fractured controls. This was associated with a significantly reduced stride length. Because stride frequency was slightly increased and, thus, stride time diminished, stride velocity was not significantly reduced compared to controls. Thus, our study demonstrates distinct alterations of the gait of mice at 2 weeks after femur fracture and stabilization. Our results support the need of gait analysis in fracture healing studies to assess the animals’ well-being.     

Development of a locking femur nail for mice

We herein report on a novel locking intramedullary nail system in a murine closed femur fracture model. The nail system consists of a modified 24-gauge injection needle and a 0.1-mm-diameter tungsten guide wire. Rotation stability was accomplished by flattening the proximal and distal end of the needle. Torsional mechanical testing of the implants in osteotomized cadaveric femora revealed a superiority of the locking nail (3.9+/-1.0 degrees rotation at a torque of 0.9 Nmm, n=10) compared to the unmodified injection needle (conventional nail; 52.4+/-3.2 degrees, n=10, p<0.05). None of the implants, however, achieved the rotation stability of unfractured femora (0.3+/-0.5 degrees, n=10). In a second step, we tested the feasibility of the in vivo application of the locking nail to stabilize a closed femoral midshaft fracture in C57BL/6 mice. Of interest, none of the 10 animals showed a dislocation of the locking nail over a 5-week period, while 3 of 4 animals with conventional nail fracture stabilization showed a significant pin dislocation within the first 3 days (p<0.05). Mechanical testing after 5-weeks stabilization with the locking nail revealed an appropriate bone healing with a torque at failure of 71.6+/-3.4% and a peak rotation before failure of 68.4+/-5.3% relative to the unfractured contralateral femur. With the advantage that closed fractures can be fixed with rotation stability, the herein introduced model may represent an ideal tool to study bone healing in transgenic and knockout mice.     

Erythropoietin (EPO): EPO-receptor signaling improves early endochondral ossification and mechanical strength in fracture healing

Beyond its role in the regulation of red blood cell proliferation, the glycoprotein erythropoietin (EPO) has been shown to promote cell regeneration and angiogenesis in a variety of different tissues. In addition, EPO has been indicated to share significant functional and structural homologies with the vascular endothelial growth factor (VEGF), a cytokine essential in the process of fracture healing. However, there is complete lack of information on the action of EPO in bone repair and fracture healing. Therefore, we investigated the effect of EPO treatment on bone healing in a murine closed femur fracture model using radiological, histomorphometric, immunohistochemical, biomechanical and protein biochemical analysis. Thirty-six SKH1-hr mice were treated with daily i.p. injections of 5000 U/kg EPO from day 1 before fracture until day 4 after fracture. Controls received equivalent amounts of the vehicle. After 2 weeks of fracture healing, we could demonstrate expression of the EPO-receptor (EPOR) in terminally differentiating chondrocytes within the callus. At this time point EPO-treated animals showed a higher torsional stiffness (biomechanical analysis: 39.6+/-19.4% of the contralateral unfractured femur) and an increased callus density (X-ray analysis (callus density/spongiosa density): 110.5+/-7.1%) when compared to vehicle-treated controls (14.3+/-8.2% and 105.9+/-6.6%; p<0.05). Accordingly, the histomorphometric examination revealed an increased fraction of mineralized bone and osteoid (33.0+/-3.0% versus 28.5+/-3.6%; p<0.05). Of interest, this early effect of the initial 6-day EPO treatment had vanished at 5 weeks after fracture. We conclude that EPO-EPOR signaling is involved in the process of early endochondral ossification, enhancing the transition of soft callus to hard callus.     

The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses.     

Hyperbaric Hyperoxia Accelerates Fracture Healing in Mice

Increased oxygen tension influences bone metabolism. This study comprised two main experiments: one aimed to determine the bone mineral apposition and bone formation rates in vivo under hyperbaric hyperoxia (HBO), and the other aimed to evaluate the effects of exposure to HBO on fracture healing. In experiment 1, male mice were exposed to HBO [90 min/day at 90% O₂ at 2 atmospheres absolute (ATA) for 5 days]. In experiment 2, an open femur fracture model was created in mice, followed by exposure to HBO 5 times/week (90 min/day at 90% O₂ at 2 ATA) for 6 weeks after surgery. In experiment 1, HBO treatment significantly increased the mineral apposition and bone formation rates in the lumbar vertebra and femur and type 1 collagen alpha 1 and alkaline phosphatase mRNA expression in the lumbar vertebra. In experiment 2, at 2 weeks after fracture, the fracture callus was significantly larger in the HBO group than in the non-HBO group. Furthermore, at 4 and 6 weeks after fracture, radiographic findings showed accelerated fracture healing in the HBO group. At 6 weeks after fracture, femur stiffness and maximum load were significantly higher in the HBO group than in the non-HBO group. Urinary 8-hydroxy-2′-deoxyguanosine and plasma calcium concentrations were not significantly different between groups. These results suggest that exposure to HBO enhances bone anabolism and accelerates fracture healing without causing oxidative DNA damage or disruption of plasma calcium homeostasis.     

Complement C3 and C5 Deficiency Affects Fracture Healing

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3^-/- and C5^-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3^-/-: -25%, p=0.02; C5^-/-: -20% p=0.052) and newly formed bone (C3^-/-: -38%, p=0.01; C5^-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3^-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.     

Modelling the effects of different fracture geometries and healing stages on ultrasound signal loss across a long bone fracture

The effect on the signal amplitude of ultrasonic waves propagating along cortical bone plates was modelled using a 2D Finite Difference code. Different healing stages, represented by modified fracture geometries were introduced to the plate model. A simple transverse and oblique fracture filled with water was introduced to simulate the inflammatory stage. Subsequently, a symmetric external callus surrounding a transverse fracture was modelled to represent an advanced stage of healing. In comparison to the baseline (intact plate) data, a large net loss in signal amplitude was produced for the simple transverse and oblique cases. Changing the geometry to an external callus with different mechanical properties caused the net loss in signal amplitude to reduce significantly. This relative change in signal amplitude as the geometry and mechanical properties of the fracture site change could potentially be used to monitor the healing process.     

Modelling the effects of different fracture geometries and healing stages on ultrasound signal loss across a long bone fracture

The effect on the signal amplitude of ultrasonic waves propagating along cortical bone plates was modelled using a 2D Finite Difference code. Different healing stages, represented by modified fracture geometries were introduced to the plate model. A simple transverse and oblique fracture filled with water was introduced to simulate the inflammatory stage. Subsequently, a symmetric external callus surrounding a transverse fracture was modelled to represent an advanced stage of healing. In comparison to the baseline (intact plate) data, a large net loss in signal amplitude was produced for the simple transverse and oblique cases. Changing the geometry to an external callus with different mechanical properties caused the net loss in signal amplitude to reduce significantly. This relative change in signal amplitude as the geometry and mechanical properties of the fracture site change could potentially be used to monitor the healing process.     

Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice

Vascular and cellular invasion into the cartilage is a critical step in the fracture healing. Matrix metalloproteinase-13 (MMP-13) is a member of the zinc-dependent endopeptidase family and plays an important role in remodeling of extracellular matrix. Therefore we investigated the possible involvement of MMP-13 in a murine model of stabilized bone fracture healing. Repair of the fracture in MMP-13 deficient (MMP-13(-/-)) mice was significantly delayed and characterized by a retarded cartilage resorption in the fracture callus. Immunohistochemistry indicated severe defects in vascular penetration and chondroclast recruitment to the fracture callus in MMP-13(-/-) mice. Consistent with the observations, the chondrocyte pellets cultured from the MMP13(-/-) mice exhibited diminished angiogenic activities when the pellets were co-cultured with endothelial cells. These results suggest that MMP-13 is crucial to the process of angiogenesis during healing of fracture, especially in the cartilage resorption process.     

An analysis of the effect of lower extremity strength on impact severity during a backward fall.

At least 280 000 hip fractures occur annually in the U.S. at an estimated cost of $9 billion. While over 90 percent of these are caused by falls, only about 2 percent of all falls result in hip fracture. Evidence suggests that the most important determinants of hip fracture risk during a fall are the body's impact velocity and configuration. Accordingly, protective responses for reducing impact velocity and the likelihood for direct impact to the hip, strongly influence fracture risk. One method for reducing the body's impact velocity and kinetic energy during a fall is to absorb energy in the lower extremity muscles during descent, as occurs during sitting and squatting. In the present study, we employed a series of in verted pendulum models to determine: (a) the theoretical effect of this mechanism on impact severity during a backward fall, and (b) the effect on impact severity of age-related declines (or exercise-induced enhancements) in lower extremity strength. Compared to the case of a fall with zero energy absorption in the lower extremity joints, best-case falls (which involved 81 percent activation of ankle and hip muscles, but only 23 percent activation of knees muscles) involved 79 percent attenuation (from 352 J to 74 J) in the body's vertical kinetic energy at impact (KEv), and 48 percent attenuation (from 3.22 to 1.68 m/s) in the downward velocity of the pelvis at impact (v(v)). Among the mechanisms responsible for this were: (1) eccentric contraction of lower extremity muscles during descent, which resulted in up to 150 J of energy absorption; (2) impact with the trunk in an upright configuration, which reduced the change in potential energy associated with the fall by 100 J; and (3) knee extension during the final stage of descent, which "transferred" up to 90 J of impact energy into horizontal (as opposed to vertical) kinetic energy. Declines in joint strength reduced the effectiveness of mechanisms (1) and (3), and thereby increased impact severity However, even with reductions of 80 percent in available torques, KEv was attenuated by 50 percent. This indicates the importance of both technique and strength in reducing impact severity. These results provide motivation for attempts to reduce elderly individuals' risk for fall-related injury through the combination of instruction in safe falling techniques and exercises that enhance lower extremity strength.     

Midkine-Deficiency Delays Chondrogenesis during the Early Phase of Fracture Healing in Mice

The growth and differentiation factor midkine (Mdk) plays an important role in bone development and remodeling. Mdk-deficient mice display a high bone mass phenotype when aged 12 and 18 months. Furthermore, Mdk has been identified as a negative regulator of mechanically induced bone formation and it induces pro-chondrogenic, pro-angiogenic and pro-inflammatory effects. Together with the finding that Mdk is expressed in chondrocytes during fracture healing, we hypothesized that Mdk could play a complex role in endochondral ossification during the bone healing process. Femoral osteotomies stabilized using an external fixator were created in wildtype and Mdk-deficient mice. Fracture healing was evaluated 4, 10, 21 and 28 days after surgery using 3-point-bending, micro-computed tomography, histology and immunohistology. We demonstrated that Mdk-deficient mice displayed delayed chondrogenesis during the early phase of fracture healing as well as significantly decreased flexural rigidity and moment of inertia of the fracture callus 21 days after fracture. Mdk-deficiency diminished beta-catenin expression in chondrocytes and delayed presence of macrophages during early fracture healing. We also investigated the impact of Mdk knockdown using siRNA on ATDC5 chondroprogenitor cells in vitro. Knockdown of Mdk expression resulted in a decrease of beta-catenin and chondrogenic differentiation-related matrix proteins, suggesting that delayed chondrogenesis during fracture healing in Mdk-deficient mice may be due to a cell-autonomous mechanism involving reduced beta-catenin signaling. Our results demonstrated that Mdk plays a crucial role in the early inflammation phase and during the development of cartilaginous callus in the fracture healing process.     

Testosterone Delivered with a Scaffold Is as Effective as Bone Morphologic Protein-2 in Promoting the Repair of Critical-Size Segmental Defect of Femoral Bone in Mice

Loss of large bone segments due to fracture resulting from trauma or tumor removal is a common clinical problem. The goal of this study was to evaluate the use of scaffolds containing testosterone, bone morphogenetic protein-2 (BMP-2), or a combination of both for treatment of critical-size segmental bone defects in mice. A 2.5-mm wide osteotomy was created on the left femur of wildtype and androgen receptor knockout (ARKO) mice. Testosterone, BMP-2, or both were delivered locally using a scaffold that bridged the fracture. Results of X-ray imaging showed that in both wildtype and ARKO mice, BMP-2 treatment induced callus formation within 14 days after initiation of the treatment. Testosterone treatment also induced callus formation within 14 days in wildtype but not in ARKO mice. Micro-computed tomography and histological examinations revealed that testosterone treatment caused similar degrees of callus formation as BMP-2 treatment in wildtype mice, but had no such effect in ARKO mice, suggesting that the androgen receptor is required for testosterone to initiate fracture healing. These results demonstrate that testosterone is as effective as BMP-2 in promoting the healing of critical-size segmental defects and that combination therapy with testosterone and BMP-2 is superior to single therapy. Results of this study may provide a foundation to develop a cost effective and efficient therapeutic modality for treatment of bone fractures with segmental defects.     

Methyltransferase-like 3-mediated N6-methyladenosine modification of miR-7212-5p drives osteoblast differentiation and fracture healing

N6-methyladenosine (m6A) modification has been reported in various diseases and implicated in increasing numbers of biological processes. However, previous studies have not focused on the role of m6A modification in fracture healing. Here, we demonstrated that m6A modifications are decreased during fracture healing and that methyltransferase-like 3 (METTL3) is the main factor involved in the abnormal changes in m6A modifications. Down-regulation of METTL3 promotes osteogenic processes both in vitro and in vivo, and this effect is recapitulated by the suppression of miR-7212-5p maturation. Further studies have shown that miR-7212-5p inhibits osteoblast differentiation in MC3T3-E1 cells by targeting FGFR3. The present study demonstrated an important role of the METTL3/miR-7212-5p/FGFR3 axis and provided new insights on m6A modification in fracture healing.     

Chondrocyte BMP2 signaling plays an essential role in bone fracture healing

The specific role of endogenous Bmp2 gene in chondrocytes and in osteoblasts in fracture healing was investigated by generation and analysis of chondrocyte- and osteoblast-specific Bmp2 conditional knockout (cKO) mice. The unilateral open transverse tibial fractures were created in these Bmp2 cKO mice. Bone fracture callus samples were collected and analyzed by X-ray, micro-CT, histology analyses, biomechanical testing and gene expression assays. The results demonstrated that the lack of Bmp2 expression in chondrocytes leads to a prolonged cartilage callus formation and a delayed osteogenesis initiation and progression into mineralization phase with lower biomechanical properties. In contrast, when the Bmp2 gene was deleted in osteoblasts, the mice showed no significant difference in the fracture healing process compared to control mice. These findings suggest that endogenous BMP2 expression in chondrocytes may play an essential role in cartilage callus maturation at an early stage of fracture healing. Our studies may provide important information for clinical application of BMP2.     

微创经皮LCP 接骨术治疗老年股骨近端粉碎骨折

目的:本研究通过观察微创锁定钢板接骨术治疗老年股骨近端粉碎骨折临床效果,旨在找出最佳治疗方式。方法:自2007年12月～2010年03月,应用股骨近端锁定加压钢板治疗老年股骨近端粉碎骨折23例。记录术中出血量、手术时间,术后并发症、骨折愈合时间及最后一次随访时功能恢复情况。结果:骨折临床愈合时间为12～28周,平均16周。除1例患者髋内翻畸形,1例锁定加压钢板断裂外,其他患者均达到骨性愈合。结论:股骨近端锁定钢板具有创伤小、固定可靠、骨折愈合快、功能恢复满意的特点,尤其适用于老年股骨近端粉碎骨折。     

Mineralized collagen fibril network spatial arrangement influences cortical bone fracture behavior

Bone is a hierarchical material exhibiting different fracture mechanisms at each length scale. At the submicroscale, the bone is composed of mineralized collagen fibrils (MCF). At this scale, the fracture processes in cortical bone have not been extensively studied in the literature. In this study, the influence of MCF size and orientation on the fracture behavior of bone under both transverse and longitudinal loading was investigated using novel 3D models of MCF networks with explicit representation of extra-fibrillar matrix. The simulation results showed that separation between MCFs was the main cause of damage and failure under transverse loading whereas under longitudinal loading, the main damage and failure mechanism was MCF rupture. When the MCF network was loaded in the transverse direction the mechanical properties increased as the orientation of fibrils deviated farther from the main fibril orientation whereas the opposite trend was observed under longitudinal loading. The fracture energy was much larger in longitudinal than transverse loading. MCF diameter variation did not affect the mechanical properties under longitudinal loading but led to higher mechanical properties with increasing MCF diameter under transverse loading. The new modeling framework established in this study generate unique information on the effect of MCF network spatial arrangement on the fracture behavior of bone at the submicroscale which is not currently possible to measure via experiments. This unique information may improve the understanding of how structural alterations at the submicroscale due to disease, age-related changes, and treatments affect the fracture processes at larger length scales.     

An experimental study in bio-ballistics: Femoral fractures produced by projectiles—II Shaft impacts

To determine the response of human cortical bone to projectile impact, 364 projectile impact tests were conducted on the shafts of embalmed human femurs. Chrome steel spherical projectiles in two diameters, 0·250 and 0·406 in., were employed to differentiate the effects of projectiles of varied sizes and masses in impacts at the same velocity. It was found that the larger projectiles expended significantly more energy in fracturing a femur than the smaller projectiles did at an identical impact velocity. Also, when impacts in which larger and smaller spheres possessed identical kinetic energies were compared, it was found that the larger spheres still expended more energy in fracturing the femur. Finally, it was clearly demonstrated by these experiments that impacts to cortical bone of the femoral shaft by either size projectile caused greater energy expenditure than impacts to the distal end of the femur, which is composed almost entirely of cancellous bone.     

A numerical approach to the biomechanical analysis of bone fracture healing

Investigations are reported in the literature, by means of experimental, analytical and numerical methods, concerning the biomechanical properties of bone. However, the evolutionary phenomena of bone fracture healing does not have a large reference literature. This work investigates and describes the behaviour of inclined human femur fractures with external fixation up to complete healing. A numerical formulation based on the finite element method has been adopted. Geometric configuration is defined using data from a magnetic resonance process applied to a femur in vivo. A three dimensional model has been developed by adopting an orthotropic material law for cortical bone and an isotropic law for the fracture gap zone. Stress and strain reponses of the bone and fixation device are investigated with reference to the evolutionary behaviour of the healing tissue.     

The haploinsufficient hematopoietic microenvironment is critical to the pathological fracture repair in murine models of neurofibromatosis type 1

Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a complex genetic disorder with a high predisposition of numerous skeletal dysplasias including short stature, osteoporosis, kyphoscoliosis, and fracture non-union (pseudoarthrosis). We have developed murine models that phenocopy many of the skeletal dysplasias observed in NF1 patients, including reduced bone mass and fracture non-union. We also show that the development of these skeletal manifestations requires an Nf1 haploinsufficient background in addition to nullizygous loss of Nf1 in mesenchymal stem/progenitor cells (MSCs) and/or their progenies. This is replicated in two animal models of NF1, PeriCre(+);Nf1(flox/-) and Col2.3Cre(+);Nf1(flox/-) mice. Adoptive transfer experiments demonstrate a critical role of the Nf1+/- marrow microenvironment in the impaired fracture healing in both models and adoptive transfer of WT bone marrow cells improves fracture healing in these mice. To our knowledge, this is the first demonstration of a non-cell autonomous mechanism in non-malignant NF1 manifestations. Collectively, these data provide evidence of a combinatory effect between nullizygous loss of Nf1 in osteoblast progenitors and haploinsufficiency in hematopoietic cells in the development of non-malignant NF1 manifestations.