Disruption of thrombospondin‐2 accelerates ischemic fracture healing

Thrombospondin‐2 (TSP2) is a matricellular protein that is highly up‐regulated during fracture healing. TSP2 negatively regulates vascularity, vascular reperfusion following ischemia, and cutaneous wound healing. As well, TSP2‐null mice show increased endocortical bone formation due to an enhanced number of mesenchymal progenitor cells and show increased cortical thickness. Mice deficient in TSP2 (TSP2‐null) show an alteration in fracture healing, that is unrelated to their cortical bone phenotype, which is characterized by enhanced vascularization with a shift towards an intramembranous healing phenotype; thus, we hypothesized that there would be enhanced ischemic fracture healing in the absence of TSP2. We investigated whether an absence of TSP2 would enhance ischemic fracture healing utilizing Laser doppler, µCT and histological analysis. Ischemic tibial fractures were created in wildtype (WT) and TSP2‐null mice and harvested 10, 20, or 40 days post‐fracture. TSP2‐null mice show enhanced vascular perfusion following ischemic fracture. At day 10 post‐fracture, TSP2‐null mice have 115% greater bone volume than WT mice. This is associated with a 122% increase in vessel density, 20% increase in cell proliferation, and 15% decrease in apoptosis compared to WT. At day 20, TSP2‐null mice have 34% more bone volume, 51% greater bone volume fraction, and 37% more bone tissue mineral density than WT. By 40 days after fracture the TSP2‐null mice have a 24% increase in bone volume fraction, but other parameters show no significant differences. These findings indicate TSP2 is a negative regulator of ischemic fracture healing and that in the absence of TSP2 bone regeneration is enhanced. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 935–943, 2013     

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.     

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.     

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.

     

The role of oxygen as a regulator of stem cell fate during fracture repair in TSP2‐null mice

It is often difficult to decouple the relative importance of different factors in regulating MSC differentiation. Genetically modified mice provide model systems whereby some variables can be manipulated while others are kept constant. Fracture repair in thrombospondin‐2 (TSP2)‐null mice is characterized by reduced endochondral ossification and enhanced intramembranous bone formation. The proposed mechanism for this shift in MSC fate is that increased vascular density and hence oxygen availability in TSP2‐null mice regulates differentiation. However, TSP2 is multifunctional and regulates other aspects of the regenerative cascade, such as MSC proliferation. The objective of this study is to use a previously developed computational model of tissue differentiation, in which substrate stiffness and oxygen tension regulate stem cell differentiation, to simulate potential mechanisms which may drive alterations in MSC fate in TSP2‐null mice. Four models (increased cell proliferation, increased numbers of MSCs in the marrow decreased cellular oxygen consumption, and an initially stiffer callus) were not predictive of experimental observations in TSP2‐null mice. In contrast, increasing the rate of angiogenic progression led to a prediction of greater intramembranous ossification, diminished endochondral ossification, and a reduced region of hypoxia in the fracture callus similar to that quantified experimentally by the immunohistochemical detection of pimonidazole adducts that develop with hypoxia. This study therefore provides further support for the hypothesis that oxygen availability during early fracture healing is a key regulator of MSC bipotential differentiation, and furthermore, it highlights the advantages of integrating computational models with genetically modified mouse studies for further elucidating mechanisms regulating stem cell fate. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1585–1596, 2013.     

Low dose erythropoietin stimulates bone healing in mice

Beyond its classical role in regulation of erythropoiesis, erythropoietin (EPO) has been shown to exert protective and regenerative actions in a variety of non‐hematopoietic tissues. However, little is known about potential actions in bone regeneration. To analyze fracture healing in mice, a femoral 0.25 mm osteotomy gap was stabilized with a pin‐clip technique. Animals were treated with 500 U EPO/kg bw per day or with vehicle only. After 2 and 5 weeks, fracture healing was analyzed biomechanically, radiologically and histologically. Expression of PCNA and NFκB was examined by Western blot analysis. Vascularization was analyzed by immunohistochemical staining of PECAM‐1. Circulating endothelial progenitor cells were measured by flow‐cytometry. Herein, we demonstrate that EPO‐treatment significantly accelerates bone healing in mice. This is indicated by a significantly greater biomechanical stiffness and a higher radiological density of the periosteal callus at 2 and 5 weeks after fracture and stabilization. Histological analysis demonstrated significantly more bone and less cartilage and fibrous tissue in the periosteal callus. Endosteal vascularization was significantly increased in EPO‐treated animals when compared to controls. The number of circulating endothelial progenitor cells was significantly greater in EPO‐treated animals. The herein shown acceleration of healing by EPO may represent a promising novel treatment strategy for fractures with delayed healing and non‐union formation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:165–172, 2011     

Bax deficiency in mice increases cartilage production during fracture repair through a mechanism involving increased chondrocyte proliferation without changes in apoptosis

This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had approximately 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and "Bik-like" pro-apoptotic gene increased approximately 1.5-fold and approximately 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2alpha1 and 9alpha1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair.     

Thrombospondin‐2 spatiotemporal expression in skeletal fractures

Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC). Identification of new markers for periosteal MSCs is essential for the development of fracture therapeutics. Expression of the matricellular protein thrombospondin‐2 (TSP2) increases during early fracture healing; however, it is currently unknown what cell population expresses TSP2. Using a TSP2 GFP reporter mouse and a stabilized murine fracture model, we characterized the expression of TSP2 during the inflammatory, soft callus formation, and hard callus formation phases of fracture healing. In addition, using TSP2 GFP positive cells harvested from reporter mouse cells, we characterized the cell population using flow cytometry and colony formation assays. In uninjured diaphyseal bone, we observed TSP2 expression in the cells located along the inner periosteum. We also observed a population of TSP2 expressing cells in undifferentiated regions of early fracture callus and along the periphery of the callus. Later in callus development, TSP2 cells were broadly distributed in the undifferentiated callus, but GFP was not expressed by chondrocytes. Flow cytometry confirmed that the majority of TSP2 expressing cells were positive for traditional murine MSC markers. Our in vitro assays further supported these findings by demonstrating all adherent and colony‐forming cells expressed TSP2. Taken together, our results suggest that TSP2 is expressed by undifferentiated MSCs, but downregulated in chondrocytes. Clinical significance: expression of the matricellular protein TSP2 is a promising new marker to identify MSCs in early fracture healing.     

抑制Wnt/β-catenin信号通路对骨折愈合的影响

目的 探讨Wnt/β-catenin信号通路在骨折愈合中所起的作用. 方法采用8周龄Col2al-ICAT转基因小鼠为实验组(转基因在软骨细胞中特异性表达后可竞争性阻断β-catenin信号)和同窝出生WT小鼠(对照组)作为实验动物,分别制备右下肢胫骨中段截断骨折模型.于骨折后7、9、14、21、28 d取材进行分析,通过X线片、组织学观察和组织形态计量学分析,比较两组软骨痂和骨性骨痂在骨折愈合不同时期所占的比例. 结果通过X线片观察发现,骨折后第21天,WT小鼠骨折线已消失,而Col2al-ICAT转基因小鼠骨折线仍可见.组织学观察发现,与WT小鼠相比,Col2al-ICAT转基因小鼠软骨痂出现延迟,软骨内骨化受阻,骨折的塑形改造延迟.组织形态计量学结果显示:Col2al-ICAT转基因小鼠软骨痂较晚出现高峰期,骨折后第14和第21天时骨性骨痂占总骨痂的比例明显低于WT小鼠,差异有统计学意义(P<0.05).结论 抑制Wnt/β-catenin信号通路将抑制骨折愈合的软骨内骨化过程,最终导致骨折愈合延迟.     

Diminished bone formation during diabetic fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity.

Histological and molecular analysis of fracture healing in normal and diabetic animals showed significantly enhanced removal of cartilage in diabetic animals. Increased cartilage turnover was associated with elevated osteoclast numbers, a higher expression of genes that promote osteoclastogenesis, and diminished primary bone formation. INTRODUCTION: Diminished bone formation, an increased incidence of nonunions, and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed and then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. MATERIALS AND METHODS: A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce a type 1 diabetic condition. After mice were hyperglycemic for 3 weeks, controlled closed simple transverse fractures of the tibia were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16, and 22 days after fracture was performed across the fracture callus at 0.5 mm proximal and distal increments using computer-assisted image analysis. Another group of 16-day samples were examined by microCT. RNA was isolated from a separate set of animals, and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real-time PCR. RESULTS: Molecular analysis of collagen types II and X mRNA expression showed that cartilage formation was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In contrast, on day 16, callus size, cartilage tissue, and new bone area were 2.0-, 4.4-, and 1.5-fold larger, respectively, in the normoglycemic compared with the diabetic group (p < 0.05). Analysis of microCT images indicated that the bone volume in the normoglycemic animals was 38% larger than in diabetic animals. There were 78% more osteoclasts in the diabetic group compared with the normoglycemic group (p < 0.05) on day 16, consistent with the reduction in cartilage. Real-time PCR showed significantly elevated levels of mRNA expression for TNF-alpha, macrophage-colony stimulating factor, RANKL, and vascular endothelial growth factor-A in the diabetic group. Similarly, the mRNA encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, was also elevated in diabetic animals. CONCLUSIONS: These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and contributes to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing.     

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.     

Increased callus mass and enhanced strength during fracture healing in mice lacking the sclerostin gene

Humans with inherited sclerostin deficiency have high bone mass. Targeted deletion of the sclerostin gene in mice (SOST-KO) causes increases in bone formation, bone mass and bone strength. Inhibition of sclerostin by a monoclonal antibody increases bone formation and enhances fracture healing in rodent and primate models. In this study, we describe the temporal progression of femoral fracture healing in SOST-KO mice compared with wild type (WT) control mice to further characterize the role of sclerostin in fracture healing. Sixty-seven male 9-10 week-old SOST-KO (N=37) and WT (N=30) mice underwent a closed femoral fracture. Weekly radiography was used to monitor the progress of healing. Histologic sections were used to characterize callus composition, evaluate callus bridging, and quantify lamellar bone formation on days 14 and 28. Densitometry and biomechanical testing were utilized to characterize bone mass and strength at the fractured and contralateral femurs on day 45. A significant improvement in time to radiographic healing (no discernible fracture line) was observed in SOST-KO mice, which corresponded to an increase in histologic bony bridging at 14 days (38% versus 0% in WT). Both genotypes appeared to be nearly fully bridged at 28 days post-fracture. The increased bridging at 14 days was associated with 97% greater bone area and 40% lower cartilage area in the callus of SOST-KO mice as compared to WT mice. Bone formation-related endpoints were higher in SOST-KO mice at both 14 and 28 days. At 45 days post-fracture, peak load and bone mass were significantly greater in the fractured femurs of SOST-KO mice as compared to WT mice. In conclusion, fractures in mice lacking sclerostin showed accelerated bridging, greater callus maturation, and increased bone formation and strength in the callus.     

A standardized experimental fracture in the mouse tibia

The increased use of transgenic mice as experimental animals provides new opportunities to study the biology of fracture repair. We have developed a technique for the production of a standard closed experimental fracture in the mouse tibia. A 0.2 mm stainless-steel rod was introduced into the medullary cavity and the pre-nailed tibial shaft was fractured by an impact device, which resulted in a reproducible transverse or slightly oblique fracture pattern. The intramedullary rod maintained axial alignment, and the fractures united without displacement. On the basis of measurements of callus geometry, four-point bending tests, biochemical analyses, and quantitative histology, the progress of callus formation and remodeling occurred in a predictable sequence of healing phases. The ultimate bending loads of the fractures increased with time, reaching 74% of the strength of intact control tibias in 4 weeks. The stiffness values of the fractures returned to normal levels and, as determined radiographically, the fractures united by external callus in 4 weeks. Radiographically, callus size, cross-sectional callus area, and callus mass peaked at 2 weeks and decreased thereafter, indicating the start of external remodeling. Histologically, the amount of mesenchymal tissue was maximal at days 5 and 7. The callus cartilage area peaked at day 9; at its maximum, it accounted for 46% of the total callus area. Early periosteal formation of membranous new bone, followed by endochondral ossification, resulted in a linear increase of callus bone during the healing process. The healing sequence of the mouse tibial fracture was similar to that seen in the rat tibia. The major difference is the small size of the mouse, which makes the surgical technique and anesthetic procedures more demanding.     

Fracture Healing in Mice Deficient in Plasminogen Activator Inhibitor-1

To evaluate the role of plasminogen activator inhibitor (PAI)-1, a key negative regulator of the plasmin system of extracellular matrix proteases in developmental bone growth and fracture repair, the bone phenotype of male adult PAI-1-deficient mice was determined and femoral fracture healing was compared with that of age- and sex-matched wild-type C57BL/6J control mice. Regarding bone phenotype, the length and size (but not cortical thickness) of the femur of male PAI-1-deficient mice were smaller than those of wild-type controls. Although the total bone mineral content of PAI-1-deficient mice was not significantly different from that of wild-type mice, the total bone area in PAI-1-deficient mice was smaller, leading to an increase in total bone mineral density. With respect to fracture healing, PAI-1-deficient mice developed fracture calluses that were larger and more mineralized than those of wild-type mice but only at 14 days postfracture. These changes were even greater given the smaller size of the normal femur in PAI-1-deficient mice. Surprisingly, the larger fracture callus remodeled rapidly to normal size and mineral content by 21 days postfracture. Examination of fracture histology revealed that these changes were associated with a dramatic increase followed by a rapid remodeling of the fracture callus cartilage. The remodeling of fracture callus cartilage in PAI-1-deficient mice also displayed an abnormal pattern. These findings demonstrate for the first time that PAI-1 (and potentially the plasminogen extracellular matrix protease system) is an important regulator of bone size during developmental growth and plays a regulatory role in the determination of fracture callus size, cartilage formation, and resorption during bone fracture repair.     

Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling

In this study we investigated if Wnt/β-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of β-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in β-catenin^Prx1ER conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (μCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the β-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through β-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in β-catenin^Prx1ER conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.     

Assessment of contrast‐enhanced computed tomography for imaging of cartilage during fracture healing

Assessment of the early stages of fracture healing via X‐rays and computed tomography is limited by the low radio‐opacity of cartilage. We validated a method of contrast‐enhanced computed tomography (CECT) for non‐destructive identification of cartilage within a healing fracture callus. Closed, stabilized fractures in femora of C57BL/6 mice were harvested on post‐operative day 9.5 and imaged ex vivo with micro‐computed tomography (µCT) before and after incubation in a cationic contrast agent that preferentially accumulates in cartilage due to the high concentration of sulfated glycosaminoglycans in the tissue. Co‐registration of the pre‐ and post‐incubation images, followed by image subtraction, enabled two‐ and three‐dimensional delineation of mineralized tissue, soft callus, and cartilage. The areas of cartilage and callus identified with CECT were compared to those identified with the gold‐standard method of histomorphometry. No difference was found between the areas of cartilage measured by the two methods (p = 0.999). Callus area measured by CECT was smaller than, but strongly predictive of (R² = 0.80, p < 0.001), the corresponding histomorphometric measurements. CECT also enabled qualitative identification of mineralized cartilage. These findings indicate that the CECT method provides accurate, quantitative, and non‐destructive visualization of the shape and composition of the fracture callus, even during the early stages of repair when little mineralized tissue is present. The non‐destructive nature of this method would allow subsequent analyses, such as mechanical testing, to be performed on the callus, thus enabling higher‐throughput, comprehensive investigations of bone healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 567–573, 2013     

An externally fixed femoral fracture model for mice.

Transgenic and knockout mouse models can be used to help understand the molecular mechanisms of fracture repair. This study examines the feasibility of applying an external fixator to the mouse femur as one model for studying fracture repair. The external fixator consisted of two aluminum blocks connected by two rods. Four pins are used to connect the blocks to the bone. Mechanical characterization of the fixators was carried out prior to their use. Sixty-two wild type mice with bilateral femoral fractures were created using an open technique and fixed using the fixator. The progress of fracture healing was monitored radiologically before sacrifice and by mechanical testing and histology after sacrifice. Initially four mice died intraoperatively from excessive blood loss, the intraoperative mortality was subsequently reduced by subcutaneous saline infusion. The bone healed between 14 and 21 days after fracture and remodeled by 60 days. Both radiological and mechanical assessments showed a steady progression of bone healing. Histology demonstrated callus and endochondral bone formation. This study demonstrated that it is possible to create a mouse femoral fracture model stabilized by external fixation and will provide an additional model to the understanding of fracture healing in transgenic and knockout mice.     

Effects of delayed stabilization on fracture healing

Previous studies have revealed that delayed internal fixation can stimulate fracture callus formation and decrease the rate of nonunion. However, the effect of delayed stabilization on stem cell differentiation is unknown. To address this, we created fractures in mouse tibiae and applied external fixation immediately, at 24, 48, 72, or 96 h after injury. Fracture healing was analyzed at 10 days by histological methods for callus, bone, and cartilage formation, and the mechanical properties of the calluses were assessed at 14 days postinjury by tension testing. The results demonstrate that delaying stabilization for 24–96 h does not significantly affect the volume of the callus tissue (TV) and the new bone (BV) that formed by 10 days, or the mechanical properties of the calluses at 14 days, compared to immediate stabilization. However, delaying stabilization for 24–96 h induces 10–40× more cartilage in the fracture calluses compared with fractures stabilized immediately. These findings suggest that delaying stabilization during the early phase of fracture healing may not significantly stimulate bone repair, but may alter the mode of bone repair by directing formation of more cartilage. Fractures that are not rigidly stabilized form a significantly larger amount of callus tissue and cartilage by 10 days postinjury than fractures stabilized at 24–96 h, indicating that mechanical instability influences chondrocytes beyond the first 96 h of fracture healing. © 2007 Orthopaedic Research Society. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1552–1558, 2007