Odanacatib increases mineralized callus during fracture healing in a rabbit ulnar osteotomy model

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ～6 and 15 weeks post‐fracture in two separate studies using the unilateral transverse mid‐ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre‐treated for 3–4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice‐weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ～6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment‐related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN‐treated rabbits at week‐6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K‐(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre‐ and post‐osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post‐surgery returned callus BMD to Veh. Peak load of ODN‐ or ALN‐treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 34:72–80, 2016.     

Effects of local vanadium delivery on diabetic fracture healing

This study evaluated the effect of local vanadyl acetylacetonate (VAC), an insulin mimetic agent, upon the early and late parameters of fracture healing in rats using a standard femur fracture model. Mechanical testing, and radiographic scoring were performed, as well as histomorphometry, including percent bone, percent cartilage, and osteoclast numbers. Fractures treated with local 1.5 mg/kg VAC possessed significantly increased mechanical properties compared to controls at 6 weeks post‐fracture, including increased torque to failure (15%; p = 0.046), shear modulus (89%; p = 0.043), and shear stress (81%; p = 0.009). The radiographic scoring analysis showed increased cortical bridging at 4 weeks and 6 weeks (119%; p = 0.036 and 209%; p = 0.002) in 1.5 mg/kg VAC treated groups. Histomorphometry of the fracture callus at days 10 and 14 showed increased percent cartilage (121%; p = 0.009 and 45%; p = 0.035) and percent mineralized tissue (66%; p = 0.035 and 58%; p = 0.006) with local VAC treated groups compared to control. Additionally, fewer osteoclasts were observed in the local VAC treated animals as compared to controls at day 14 (0.45% ± 0.29% vs. 0.83% ± 0.36% of callus area; p = 0.032). The results suggest local administration of VAC acts to modulate osteoclast activity and increase percentage of early callus cartilage, ultimately enhancing mechanical properties comparably to non‐diabetic animals treated with local VAC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2174–2180, 2017.

     

Quantitative Assessment of Experimental Fracture Repair by Peripheral Computed Tomography

An experimental fracture model was used to assess bone mineral density at the fracture site by peripheral computed tomography and to compare the model with biomechanical, histological, and radiographic methods for the quantification of the fracture repair process. Transverse osteotomies in the mid-diaphysis of 28 tibia of sheep were externally fixed and mineral densities, cross-sectional areas, flexural rigidities, tissue composition, and projected callus area were calculated after 9 weeks of healing time. BMD measured by pQCT was strongly correlated with histologically determined percentages of mineralized tissue in the osteotomy gap (R ²= 0.71) and in the periosteal callus (R ²= 0.62). The percentage of mineralized tissue in the osteotomy gap was the best predictor of the flexural rigidity of the tibiae (R ²= 0.74). Because of high correlations with the histological findings, the volumetric BMD at the level of the osteotomy gap was also strongly correlated with the biomechanical findings (R ²= 0.70). Neither the cross-sectional area in pQCT nor the projected callus area in plane film radiography were positively correlated to the flexural rigidity of the tibiae. Quantitative computed tomography proved to be a successful estimator for the prediction of the mechanical stability of healing bones. The noninvasive procedure is a reliable tool for the quantification of the fracture repair process in experimental studies and may be useful for treatment decisions in particular clinical situations. Received: 2 May 1996 / Accepted: 24 June 1996     

Effects of local insulin delivery on subperiosteal angiogenesis and mineralized tissue formation during fracture healing

Local insulin delivery has been shown to improve osseous healing in diabetic animals. The purpose of this study was to quantify the effects of local intramedullary delivery of saline or Ultralente insulin (UL) on various fracture healing parameters using an in vivo non‐diabetic BB Wistar rat model. Quantitation of local insulin levels showed a rapid release of insulin from the fractured femora, demonstrating complete release at 2 days. RT‐PCR analysis revealed that the expression of early osteogenic markers (Col1α2, osteopontin) was significantly enhanced with UL treatment when compared with saline controls (p < 0.05). Significant differences in VEGF + cells and vascularity were evident between the treatment and control groups at day 7 (p < 0.05). At day 21, histomorphometric analysis demonstrated a significant increase in percent mineralized tissue in the UL‐treated animals compared with controls (p < 0.05), particularly within the subperiosteal region of the fracture callus. Mechanical testing at 4 weeks showed significantly greater mechanical strength for UL‐treated animals (p < 0.05), but healing in control animals caught up at 6 weeks post‐fracture. These results suggest that the primary osteogenic effect of UL during the early stages of fracture healing (1–3 weeks) is through an increase in osteogenic gene expression, subperiosteal angiogenesis, and mineralized tissue formation. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 783–791, 2013     

Thymosin β4 administration enhances fracture healing in mice

Thymosin β₄ (Tβ₄) is a regenerative peptide that we hypothesized would promote healing of fractured bone. Mice received a bilateral fibular osteotomy and were given i.p. injections of either Tβ₄ (6 mg/kg) or saline. Calluses from saline‐ and Tβ₄‐treated mice were analyzed for: (1) biomechanical properties and (2) composition using micro‐computed tomography (µCT) and histomorphometry. Biomechanical analysis showed that Tβ₄‐treated calluses had a 41% increase in peak force to failure (p < 0.01) and were approximately 25% stiffer (p < 0.05) than saline‐treated controls. µCT analysis at 21 days post‐fracture showed that the fractional volume of new mineralized tissue and new highly mineralized tissue were respectively 18% and 26% greater in calluses from Tβ₄‐treated mice compared to controls (p < 0.01; p < 0.05, respectively). Histomorphometry complemented the µCT data; at 21 days post‐fracture, Tβ₄‐treated calluses were almost 23% smaller (p < 0.05), had nearly 47% less old cortical bone (p < 0.05) and had a 31% increase in new trabecular bone area/total callus area fraction compared with controls (p < 0.05). Our finding of enhanced biomechanical properties of fractures in mice treated with Tβ₄ provides novel evidence of the therapeutic potential of this peptide for treating bone fractures. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1277–1282, 2014.     

Local vanadium release from a calcium sulfate carrier accelerates fracture healing

This study evaluated the efficacy of using calcium sulfate (CaSO₄) as a carrier for intramedullary delivery of an organic vanadium salt, vanadyl acetylacetonate (VAC) after femoral fracture. VAC can act as an insulin‐mimetic and can be used to accelerate fracture healing in rats. A heterogenous mixture of VAC and CaSO₄ was delivered to the fracture site of BB Wistar rats, and mechanical testing, histomorphometry, micro‐computed tomography (micro‐CT) were performed to measure healing. At 4 weeks after fracture, maximum torque to failure, effective shear modulus, and effective shear stress were all significantly higher (p < 0.05) in rats treated with 0.25 mg/kg VAC–CaSO₄ as compared to carrier control rats. Histomorphometry found a 71% increase in percent cartilage matrix (p < 0.05) and a 64% decrease in percent mineralized tissue (p < 0.05) at 2 weeks after fracture in rats treated with 0.25 mg/kg of VAC–CaSO₄. Micro‐CT analyses at 4 weeks found a more organized callus structure and higher trending maximum connected z‐ray. fraction for VAC–CaSO₄ groups. Evaluation of radiographs and serial histological sections at 12 weeks did not show any evidence of ectopic bone formation. As compared to previous studies, CaSO₄ was an effective carrier for reducing the dose of VAC required to accelerate femoral fracture healing in rats. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:727–734, 2014.     

The effects of local vanadium treatment on angiogenesis and chondrogenesis during fracture healing

This study quantified the effects of local intramedullary delivery of an organic vanadium salt, which may act as an insulin‐mimetic on fracture healing. Using a BB Wistar rat femoral fracture model, local vanadyl acetylacetonate (VAC) was delivered to the fracture site and histomorphometry, mechanical testing, and immunohistochemistry were performed. Callus percent cartilage was 200% higher at day 7 (p < 0.05) and 88% higher at day 10 (p < 0.05) in the animals treated with 1.5 mg/kg of VAC. Callus percent mineralized tissue was 37% higher at day 14 (p < 0.05) and 31% higher at day 21 (p < 0.05) in the animals treated with 1.5 mg/kg of VAC. Maximum torque to failure was 104% and 154% higher at 4 weeks post‐fracture (p < 0.05) for the healing femurs from the VAC‐treated (1.5 and 3.0 mg/kg) animals. Animals treated with other VAC doses demonstrated increased mechanical parameters at 4 weeks (p < 0.05). Immunohistochemistry detected 62% more proliferating cells at days 7 (p < 0.05) and 94% more at day 10 (p < 0.05) in the animals treated with 1.5 mg/kg VAC. Results showed 100% more vascular endothelial growth factor‐C (VEGF‐C) positive cells and 80% more blood vessels at day 7 (p < 0.05) within the callus subperiosteal region of VAC‐treated animals (1.5 mg/kg) compared to controls. The results suggest that local VAC treatment affects chondrogenesis and angiogenesis within the first 7–10 days post‐fracture, which leads to enhanced mineralized tissue formation and accelerated fracture repair as early as 3–4 weeks post‐fracture. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1971–1978, 2012     

Endostatin Inhibits Callus Remodeling during Fracture Healing in Mice

Information on the impact of endogenous anti‐angiogenic factors on bone repair is limited. The hypothesis of the present study was endostatin, an endogenous inhibitor of angiogenesis, disturbs fracture healing. We evaluated this hypothesis in a closed femoral fracture model studying two groups of mice, one that was treated by a daily injection of 10 µg recombinant endostatin subcutaneously (n = 38) and a second one that received the vehicle for control (n = 37). Histomorphometric analysis showed a significantly increased callus formation in endostatin‐treated animals at 2 and 5 weeks post‐fracture. This was associated with a significantly higher callus tissue fraction of cartilage and fibrous tissue at 2 weeks and a significantly higher fraction of bone at 5 weeks post‐fracture. Biomechanical testing revealed a significantly higher torsional stiffness in the endostatin group at 2 weeks. For both groups, we could demonstrate the expression of the endostatin receptor unit integrin alpha5 in endothelial cells, osteoblasts, osteoclasts, and chondrocytes at 2 weeks. Immunohistochemical fluorescence staining of CD31 showed a lower number of blood vessels in endostatin‐treated animals compared to controls. The results of the present study indicate endostatin promotes soft callus formation but inhibits callus remodeling during fracture healing most probably by an inhibition of angiogenesis. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1579–1584, 2013.     

Low‐dose lithium regimen enhances endochondral fracture healing in osteoporotic rodent bone

Osteoporotic bone fractures are highly prevalent and involve lengthy recovery. Lithium, commonly used in psychiatric medicine, inhibits glycogen synthase kinase‐3β in the Wnt/β‐catenin pathway, leading to up‐regulation of osteogenesis. Our recent preclinical work demonstrated that a 20 mg/kg lithium dose administered beginning 7 days post‐fracture for 14 days optimally improved femoral fracture healing in healthy rats at 4 weeks post fracture (46% higher torsional strength). In this study, lithium treatment was evaluated for healing of osteoporotic bone fractures. Six‐month‐old ovariectomized rats were subjected to closed, load‐drop induced femoral diaphyseal fracture. Two regimens involving treatment initiation on day 7 and day 10, respectively, 20 mg/kg/day oral dose and 14 days duration were evaluated. Femurs of lithium‐ vs. saline‐ treated rats were analyzed at 4 weeks (for day 7 onset regimen) or 6 weeks (for day 10 onset regimen) post‐fracture by stereology and torsional mechanical testing. Initiation on day 10 led to a significant 50% higher maximum yield torque (primary outcome measure) at 6 weeks (309 vs. 206 N‐mm, p = 0.005; n = 7, 7). Initiation on day 7 suggested a trend toward a more modest improvement in maximum yield torque (13%) evaluated at 4 weeks post‐fracture (234 vs. 206 N‐mm, p = 0.10; n = 10, 13). Qualitatively, lithium‐treated femurs demonstrated better periosteal and mineralized callus bridging in the day 10 initiation group. Lithium is a widely‐available, orally administered, low‐cost drug, which represents a feasible pharmacological intervention for both healthy and osteoporotic fracture healing. This study provides important guidelines for future clinical evaluation of lithium in osteoporotic fracture patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1783–1789, 2018.

     

Contrast‐enhanced CT using a cationic contrast agent enables non‐destructive assessment of the biochemical and biomechanical properties of mouse tibial plateau cartilage

Mouse models of osteoarthritis (OA) are commonly used to study the disease's pathogenesis and efficacy of potential treatments. However, measuring the biochemical and mechanical properties of articular cartilage in these models currently requires destructive and time‐consuming histology and mechanical testing. Therefore, we examined the feasibility of using contrast‐enhanced CT (CECT) to rapidly and non‐destructively image and assess the glycosaminoglycan (GAG) content. Using three ex vivo C57BL/6 mouse tibial plateaus, we determined the time required for the cationic contrast agent CA4+ to equilibrate in the cartilage. The whole‐joint coefficient of friction (μ) of 10 mouse knees (some digested with Chondroitenase ABC to introduce variation in GAG) was evaluated using a modified Stanton pendulum. For both the medial and lateral tibial plateau cartilage of these knees, linear regression was used to compare the equilibrium CECT attenuations to μ, as well as each side's indentation equilibrium modulus (E) and Safranin‐O determined GAG content. CA4+ equilibrated in the cartilage in 30.9 ± 0.95 min (mean ± SD, tau value of 6.17 ± 0.19 min). The mean medial and lateral CECT attenuation was correlated with μ (R² = 0.69, p < 0.05), and the individual medial and lateral CECT attenuations correlated with their respective GAG contents (R² ≥ 0.63, p < 0.05) and E (R² ≥ 0.63, p < 0.05). In conclusion, CECT using CA4+ is a simple, non‐destructive technique for three‐dimensional imaging of ex vivo mouse cartilage, and significant correlations between CECT attenuation and GAG, E, and μ are observed. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1130–1138, 2016.     

Low‐magnitude high‐frequency vibration enhances gene expression related to callus formation,mineralization and remodeling during osteoporotic fracture healing in rats

Low magnitude high frequency vibration (LMHFV) has been shown to improve anabolic and osteogenic responses in osteoporotic intact bones and during osteoporotic fracture healing; however, the molecular response of LMHFV during osteoporotic fracture healing has not been investigated. It was hypothesized that LMHFV could enhance osteoporotic fracture healing by regulating the expression of genes related to chondrogenesis (Col‐2), osteogenesis (Col‐1) and remodeling (receptor activator for nuclear factor‐ κ B ligand (RANKL) and osteoproteger (OPG)). In this study, the effects of LMHFV on both osteoporotic and normal bone fracture healing were assessed by endpoint gene expressions, weekly radiographs, and histomorphometry at weeks 2, 4 and 8 post‐treatment. LMHFV enhanced osteoporotic fracture healing by up‐regulating the expression of chondrogenesis‐, osteogenesis‐ and remodeling‐related genes (Col‐2 at week 4 (p = 0.008), Col‐1 at week 2 and 8 (p < 0.001and p = 0.008) and RANKL/OPG at week 8 (p = 0.045)). Osteoporotic bone had a higher response to LMHFV than normal bone and showed significantly better results as reflected by increased expression of Col‐2 and Col‐1 at week 2 (p < 0.001 for all), larger callus width at week 2 (p = 0.001), callus area at week 1 and 5(p < 0.05 for all) and greater relative area of osseous tissue (p = 0.002) at week 8. This study helps to understand how LMHFV regulates gene expression of callus formation, mineralization and remodeling during osteoporotic fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1572–1579, 2014.     

BMPR1A antagonist differentially affects cartilage and bone formation during fracture healing

A soluble form of BMP receptor type 1A (mBMPR1A‐mFC) acts as an antagonist to endogenous BMPR1A and has been shown to increase bone mass in mice. The goal of this study was to examine the effects of mBMPR1A‐mFC on secondary fracture healing. Treatment consisted of 10 mg/kg intraperitoneal injections of mBMPR1A‐mFC twice weekly in male C57BL/6 mice. Treatment beginning at 1, 14, and 21 days post‐fracture assessed receptor function during endochondral bone formation, at the onset of secondary bone formation, and during coupled remodeling, respectively. Control animals received saline injections. mBMPR1A‐mFC treatment initiated on day 1 delayed cartilage maturation in the callus and resulted in large regions of fibrous tissue. Treatment initiated on day 1 also increased the amount of mineralized tissue and up‐regulated many bone‐associated genes (p = 0.002) but retarded periosteal bony bridging and impaired strength and toughness at day 35 (p < 0.035). Delaying the onset of treatment to day 14 or 21 partially mitigated these effects and produced evidence of accelerated coupled remodeling. These results indicate that inhibition of the BMPR1A‐mediated signaling has negative effects on secondary fracture healing that are differentially manifested at different stages of healing and within different cell populations. These effects are most pronounced during the endochondral period and appear to be mediated by selective inhibition of BMPRIA signaling within the periosteum. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2096–2105, 2016.     

Local ZnCl2 accelerates fracture healing

This study evaluated the effect of local zinc chloride (ZnCl₂), an insulin mimetic agent, upon the early and late parameters of fracture healing in rats using a standard femur fracture model. Mechanical testing, radiographic scoring, histomorphometry, qualitative histological scoring, PCNA immunohistochemistry, and local growth factor analysis were performed. Fractures treated with local ZnCl₂ possessed significantly increased mechanical properties compared to controls at 4 weeks post fracture. The radiographic scoring analysis showed increased cortical bridging at 4 weeks in the 1.0 (p = 0.0015) and 3.0 (p < 0.0001) mg/kg ZnCl₂ treated groups. Histomorphometry of the fracture callus at day 7 showed 177% increase (p = 0.036) in percent cartilage and 133% increase (p = 0.002) in percent mineralized tissue with local ZnCl₂ treatment compared to controls. Qualitative histological scoring showed a 2.1× higher value at day 7 in the ZnCl₂ treated group compared to control (p = 0.004). Cell proliferation and growth factors, VEGF and IGF‐I, within fracture calluses treated with local ZnCl₂ were increased at day 7. The results suggest local administration of ZnCl₂ increases cell proliferation, causing increased growth factor production which yields improved chondrogenesis and endochondral ossification. Ultimately, these events lead to accelerated fracture healing as early as 4 weeks post fracture. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:834–841, 2014.     

Local manganese chloride treatment accelerates fracture healing in a rat model

This study investigated the effects of local delivery of manganese chloride (MnCl₂), an insulin‐mimetic compound, upon fracture healing using a rat femoral fracture model. Mechanical testing, histomorphometry, and immunohistochemistry were performed to assess early and late parameters of fracture healing. At 4 weeks post‐fracture, maximum torque to failure was 70% higher (P < 0.05) and maximum torsional rigidity increased 133% (P < 0.05) in animals treated with 0.125 mg/kg MnCl₂ compared to saline controls. Histological analysis of the fracture callus revealed percent new mineralized tissue was 17% higher (P < 0.05) at day 10. Immunohistochemical analysis of the 0.125 mg/kg MnCl₂ treated group, compared to saline controls, showed a 379% increase in the density of VEGF‐C+ cells. In addition, compared to saline controls, the 0.125 mg/kg MnCl₂ treated group showed a 233% and 150% increase in blood vessel density in the subperiosteal region at day 10 post‐fracture as assessed by detection of PECAM and smooth muscle α actin, respectively. The results suggest that local MnCl₂ treatment accelerates fracture healing by increasing mechanical parameters via a potential mechanism of amplified early angiogenesis leading to increased osteogenesis. Therefore, local administration of MnCl₂ is a potential therapeutic adjunct for fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:122–130, 2015.     

Hypochlorhydria‐induced calcium malabsorption does not affect fracture healing but increases post‐traumatic bone loss in the intact skeleton

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post‐traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr?/? mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr?/? and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium‐enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro‐computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme‐linked immunosorbent assay. Fracture healing was unaffected in Cckbr?/? mice. However, Cckbr?/? mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr?/? mice. Therefore, under conditions of hypochlorhydria‐induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914–1921, 2016.

     

Fracture healing is accelerated in the absence of the adaptive immune system

Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild‐type (WT) and recombination activating gene 1 knockout (RAG1^?/?) mice lacking the adaptive immune system. Healing was investigated using micro–computed tomography (µCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1^?/? mice compared to the WT group. µCT evaluation of RAG1^?/? specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1^?/? compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1^?/? mice than in the WT mice. Endochondral ossification was accelerated in the RAG1^?/? mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1^?/? mice. In contrast, expression of anti‐inflammatory interleukin 10 (IL‐10) was strongly upregulated in RAG1^?/? mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1^?/? mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti‐inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair. © 2011 American Society for Bone and Mineral Research.     

Implantable microelectromechanical sensors for diagnostic monitoring and post‐surgical prediction of bone fracture healing

The relationship between modern clinical diagnostic data, such as from radiographs or computed tomography, and the temporal biomechanical integrity of bone fracture healing has not been well‐established. A diagnostic tool that could quantitatively describe the biomechanical stability of the fracture site in order to predict the course of healing would represent a paradigm shift in the way fracture healing is evaluated. This paper describes the development and evaluation of a wireless, biocompatible, implantable, microelectromechanical system (bioMEMS) sensor, and its implementation in a large animal (ovine) model, that utilized both normal and delayed healing variants. The in vivo data indicated that the bioMEMS sensor was capable of detecting statistically significant differences (p‐value <0.04) between the two fracture healing groups as early as 21 days post‐fracture. In addition, post‐sacrifice micro‐computed tomography, and histology data demonstrated that the two model variants represented significantly different fracture healing outcomes, providing explicit supporting evidence that the sensor has the ability to predict differential healing cascades. These data verify that the bioMEMS sensor can be used as a diagnostic tool for detecting the in vivo course of fracture healing in the acute post‐treatment period. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1439–1446, 2015.     

Early dynamization by reduced fixation stiffness does not improve fracture healing in a rat femoral osteotomy model

Dynamization of fracture fixation is used clinically to improve the bone healing process. However, the effect of early dynamization remains controversial. This study evaluated the effect of early dynamization, by reduced stiffness of fixation on callus stiffness and size after 5 weeks of healing in a rat diaphyseal femoral osteotomy. An external unilateral fixator allowed either a rigid (R‐group; n = 8) or a flexible (F‐group; n = 8) fixation. The dynamized group (D‐group: n = 8) had a rigid fixation for 1 week, and then a flexible fixation for the remaining 4 weeks. The pre‐ and postoperative activity of the rats was measured. After 5 weeks, the rats were sacrificed, and healing was evaluated by biomechanical and densitometric methods. The R‐group had a higher activity more closely approaching preoperative levels, compared to the D‐group throughout all time points measured. This difference was significant after 14 days and 21 days. The flexural rigidity of the R‐group was 82% (tested in the anterior‐posterior direction; p = 0.01) and 93% (tested in the medial‐lateral direction; p = 0.002) greater than the flexural rigidity of the D‐group. The rigid fixation led to a stiffer callus with a smaller callus volume, but better mineralized tissue in the whole callus and at the level of the osteotomy gap than the flexible or the dynamized fixation. Early dynamization did not improve healing compared to rigid or flexible fixation in a rat femoral osteotomy model. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:22–27, 2009     

Effect of mechanical convection on the partitioning of an anionic iodinated contrast agent in intact patellar cartilage

To determine if mechanical convection accelerates partitioning of an anionic contrast agent into cartilage while maintaining its ability to reflect the glycosaminoglycan (GAG) content in contrast‐enhanced computed tomography (CECT) of cartilage. Bovine patellae (N = 4) were immersed in iothalamate and serially imaged over 24 h of passive diffusion at 34°C. Following saline washing for 14 h, each patella was serially imaged over 2.5 h of mechanical convection by cyclic compressive loading (120N, 1 Hz) while immersed in iothalamate at 34°C. After similar saline washing, each patella was sectioned into 15 blocks (n = 60) and contrast concentration per time point as well as GAG content were determined for each cartilage block. Mechanical convection produced 70.6%, 34.4%, and 16.4% higher contrast concentration at 30, 60, and 90 min, respectively, compared to passive diffusion (p < 0.001) and boosted initial contrast flux 330%. The correlation between contrast concentration and GAG content was significant at all time points and correlation coefficients improved with time, reaching R² = 0.60 after 180 min of passive diffusion and 22.5 min of mechanical convection. Mechanical convection significantly accelerated partitioning of a contrast agent into healthy cartilage while maintaining strong correlations with GAG content, providing an evidence‐based rationale for adopting walking regimens in CECT imaging protocols. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1333–1340, 2014.     

Changes of microstructure and mineralized tissue in the middle and late phase of osteoporotic fracture healing in rats

BACKGROUND: With osteoporosis emerged as one of the most important health issues, more and more investigations are focusing on osteoporotic fracture healing. However, there are few studies on the changes of microstructure and mineralized tissue of newly formed callus. OBJECTIVE: We established an osteoporotic fracture rat model to evaluate the changes of microstructure and mineralized tissue during osteoporotic fracture healing. MATERIALS AND METHODS: A mid-shaft femur fracture model was established 12 weeks after ovariectomy as an osteoporotic fracture group (OPF group). Femurs were then harvested at 4 weeks, 8 weeks and 12 weeks after fracture for peripheral quantitative computed tomography (pQCT), micro-computed tomography (MicroCT), histology and biomechanical test. A sham-operated group was used for comparison, i.e. the normal fracture group (NF group). RESULTS: The pQCT-derived total external callus area in the OPF group was smaller than that in the NF group at 4 weeks after fracture (P<0.05), whereas it was 21% larger in the OPF group than that in the NF group at 12 weeks after fracture (P<0.01). The pQCT-derived bone mineral density in the OPF group was significantly inferior to the NF group at all the time points (P<0.05 for all the time points, respectively). MicroCT data, at 12 weeks after fracture, showed the total callus, bony callus, and newly formed bone was approximately 20% lower in the OPF group than that in the NP group, and the total connectivity was 56% lower in the OPF group as compared to the NF group. Biomechanical test data, at 12 weeks after fracture, showed that the failure load of the left femur of OPF group was 17% less compared to that of the NF group (P<0.01), and 15% lower bending stiffness (P<0.05), 20% lower bending stress (P<0.01), and 28% lower energy at failure (P<0.01) were observed in the OPF group as compared to the NF group. CONCLUSION: The decrease in mineralized tissue and the not well connected microstructure in newly formed callus may explain the decline of mechanical impairment of fracture healing in the ovariectomized rats.