Ligamentous versus physeal failure in murine medial collateral ligament biomechanical testing

This study examines the age at which a femoral physeal failure ceased to occur in a mouse model of medial collateral ligament (MCL) testing. Biomechanical testing of the MCL with load to failure can result in physeal failure rather than MCL failure in skeletally immature animals. Failure mode depended significantly on age (p<0.05). Sixty percent of the knees tested at 4 months failed at the physis rather than at the ligament, whereas, only ten percent of the knees tested at 5 and 6 months failed at the physis. The mean ultimate force to failure for the specimens in which the failure occurred at the ligament was 8.1 N with a higher values for the right side versus the left (p<0.05). For the specimens in which the failure occurred at the physis, the mean ultimate force to failure was 11.2 N. We now consider that 5 month old mice are functionally skeletally mature and old enough to be tested biomechanically with few failures at the physis.     

Relationship between the chondrocyte maturation cycle and the endochondral ossification in the diaphyseal and epiphyseal ossification centers

The chondrocyte maturation cycle and endochondral ossification were studied in human, fetal cartilage Anlagen and in postnatal meta‐epiphyses. The relationship between the lacunar area, the inter‐territorial fibril network variations, and calcium phosphorus nucleation in primary and secondary ossification centers were assessed using light microscopy and scanning electron microscopy (SEM) morphometry. The Anlage topographic, zonal classification was derived from the anatomical nomenclature of the completely developed long bone (diaphysis, metaphyses and epiphyses). A significant increase in the chondrocyte lacunar area was documented in the Anlage of epiphyseal zones 4 and 3 to zone 2 (metaphysis) and zone 1 (diaphysis), with the highest variation from zone 2 to zone 1. An inverse reduction in the intercellular matrix area and matrix interfibrillar empty space was also documented. These findings are consistent with the osmotic passage of free cartilage water from the interfibrillar space into the swelling chondrocytes, which increased the ion concentrations to a critical threshold for mineral precipitation in the matrix. The mineralized cartilage served as a scaffold for osteoblast apposition both in primary and secondary ossification centers and in the metaphyseal growth plate cartilage, though at different periods of bone Anlage development and with distinct patterns for each zone. All developmental processes shared a common initial pathway but progressed at different rates, modes and organization in diaphysis, metaphysis and epiphysis. In the ossification phase the developing vascular supply appeared to play a key role in determining the cortical or trabecular structure of the long bones. J. Morphol. 277:1187–1198, 2016. © 2016 Wiley Periodicals, Inc.     

ABC of emergency radiology. The ankle.

The os trigonum is a common normal variant of the talus and is due to a separate ossification centre arising from the posterior tubercle. The appearance may resemble an old ununited fracture fragment. However, it is triangular, well corticated, in a classic location, and usually bilateral, which enables it to be distinguished from a fracture. Transverse, sclerotic, linear lines located at the metaphysis of growing long bones are due to short periods of growth arrest and have no clinical importance (fig 5). They may be confused with compression fractures, but again these lines are usually bilateral. Fibrous cortical defects are the most commonly seen benign lesions of long bones and are usually identified incidentally in radiographs taken for another reason. The defect is limited to the cortex, commonly found at the metaphysis, but may be located in the diaphysis as the bone grows. The lesion is well corticated (sclerotic margins) and usually does not produce signs or symptoms.     

Chondrocyte differentiation and bone growth during the development of the cartilaginous skeleton of chickens]

Certain local alterations in functional and reproductive activity of chondrocytes were stated at the development of the cartilage skeleton. In epiphyses chondrocytes gradually pass into the phase of rest (G0) with subsequent multiplication during the process of skeletal development. In these structures biosynthesis of nonsulfated proteoglycans predominate, in time, while in other cartilage zones--that of sulfated ones. Proofs are furnished on gradual transition of epiphyseal chondrocytes into the state peculiar for cells of the proliferative zone accompanied by an intensified biosynthesis of sulfated proteoglycans and collagenous proteins. Owing to these peculiarities they can be compared with the cells of the reserve zone in the mammalian metaepiphyseal cartilage. It was stated that intensity of chondrogenesis and growth of bones are affected by several processes: intensity of chondrocyte multiplication, the rate of their repeated division in the proliferative zone, the velosity with which the cells transfer into the state of hypertrophy and the rate of the periostal bone formation at the border-line of metaphysis and diaphysis.     

In vitro replication of spontaneous fractures of the proximal human femur

Spontaneous fractures (i.e. caused by sudden loading and muscle contraction, not by trauma) represent a significant percentage of proximal femur fractures. They are particularly relevant as may occur in elderly (osteoporotic) subjects, but also in relation to epiphyseal prostheses. Despite its clinical and legal relevance, this type of fracture has seldom been investigated. Studies concerning spontaneous fractures are based on a variety of loading scenarios. There is no evidence, nor consensus on the most relevant loading scenario. The aim of this work was to develop and validate an experimental method to replicate spontaneous fractures in vitro based on clinically relevant loading. Primary goals were: (i) repeatability and reproducibility, (ii) clinical relevance. A validated numerical model was used to identify the most critical loading scenario that can lead to head-neck fractures, and to determine if it is necessary to include muscle forces when the head-neck region is under investigation. The numerical model indicated that the most relevant loading scenario is when the resultant joint force is applied to the head at 8 degrees from the diaphysis. Furthermore, it was found that it is not essential to include the muscles when investigating head-neck fractures. The experimental setup was consequently designed. The procedure included high-speed filming of the fracture event. Clinically relevant fracture modes were obtained on 10 cadaveric femurs. Failure load should be reported as a fraction of donor's body-weight to reduce variability. The proposed method can be used to investigate the reason and mechanism of failure of natural and operated proximal femurs.     

Cellular and subcellular distribution of galectin-3 in the epiphyseal cartilage and bone of fetal and neonatal mice.

Galectin-3 is a 30 kDa beta-galactoside binding protein that belongs to the galectin family of animal lectins. By immunocytochemistry we show the presence of galectin-3 protein in the differentiated chondrocytes of the epiphyseal plate cartilage of long bones of both fetal and neonatal mice. The highest concentrations of galectin-3 are found in the cytoplasm of mature and early hypertrophic chondrocytes. Very little protein is detected in the late hypertrophic chondrocytes undergoing terminal maturation and cell death. Galectin-3 has also been found in osteoblasts and osteocytes of the woven bone of the metaphysis and the cortical bone of the diaphysis, as well as in osteoclasts and mononuclear cells within bone marrow cavities. Galectin-3 is never detected extracellularly, the protein seems restricted to the cytoplasm of chondrocytes and bone cells, although it is occasionally detected in the nuclei of dense non-hypertrophic chondrocytes in the zone of calcification and in young osteoblasts. The results indicate that galectin-3 is a marker of both chondrogenic and osteogenic cell lineages. They also suggest that galectin-3 could be involved in the process of endochondral bone formation, possibly as a regulator of chondrocyte survival.     

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18- 19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.     

Atypical subtrochanteric femoral shaft fractures: role for mechanics and bone quality

Bisphosphonates are highly effective agents for reducing osteoporotic fractures in women and men, decreasing fracture incidence at the hip and spine up to 50%. In a small subset of patients, however, these agents have recently been associated with ''atypical femoral fractures'' (AFFs) in the subtrochanteric region or the diaphysis. These fractures have several atypical characteristics, including occurrence with minimal trauma; younger age than typical osteoporotic fractures; occurrence at cortical, rather than cancellous sites; early radiographic appearance similar to that of a stress fracture; transverse fracture pattern rather than the familiar spiral or transverse-oblique morphologies; initiation on the lateral cortex; and high risk of fracture on the contralateral side, at the same location as the initial fracture. Fracture is a mechanical phenomenon that occurs when the loads applied to a structure such as a long bone exceed its load-bearing capacity, either due to a single catastrophic overload (traumatic failure) or as a result of accumulated damage and crack propagation at sub-failure loads (fatigue failure). The association of AFFs with no or minimal trauma suggests a fatigue-based mechanism that depends on cortical cross-sectional geometry and tissue material properties. In the case of AFFs, bisphosphonate treatment may alter cortical tissue properties, as these agents are known to alter bone remodeling. This review discusses the use of bisphosphonates, their effects on bone remodeling, mechanics and tissue composition, their significance as an effective therapy for osteoporosis, and why these agents may increase fracture risk in a small population of patients.     

The physical and mechanical effects of suspension-induced osteopenia on mouse long bones.

The present investigation addresses the extent of tail-suspension effects on the long bones of mice. The effects are explored in both sexes, in both forelimb and hindlimb bones, and in both diaphyseal and metaphyseal/epiphyseal bones. Two weeks of suspension provided unloading of the femora and tibiae and an altered loading of the humeri. Whole-bone effects included lower mass (approximately 10%) and length (approximately 4%) in the bones of suspended mice compared to controls. The geometric and material properties of the femora were considered along the entire length of the diaphysis and in the metaphysis/epiphysis portions as a unit. Geometric effects included lower cross-sectional cortical area (16%), cortical thickness (25%) and moment of inertia (21%) in the femora of suspended mice; these differences were observed in both distal and proximal portions of the femur diaphysis. The relative amount of bone comprising the middle 8 mm of the diaphysis was greater (3%) in the control mice than in the suspended mice. Significant mass differences between the group in the metaphysis/epiphysis were not observed. Material effects included lower %ash (approximately 2%) in the femora and tibiae as well as in the humeri of suspended mice compared to controls. With respect to the measured physical and material properties, suspension produced similar bone responses in male and female mice. The effects of suspension are manifested largely through geometric rather than through material changes.     

Features of development of fetal bone organ culture in space flight

Fetal mouse metatarsals cultured for 4 days onboard international space laboratory IML-1 (STS-42) were investigated using light microscopy and electron microscopy combined with X-ray microanalysis. Bones cultured in microgravity were equal in length to both ground and inflight (1 g) controls. Three zones: epiphyseal, proliferative, and hypertrophic chondrocytes were distinguished and measured in metatarsals isolated from 16-day-old fetuses. In bone cultures exposed to microgravity, hypertrophic zone tended to decrease and epiphyseal area was increased compared to controls. Proliferative zone has equal length both in bones cultured under microgravity and in controls. The same tendency was observed in bone cultures from 17-day-old fetuses. Metatarsals cultured in microgravity have less spreading calcification zone of diaphysis in comparison with both controls. The results suggest that maturation of chondrocytes and calcification of cartilage, but not cell proliferation, are microgravity sensitive processes in developing bones isolated from the organism.     

Structural study on the effect of 24,25-dihydroxyvitamin D3 on epiphyseal growth plate in suckling mice

The in vivo effects of 24,25(OH)2D3 on cellular structure and organization, matrix metachromasia and mineralization were studied in epiphyseal growth plate of normal neonatal mice. A relatively low dose of the metabolite, 40 ng/kg body weight, significantly increased the overall size of humeral growth plate and the zone of cellular proliferation. By and large, the tissue's response to the metabolite did not change with the increase in dose administered except for a decrease in the number of chondroblasts. 24,25(OH)2D3 led to significant increases in the metachromatic reaction of the cartilaginous matrix, but appeared to depress the mineralization process. Qualitative structural changes were noted in chondroblasts and hypertrophic chondrocytes. 24,25(OH)2D3 affected the osteoblastic and osteocytic populations of cells in the metaphysis and diaphysis of the humerus. High doses of 24,25(OH)2D3 brought about distinct atrophic changes in the above cells. These findings indicate that excessive doses of 24,25(OH)2D3 in an intact animal may lead to retardative effects upon bone growth.     

Chondrocyte apoptosis enhanced at the growth plate: a physeal response to a diaphyseal fracture

Post-traumatic overgrowth of growing long bones is a common clinical phenomenon in paediatric traumatology and is the result of an enhanced stimulation of the nearby growth plate after fracture. To date, the exact post-fractural reactions of the growth plate are poorly understood. The aim of this study has been to determine the impact of fracture on the frequency of chondrocyte apoptosis of the growth plate. Rats sustained a mid-diaphyseal closed fracture of the left tibia or were left untreated. All animals were killed 3, 10, 14 or 29 days after trauma. The left and right tibiae were harvested and apoptotic chondrocytes of the proximal tibial growth plate were detected by TUNEL staining. The apoptosis percentage of physeal chondrocytes was statistically compared among fractured bones, intact contra-lateral bones and control bones. The physeal apoptosis rate of the fractured bone was significantly higher than that of the contra-lateral intact bone (valid for all evaluated days) and the control bone (valid from day 10 onwards). Contra-lateral intact tibiae never showed significantly higher apoptosis rates compared with control tibiae. Thus, mid-diaphyseal fracture influences the nearby growth plate by stimulating chondrocyte programmed cell death, which is associated with cartilage resorption and bone replacement. The lack of a significant difference between the intact contra-lateral and the intact control bone suggests that fracture only has a local effect that contributes to the greater apoptosis rate of the adjacent physis.     

Effect of age on bone mineral density and micro architecture in the radius and tibia of horses: An Xtreme computed tomographic study

Background

The effect of age on the bone mineral density and microarchitecture of the equine radius and tibia was investigated. Fifty-six bones from 15 horses aged four to 21 years were used. There were nine geldings and six mares, and none of the horses had any disease influencing bone properties. Xtreme computed tomography was used to evaluate a 9-mm segment of the diaphysis and metaphysis of each bone. The following variables were determined: length of the bone, circumference and diameter in the frontal and sagittal planes in the middle of the bone.Diaphysis: total volume, bone volume, bone volume ratio, slice area, bone area, marrow area, cortical and marrow thickness, bone mineral density, polar moment of inertia of the cortex.Metaphysis: total area, bone area, cortical bone area, cortical thickness, bone mineral density, bone mineral density in the cortex, bone mineral density in the trabecular region, trabecular number, trabecular thickness, trabecular separation, polar moment of inertia of the metaphysis, polar moment of inertia of the cortex of the metaphysis.

Results

Bone density and microarchitecture were not affected by breed or gender. However, the microarchitecture varied with the age of the horse; the number of trabeculae decreased significantly and the distance between trabeculae increased significantly with increasing age. There were no significant differences between bones of the left and right limbs or between the radius and tibia.

Conclusion

The variables investigated did not differ between geldings and mares. However, there were age-related changes in the microstructure of the bones. Further experimental studies are necessary to determine whether these changes reduce bone strength. Age-related changes in the bones were seen and may explain the higher incidence of fractures and fissures in older horses.

     

Comparative effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats.

The purpose of the present study was to compare the effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats. Twenty-six female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into four groups: the sham-operated control (Sham) group and the three ovariectomy (OVX) groups, namely, OVX + vehicle, OVX + alendronate (2.5 mg/kg, p.o., daily), and OVX + alfacalcidol (0.5 mug/kg, p.o., daily). At the end of the 8-week experimental period, bone histomorphometric analyses of cancellous bone at the proximal tibial metaphysis and cortical bone at the tibial diaphysis were performed, and the mechanical properties of the femoral distal metaphysis and femoral diaphysis were evaluated. OVX decreased cancellous bone volume per total tissue volume (BV/TV), and the maximum load of the femoral distal metaphysis, as a result of increases in serum osteocalcin (OC) levels, and also the number of osteoclasts (N.Oc), osteoclast surface (OcS) and bone formation rate (BFR) per bone surface (BS), and BFR/BV, without any effect on cortical area (Ct Ar), or maximum load of the femoral diaphysis. Alendronate prevented this decrease in cancellous BV/TV by suppressing increases in N.Oc/BS, OcS/BS, BFR/BS, and BFR/BV, without any apparent effect on Ct Ar, or maximum load of the femoral distal metaphysis and femoral diaphysis. On the other hand, alfacalcidol increased cancellous BV/TV, Ct Ar, and the maximum load of the femoral distal metaphysis and femoral diaphysis, by mildly decreasing trabecular BFR/BV, maintaining trabecular mineral apposition rate and osteoblast surface per BS, increasing periosteal and endocortical BFR/BS, and preventing an increase in endocortical eroded surface per BS. The present study clearly showed the differential skeletal effects of alendronate and alfacalcidol in ovariectomized rats. Alendronate prevented OVX-induced cancellous bone loss by suppressing bone turnover, while alfacalcidol improved cancellous and cortical bone mass and bone strength by suppressing bone resorption and maintaining or even increasing bone formation.     

The spatial and temporal expression of VEGF and its receptors 1 and 2 in post-traumatic bone bridge formation of the growth plate

Injuries to growth plates may initiate the formation of reversible or irreversible bone-bridges, may leading to bone length discrepancy or axis deviation. As vascular invasion is essential for the formation of bone tissue, the aim of our study was to investigate the kinetic expression of Vascular Endothelial Growth Factor (VEGF) and its receptors R1 and R2 and the ingrowth of vessels in the formation of bone bridges in a rat physeal injury model. Quantitative Real-Time Polymerase Chain Reaction was performed for VEGF and its receptors. Samples from the proximal physis of the tibial bone were immunohistochemically evaluated for the expression of VEGF and its R1 and R2 receptors and Laminin. Morphologically, physeal bone bridge formation was validated by means of Magnetic Resonance Imaging. Kinetic expression of VEGF and VEGF-R1 mRNA documented a tendency towards an increase in expression on day 7. Histological analyses showed a hematoma containing bone debris on day 1 which was replaced with bony trabeculae by day 14, forming a bone bridge by day 28 which was preceded and accompanied by angiogenesis and consistent with MRI data. VEGF and VEGF-R2 was expressed on the debris within the hematoma and bone trabeculae from days 1 to 28. VEGF-R1 expression was only noted until day 14. The findings of our study suggest that physeal bone bridge formation is in part triggered by VEGF expression and associated with angiogenesis, which was shown to precede bone bridge formation and may be further stimulated through VEGF-positive bone debris.     

Effect of ipriflavone on glucocorticoid-induced osteoporosis in rats

Ipriflavone, 7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one, was administered orally for 12 weeks to male rats with prednisolone-induced osteoporosis. Microdensitometric analysis of a roentgenograph of the femurs revealed that ipriflavone increased the density of the distal metaphysis dose-dependently and tended to increase the density of the diaphysis. It also inhibited dose-dependently the decrease in the mechanical strength of the tibia, breaking strain and breaking energy, and the fractional content of ash in femurs. These results indicate that ipriflavone markedly suppresses bone resorption at the metaphysis where the content of trabecular bone with a rapid turnover rate is high, and possibly inhibits bone reduction at the diaphysis.     

Biodegradable Implants in the Fixation of Physeal Fractures in Cats and Dogs

In a preclinical and a clinical study physeal fractures of cats and dogs were fixated with biodegradable implants. The preclinical part consisted of 4 cats with experimental physeal fractures of the distal femurs and the clinical part of 6 cats and 8 dogs with different physeal fractures. All fractures were fixated with selfreinforced polyglycolic acid (PGA) implants of different sizes. No external support was applied after the fixation. All cats and dogs used their operated legs during the first postoperative week and they could walk without lameness in 6 weeks. The fracture healed without delay or malformations. The retardations of the growth of the physeal regions were considered minimal.     

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers

The non-mineral component of bone matrix consists of 90% collagenous, 10% non-collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass-spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty-one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states.