Cortical Bone Deposition Research Articles

Ontogenetic changes in cortical bone vascular microstructure in the domestic duck (Anas platyrhynchos) and ring-necked pheasant (Phasianus colchicus).

Age-related changes in bone microstructure can inform our understanding the biology of both extant and fossil birds, but to date, histological work in birds, and particularly work using high-resolution 3D imaging, has largely been restricted to limited growth stages. We used minimally destructive synchrotron radiation-based X-ray computed tomography to visualise and measure key morphological and histological traits in 3D across development in the domestic duck and ring-necked pheasant. We use these measurements to build on the database of key reference material for interpreting bone histology. We found that growth patterns differed between the two species, with the ducks showing rapid growth in their lower limbs and early lower limb maturation, while pheasants grew more slowly, reflecting their later age at maturity. In the pheasant, both walking and flight occur early and their upper and lower limbs grew at similar rates. In the duck, flight and wing development are delayed until the bird is almost at full body mass. Through juvenile development, the second moment of area for the duck wing was low but increased rapidly towards the age of flight, at which point it became significantly greater than that of the lower limb, or the pheasant. On a microstructural level, both cortical porosity and canal diameter were related to cortical bone deposition rate. In terms of orientation, vascular canals in the bone cortex were more laminar in the humerus and femur compared with the tibiotarsus, and laminarity increased through juvenile development in the humerus, but not the tibiotarsus, possibly reflecting torsional vs compressive loading. These age-related changes in cortical bone vascular microstructure of the domestic duck and pheasant will help understanding the biology of both extant and fossil birds, including age estimation, growth rate and growth patterns, and limb function.

Mesenchymal stem cell seeded, biomimetic 3D printed scaffolds induce complete bridging of femoral critical sized defects.

No current clinical treatments provide an ideal long-term solution for repair of long bone segment defects. Incomplete healing prevents patients from returning to preinjury activity and ultimately requires additional surgery to induce healing. Obtaining autologous graft material is costly, incurs morbidity, requires surgical time, and quality material is finite. In this pilot study, 3D printed biomimetic scaffolds were used to facilitate rapid bone bridging in critical sized defects in a sheep model. An inverse trabecular pattern based on micro-CT scans of sheep trabecular bone was printed in polybutylene terephthalate. Scaffolds were coated with micron-sized tricalcium phosphate particles to induce osteoconductivity. Mesenchymal stem cells (MSCs) were isolated from sheep inguinal and tail fat, in one group of sheep and scaffolds were infiltrated with MSCs in a bioreactor. Controls did not undergo surgery for cell extraction. Scaffolds were implanted into two experimental and two control adult sheep, and followed for either 3 or 6 months. Monthly radiographs and post explant micro-CT scanning demonstrated bone formation on the lateral, anterior, medial, and posterior-medial aspects along the entire length of the defect. Bone formation was absent on the posterior-lateral aspect where a muscle is generally attached to the bone. The 3-month time point showed 15.5% more cortical bone deposition around the scaffold circumference while the 6-month time point showed 40.9% more bone deposition within scaffold pores. Control sheep failed to unite. Serum collagen type-1C-terminus telopeptides (CTX-1) showed time-dependent levels of bone resorption, and calcein labeling demonstrated an increase in bone formation rate in treated animals compared with controls. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 242-252, 2019.

Measures of Relative Dentary Strength in Rancho La Brea Smilodon fatalis over Time.

The late Pleistocene megafaunal extinction of approximately 12,000 years ago, included the demise of Smilodon fatalis, a hypercarnivore from the Rancho La Brea deposits, which has been studied across time by looking at different deposits or pits to determine morphological size and shape changes and trends during this time. To better understand functional aspects of these changes, this study focused on a measure of jaw strength over time, which can give an indication of morphological changes within the jaw that cannot be seen using surface morphometrics. By radiographing dentaries, cortical bone can be seen, which provides an estimate of resistance to bending forces while biting, and can be measured and used as an indicator of jaw strength. Measurements were taken at repeatable locations on the dentary of the depth of the cortical bone, and of a standardized measure of cortical bone, which allows for the comparison between different individuals. Specimens included those of five different pits ranging from about 37 Kybp to 13 Kybp (just before the extinction of S. fatalis). No significant difference was found in the depth of jaws at any of the measurement points from any of the pits. However, significant differences were found in both the actual thickness of cortical bone, and the standardized thickness of cortical bone at the lower P4 between pit 13 (which had the lowest amount of bone) and pit 61/67 (which had the highest). These conclusions support other studies that have shown that individuals in pit 13 were under physiological and perhaps dietary stress, which may be reflected in the deposition of cortical bone, while the opposite trend is seen in the individuals in pit 61/67. Our results further support findings suggesting Smilodon did not appear to be morphologically most vulnerable right before its extinction.

Non-alcoholic fatty liver, adipose tissue, and the bone: a new triumvirate on the block

In the latest years, a growing body of evidence has connected metabolic syndrome and obesity to bone loss and osteoporosis [1], thereby challenging the conventional view that excessive body weight protects against osteoporosis through increased mechanical load to the skeleton and enhanced cortical bone deposition [2]. In a parallel way, available data suggest that excess body weight may be detrimental to the skeleton, and that excessive adipose tissue plays a key role in this relationship [3]. Within this frame of research, recent studies focused on the relationship between bone health and non-alcoholic fatty liver (NAFLD), the hepatic manifestation of the metabolic syndrome, which recognizes in altered adipose tissue adipokine secretion a key pathogenic factor [4]. The two published studies on endocrine add importantly to the existing literature on the association between NAFLD and osteoporosis. First, ultrasonographic NAFLD was linked to low bone mass in a large cohort of Korean postmenopausal women, independently of age, BMI, smoking status, alcohol intake, and metabolic syndrome [5]. Second, the effect of a multidisciplinary weight-losing regimen on NAFLD, bone mineral density (BMD), and adipokine profile was assessed in post-puberal obese adolescents: weight loss improved NAFLD, BMD, insulin resistance, and plasma lipids. Importantly, bone mineral content changes were related to a reduction in insulin resistance and plasma leptin levels [6]. Consistent with these data, a recent crosssectional study, connected the histological severity of NAFLD to the degree of BMD changes in obese children [7]. Together with existing literature, these data strengthen the view that adipose tissue, the bone, and the liver are functionally interrelated organs and cooperate in regulating bone remodeling, glucose homeostasis, lipid and energy metabolism, through hormonal mediators that are only lately being unraveled (Table 1). As an example, insulin activates the osteoblast insulin receptor to increase bone formation and resorption and release of undercarboxylated osteocalcin into the systemic circulation, and adipose tissue-derived adipokines leptin and adiponectin can alter bone remodeling by either stimulating resorption or suppressing bone formation. Reciprocally, the bone may be not only a target of visceral adipose tissue-released mediators, but also a metabolically active regulator of energy balance through the secretion of hormone and peptide signals, including undercarboxylated osteocalcin, a bone-specific secreted protein that enhances pancreatic b-cell viability and function, increases insulin and adiponectin secretion, and reduces fat mass and lipolysis [3, 8, 9] (Table 1). Perhaps the best evidence of the complex relationship between bone health and energy metabolism is provided by two lines of data: the first is represented by the experiments of Fulzele et al. [10], where mice harboring selective osteoblast deletion of the insulin receptor developed insulin resistance, obesity, fatty liver, and osteopenia, alterations that were all reversed by the administration of osteocalcin. The second line of evidence is exemplified by the thiazolidinedione (TZD) paradigm. TZDs are peroxisome proliferator-activated receptor(PPAR)-c agonists that effectively ameliorated liver histology, glycemic control, and insulin resistance in NASH and diabetes [11], but are associated with bone loss and increased risk of fractures, particularly in diabetic females [12, 13]. In vitro studies demonstrated that PPAR-c agonists activators reallocate the fate of bone marrow G. Musso (&) Gradenigo Hospital, C.so Regina Margherita 8, 10132 Turin, Italy e-mail: giovanni_musso@yahoo.it

Chronic exercise does not alter limb bone morphology or microstructure in the American alligator

In contrast to mammals and birds, effects of exercise on bone microstructure in reptiles have received scant attention. We investigated the effects of long‐term exercise on a treadmill or in a flume on limb bones of the American alligator. Juvenile alligators were run or swum to exhaustion every other day for 17 months, and received fluorescent dye injections to determine bone deposition rates. We found no significant differences in whole bone morphology and cortical bone deposition rates in the alligator humeral or femoral midshaft, regardless of exercise regimen. Similarly, we found no effects of either exercise regimen on trabecular number, thickness or density in the distal femur. In addition, bone mineral content was similar across exercise groups. Altogether, this suggests that long‐term exercise has no discernible effect on bone microstructure in alligators. These results stand in contrast to studies on endothermic vertebrates. The disparity could be due to metabolic differences between ectothermic and endothermic vertebrates. An alternative explanation is that alligator limb bones are subjected to insufficient strain levels or load cycles during short exercise bouts, which may account for lack of bone remodelling in response to exercise in alligators. Funded by the NSF IOB 04445680 to JWH.

Betacellulin inhibits osteogenic differentiation and stimulates proliferation through HIF-1α

Cellular signaling via epidermal growth factor (EGF) and EGF-like ligands can determine cell fate and behavior. Osteoblasts, which are responsible for forming and mineralizing osteoid, express EGF receptors and alter rates of proliferation and differentiation in response to EGF receptor activation. Transgenic mice over-expressing the EGF-like ligand betacellulin (BTC) exhibit increased cortical bone deposition; however, because the transgene is ubiquitously expressed in these mice, the identity of cells affected by BTC and responsible for increased cortical bone thickness remains unknown. We have therefore examined the influence of BTC upon mesenchymal stem cell (MSC) and pre-osteoblast differentiation and proliferation. BTC decreases the expression of osteogenic markers in both MSCs and pre-osteoblasts; interestingly, increases in proliferation require hypoxia-inducible factor-alpha (HIF-α), as an HIF antagonist prevents BTC-driven proliferation. Both MSCs and pre-osteoblasts express EGF receptors ErbB1, ErbB2, and ErbB3, with no change in expression under osteogenic differentiation. These are the first data that demonstrate an influence of BTC upon MSCs and the first to implicate HIF-α in BTC-mediated proliferation.

Contrasting Skeletal Phenotypes in Mice with an Identical Mutation Targeted to Thyroid Hormone Receptor α1 or β

Thyroid hormone (T(3)) regulates bone turnover and mineralization in adults and is essential for skeletal development. Surprisingly, we identified a phenotype of skeletal thyrotoxicosis in T(3) receptor beta(PV) (TRbeta(PV)) mice in which a targeted frameshift mutation in TRbeta results in resistance to thyroid hormone. To characterize mechanisms underlying thyroid hormone action in bone, we analyzed skeletal development in TRalpha1(PV) mice in which the same PV mutation was targeted to TRalpha1. In contrast to TRbeta(PV) mice, TRalpha1(PV) mutants exhibited skeletal hypothyroidism with delayed endochondral and intramembranous ossification, severe postnatal growth retardation, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed closure of the skull sutures. Skeletal hypothyroidism in TRalpha1(PV) mutants was accompanied by impaired GH receptor and IGF-I receptor expression and signaling in the growth plate, whereas GH receptor and IGF-I receptor expression and signaling were increased in TRbeta(PV) mice. These data indicate that GH receptor and IGF-I receptor are physiological targets for T(3) action in bone in vivo. The divergent phenotypes observed in TRalpha1(PV) and TRbeta(PV) mice arise because the pituitary gland is a TRbeta-responsive tissue, whereas bone is TRalpha responsive. These studies provide a new understanding of the complex relationship between central and peripheral thyroid status.

Changes in bone mineral density in survivors of childhood acute lymphoblastic leukemia

There is little information about factors modulating bone mineral density (BMD) in survivors of childhood acute lymphoblastic leukemia (ALL). We analyzed data from 57 survivors (26 male, 52 Caucasian) who underwent two serial quantitative computed tomography (QCT) studies of BMD. Using multiple linear regression, we evaluated the association of BMD change with demographic variables, treatment history, hormone therapy, exercise, and tobacco and alcohol use. The median age was 3.4 years (range, 0.9-17.4 years) at diagnosis of ALL; the median age at the first QCT (Study I) was 15.0 years (range, 10.6-31.0 years) and at the second QCT (Study II) was 18.2 years (range, 14.2-35.3 years). Mean height increased 4.7 cm and mean weight increased 8.8 kg between Studies I and II. While the mean BMD increased 9.33 mg/cc (P = 0.003), the BMD Z-score increased only slightly (0.21 SD, P = 0.035). Cortical bone density increased significantly (approximately 25.3 mg/cc; P = 0.001), but the ratio of trabecular to cortical BMD decreased significantly (P = 0.045). Factors independently associated with unfavorable BMD changes included older age at diagnosis (P = 0.001), female sex (P = 0.018), and nutritional supplementation (0.032). Alcohol (P = 0.009) was an unfavorable factor in a univariable analysis. Bone mineral accretion during adolescence is attenuated in childhood ALL survivors by a comparative deficit in trabecular versus cortical bone deposition. BMD is influenced favorably by exercise in early adolescence and unfavorably by the use of nutritional supplements and alcohol. These results provide new information about behavioral factors that affect bone accrual in survivors of childhood ALL and warrant definitive evaluation in a larger cohort.

Femoral morphology and cross-sectional geometry of adult myostatin-deficient mice

GDF-8, also known as myostatin, is a member of the transforming growth factor-β (TGF-β) superfamily of secreted growth and differentiation factors that is expressed in vertebrate skeletal muscle. Myostatin functions as a negative regulator of skeletal muscle growth and myostatin null mice show a doubling of muscle mass compared with normal mice. We examined femoral morphology of adult myostatin-deficient mice to assess the effects of muscle fiber hypertrophy and hyperplasia on bone shape and cross-sectional geometry. Femora of age- and weight-matched adult mice homozygous for the disrupted myostatin sequence were compared with those of wild-type controls (n = 8 per group). Results show that, as was the case in previous studies, myostatin null mice have hindlimb muscle masses that are approximately double those of controls. Myostatin-deficient mice exhibit third trochanters that are significantly larger than those of controls, whereas the femoral midshafts of the control and experimental mice do not differ significantly from one another in cortical area, bending moment of inertia, and polar moment of inertia. Our findings indicate that the increased muscle mass of myostatin-deficient mice primarily affects sites of muscle insertion, but does not induce additional cortical bone deposition in the diaphysis relative to controls. We therefore conclude that the expanded third trochanters of myostatin-deficient subjects result from tendon and Sharpey fiber expansion associated with muscle growth rather than cortical bone deposition in response to increased levels of mechanical stress.

Human growth hormone transgene expression increases the biomechanical structural properties of mouse vertebrae.

Caudal vertebrae were obtained from male and female mice from two transgenic lines expressing an erythroid-specific human growth hormone transgene construct, and gender-matched, age-matched, non-transgenic control mice. To characterize the effect of human growth hormone transgene expression on the biomechanical structural properties of caudal vertebrae in compression. An increase in trabecular and cortical bone deposition caused by erythroid-specific human growth hormone transgene expression was demonstrated previously. Compression tests were performed on individual caudal vertebrae (Ca4, Ca5, Ca6) obtained from male and female mice from two transgenic lines (TG420 and TG450) and nontransgenic control mice. Two age groups were evaluated: 12 weeks old and 6 months old. The data were used to obtain axial stiffness, maximum load, and energy to failure. Vertebrae from male TG420 transgenic mice produced significantly larger values for maximum load, energy to failure, and axial stiffness at both 12 weeks and 6 months in comparison with their age-matched non-transgenic male controls. Vertebrae from female TG420 transgenic mice produced similar responses at 6 months. Vertebrae from male TG450 transgenic mice showed significant increases in maximum load and energy to failure at 6 months. In general, the biomechanical properties of vertebrae were significantly larger in the 6-month age group than in the 12-week age group, and this increase was significantly greater in the transgenic mice than in the gender-matched control mice during the same time span. This process was also influenced by transgenic genotype and gender. Erythroid-specific production of human growth hormone in transgenic mice resulted in significant increases in biomechanical properties of their caudal vertebrae in compression. The changes in the biomechanical properties were influenced by genotype, age, and gender.

Patterns of growth of the mandibular corpus in spotted hyenas (Crocuta crocuta) and cougars (Puma concolor)

Abstract Differences in jaw morphology among adult carnivorans are well established, but the ontogenetic mechanisms by which these differences arise are largely unexplored. Mandibular ontogeny in Crocuta crocuta and Puma concolor is analysed biomechanically using principles of beam theory. In each species, the development of cross-sectional properties of the mandibular corpus associated with rigidity under loading follows a biphasic pattern of growth. In early postnatal growth, deposition of cortical bone appears to be constrained by the overall weaker tissue with which juvenile skeletons are constructed and by the need to volumetrically accommodate the developing teeth within their bony crypts. Thus, this stage of growth is characterized by a net periosteal deposition of bone and a swelling of the medullary cavity. In late postnatal growth, the constraints on endosteal deposition of bone are relieved as the permanent teeth erupt; thus, cortical thicknesses increase sharply by periosteal expansion as well as medullary contraction. Finally, it is noted that basic differences in jaw construction between Crocuta and Puma appear to develop prenatally as they are largely in place at birth. Hence, postnatal development enhances, but does not soley contribute to, the biomechanical differences in the jaws of these species.

Patterns of growth of the mandibular corpus in spotted hyenas (Crocuta crocuta) and cougars (Puma concolor)

Differences in jaw morphology among adult carnivorans are well established, but the ontogenetic mechanisms by which these differences arise are largely unexplored. Mandibular ontogeny inCrocuta crocutaandPuma concoloris analysed biomechanically using principles of beam theory. In each species, the development of cross-sectional properties of the mandibular corpus associated with rigidity under loading follows a biphasic pattern of growth. In early postnatal growth, deposition of cortical bone appears to be constrained by the overall weaker tissue with which juvenile skeletons are constructed and by the need to volumetrically accommodate the developing teeth within their bony crypts. Thus, this stage of growth is characterized by a net periosteal deposition of bone and a swelling of the medullary cavity. In late postnatal growth, the constraints on endosteal deposition of bone are relieved as the permanent teeth erupt; thus, cortical thicknesses increase sharply by periosteal expansion as well as medullary contraction. Finally, it is noted that basic differences in jaw construction betweenCrocutaandPumaappear to develop prenatally as they are largely in place at birth. Hence, postnatal development enhances, but does not soley contribute to, the biomechanical differences in the jaws of these species.

The influence of growth hormone on cancellous and cortical bone of the vertebral body in aged rats.

The influence of growth hormone administration on cancellous and cortical bone of the vertebral body in 2-year-old male rats has been investigated. All rats were injected for 80 days, then killed. Controls were given saline, and three recombinant human growth hormone (rhGH) injected groups were given either rhGH (2.7 mg/kg/day) for the first 20 or 40 days, followed by saline injection, or rhGH for all 80 days. Tetracycline labeling was performed on days 41 and 69. In all groups given rhGH, an increase in the cortical bone volume was found. In the rhGH 40-day group, single labeling corresponding to injection on day 41 was seen all around the anterior surface of the vertebral body wall (toward the abdominal cavity). In the rhGH 80-day group, double labeling was seen all around the anterior surface of the vertebral body, and a substantial increase in the mineralizing surface/total surface, mineral apposition rate, and mineralized bone formation rate was found. In the cortical bone of the anterior wall, cavities had developed in the rhGH 40- and 80-day groups. In the cancellous bone, no differences in bone volume, bone volume/total volume, or bone surface/bone volume were seen, but in the middle part of the vertebral body a decrease in the mineralizing surface/total surface was found in the rhGH 80-day group. The height of the vertebral body was not influenced by rhGH administration, whereas the transversal and midsaggital diameters were increased in the rhGH 80-day group. The compressive mechanical strength of the vertebral body specimens was increased in the rhGH 80-day group, and this increase most likely could be explained by formation and deposition of cortical bone.

Qualitative and morphometric radiographic findings in the distal phalanx and digital soft tissues of sound Thoroughbred racehorses

SUMMARY Lameness examinations and radiography of the distal phalanx and associated soft-tissue structures of the front feet of 103 Thoroughbred racehorses, 4 to 9 years old, were performed to determine normal radiographic appearance and morphometry. Of 103 horses examined, 41 were used in the study that were without clinical signs of foot problems or lameness, had raced at least twice prior to radiography, and had raced at least twice more in the 6 months after radiography. Lateromedial and dorsoproximal-palmarodistal radiographic views of each front distal phalanx were used to measure 28 bone and soft-tissue structures, and to evaluate 14 radiographic findings. Significant differences were not observed between left and right digits for any radiographic determination. Mean thickness of the soft tissues dorsal to the distal phalanx, which provides an evaluation of the epidermal laminae, was 14.6 ± 1.0 mm when measured adjacent to the distal aspect of the distal phalanx. Most horses had straight, smooth hoof walls that were parallel to the dorsal cortex of the distal phalanx. The mean degree of palmar rotation of the distal phalanx was −0.5 ± 1.3, and none was rotated more than 4°. The dorsal cortex was smooth and straight, without bone deposition or reaction in either digit for only 5 of the 41 horses. Active bone formation was seen unilaterally along the middle portion of the dorsal cortex in 7 horses, and along the distal portion of the dorsal cortex in 4 of the phalanges from 3 horses. New bone formation along the distal dorsal cortex was often accompanied by resorption of the palmar cortex. For 26 of the 31 horses without active bone deposition, smooth inactive bone formation along the midportion of the dorsal cortex was identified in 1 or both distal phalanges. Bone at the solar margin of the distal phalanx was uniformly dense and finely trabeculated, without evidence of resorption or fractures. Severe irregularity of the solar margin was not found in any digit, and the margin of both phalanges was smooth in 8 horses. Various degrees of solar margin irregularity were observed in the other 33 horses. The mean number of vascular canals within the distal phalanx was 8.4 ± 1.7, and the diameter of the largest canal was 3.4 ± 0.6 mm. A mean number of 2.0 ± 1.2 vascular canals was oriented parallel to the radiographic beam on the dorsoproximal-palmarodistal view, and these were termed end-on vessels, because they were visualized as radiolucent dots ≥ 1 mm in diameter in the central portion of the distal phalanx. Racing performance of horses with subtle radiographic signs of laminitis (palmar rotation, hoof wall curvature or undulations, palmar cortical resorption, distal dorsal cortical bone deposition) was poorer than that of horses without these signs. These findings are suggestive of a subclinical laminitis condition, which may influence performance without causing overt clinical signs.

Effect of antithyroid treatment on calcium-phosphorus metabolism in hyperthyroidism. II: Bone histomorphometry.

Histomorphometric analysis of iliac crest biopsies was performed after tetracycline double-labelling in 22 hyperthyroid patients before and after medical antithyroid treatment for an average period of 4 months. The initially increased cortical porosity was normalized during treatment whereas the amount of trabecular bone was unchanged. The osteoclastic resorption in cortical bone decreased but was still elevated. The osteocytic osteolysis remained slightly increased. In trabecular bone, however, the bone turn-over decreased to a subnormal level following treatment and the surfaces were inactive in bone resorption and bone formation. An increase was observed in the amount, extent and width of osteoid seams due to an increase in the lifespan of bone forming sites and a prolongation of the maturity period of osteoid. The observed increased deposition of cortical bone after antithyroid treatment may explain the positive calcium balance in this period.