The effect of muscle dysfunction on bone mass and morphology.

There is little doubt that skeletal development and subsequent maintenance of bone mass and morphology during adulthood is greatly influenced by viable muscle function. In this review, we will summarize human observations that support this concept, then focus on models that have enabled (or may enable in the future) insight into the co-dependency of muscle and bone. Specifically, we will summarize data generated with three types of models: 1) spinal cord injury models, 2) transgenic mice with altered muscle function, and 3) experimental models affecting one hindlimb or a single muscle group. In sum, these data clearly support the concept that muscle function is critical for the successful development of the skeleton and is likely to play an important role in mediating bone health through life. The specific signaling pathways by which this interdependency is achieved, however, remain to be clarified.

Strontium ranelate improves bone strength in ovariectomized rat by positively influencing bone resistance determinants.

SUMMARY: Treatment of adult ovariectomized (OVX) rats with strontium ranelate prevented vertebral biomechanics degradation as a result of the prevention of bone loss and micro-architecture deterioration associated to an effect on intrinsic bone material quality. Strontium ranelate influenced the determinants of bone strength by prevention of ovariectomy-induced changes which contribute to explain strontium ranelate antifracture efficacy. INTRODUCTION: Strontium ranelate effects on the determinants of bone strength in OVX rats were evaluated. METHODS: Adult female Sprague-Dawley rats were OVX, then treated daily for 52 weeks with 125, 250, or 625 mg strontium ranelate/kg. Bone strength, mass, micro-architecture, turnover, and intrinsic quality were assessed. RESULTS: Strontium ranelate prevented ovariectomy-induced deterioration in mechanical properties with energy necessary for fracture completely maintained vs. SHAM at 625 mg/kg/day, which corresponds to the clinical dose. This was related to a dose-dependent effect on bone volume, higher trabeculae number, and lower trabecular separation in strontium ranelate vs. OVX. Load and energy required to induce lamella deformation were higher with strontium ranelate than in OVX and in SHAM, indicating that the bone formed with strontium ranelate is able to withstand greater damage before fracture. Bone formation was maintained high or even increased in strontium ranelate as shown by mineralizing surfaces and alkaline phosphatase while strontium ranelate led to reductions in deoxypyridinoline. CONCLUSION: Strontium ranelate administered at 625 mg/kg/day for 52 weeks prevented OVX-induced biomechanical properties deterioration by influencing the determinants of bone strength: it prevented bone loss and micro-architecture degradation in association with an effect on intrinsic bone quality. These beneficial effects on bone contribute to explain strontium ranelate antifracture efficacy.

Estrogen modulates estrogen receptor alpha and beta expression, osteogenic activity, and apoptosis in mesenchymal stem cells (MSCs) of osteoporotic mice.

In the mouse, ovariectomy (OVX) leads to significant reductions in cancellous bone volume while estrogen (17beta-estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2-dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) alpha and beta expression of primary culture MSCs isolated from OVX and sham-operated mice. MSCs, treated in vitro with 10(-7) M E2, displayed a significant increase in ERalpha mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERbeta mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up-regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF-beta1, BMP-2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERalpha was the predominant form expressed in MSCs obtained from both OVX and sham-operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERalpha expression, osteogenic gene expression and, inhibits apoptosis and ERbeta expression in MSCs obtained from OVX and sham-operated mice. Co-expression of ERalpha, but not ERbeta, and osteogenic differentiation markers might indicate that ERalpha function as an activator and ERbeta function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERalpha activation. J. Cell. Biochem. Suppl. 36: 144-155, 2001.

Increased marrow-derived osteoprogenitor cells and endosteal bone formation in mice lacking thrombospondin 2.

The phenotype of thrombospondin 2 (TSP2)-null mice includes abnormalities in collagen fibrils and increases in ligamentous laxity, vascular density, and bleeding time. In this study, analyses by computerized tomography (CT) revealed that cortical density was increased in long bones of TSP2-null mice. Histomorphometric analysis showed that the mid-diaphyseal endosteal bone formation rate (BFR) of TSP2-null mice was increased in comparison with that of wild-type (WT) animals. Although microgeometric analysis showed that periosteal and endosteal radii were reduced, the mechanical properties of femurs from TSP2-null mice were not significantly different from those of controls, presumably because of the concomitant increase in endosteal bone mass. Bone loss in ovariectomized mice was equivalent for WT and mutant mice, a finding that indicates that TSP2-null animals are capable of normal bone resorption. To further explore the cellular basis for the increased endosteal BFR in TSP2-null mice, marrow stromal cells (MSCs) were isolated and examined in vitro. These cells were found to be present in increased numbers in a colony forming unit (CFU) assay and showed an increased rate of proliferation in vitro. We conclude that TSP2 regulates the proliferation of osteoblast progenitors, directly or indirectly, and that in its absence endosteal bone formation is increased.

Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis.

Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2-/- mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration.

Dietary lipids modulate bone prostaglandin E2 production, insulin-like growth factor-I concentration and formation rate in chicks.

This study examined the effects of dietary fat on the fatty acid composition of liver and bone, and on the concentration of insulin-like growth factor-I (IGF-I) in liver and bone, as well as the relationship of these factors to bone metabolism. Day-old male broiler chicks were given a semipurified diet containing one of four lipid sources: soybean oil (SBO), butter+corn oil (BC), margarine+corn oil (MAC), or menhaden oil+corn oil (MEC) at 70 g/kg of the diet. At 21 and 42 d of age, chicks fed MEC had the highest concentration of (n-3) fatty acids [20:5(n-3), 22:5(n-3) and 22:6(n-3)] in polar and neutral lipids of cortical bone but the lowest amount of 20:4(n-6) in polar lipids. Diets containing t-18:1 fatty acids (MAC and BC) resulted in t18:1 accumulation in bone and liver. Bone IGF-I concentration increased from 21 to 42 d in chicks given the SBO and BC diets. Tibial periosteal bone formation rate (BFR) was higher in chicks given BC compared with those consuming SBO and MEC at 21 d. The higher BFR and concentrations of hexosamine in serum and IGF-I in cartilage, but lower 20:4(n-6) content in bone polar lipids in chicks given BC compared with those given SBO suggest that BC optimized bone formation by altering the production of bone growth factors. A second study confirmed that dietary butter fat lowered ex vivo prostaglandin E2 production and increased trabecular BFR in chick tibia. These studies showed that dietary fat altered BFR perhaps by controlling the production of local regulatory factors in bone.

Effect of controlled release of platelet-derived growth factor from a porous hydroxyapatite implant on bone ingrowth.

Platelet-derived growth factor (PDGF) is one of several osteogenic factors which affect bone growth and fracture healing. This study examined the potential of hydroxyapatite (HA) rods with interconnected pores of mean diameter 200 microns to be used as a matrix for the release of PDGF to enhance bone ingrowth into the implant. In the initial phase of the study the sustained release of PDGF from the HA rods was characterized in vitro for two different PDGF loadings, 10 and 100 micrograms per implant. The second phase of the study examined bone ingrowth in HA implants placed into the medullary canals of rabbit femora. The specimens were dumb-bell shaped, with a reduced central diameter so that bone growth across a gap could also be determined. Bone ingrowth into HA implants was compared with growth into HA implants loaded with 100 micrograms of PDGF. Pushout measurements were made of average shear strength across the bone-implant interface and backscatter scanning electron microscopy of thick sections was used to quantify the amount of bone ingrowth into the implant. Although greater interfacial shear strength and area of ingrowth were observed, especially across gap sites, in specimens loaded with PDGF, no difference was statistically significant.

Morphologic stages in lamellar bone formation stimulated by a potent mechanical stimulus.

The temporal stages of lamellar bone formation were studied using an animal model subject to up to 16 weeks of a controlled, externally applied load. The left ulnae of 15 adult male turkeys were functionally isolated via transverse metaphyseal osteotomies, while transcutaneous Steinmann pins permitted in vivo loading of the preparation via a servo-hydraulic actuator. For 5 days per week, the ulnae were exposed to 100 cycles per day of an applied load sufficient to cause a peak strain normal to the bone's longitudinal axis of 2000 microstrain (mu E). The contralateral limb was left surgically undisturbed and served as a baseline control. Following a loading period of 4, 8 or 16 weeks, ulnae were harvested and prepared for quantitative bone histomorphometry. Compared with each animal's contralateral ulna, the area of the experimental ulnae increased by 12.5% (+/- 5.6%) at 16 weeks. Periosteal mineral apposition rates in the loaded ulnae were significantly increased compared with control values, with a maximum rate of 6.0 +/- 3.4 microns/day at 5 weeks, slowing to 2.0 +/- 0.3 microns/day by 15 weeks. At 16 weeks, new bone was composed of primary and secondary osteons as well as circumferential lamellae, with osteocyte density and organization indistinguishable from that of the original cortex. Remnants of the initial woven bone response seen at 4 weeks remained clearly visible at both 8 and 16 weeks as diffusely labeled interstitial elements within the newly formed lamellar construct.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramembranous bone matrix is osteoinductive.

All known bone-derived osteoinductive factors have been isolated from endochondral (EC) bones and all initiate bone induction via EC ossification. However, to date no attempt has been made to isolate comparable factors from bones which form initially and completely via intramembranous (IM) ossification. The purpose of this work was to isolate osteoinductive proteins from IM bones. To accomplish this, we extracted proteins from bovine frontal bone matrix (intramembranous origin) using methods previously described for endochondral (EC) bone matrix (i.e., femur). Bone powder (< 1 mm) was decalcified and proteins extracted with 4 M guanidine hydrochloride. Ultrafiltration was used to isolate and concentrate a 10-100 kilodalton (kDa) fraction, upon which heparin-Sepharose (HS) affinity chromatography was performed. HS-binding (HS-B) and non-binding proteins (HS-NB) were lyophilized with bovine type I collagen (Vitrogen) to form pellets which were implanted subcutaneously in rats. Radiology as well as brightfield, fluorescent, and polarizing microscopy were used to assess the formation of ectopic bone at the site of pellet implantation. In this report we demonstrate that a heparin-Sepharose binding, osteoinductive factor can be extracted and partially purified from bovine intramembranous bone matrix. This factor has a different sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern than a comparable osteoinductive/chondroinductive factor isolated from EC bone.

High-dose estrogen inhibits bone resorption and stimulates bone formation in the ovariectomized mouse.

In this study, we have investigated estrogen's capacity to regulate bone formation and resorption in the ovariectomized mouse, evaluating the dose and site dependence of estrogen action on bone modeling and remodeling surfaces. To quantitate bone resorption, the skeletons of fifty 8-week-old Swiss-Webster mice were prelabeled with [3H]tetracycline (3H-T) before initiation of treatment protocols. Ovariectomies (OVX) and sham surgeries were performed 3 days after the final 3H-T injection, and the animals were assigned to treatment groups and injected once per week for 4 weeks with one of the following doses of 17 beta-estradiol (E2): sham/oil vehicle (SV), OVX/oil vehicle, OVX/50 micrograms E2, OVX/250 micrograms E2, and OVX/500 micrograms E2. To assess bone formation, fluorochrome labels were administered 9 and 2 days before sacrifice. At the conclusion of the 4 week protocol, the femora and thoracic vertebrae were removed to quantitate the levels of bone resorption based on the skeletal retention of 3H-T. The tibiae were excised for histomorphometric evaluation of the proximal metaphyses and middiaphyses. Indicative of increased bone resorption, vehicle-treated OVX animals had significantly reduced levels of 3H-T in femora and vertebrae compared to SV mice. This result was consistent with histomorphometric data showing a 49% decrease in cancellous bone area of the proximal tibiae in the OVX/oil-treated group. Treatment of OVX animals with 50 micrograms E2 was sufficient to maintain 3H-T levels in vertebrae at SV values, with higher E2 doeses leading to a dose-dependent increase in the retention of 3H-T at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

High-dose gestagens modulate bone resorption and formation and enhance estrogen-induced endosteal bone formation in the ovariectomized mouse.

To determine if gestagens of two separate classes have differing skeletal actions, we studied the effects of pharmacologic doses of norethisterone acetate (NETA), a 19-nortestosterone, and megestrol acetate (MA), a 17 alpha-hydroxyprogesterone, on bone formation and resorption in intact and in ovariectomized mice. In the same set of experiments, we also attempted to determine if these gestagens can alter the skeletal activity of 17 beta-estradiol (E2). Experimentally, the skeletons of 78 female BALB/c mice were prelabeled with [3H]tetracycline (3H-T). The animals were randomized to 13 groups of 6 mice each 3 days after the final 3H-T injection. Ovariectomies (OVX) were performed on 8 groups and sham operations (SO) on 5 groups. To study the skeletal effects of the gestagens, 4 groups each of the OVX and SO mice were treated with controlled-release pellet implants calculated to deliver 80 or 250 micrograms of NETA or MA per day. To study gestagen interactions with E2, 3 groups of OVX mice were treated with either 40 micrograms/day of E2 or 40 micrograms/day of E2 plus 250 micrograms/day of NETA or MA. One group of OVX and one group of SO animals received placebo pellets. Fluorochrome labels were administered 10 and 11 and 3 and 4 days before sacrifice to allow histomorphometric evaluation of bone formation. At the end of the 60 day protocol, tibiae and thoracic vertebrae were removed and processed for quantitating the levels of bone resorption based on the amounts of 3H-T retained in the bones. The femora were fixed and embedded for comparison of diaphyseal bone histomorphometry, and the humeri and lumbar vertebrae were prepared for bone density determinations. Reflecting an increase in bone resorption, 3H-T levels in tibiae and vertebrae were decreased in placebo-treated OVX animals compared to the placebo-treated SO group (p < 0.01). Treatment of both SO and OVX mice with NETA decreased bone resorption in a dose-dependent manner, but MA had no significant effects on vertebral bone resorption and increased bone resorption in the tibiae (p < 0.01). E2 treatment of OVX mice reduced bone resorption, but there were no significant interactions between the E2 and gestagen treatments on resorptive activity. Based on bone histomorphometry of in vivo fluorochrome labels, both gestagens increased periosteal bone formation rates but had no effect on endosteal bone formation (BFRe). In contrast, E2 treatment of the OVX mice stimulated bone formation at the endosteal surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Local modulation of skeletal growth and bone modeling in poultry.

Skeletal growth and bone modeling in poultry are regulated by complex interactions between the animal's genetic potential and a host of systemic and localized factors (growth factors and cytokines) influencing bone biology. The objective of these interactions is to orchestrate the achievement of bone architecture that balances functionally appropriate morphology with the skeleton's involvement in mineral homeostasis. Within this context, bone modeling in the growing animal represents an adaptive process that is distinct from bone remodeling, which is the term used to describe the resorption and formation of mineralized tissue that maintains skeletal mass and morphology in the adult. As many of the skeletal lesions that afflict poultry are the consequence of abnormalities in bone modeling, not bone remodeling, an appreciation of the differences between these two contrasting processes is a prerequisite for understanding the pathogenesis of skeletal lesions in poultry.

The anabolic effect of estrogen on endosteal bone formation in the mouse is attenuated by ovariohysterectomy: a role for the uterus in the skeletal response to estrogen?

In the mouse, the anabolic effect of estrogen on the uterus and its stimulatory effect on endosteal bone formation are well documented. When these observations are coupled with the recent description of uterine-derived bone cell mitogens, it raises the possibility that uterine hypertrophy in response to estrogen might lead to the production and release of factors that participate in the skeleton's anabolic response to estrogen. To determine if the stimulatory effects of estrogen on endosteal bone formation and uterine tissue in the mouse are related, we have studied this specific skeletal response to ovariectomy (OVX) and ovariohysterectomy (OHTX), and to two levels of 17 beta-estradiol (17 beta-E2). To assess treatment effects, 48 Swiss-webster mice were assigned to six groups: OHTX/oil vehicle, OVX/oil vehicle, OHTX/150 micrograms 17 beta-E2, OHTX/300 micrograms 17 beta-E2, OVX/150 micrograms 17 beta-E2, and OVX/300 micrograms 17 beta-E2. Animals were treated once per week with vehicle or the respective 17 beta-E2 dose. To quantitate bone formation, fluorochrome labels were administered at the beginning and end of the experimental period. At the conclusion of the 5-week study, tibiae were processed undecalcified for embedding in methyl methacrylate plastic. Cross-sectional areal properties and bone formation rates were quantitated from 30 microns mid-diaphyseal sections using a Bioquant Bone Morphometry system. Compared with the vehicle-treated OVX and OHTX mice, 150 micrograms of 17 beta-E2 administered once per week significantly increased cortical bone areas (P less than 0.05) but cortical bone widths and the ratio of cortical bone area to total bone area was increased only in estrogen-treated OVX mice (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of the osteogenic response in the aging skeleton.

The ability of physical stimuli demonstrated as potently osteogenic in the young adult skeleton were evaluated for their capacity to stimulate new bone formation in the aging skeleton. Using the externally loadable, functionally isolated turkey ulna preparation, the ulnae of 1-year-old (n = 5), and 3-year-old (n = 3) turkeys were subjected to 300 cycles per day of a load regimen generating a high but physiologic level of normal strain (3,000 microstrain). Following 8 weeks of loading, areal properties and histomorphometry were performed on both the experimental and intact control ulnae. Bone cross-sectional areas in the 1-year-old animal increased by 30.2% (+/- 7.8%) as compared with the intact contralateral control ulnae, whereas the areal properties of the older skeleton remained essentially unchanged (-3.3 +/- 7.5%). Renewed bone formation in the experimental ulnae of the 1-year-old animals was characterized by the activation of periosteal bone apposition (4.0 +/- 0.4 microns/day). In comparison, periosteal bone formation in the 3-year-old males was activated in only 1 animal, and this at a significantly attenuated level (less than 0.8 micron/day). The histomorphometric evaluation of intracortical bone remodeling revealed no significant differences between the control and experimental ulnae in either age group. However, osteon mean wall thickness and bone formation sigma were significantly increased in the 3-year-old males (P less than 0.05). In conclusion, these data suggest that a physical signal that is clearly osteogenic in the young adult skeleton is hardly acknowledged in older bone tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

17 beta estradiol stimulation of endosteal bone formation in the ovariectomized mouse: an animal model for the evaluation of bone-targeted estrogens.

To evaluate the potential of an animal model as a means to identify bone-targeted estrogens, we have studied the response of the skeleton of ovariectomized mice to prolonged estrogen treatment. Seventy female Swiss-Webster mice were randomly divided into ten groups, with nine groups undergoing bilateral ovariectomy and one group a sham procedure. Mice were injected subcutaneously once per week for nine weeks with one of the following doses of estrogen (17 beta-E2) in oil vehicle: 19.3, 38.5, 75, 150, 300, 500, 1000, or 3000 micrograms. One group of ovariectomized (OVX) mice and the sham operated animals received vehicle injections only. At the end of the nine-week experimental protocol, there were no significant differences in body weights among any treatment groups. However, when compared to control values, spleen weights in vehicle-treated OVX mice and in mice treated with 1000 micrograms or 3000 micrograms of 17 beta-E2 were significantly elevated (p less than .01). Liver weights in the OVX mice treated with 1000 or 3000 micrograms 17 beta-E2 were also increased significantly (p less than .05). Comparisons of uterine weights and cortical bone areas were strongly correlated with 17 beta-E2 dose (r2 = .86 and .94, respectively), with maximal increases observed at estradiol doses in excess of 500 micrograms per week. Furthermore, based on bone histomorphometry of in vivo fluorochrome labels, increases in cortical bone area could be attributed to accelerated rates of endosteal mineral apposition and bone formation. These results indicate that the comparison of the response of endosteal bone and uterine tissue in the OVX mice to chronic estrogen treatment offers the potential to identify estrogen and/or estrogen-like compounds with bone-specific activity.

Metabolic modulation of disuse osteopenia: endocrine-dependent site specificity of bone remodeling.

The remodeling response of bone tissue to disuse in four normal adult male turkeys and four adult males metabolically altered by castration was compared by functionally isolating the left ulna of each animal via transverse epiphyseal osteotomies. The right ulna in each animal was left intact and served as a control. After 8 weeks, the animals were euthanized, the ulnae harvested, and 100 microns undecalcified cross sections of the midshaft microradiographed. Areal properties, osteon mineral apposition rates from in vivo fluorochrome labels, and the number and ratios of bone-forming and bone-resorbing foci were quantitated. Compared to their control ulnae, the magnitude of bone resorbed from the functionally isolated ulnae of normal versus castrated males was not significantly different (-12.8 +/- 3.7 versus -10.7 +/- 3.5%, respectively). However, in the functionally isolated ulnae of normal birds, 94% of the total bone loss resulted from expansion of the corticoendosteal envelope, and 97% of the decrease in cross-sectional areas of the ulnae in the castrated birds was due to intracortical porosity. Furthermore, there was a significant interaction between disuse and castration, increasing the total number of intracortical remodeling events (9.4 +/- 0.9) when compared to disuse alone (4.7 +/- 1.4, p less than 0.01), or to the intact ulnae of castrated (2.1 +/- 0.5) and normal adult males (2.0 +/- 1.1). This work emphasizes that the manner in which the bone tissue responds to local changes in its physical environment is directly dependent on the status of the organism's metabolic milieu.

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.

Functional strains and cortical bone adaptation: epigenetic assurance of skeletal integrity.

The skeleton's ability to withstand the extremes of physical activity is achieved in large part by its capacity to perceive and respond to small changes in its mechanical environment. Strains generated by functional activity would represent an efficient, epigenetic parameter by which the bone cell population could assess the skeleton's structural effectiveness, and subsequently use this information to influence its morphology. However, contrary to our normal interpretation of Wolff's Law, minimizing strain does not appear to be the paramount goal of adaptation, but rather skeletal morphology interacts with functional activity to generate a certain, perhaps cytologically beneficial, type of strain. Three sections are used to address the epigenetic impact on bone morphology: (a) at the level of the organ, the strains generated by functional activity; (b) at the level of the tissue, osteoregulatory parameters of the strain environment; and (c) at the level of the cell, the mechanisms by which physical information is translated to an adaptive response. Only when we understand the mechanisms and objectives of tissue adaptation in the normal skeleton can our perspective and treatment of functionally influenced skeletal pathologies (e.g. osteopenias, fractures) be enhanced.

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.

Eicosanoic fatty acid reduction in the tibiotarsus of biotin-deficient chicks.

Day-old male broiler chicks (Hubbard x Hubbard) were fed a purified diet containing biotin at 0 microgram/kg of diet (biotin-deficient) or 500 micrograms/kg of diet (biotin-adequate). Biotin-deficient (BD) chicks had decreased growth and feed efficiency and greater twisted leg and dermatitis symptoms than biotin-adequate (BA) chicks. Lipids in cortical bone of the tibiotarsi in BD chicks contained higher levels of linoleate, gamma-linolenate, and alpha-linolenate. Prostaglandin precursors, dihomo-gamma-linolenate (20:3 omega 6), arachidonate (20:4 omega 6), and eicosapentaenoate (20:5 omega 3) were all lower in BD chicks compared with BA chicks. Periosteal bone appositional and bone formation rates, and percent new bone formation were reduced in the tibiotarsi of BD chicks. Anatomically there were two different bone modeling patters at the mid-diaphysis. The cortex was thickest laterally in chicks fed the BA diet and thickest medially in chicks fed the BD diet. The quantitative differences in bone growth and the distinct bone modeling patterns, coupled with corresponding decreases in PG precursors, suggest that biotin deficiency may alter bone growth and modeling via a PG-dependent mechanism.