Peak power and body mass as predictors of tibial bone strength in healthy male and female adults.

OBJECTIVE: The purpose of this study was to examine whether a non-invasive, muscular fitness field test was a better predictor of bone strength compared to body mass in healthy adults. METHODS: Hierarchical multiple regression analyses were used to determine the amount of variance that peak power explained for tibial bone strength compared to body mass. Peak power was estimated from maximal vertical jump height using the Sayer's equation. Peripheral quantitative computed tomography scans were used to assess bone strength measures. RESULTS: Peak power (ß=0.541, p<0.001) contributed more to the unique variance in bone strength index for compression (trabecular bone) compared to body mass (ß=-0.102, p=0.332). For polar strength strain index (cortical bone), the beta coefficient for body mass remained significant (ß=0.257, p<0.006), however peak power's contribution was similar (ß=0.213, p=0.051). CONCLUSION: Compared to body mass, peak power was a better predictor for trabecular bone strength but similar to body mass for cortical bone strength. These data provide additional support for the development of a vertical jump test as an objective, valid and reliable measure to monitor bone strength among youth and adult populations.

L-Plastin deficiency produces increased trabecular bone due to attenuation of sealing ring formation and osteoclast dysfunction.

Bone resorption requires the formation of complex, actin-rich cytoskeletal structures. During the early phase of sealing ring formation by osteoclasts, L-plastin regulates actin-bundling to form the nascent sealing zones (NSZ). Here, we show that L-plastin knockout mice produce osteoclasts that are deficient in the formation of NSZs, are hyporesorptive, and make superficial resorption pits in vitro. Transduction of TAT-fused full-length L-plastin peptide into osteoclasts from L-plastin knockout mice rescued the formation of nascent sealing zones and sealing rings in a time-dependent manner. This response was not observed with mutated full-length L-plastin (Ser-5 and -7 to Ala-5 and -7) peptide. In contrast to the observed defect in the NSZ, L-plastin deficiency did not affect podosome formation or adhesion of osteoclasts in vitro or in vivo. Histomorphometry analyses in 8- and 12-week-old female L-plastin knockout mice demonstrated a decrease in eroded perimeters and an increase in trabecular bone density, without a change in bone formation by osteoblasts. This decrease in eroded perimeters supports that osteoclast function is attenuated in L-plastin knockouts. Micro-CT analyses confirmed a marked increase in trabecular bone mass. In conclusion, female L-plastin knockout mice had increased trabecular bone density due to impaired bone resorption by osteoclasts. L-plastin could be a potential target for therapeutic interventions to treat trabecular bone loss.

Muscle Power Predicts Bone Strength in Division II Athletes.

Yingling, VR, Webb, SL, Inouye, C, O, J, and Sherwood, JJ. Muscle power predicts bone strength in Division II athletes. J Strength Cond Res 34(6): 1657-1665, 2020-The relationship between muscle fitness measures and tibial bone strength in collegiate level athletes was investigated. Eighty-six Division II collegiate athletes (mean ± SD: age: [18-29 years], height: 1.71 m [0.09], mass: 66.7 kg [10.5], 56 female: 30 male) participated in this cross-sectional study. Maximum grip strength (GS), 1 repetition maximum leg press, and vertical jump peak power (PP) tests were measured. Cortical area, cortical bone mineral density (cBMD), moment of inertia, and bone strength (polar strength-strain index) were measured using peripheral quantitative computed tomography at 50% tibia length. For each bone strength parameter, a hierarchical multiple regression analysis was performed to examine the contribution of sex and the 3 muscle fitness parameters (muscle power, relative 1 repetition leg extensor strength, and relative GS) to bone parameters. Vertical jump PP explained 54-59% of the variance in bone strength parameters, and relative leg extensor and GS were not predictive of bone strength parameters. Muscle power correlated with bone mass and architecture variables but not cBMD values. Cortical bone mineral density was also not predicted by relative leg extensor strength or relative GS. Muscular fitness assessment, specifically PP calculated from vertical jump height assessments, provides a simple, objective, valid, and reliable measure to identify and monitor bone strength in collegiate athletes.

Tibia functionality and Division II female and male collegiate athletes from multiple sports.

BACKGROUND: Bone strength is developed through a combination of the size and shape (architecture) of a bone as well as the bone's material properties; and therefore, no one outcome variable can measure a positive or negative adaptation in bone. Skeletal robusticity (total area/ bone length) a measure of bones external size varies within the population and is independent of body size, but robusticity has been associated with bone strength. Athletes may have similar variability in robusticity values as the general population and thus have a wide range of bone strengths based on the robustness of their bones. Therefore, the purpose of this study was to determine if an athlete's bone strength and cortical area relative to body size was dependent on robusticity. The second aim was to determine if anthropometry or muscle function measurements were associated with bone robusticity. METHODS: Bone variables contributing to bone strength were measured in collegiate athletes and a reference group using peripheral quantitative computed tomography (pQCT) at the 50% tibial site. Bone functionality was assessed by plotting bone strength and cortical area vs body size (body weight x tibial length) and robustness (total area/length) vs body size. Bone strength was measured using the polar strength-strain index (SSIp). Based on the residuals from the regression, an athlete's individual functionality was determined, and two groups were formed "weaker for size" (WS) and "stronger for size" (SS). Grip strength, leg extensor strength and lower body power were also measured. RESULTS: Division II athletes exhibited a natural variation in (SSIp) relative to robusticity consistent with previous studies. Bone strength (SSIp) was dependent on the robusticity of the tibia. The bone traits that comprise bone strength (SSIp) were significantly different between the SS and WS groups, yet there were minimal differences in the anthropometric data and muscle function measures between groups. A lower percentage of athletes from ball sports were "weaker for size" (WS group) and a higher percentage of swimmers were in the WS group. DISCUSSION: A range of strength values based on robusticity occurs in athletes similar to general populations. Bones with lower robusticity (slender) were constructed with less bone tissue and had less strength. The athletes with slender bones were from all sports including track and field and ball sports but the majority were swimmers. CONCLUSIONS: Athletes, even after optimal training for their sport, may have weaker bones based on robusticity. Slender bones may therefore be at a higher risk for fracture under extreme loading events but also yield benefits to some athletes (swimmers) due to their lower bone mass.

The reliability of vertical jump tests between the Vertec and My Jump phone application.

BACKGROUND: The vertical jump is used to estimate sports performance capabilities and physical fitness in children, elderly, non-athletic and injured individuals. Different jump techniques and measurement tools are available to assess vertical jump height and peak power; however, their use is limited by access to laboratory settings, excessive cost and/or time constraints thus making these tools oftentimes unsuitable for field assessment. A popular field test uses the Vertec and the Sargent vertical jump with countermovement; however, new low cost, easy to use tools are becoming available, including the My Jump iOS mobile application (app). The purpose of this study was to assess the reliability of the My Jump relative to values obtained by the Vertec for the Sargent stand and reach vertical jump (VJ) test. METHODS: One hundred and thirty-five healthy participants aged 18-39 years (94 males, 41 females) completed three maximal Sargent VJ with countermovement that were simultaneously measured using the Vertec and the My Jump. Jump heights were quantified for each jump and peak power was calculated using the Sayers equation. Four separate ICC estimates and their 95% confidence intervals were used to assess reliability. Two analyses (with jump height and calculated peak power as the dependent variables, respectively) were based on a single rater, consistency, two-way mixed-effects model, while two others (with jump height and calculated peak power as the dependent variables, respectively) were based on a single rater, absolute agreement, two-way mixed-effects model. RESULTS: Moderate to excellent reliability relative to the degree of consistency between the Vertec and My Jump values was found for jump height (ICC = 0.813; 95% CI [0.747-0.863]) and calculated peak power (ICC = 0.926; 95% CI [0.897-0.947]). However, poor to good reliability relative to absolute agreement for VJ height (ICC = 0.665; 95% CI [0.050-0.859]) and poor to excellent reliability relative to absolute agreement for peak power (ICC = 0.851; 95% CI [0.272-0.946]) between the Vertec and My Jump values were found; Vertec VJ height, and thus, Vertec calculated peak power values, were significantly higher than those calculated from My Jump values (p < 0.0001). DISCUSSION: The My Jump app may provide a reliable measure of vertical jump height and calculated peak power in multiple field and laboratory settings without the need of costly equipment such as force plates or Vertec. The reliability relative to degree of consistency between the Vertec and My Jump app was moderate to excellent. However, the reliability relative to absolute agreement between Vertec and My Jump values contained significant variation (based on CI values), thus, it is recommended that either the My Jump or the Vertec be used to assess VJ height in repeated measures within subjects' designs; these measurement tools should not be considered interchangeable within subjects or in group measurement designs.

Acute hypothalamic suppression significantly affects trabecular bone but not cortical bone following recovery and ovariectomy surgery in a rat model.

Background. Osteoporosis is "a pediatric disease with geriatric consequences." Bone morphology and tissue quality co-adapt during ontogeny for sufficient bone stiffness. Altered bone morphology from hypothalamic amenorrhea, a risk factor for low bone mass in women, may affect bone strength later in life. Our purpose was to determine if altered morphology following hypothalamic suppression during development affects cortical bone strength and trabecular bone volume (BV/TV) at maturity. Methods. Female rats (25 days old) were assigned to a control (C) group (n = 45) that received saline injections (.2 cc) or an experimental group (GnRH-a) (n = 45) that received gonadotropin releasing hormone antagonist injections (.24 mg per dose) for 25 days. Fifteen animals from each group were sacrificed immediately after the injection protocol at Day 50 (C, GnRH-a). The remaining animals recovered for 135 days and a subset of each group was sacrificed at Day 185 ((C-R) (n = 15) and (G-R) (n = 15)). The remaining animals had an ovariectomy surgery (OVX) at 185 days of age and were sacrificed 40 days later (C-OVX) (n = 15) and (G-OVX) (n = 15). After sacrifice femurs were mechanically tested and scanned using micro CT. Serum C-terminal telopeptides (CTX) and insulin-like growth factor 1 (IGF-1) were measured. Two-way ANOVA (2 groups (GnRH-a and Control) X 3 time points (Injection Protocol, Recovery, post-OVX)) was computed. Results. GnRH-a injections suppressed uterine weights (72%) and increased CTX levels by 59%. Bone stiffness was greater in the GnRH-a groups compared to C. Ash content and cortical bone area were similar between groups at all time points. Polar moment of inertia, a measure of bone architecture, was 15% larger in the GnRH-a group and remained larger than C (19%) following recovery. Both the polar moment of inertia and cortical area increased linearly with the increases in body weight. Following the injection protocol, trabecular BV/TV was 31% lower in the GnRH-a group compared to C, a similar deficit in BV/TV was also measured following recovery and post-OVX. The trabecular number and thickness were lower in the GnRH-a group compared to control. Conclusion. These data suggest that following a transient delay in pubertal onset, trabecular bone volume was significantly lower and no restoration of bone volume occurred following recovery or post-OVX surgery. However, cortical bone strength was maintained through architectural adaptations in the cortical bone envelope. An increase in the polar moment of inertia offset increased bone resorption. The current data are the first to suppress trabecular bone during growth, and then add an OVX protocol at maturity. Trabecular bone and cortical bone differed in their response to hypothalamic suppression during development; trabecular bone was more sensitive to the negative effects of hypothalamic suppression.

Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo.

Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-ß1 and TGF-ß receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo.

The effects of short-term jump training on bone metabolism in females using oral contraceptives.

The purpose of this study was to determine the effect of oral contraceptive use on bone serum markers following a 3-week jumping protocol. Twenty-three females (18-25 years) were grouped as oral contraceptive users (OC+) or non-users (OC-). Following a 3-week observation period, participants completed a 3-week (15-day) jump protocol. Jump sessions consisting of ten 42 cm drop jumps with a 30 s rest interval between jumps were completed each day, 5 days per week. Peak vertical ground reaction force and loading rate were measured and the osteogenic index was calculated. Serum markers for bone formation, bone alkaline phosphatase (BAP) and bone resorption, C-terminal telopeptides of type I collagen (CTX) were measured at three time points (pre-, mid-, post-jump). BAP and CTX increased significantly (P = 0.0017, 0.0488) in both groups post-jump; however, bone metabolic markers were not different between the OC+ and OC- groups. Osteogenic index, ground reaction force and vertical jump height were similar between groups. Correlations between markers of bone metabolism and participants' age at menarche, weight, loading rate and years on OC were not significant. A 3-week jumping protocol was found to be effective in stimulating bone metabolism in both OC+ and OC- groups.

Bioinformatic identification of connective tissue growth factor as an osteogenic protein within skeletal muscle.

Aging is associated with increasing incidence of osteoporosis; a skeletal disorder characterized by compromised bone strength that may predispose patients to an increased risk of fracture. It is imperative to identify novel ways in which to attenuate such declines in the functional properties of bone. The purpose of this study was to identify, through in silico, in vitro, and in vivo approaches, a protein secreted from skeletal muscle that is putatively involved in bone formation. We performed a functional annotation bioinformatic analysis of human skeletal muscle-derived secretomes (n = 319) using DAVID software. Cross-referencing was conducted using OMIM, Unigene, UniProt, GEO, and CGAP databases. Signal peptides and transmembrane residues were analyzed using SignalP and TMHMM software. To further investigate functionality of the identified protein, L6 and C2C12 myotubes were grown for in vitro analysis. C2C12 myotubes were subjected to 16 h of glucose deprivation (GD) prior to analysis. In vivo experiments included analysis of 6-week calorie restricted (CR) rat muscle samples. Bioinformatic analysis yielded 15 genes of interest. GEO dataset analysis identified BMP5, COL1A2, CTGF, MGP, MMP2, and SPARC as potential targets for further processing. Following TMHMM and SignalP processing, CTGF was chosen as a candidate gene. CTGF expression level was increased during L6 myoblast differentiation (P < 0.01). C2C12 myotubes showed no change in response to GD. Rat soleus muscle samples exhibited an increase in CTGF expression (n = 16) in response to CR (35%) (P < 0.05). CTGF was identified as a skeletal muscle expressed protein through bioinformatic analysis of skeletal muscle-derived secretomes and in vitro/in vivo analysis. Future study is needed to determine the role of muscle-derived CTGF in bone formation and remodeling processes.

The effects of delayed puberty on the growth plate.

BACKGROUND: Many athletes are beginning intense training before puberty, a time of increased bone accrual when up to 25% of total bone mineral accrual occurs. Female athletes experiencing late or delayed pubertal onset may have open epiphyseal plates that are vulnerable to injury. This investigation's purpose was to determine whether a delay in puberty (primary amenorrhea) affects the growth plate immediately postpuberty and at maturity. METHODS: Forty-eight female Sprague-Dawley rats (23 d old) were randomly assigned to 4 groups (n=12); short-term control (C-ST), long-term control (C-LT), short-term GnRH antagonist (G-ST), and long-term GnRH antagonist (G-LT). At 25 days of age, daily gonadotropin-releasing hormone antagonist (GnRH-a) injections were administered delaying pubertal onset. Left tibias were analyzed. Stained frontal slices of proximal tibia (5 µm thick) were analyzed in hypertrophic, proliferative, and reserve zones for total height, zone height, and cell/column counts. All procedures were approved by Institutional Animal Care and Use Committee at Brooklyn College. RESULTS: Growth plate height was 19.7% wider in delayed puberty (G-ST) group and at maturity was 27.9% greater in G-LT group compared with control (C-LT) (P<0.05). No significant differences were found in short-term or long-term growth plate zone heights or cell/column counts between groups (P>0.05). Growth plate zone height normalized to total height resulted in 28.7% larger reserve zone in the short-term GnRH-a group but the proliferative zone was 8.5% larger in the long-term group compared with the control group (P<0.05). Normalized to growth plate height a significant decrease was found in column counts in proliferative zones of the short-term and long-term GnRH-a groups. CONCLUSIONS: Current data illustrate that delayed puberty using GnRH-a injections results in significant growth plate height and decreases proliferative column counts and zone height, thus potentially contributing to decreases in bone mass at maturity. CLINICAL RELEVANCE: Growth plate height increases indicate increased potential for growth and bone accrual. However, previous models report decreased bone volume following delayed puberty via GnRH-a injections that may have detrimental effects in the long term.

Short-term jump activity on bone metabolism in female college-aged nonathletes.

There have been few studies examining the short-term effect of high-impact activities on bone metabolism measured by bone serum marker concentrations. The purpose of this study was to examine the effect of short-term high-impact jump activity on bone turnover in female college-aged non-athletes. Twenty six healthy females were randomly assigned to a control or jump group. The subjects jumped 5 days per week for 2 weeks. The participants completed 10 jumps per session. A general health questionnaire and a bone-specific physical activity assessment instrument (BPAQ) were completed. BPAQ scores were calculated based on the past history of exercise. Blood draws were taken in both groups before and after the two-week experimental period. The vertical ground reaction force (VGRF) of all jumps and jump height were measured for each subject daily and the osteogenic index (OI) was measured. Concentrations of serum osteocalcin (OC), Bone Specific Alkaline Phosphatase (BAP), C-Terminal Telopeptides of Type I Collagen (CTX) and plasma Tartrate-Resistant Acid Phosphatase (TRAP5b) were assessed pre and post jump protocol to measure bone formation and resoprtion respectively. A significant interaction (time x group) was found in TRAP5b, and BAP values (p < 0.05). There was a significant decrease in CTX and BAP values in the jump group (p < 0.05) after the two week jump protocol. No significant interactions or changes were observed in OC values for either the jump or the control group. Two weeks of jump activity consisting of 10 jumps/day for 5 days/week with a weekly osteogenic index of 52.6 significantly decreased markers of bone resorption (TRAP5b and CTX) and bone formation (BAP) in young female non-athletes.

Abrogation of Cbl-PI3K interaction increases bone formation and osteoblast proliferation.

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl-PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (Cbl(YF/YF), YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745-36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl-PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation.

Hypothalamic suppression during adolescence varies by bone envelope.

PURPOSE: The purposes of this study were to suppress estradiol levels in adolescent (postpubertal rats) using gonadotropin-releasing hormone antagonist (GnRH-a) injections and to determine the changes in bone structure and mechanical strength. METHODS: In an Institutional Animal Care and Use Committee-approved study, female rats at 23 d of age were assigned to a baseline group (BL65; n = 10) sacrificed on day 65, a control group (Control; n = 15) sacrificed on day 90, or an experimental group (AMEN; n = 9) sacrificed on day 90 that received daily injections of GnRH-a for a 25-d period from 65 to 90 d of age (2.5 mg·kg(-1) per dose). RESULTS: Body weights were similar on day 65; however, the AMEN group was significantly heavier than the Control group (17%, P = 0.001) on day 90. In the AMEN rats relative to the Control group, plasma estradiol levels were reduced by 36% (P = 0.0001) and plasma insulin-like growth factor 1 levels were 24% higher (P = 0.003). In the femur, there was no change in periosteal bone apposition or total cross-sectional area. The marrow area increased by 13.7% (P = 0.05) resulting in a 7.8% decrease in relative cortical area (P = 0.012), and endocortical bone formation rate increased by 39.4% (P = 0.04). Trabecular volume and number decreased by 51.5% (P = 0.0003) and 49.5% (P = 0.0003), respectively. The absolute peak moments of the tibiae and femurs were unchanged in the AMEN group relative to the Control group, but these were reduced by 8.8% (P = 0.03) and 7.5% (P = 0.09), respectively, when normalized by body weight. CONCLUSIONS: Suppression of estradiol by 25 d of GnRH-a administration to 65-d-old (postpubertal) rats reduced trabecular volume and number by about 50%, increased endocortical bone turnover, and reduced relative cortical thickness without changing tibial and femoral total area. These changes in bone structure were associated with no change in absolute mechanical strength possibly because of increases in body weight or in insulin-like growth factor 1 concentrations.

Hypothalamic suppression decreases bone strength before and after puberty in a rat model.

The incidence of menstrual irregularities, both primary and secondary amenorrhea, has been reported to be as high as 60%, with the highest incidence in younger athletes, suggesting possible adverse effects on bone development. It was hypothesized that in a rat model, suppressed hypothalamic activity via a gonadotropin-releasing hormone antagonist (GnRH-a) before onset of puberty would result in a relatively larger bone strength deficit compared with suppression after puberty. Hypothalamic suppression was achieved by providing GnRH injections. Animals received injections for 25 days either before puberty (pre group) (age 23-46 days) or after puberty (post group) (age 65-90 days). Body weights and uterine weights were measured. Serum estradiol was assayed. Mechanical strength of the right femora and histomorphometry of the left femur were measured. Suppression of the hypothalamic-pituitary-gonadal axis was confirmed by significant atrophy of uterine tissue and suppressed estradiol levels. The peak moment was significantly lower in the pre and post GnRH-a groups compared with control. The percentage difference of the average peak moment and stiffness values from the respective age-matched control groups yielded a greater percentage difference in the pre group. The cortical area was less in the GnRH-a-treated groups, but no significant difference between the relative deficits between pre and post groups were found. Hypothalamic-pituitary-gonadal axis suppression before puberty resulted in a significantly larger deficit in mechanical strength compared with postpubertal animals. The time before puberty may represent a time when skeletal strength is more compromised. Women experience both primary and secondary amenorrhea; however, the treatment may need to be different for each condition.

A delay in pubertal onset affects the covariation of body weight, estradiol, and bone size.

The skeletal system functions as a locomotive organ and a mineral reservoir and combinations of genetic and environmental factors affect the skeletal system. Although delayed puberty is associated with compromised bone mass, suppression of estrogen should be beneficial to cortical strength. The purpose was to employ path analysis to study bone strength and delayed puberty. Forty-five female rats were randomly assigned to a control group (n = 15) and an experimental group (n = 30) that received injections of gonadotropin releasing hormone antagonist (GnRH-a). Causal models were constructed by specifying directed paths between bone traits. The first model tested the hypothesis that the functional relationships between bone traits and body weight were altered by a delay in pubertal onset. GnRH-a injections during puberty altered the covariation between body weight and bone size. The second model was constructed to test the hypothesis that variability in stiffness was causally related to variability in body weight. The model also tested the relationship between the periosteal and endocortical surfaces and their relationship to stiffness. There was no change in the relationship between the surfaces in the GnRH-a group. The third model determined the effect of estradiol on both total area and relative cortical area in both groups. The relationship between periosteal surface and serum estradiol levels was only significant during estrogen suppression. These data suggest that increases in body weight during or prior to puberty may not be protective of bone strength.

Delayed pubertal development by hypothalamic suppression causes an increase in periosteal modeling but a reduction in bone strength in growing female rats.

The timing of the pubertal growth is a critical event in skeletal development. A delay in the onset of puberty has been correlated with increased stress fracture incidence in young women and as a result, suboptimal skeletal development may affect long-term bone strength. Gonadotropin releasing hormone antagonist (GnRH-a) injections were used to delay the onset of puberty in growing female rats. 23-day-old female rats were injected with a GnRH-antagonist at 2 dosage levels (n=15/group). The Low Dose group (1.25 mg/kg/dose) received daily injections for 27 days (sacrifice 49 days). The High Dose group received (5.0 mg/kg/dose) only 5 days per week over a 26 day period (sacrifice 48 days). Calcein injections measured bone formation activity on the periosteal and endocortical surfaces. Standard histomorphometric and biomechanical analyses were performed on the femora and ash content was measured on the tibiae of all animals. Serum estradiol and insulin-like growth factor (IGF)-1 levels were assayed. Significant delays in pubertal development occurred in the two GnRH-a groups as evidenced by delayed vaginal openings, decreased uterine and ovarian weights and suppressed estradiol levels compared to control. Femoral lengths were significantly shorter in the experimental groups and serum IGF-1 levels were higher than control. Bone strength and stiffness were significantly lower in the GnRH-a groups. Cortical bone area was decreased and total area was not different between groups. There was a significant decrease in % Ct.Ar/T.Ar. The decreased bone strength may have resulted from a decrease in the amount and distribution of bone, however, stress and Young's modulus were also decreased. There was a different response between endocortical formation indices and periosteal formation indices to the GnRH-a protocol. Endocortical bone formation rates decreased and there was an increase in periosteal labeled surface. A dose response between bone strength and GnRH-a dosage was found. The data suggest that hypothalamic suppression during pubertal development resulted in decreased bone strength which may result in fracture development.

The effect of a short-term delay of puberty on trabecular bone mass and structure in female rats: a texture-based and histomorphometric analysis.

Accrual of bone mass and strength during development is imperative in order to reduce the risk of fracture later in life. Although delayed pubertal onset is associated with an increased incidence of stress fracture, evidence supports the concept of "catch up" growth. It remains unclear if deficits in bone mass associated with delayed puberty have long-term effects on trabecular bone structure and strength. The purpose of this study was to use texture-based analysis and histomorphometry to investigate the effect of a delay in puberty on trabecular bone mass and structure immediately post-puberty and at maturity in female rats. Forty-eight female Sprague-Dawley rats (25 days) were randomly assigned to one of four groups; (1) short-term control (C-ST), (2) long-term control (C-LT), (3) short-term GnRH antagonist (G-ST) and (4) long-term GnRH antagonist (G-LT). Injections of either saline or gonadotropin-releasing hormone antagonist (GnRH-a) (100 microg/day) (Cetrotide, Serono, Inc.) were given intraperitoneally for 18 days (day 25-42) to both ST and LT. The ST groups were sacrificed after the last injection (day 43) and the LT groups at 6 months of age. Pubertal and gonadal development was retarded by the GnRA antagonist injections as indicated by a delay in vaginal opening, lower ovarian and uterine weights and suppressed estradiol levels in the short-term experimental animals (G-ST). Delayed puberty caused a transient reduction in trabecular bone area as assessed by histomorphometry. Specifically, the significant deficit in bone area resulted from a decreased trabecula number and an increase in trabecular separation. Texture analysis, a new method to assess bone density and structural anisotropy, correlated well with the standard histomorphometry and measured significant deficits in the density measure (M(Density)) in the G-ST group that remained at maturity (6 months). The texture energy deficit in the G-ST group was primarily in the 0 degrees orientation (-13.2%), which measures the longitudinal trabeculae in the proximal tibia. However, the deficit in the G-LT group was in the 45 degrees and 135 degrees orientations. These results suggest that any "catch-up" growth following the cessation of the GnRH-antagonist injection protocol may be directed in trabeculae oriented perpendicular to 0 degrees at the expense of trabeculae in other orientations.

Comparative assessment of bone mass and structure using texture-based and histomorphometric analyses.

The purpose of this study was to develop a methodology for quantitatively assessing bone quantity and anisotropy based on texture analysis using Gabor wavelets. The wavelet approach has the capability to simultaneously examine the images at low and high resolutions to gain information on both global and detailed local features of the bone image. The program that implemented the texture analysis gave measures of density (M(Density)) and anisotropy (M(Anisotropy)). It also allowed us to examine the texture energy at four orientations (0 degrees , 45 degrees , 90 degrees , 135 degrees) to gain insight about the details of the anisotropy. Analysis of templates of four simulated patterns, which had same number of dots but with differing orientations, demonstrated how the texture-based analysis differentiated between these templates. The measures of M(Anisotropy) discriminated between the four simulated patterns. The M(Density) measures were similar across all patterns. These outcomes matched the design intent of the simulated patterns. We also compared the trabecular bone images obtained from a previous study, in which the right forelimbs of normal female retired breeder beagle dogs (5-7 years old) were cast for 12 months to induce bone loss, using both histomorphometry and texture analysis. Both histomorphometry and the texture analysis detected significant differences in the trabecular bone of the distal metatarsal between the control and disuse groups. Percent trabecular bone (Tb.Ar/T.Ar) and the textural density parameter (M(Density)) were highly correlated (r=0.962). M(Anisotropy) was decreased (3.9%) after the 12-month disuse protocol, but was not significantly different from normal. However, the texture energy values at all orientations (0 degrees , 45 degrees , 90 degrees and 135 degrees) were significantly decreased in the disuse group. Therefore, texture analysis was able to assess anisotropy, which could not be extracted from histomorphometric parameters. We conclude that texture analysis is an effective tool for assessing 2D bone images that yields information regarding the quantity of bone as well as the orientation of the trabecular structure that can augment our ability to discriminate between normal and pathological bone tissue.

Short-term delay of puberty causes a transient reduction in bone strength in growing female rats.

Multiple factors affect the structural development of the skeleton; in particular, estrogen levels during growth are an important factor in the pathogenesis of bone fragility. The delay of menarche and infrequent menstrual cycles decrease estrogen levels during adolescence and decrease peak bone mass. The aim of this study was to determine if delayed puberty through administration of a GnRH antagonist initiated prior to the onset of the first estrus cycle would delay the increase in estrogen levels and impede bone strength development in female rats. Twenty-three-day-old female Sprague-Dawley rats were randomly assigned to one of four groups; 1) short-term control group (C-ST) (n = 12), 2) long-term control (C-LT) (n = 12), 3) short-term GnRH antagonist group (G-ST) (n = 12) and 4) long-term GnRH antagonist group (G-LT) (n = 12). Injections (0.2 ml) of either saline or GnRH antagonist (100 microg/day) (Cetrotide, Serono, Inc) were given intraperitoneally for a duration of 18 days. Pubertal and gonadal development was retarded as indicated by a delay in vaginal opening (an indicator of pubertal onset), lower ovarian and uterine weights and lower estradiol levels in the short-term experimental animals (G-ST). However, at maturity (G-LT), there were no significant differences found in these measures. A delay in the timing of puberty significantly attenuated the development of femoral bone strength at 6 weeks of age. Peak moment, yield moment and stiffness in the G-ST group were all significantly less than the C-ST group. Cortical width was significantly attenuated due to the increased percentage of marrow area per total bone area in the G-ST group. However, femoral bone strength was recovered at maturity (G-LT). In summary, a transient delay in pubertal timing has short-term effects on bone strength development. In the current animal model of delaying puberty through GnRH antagonist injections, there appears to be no long-term effects on bone strength.