Intraspecific variation of long bone cross-sectional properties in Pan troglodytes troglodytes and Gorilla gorilla gorilla.

OBJECTIVES: Morphological intraspecific variation is due to the balance between skeletal plasticity and genetic constraint on the skeleton. Osteogenic responses to external stimuli, such as locomotion, have been well documented interspecifically across the primate order, but less so at the intraspecific level. Here, we examine the differences in cross-sectional variability of the femur, humerus, radius, and tibia in Pan troglodytes troglodytes versus Gorilla gorilla gorilla. We investigate whether there are sex, species, bone, and trait differences in response to variable body size and locomotion. MATERIALS AND METHODS: Adult male and female P. t. troglodytes and G. g. gorilla long bones from the Cleveland Museum of Natural History were scanned with a peripheral quantitative computer tomography system. Scans were taken at the midshaft of each bone according to functional bone length. Coefficients of variation were used to provide a size-independent measure of variation. We applied a Bonferroni correction to account for the multiple pairwise tests. RESULTS: There were limited significant differences between males and females, however, females tended to be more variable than males. Variation in Gorilla, when significant, was greater than in Pan, although significant differences were limited. There were no differences between bone variability in male and female Gorilla, and female Pan. DISCUSSION: Increased female variability may be due to more variable locomotor behavior, particularly during periods of pregnancy, lactation, and caring for an offspring compared to consistent locomotion over the life course by males. Body size may be a contributing factor to variability; more work is needed to understand this relationship.

Acute Bone Loss and Infrapatellar Fat Pad Fibrosis in the Knee After an In Vivo ACL Injury in Adolescent Mice.

BACKGROUND: Young patients are 6 times more likely than adults to have a primary anterior cruciate ligament (ACL) graft failure. Biological factors (ie, tunnel osteolysis) may account for up to a third of these failures. Previous evaluations of patient ACL explants indicated significant bone loss within the entheseal regions. However, it remains unknown if the degree of bone loss within the ACL insertion regions, wherein ACL grafts are fixated, exceeds that of the femoral and tibial condylar bone. HYPOTHESIS: Bone loss in the mineralized matrices of the femoral and tibial ACL entheses is distinct from that clinically reported across the whole knee after injury. STUDY DESIGN: Controlled laboratory study. METHODS: We developed a clinically relevant in vivo mouse ACL injury model to cross-sectionally track the morphological and physiological postinjury changes within the ACL, femoral and tibial entheses, synovial joint space, and load-bearing epiphyseal cortical and trabecular bone components of the knee joint. Right ACLs of 10-week-old C57BL/6J female mice (N = 75) were injured in vivo with the contralateral ACLs serving as controls. Mice were euthanized at 1, 3, 7, 14, or 28 days after injury (n = 12/cohort). Downstream analyses included volumetric cortical and trabecular bone analyses and histopathologic assessments of the knee joint after injury. Gait analyses across all time points were also performed (n = 15 mice). RESULTS: The majority of the ACL injuries in mice were partial tears. The femoral and tibial cortical bone volumes were 39% and 32% lower, respectively, at 28 days after injury than those of the uninjured contralateral knees (P < .01). Trabecular bone measures demonstrated little difference between injured and control knees after injury. Across all bone measures, bone loss was similar between the injured knee condyles and ACL entheses. There was also significant inflammatory activity within the knee after injury. By 7 days after injury, synovitis and fibrosis were sigificantly elevated in the injured knee compared with the controls (P < .01), which corresponded with significantly higher osteoclast activity in bone at this time point compared with the controls. This inflammatory response signficantly persisted throughout the duration of the study (P < .01). The hindlimb gait after injury deviated from normal, but mice habitually loaded their injured knee throughout the study. CONCLUSION: Bone loss was acute and persisted for 4 weeks after injury in mice. However, the authors' hypothesis was not confirmed, as bone quality was not significantly lower in the entheses compared with the condylar bone regions after injury. With relatively normal hindlimb loading but a significant physiological response after injury, bone loss in this model may be driven by inflammation. CLINICAL RELEVANCE: There is persistent bone resorption and fibrotic tissue development after injury that is not resolved. Inflammatory and catabolic activity may have a significant role in the postinjury decline of bone quality in the knee.

Fatigue-driven compliance increase and collagen unravelling in mechanically tested anterior cruciate ligament.

Approximately 300,000 anterior cruciate ligament (ACL) tears occur annually in the United States, half of which lead to the onset of knee osteoarthritis within 10 years of injury. Repetitive loading is known to result in fatigue damage of both ligament and tendon in the form of collagen unravelling, which can lead to structural failure. However, the relationship between tissue's structural, compositional, and mechanical changes are poorly understood. Herein we show that repetitive submaximal loading of cadaver knees causes an increase in co-localised induction of collagen unravelling and tissue compliance, especially in regions of greater mineralisation at the ACL femoral enthesis. Upon 100 cycles of 4× bodyweight knee loading, the ACL exhibited greater unravelled collagen in highly mineralized regions across varying levels of stiffness domains as compared to unloaded controls. A decrease in the total area of the most rigid domain, and an increase in the total area of the most compliant domain was also found. The results highlight fatigue-driven changes in both protein structure and mechanics in the more mineralized regions of the ACL enthesis, a known site of clinical ACL failure. The results provide a starting point for designing studies to limit ligament overuse injury.

An Adolescent Murine In Vivo Anterior Cruciate Ligament Overuse Injury Model.

BACKGROUND: Overuse ligament and tendon injuries are prevalent among recreational and competitive adolescent athletes. In vitro studies of the ligament and tendon suggest that mechanical overuse musculoskeletal injuries begin with collagen triple-helix unraveling, leading to collagen laxity and matrix damage. However, there are little in vivo data concerning this mechanism or the physiomechanical response to collagen disruption, particularly regarding the anterior cruciate ligament (ACL). PURPOSE: To develop and validate a novel in vivo animal model for investigating the physiomechanical response to ACL collagen matrix damage accumulation and propagation in the ACL midsubstance, fibrocartilaginous entheses, and subchondral bone. STUDY DESIGN: Controlled laboratory study. METHODS: C57BL/6J adolescent inbred mice underwent 3 moderate to strenuous ACL fatigue loading sessions with a 72-hour recovery between sessions. Before each session, randomly selected subsets of mice (n = 12) were euthanized for quantifying collagen matrix damage (percent collagen unraveling) and ACL mechanics (strength and stiffness). This enabled the quasi-longitudinal assessment of collagen matrix damage accrual and whole tissue mechanical property changes across fatigue sessions. Additionally, all cyclic loading data were quantified to evaluate changes in knee mechanics (stiffness and hysteresis) across fatigue sessions. RESULTS: Moderate to strenuous fatigue loading across 3 sessions led to a 24% weaker (P = .07) and 35% less stiff (P < .01) ACL compared with nonloaded controls. The unraveled collagen densities within the fatigued ACL and entheseal matrices after the second and third sessions were 38% (P < .01) and 15% (P = .02) higher compared with the nonloaded controls. CONCLUSION: This study confirmed the hypothesis that in vivo ACL collagen matrix damage increases with tissue fatigue sessions, adversely impacting ACL mechanical properties. Moreover, the in vivo ACL findings were consistent with in vitro overloading research in humans. CLINICAL RELEVANCE: The outcomes from this study support the use of this model for investigating ACL overuse injuries.

Telehealth in Orthopedic Sports Medicine: A Survey Study on Patient Satisfaction and Experience.

Background: Telehealth has seen breakthroughs in many fields of medicine, but utilization remains limited in orthopedic sports medicine. The purpose of this investigation was to compare patient satisfaction, duration of care, and overall patient experiences with telehealth and in-person clinical visits for sports-related injuries. Methods: A cross-sectional survey study was conducted at an orthopedic sports medicine clinic during the peak of the COVID-19 pandemic between March and November 2020. Anonymous electronic surveys were used to record patient responses and statistical comparisons were drawn through two-sample t-tests. Results: A total of 175 patients (82 telehealth vs. 93 in-person) consented to participate in this investigation, and all were included in the final analysis. The overall composite satisfaction score, when compared between the two groups, did not differ (p = 0.63). Duration of care was significantly longer in the 93 patients who had in-person clinical visits as compared with the 82 patients who had telehealth visits (61/93: >31 min vs. 75/82: <30 min; p < 0.001). Finally, of the 82 patients who had telehealth, 3 respondents said they were "very unlikely" and "unlikely" to request another virtual clinical visit and/or recommend this mode of health care delivery to friends or family. Of the 93 patients had in-person clinical visits, only 15 respondents stated they were uninterested in telehealth under any circumstance. Conclusion: Most patients presenting to an orthopedic sports medicine clinic are open to telehealth, recognize its utility, and believe it to be just as comparable with in-person clinical visits. Level of Evidence: IV.

Sex and External Size Specific Limitations in Assessing Bone Health From Adult Hand Radiographs.

Morphological parameters measured for the second metacarpal from hand radiographs are used clinically for assessing bone health during growth and aging. Understanding how these morphological parameters relate to metacarpal strength and strength at other anatomical sites is critical for providing informed decision-making regarding treatment strategies and effectiveness. The goals of this study were to evaluate the extent to which 11 morphological parameters, nine of which were measured from hand radiographs, relate to experimentally measured whole-bone strength assessed at multiple anatomical sites and to test whether these associations differed between men and women. Bone morphology and strength were assessed for the second and third metacarpals, radial diaphysis, femoral diaphysis, and proximal femur for 28 white male donors (18-89 years old) and 35 white female donors (36-89+ years old). The only morphological parameter to show a significant correlation with strength without a sex-specific effect was cortical area. Dimensionless morphological parameters derived from hand radiographs correlated significantly with strength for females, but few did for males. Males and females showed a significant association between the circularity of the metacarpal cross-section and the outer width measured in the mediolateral direction. This cross-sectional shape variation contributed to systematic bias in estimating strength using cortical area and assuming a circular cross-section. This was confirmed by the observation that use of elliptical formulas reduced the systematic bias associated with using circular approximations for morphology. Thus, cortical area was the best predictor of strength without a sex-specific difference in the correlation but was not without limitations owing to out-of-plane shape variations. The dependence of cross-sectional shape on the outer bone width measured from a hand radiograph may provide a way to further improve bone health assessments and informed decision making for optimizing strength-building and fracture-prevention treatment strategies. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Anterior cruciate ligament microfatigue damage detected by collagen autofluorescence in situ.

PURPOSE: Certain types of repetitive sub-maximal knee loading cause microfatigue damage in the human anterior cruciate ligament (ACL) that can accumulate to produce macroscopic tissue failure. However, monitoring the progression of that ACL microfatigue damage as a function of loading cycles has not been reported. To explore the fatigue process, a confocal laser endomicroscope (CLEM) was employed to capture sub-micron resolution fluorescence images of the tissue in situ. The goal of this study was to quantify the in situ changes in ACL autofluorescence (AF) signal intensity and collagen microstructure as a function of the number of loading cycles. METHODS: Three paired and four single cadaveric knees were subjected to a repeated 4 times bodyweight landing maneuver known to strain the ACL. The paired knees were used to compare the development of ACL microfatigue damage on the loaded knee after 100 consecutive loading cycles, relative to the contralateral unloaded control knee, through second harmonic generation (SHG) and AF imaging using confocal microscopy (CM). The four single knees were used for monitoring progressive ACL microfatigue damage development by AF imaging using CLEM. RESULTS: The loaded knees from each pair exhibited a statistically significant increase in AF signal intensity and decrease in SHG signal intensity as compared to the contralateral control knees. Additionally, the anisotropy of the collagen fibers in the loaded knees increased as indicated by the reduced coherency coefficient. Two out of the four single knee ACLs failed during fatigue loading, and they exhibited an order of magnitude higher increase in autofluorescence intensity per loading cycle as compared to the intact knees. Of the three regions of the ACL - proximal, midsubstance and distal - the proximal region of ACL fibers exhibited the highest AF intensity change and anisotropy of fibers. CONCLUSIONS: CLEM can capture changes in ACL AF and collagen microstructures in situ during and after microfatigue damage development. Results suggest a large increase in AF may occur in the final few cycles immediately prior to or at failure, representing a greater plastic deformation of the tissue. This reinforces the argument that existing microfatigue damage can accumulate to induce bulk mechanical failure in ACL injuries. The variation in fiber organization changes in the ACL regions with application of load is consistent with the known differences in loading distribution at the ACL femoral enthesis.

Region-specific associations among tissue-level mechanical properties, porosity, and composition in human male femora.

Region-specific differences in age-related bone remodeling are known to exist. We therefore hypothesized that the decline in tissue-level strength and post-yield strain (PYS) with age is not uniform within the femur, but is driven by region-specific differences in porosity and composition. Four-point bending was conducted on anterior, posterior, medial, and lateral beams from male cadaveric femora (n = 33, 18-89 yrs of age). Mid-cortical porosity, composition, and mineralization were assessed using nano-computed tomography (nanoCT), Raman spectroscopy, and ashing assays. Traits between bones from young and elderly groups were compared, while multivariate analyses were used to identify traits that predicted strength and PYS at the regional level. We show that age-related decline in porosity and mechanical properties varied regionally, with highest positive slope of age vs. Log(porosity) found in posterior and anterior bone, and steepest negative slopes of age vs. strength and age vs. PYS found in anterior bone. Multivariate analyses show that Log(porosity) and/or Raman 1246/1269 ratio explained 46-51% of the variance in strength in anterior and posterior bone. Three out of five traits related to Log(porosity), mineral crystallinity, 1246/1269, mineral/matrix ratio, and/or hydroxyproline/proline (Hyp/Pro) ratio, explained 35-50% of the variance in PYS in anterior, posterior and lateral bones. Log(porosity) and Hyp/Pro ratio alone explained 13% and 19% of the variance in strength and PYS in medial bone, respectively. The predictive performance of multivariate analyses was negatively impacted by pooling data across all bone regions, underscoring the complexity of the femur and that the use of pooled analyses may obscure underlying region-specific differences.

Endurance running during late murine adolescence results in a stronger anterior cruciate ligament and flatter posterior tibial slopes compared to controls.

BACKGROUND: Anterior cruciate ligament (ACL) injury rates continue to rise among youth involved in recreational and competitive athletics, requiring a better understanding of how the knee structurally and mechanically responds to activity during musculoskeletal growth. Little is understood about how anatomical risk factors for ACL injury (e.g., small ACL size, narrow intercondylar notch, and steep posterior tibial slope) develop and respond to increased physical activity throughout growth. We hypothesized that the ACL-complex of mice engaged in moderate to strenuous physical activity (i.e., endurance running) throughout late adolescence and young adulthood would positively functionally adapt to repetitive load perturbations. METHODS: Female C57BL6/J mice (8 weeks of age) were either provided free access to a standard cage wheel with added resistance (n = 18) or normal cage activity (n = 18), for a duration of 4 weeks. Daily distance ran, weekly body and food weights, and pre- and post-study body composition measures were recorded. At study completion, muscle weights, three-dimensional knee morphology, ACL cross-sectional area, and ACL mechanical properties of runners and nonrunners were quantified. Statistical comparisons between runners and nonrunners were assessed using a two-way analysis of variance and a Tukey multiple comparisons test, with body weight included as a covariate. RESULTS: Runners had larger quadriceps (p = 0.02) and gastrocnemius (p = 0.05) muscles, but smaller hamstring (p = 0.05) muscles, compared to nonrunners. Though there was no significant difference in ACL size (p = 0.24), it was 13% stronger in runners (p = 0.03). Additionally, both the posterior medial and lateral tibial slopes were 1.2 to 2.2 degrees flatter than those of nonrunners (p < 0.01). CONCLUSIONS: Positive functional adaptations of the knee joint to moderate to strenuous exercise in inbred mice offers hope that that some anatomical risk factors for ACL injury may be reduced through habitual physical activity. However, confirmation that a similar response to loading occurs in humans is needed.

State of the mineralized tissue comprising the femoral ACL enthesis in young women with an ACL failure.

Despite poor graft integration among some patients that undergo an anterior cruciate ligament (ACL) reconstruction, there has been little consideration of the bone quality into which the ACL femoral tunnel is drilled and the graft is placed. Bone mineral density of the knee decreases following ACL injury. However, trabecular and cortical architecture differences between injured and non-injured femoral ACL entheses have not been reported. We hypothesize that injured femoral ACL entheses will show significantly less cortical and trabecular mass compared with non-injured controls. Femoral ACL enthesis explants from 54 female patients (13-25 years) were collected during ACL reconstructive surgery. Control explants (n = 12) were collected from seven donors (18-36 years). Injured (I) femoral explants differed from those of non-injured (NI) controls with significantly less (p ≤ 0.001) cortical volumetric bone mineral density (vBMD) (NI: 736.1-867.6 mg/cm3 ; I: 451.2-891.9 mg/cm3 ), relative bone volume (BV/TV) (NI: 0.674-0.867; I: 0.401-0.792) and porosity (Ct.Po) (NI: 0.133-0.326; I: 0.209-0.600). Injured explants showed significantly less trabecular vBMD (p = 0.013) but not trabecular BV/TV (p = 0.314), thickness (p = 0.412), or separation (p = 0.828). We found significantly less cortical bone within injured femoral entheses compared to NI controls. Lower cortical and trabecular bone mass within patient femoral ACL entheses may help explain poor ACL graft osseointegration outcomes in the young and may be a contributor to the osteolytic phenomenon that often occurs within the graft tunnel following ACL reconstruction.

In vivo quantitative imaging biomarkers of bone quality and mineral density using multi-band-SWIFT magnetic resonance imaging.

Bone is a composite biomaterial of mineral crystals, organic matrix, and water. Each contributes to bone quality and strength and may change independently, or together, with disease progression and treatment. Even so, there is a near ubiquitous reliance on ionizing x-ray-based approaches to measure bone mineral density (BMD) which is unable to fully characterize bone strength and may not adequately predict fracture risk. Characterization of treatment efficacy in bone diseases of altered remodeling is complicated by the lack of imaging modality able to safely monitor material-level and biochemical changes in vivo. To improve upon the current state of bone imaging, we tested the efficacy of Multi Band SWeep Imaging with Fourier Transformation (MB-SWIFT) magnetic resonance imaging (MRI) as a readout of bone derangement in an estrogen deficient ovariectomized (OVX) rat model during growth. MB-SWIFT MRI-derived BMD correlated significantly with BMD measured using micro-computed tomography (µCT). In this rodent model, growth appeared to overcome estrogen deficiency as bone mass continued to increase longitudinally over the duration of the study. Nonetheless, after 10 weeks of intervention, MB-SWIFT detected significant changes consistent with estrogen deficiency in cortical water, cortical matrix organization (T1), and marrow fat. Findings point to MB-SWIFT's ability to quantify BMD in good agreement with µCT while providing additive quantitative outcomes about bone quality in a manner consistent with estrogen deficiency. These results indicate MB-SWIFT as a non-ionizing imaging strategy with value for bone imaging and may be a promising technique to progress to the clinic for monitoring and clinical management of patients with bone diseases such as osteoporosis.

Gene Expression Profile and Acute Gene Expression Response to Sclerostin Inhibition in Osteogenesis Imperfecta Bone.

Sclerostin antibody (SclAb) therapy has been suggested as a novel therapeutic approach toward addressing the fragility phenotypic of osteogenesis imperfecta (OI). Observations of cellular and transcriptional responses to SclAb in OI have been limited to mouse models of the disorder, leaving a paucity of data on the human OI osteoblastic cellular response to the treatment. Here, we explore factors associated with response to SclAb therapy in vitro and in a novel xenograft model using OI bone tissue derived from pediatric patients. Bone isolates (approximately 2 mm3) from OI patients (OI type III, type III/IV, and type IV, n = 7; non-OI control, n = 5) were collected to media, randomly assigned to an untreated (UN), low-dose SclAb (TRL, 2.5 µg/mL), or high-dose SclAb (TRH, 25 µg/mL) group, and maintained in vitro at 37°C. Treatment occurred on days 2 and 4 and was removed on day 5 for TaqMan qPCR analysis of genes related to the Wnt pathway. A subset of bone was implanted s.c. into an athymic mouse, representing our xenograft model, and treated (25 mg/kg s.c. 2×/week for 2/4 weeks). Implanted OI bone was evaluated using µCT and histomorphometry. Expression of Wnt/Wnt-related targets varied among untreated OI bone isolates. When treated with SclAb, OI bone showed an upregulation in osteoblast and osteoblast progenitor markers, which was heterogeneous across tissue. Interestingly, the greatest magnitude of response generally corresponded to samples with low untreated expression of progenitor markers. Conversely, samples with high untreated expression of these markers showed a lower response to treatment. in vivo implanted OI bone showed a bone-forming response to SclAb via µCT, which was corroborated by histomorphometry. SclAb induced downstream Wnt targets WISP1 and TWIST1, and elicited a compensatory response in Wnt inhibitors SOST and DKK1 in OI bone with the greatest magnitude from OI cortical bone. Understanding patients' genetic, cellular, and morphological bone phenotypes may play an important role in predicting treatment response. This information may aid in clinical decision-making for pharmacological interventions designed to address fragility in OI. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

A xenograft model to evaluate the bone forming effects of sclerostin antibody in human bone derived from pediatric osteogenesis imperfecta patients.

Osteogenesis imperfecta (OI) is a rare and severe skeletal dysplasia marked by low bone mass and poor bone quality which is especially burdensome during childhood. Since clinical trials for pediatric OI are difficult, there is a widespread reliance on genetically modified murine models to understand the skeletal effects of emerging therapeutics. However a common model does not yet exist to understand how patient-specific genotype may influence treatment efficacy. Recently, sclerostin antibody (SclAb) has been introduced as a novel putative anabolic therapy for diseases of low bone mass, but effects in pediatric patients remain unexplored. In this study, we aim to establish a direct xenograft approach using OI patient-derived bone isolates which retain patient-specific genetic defects and cells residing in their intrinsic extracellular environment to evaluate the bone-forming effects of SclAb as a bridge to clinical trials. OI and age matched non-OI patient bone typically discarded as surgical waste during corrective orthopaedic procedures were collected, trimmed and implanted subcutaneously (s.c.) on the dorsal surface of 4-6-week athymic mice. A subset of implanted mice were evaluated at short (1 week), intermediate (4 week), and long-term (12 week) durations to assess bone cell survival and presence of donor bone cells in order to determine an appropriate treatment duration. Remaining implanted mice were randomly assigned to a two or four-week SclAb-treated (25mg/kg s.c. 2QW) or untreated control group. Immunohistochemistry determined osteocyte and osteoblast donor/host relationship, TRAP staining quantified osteoclast activity, and TUNEL assay was used to understand rates of bone cell apoptosis at each implantation timepoint. Longitudinal changes of in vivo µCT outcomes and dynamic histomorphometry were used to assess treatment response and ex vivo µCT and dynamic histomorphometry of host femora served as a positive internal control to confirm a bone forming response to SclAb. Human-derived osteocytes and lining cells were present up to 12 weeks post-implantation with nominal cell apoptosis in the implant. Sclerostin expression remained donor-derived throughout the study. Osterix expression was primarily donor-derived in treated implants and shifted in favor of the host when implants remained untreated. µCT measures of BMD, TMD, BV/TV and BV increased with treatment but response was variable and impacted by bone implant morphology (trabecular, cortical) which was corroborated by histomorphometry. There was no statistical difference between treated and untreated osteoclast number in the implants. Host femora confirmed a systemic bone forming effect of SclAb. Findings support use of the xenograft model using solid bone isolates to explore the effects of novel bone-targeted therapies. These findings will impact our understanding of SclAb therapy in pediatric OI tissue through establishing the efficacy of this treatment in human cells prior to extension to the clinic.

An Anterior Cruciate Ligament Failure Mechanism.

BACKGROUND: Nearly three-quarters of anterior cruciate ligament (ACL) injuries occur as "noncontact" failures from routine athletic maneuvers. Recent in vitro studies revealed that repetitive strenuous submaximal knee loading known to especially strain the ACL can lead to its fatigue failure, often at the ACL femoral enthesis. HYPOTHESIS: ACL failure can be caused by accumulated tissue fatigue damage: specifically, chemical and structural evidence of this fatigue process will be found at the femoral enthesis of ACLs from tested cadaveric knees, as well as in ACL explants removed from patients undergoing ACL reconstruction. STUDY DESIGN: Controlled laboratory study. METHODS: One knee from each of 7 pairs of adult cadaveric knees were repetitively loaded under 4 times-body weight simulated pivot landings known to strain the ACL submaximally while the contralateral, unloaded knee was used as a comparison. The chemical and structural changes associated with this repetitive loading were characterized at the ACL femoral enthesis at multiple hierarchical collagen levels by employing atomic force microscopy (AFM), AFM-infrared spectroscopy, molecular targeting with a fluorescently labeled collagen hybridizing peptide, and second harmonic imaging microscopy. Explants from ACL femoral entheses from the injured knee of 5 patients with noncontact ACL failure were also characterized via similar methods. RESULTS: AFM-infrared spectroscopy and collagen hybridizing peptide binding indicate that the characteristic molecular damage was an unraveling of the collagen molecular triple helix. AFM detected disruption of collagen fibrils in the forms of reduced topographical surface thickness and the induction of ~30- to 100-nm voids in the collagen fibril matrix for mechanically tested samples. Second harmonic imaging microscopy detected the induction of ~10- to 100-µm regions where the noncentrosymmetric structure of collagen had been disrupted. These mechanically induced changes, ranging from molecular to microscale disruption of normal collagen structure, represent a previously unreported aspect of tissue fatigue damage in noncontact ACL failure. Confirmatory evidence came from the explants of 5 patients undergoing ACL reconstruction, which exhibited the same pattern of molecular, nanoscale, and microscale structural damage detected in the mechanically tested cadaveric samples. CONCLUSION: The authors found evidence of accumulated damage to collagen fibrils and fibers at the ACL femoral enthesis at the time of surgery for noncontact ACL failure. This tissue damage was similar to that found in donor knees subjected in vitro to repetitive 4 times-body weight impulsive 3-dimensional loading known to cause a fatigue failure of the ACL. CLINICAL RELEVANCE: These findings suggest that some ACL injuries may be due to an exacerbation of preexisting hierarchical tissue damage from activities known to place larger-than-normal loads on the ACL. Too rapid an increase in these activities could cause ACL tissue damage to accumulate across length scales, thereby affecting ACL structural integrity before it has time to repair. Prevention necessitates an understanding of how ACL loading magnitude and frequency are anabolic, neutral, or catabolic to the ligament.

Morphology of Mouse Anterior Cruciate Ligament-Complex Changes Following Exercise During Pubertal Growth.

Postnatal development and the physiological loading response of the anterior cruciate ligament (ACL) complex (ACL proper, entheses, and bony morphology) is not well understood. We tested whether the ACL-complex of two inbred mouse strains that collectively encompass the musculoskeletal variation observed in humans would demonstrate significant morphological differences following voluntary cage-wheel running during puberty compared with normal cage activity controls. Female A/J and C57BL/6J (B6) 6-week-old mice were provided unrestricted access to a standard cage-wheel for 4 weeks. A/J-exercise mice showed a 6.3% narrower ACL (p = 0.64), and a 20.1% more stenotic femoral notch (p < 0.01) while B6-exercise mice showed a 12.3% wider ACL (p = 0.10), compared with their respective controls. Additionally, A/J-exercise mice showed a 5.3% less steep posterior medial tibial slope (p = 0.07) and an 8.8% less steep posterior lateral tibial slope (p = 0.07), while B6-exercise mice showed a 9.8% more steep posterior medial tibial slope (p < 0.01) than their respective controls. A/J-exercise mice also showed more reinforcement of the ACL tibial enthesis with a 20.4% larger area (p < 0.01) of calcified fibrocartilage distributed at a 29.2% greater depth (p = 0.02) within the tibial enthesis, compared with their controls. These outcomes suggest exercise during puberty significantly influences ACL-complex morphology and that inherent morphological differences between these mice, as observed in their less active genetically similar control groups, resulted in a divergent phenotypic outcome between mouse strains. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1910-1919, 2019.

Differential changes in bone strength of two inbred mouse strains following administration of a sclerostin-neutralizing antibody during growth.

Administration of sclerostin-neutralizing antibody (Scl-Ab) treatment has been shown to elicit an anabolic bone response in growing and adult mice. Prior work characterized the response of individual mouse strains but did not establish whether the impact of Scl-Ab on whole bone strength would vary across different inbred mouse strains. Herein, we tested the hypothesis that two inbred mouse strains (A/J and C57BL/6J (B6)) will show different whole bone strength outcomes following sclerostin-neutralizing antibody (Scl-Ab) treatment during growth (4.5-8.5 weeks of age). Treated B6 femurs showed a significantly greater stiffness (S) (68.8% vs. 46.0%) and maximum load (ML) (84.7% vs. 44.8%) compared to A/J. Although treated A/J and B6 femurs showed greater cortical area (Ct.Ar) similarly relative to their controls (37.7% in A/J and 41.1% in B6), the location of new bone deposition responsible for the greater mass differed between strains and may explain the greater whole bone strength observed in treated B6 mice. A/J femurs showed periosteal expansion and endocortical infilling, while B6 femurs showed periosteal expansion. Post-yield displacement (PYD) was smaller in treated A/J femurs (-61.2%, p < 0.001) resulting in greater brittleness compared to controls; an effect not present in B6 mice. Inter-strain differences in S, ML, and PYD led to divergent changes in work-to-fracture (Work). Work was 27.2% (p = 0.366) lower in treated A/J mice and 66.2% (p < 0.001) greater in treated B6 mice relative to controls. Our data confirmed the anabolic response to Scl-Ab shown by others, and provided evidence suggesting the mechanical benefits of Scl-Ab administration may be modulated by genetic background, with intrinsic growth patterns of these mice guiding the location of new bone deposition. Whether these differential outcomes will persist in adult and elderly mice remains to be determined.

External Bone Size Is a Key Determinant of Strength-Decline Trajectories of Aging Male Radii.

Given prior work showing associations between remodeling and external bone size, we tested the hypothesis that wide bones would show a greater negative correlation between whole-bone strength and age compared with narrow bones. Cadaveric male radii (n = 37 pairs, 18 to 89 years old) were evaluated biomechanically, and samples were sorted into narrow and wide subgroups using height-adjusted robustness (total area/bone length). Strength was 54% greater (p < 0.0001) in wide compared with narrow radii for young adults (<40 years old). However, the greater strength of young-adult wide radii was not observed for older wide radii, as the wide (R2 = 0.565, p = 0.001), but not narrow (R2 = 0.0004, p = 0.944) subgroup showed a significant negative correlation between strength and age. Significant positive correlations between age and robustness (R2 = 0.269, p = 0.048), cortical area (Ct.Ar; R2 = 0.356, p = 0.019), and the mineral/matrix ratio (MMR; R2 = 0.293, p = 0.037) were observed for narrow, but not wide radii (robustness: R2 = 0.015, p = 0.217; Ct.Ar: R2 = 0.095, p = 0.245; MMR: R2 = 0.086, p = 0.271). Porosity increased with age for the narrow (R2 = 0.556, p = 0.001) and wide (R2 = 0.321, p = 0.022) subgroups. The wide subgroup (p < 0.0001) showed a significantly greater elevation of a new measure called the Cortical Pore Score, which quantifies the cumulative effect of pore size and location, indicating that porosity had a more deleterious effect on strength for wide compared with narrow radii. Thus, the divergent strength-age regressions implied that narrow radii maintained a low strength with aging by increasing external size and mineral content to mechanically offset increases in porosity. In contrast, the significant negative strength-age correlation for wide radii implied that the deleterious effect of greater porosity further from the centroid was not offset by changes in outer bone size or mineral content. Thus, the low strength of elderly male radii arose through different biomechanical mechanisms. Consideration of different strength-age regressions (trajectories) may inform clinical decisions on how best to treat individuals to reduce fracture risk. © 2019 American Society for Bone and Mineral Research.

The relationship between whole bone stiffness and strength is age and sex dependent.

Accurately estimating whole bone strength is critical for identifying individuals that may benefit from prophylactic treatments aimed at reducing fracture risk. Strength is often estimated from stiffness, but it is not known whether the relationship between stiffness and strength varies with age and sex. Cadaveric proximal femurs (44 Male: 18-78 years; 40 Female: 24-95 years) and radial (36 Male: 18-89 years; 19 Female: 24-95 years) and femoral diaphyses (34 Male: 18-89 years; 19 Female: 24-95 years) were loaded to failure to evaluate how the stiffness-strength relationship varies with age and sex. Strength correlated significantly with stiffness at all sites and for both sexes, as expected. However, females exhibited significantly less strength for the proximal femur (58% difference, p < 0.001). Multivariate regressions revealed that stiffness, age and PYD were significant negative independent predictors of strength for the proximal femur (Age: M: p = 0.005, F: p < 0.001, PYD: M: p = 0.022, F: p = 0.025), radial diaphysis (Age: M = 0.055, PYD: F = 0.024), and femoral diaphysis (Age: M: p = 0.014, F: p = 0.097, PYD: M: p = 0.003, F: p = 0.091). These results indicated that older bones tended to be significantly weaker for a given stiffness than younger bones. These results suggested that human bones exhibit diminishing strength relative to stiffness with aging and with decreasing PYD. Incorporating these age- and sex-specific factors may help to improve the accuracy of strength estimates.

Differential Adaptive Response of Growing Bones From Two Female Inbred Mouse Strains to Voluntary Cage-Wheel Running.

The phenotypic response of bones differing in morphological, compositional, and mechanical traits to an increase in loading during growth is not well understood. We tested whether bones of two inbred mouse strains that assemble differing sets of traits to achieve mechanical homeostasis at adulthood would show divergent responses to voluntary cage-wheel running. Female A/J and C57BL6/J (B6) 4-week-old mice were provided unrestricted access to a standard cage-wheel for 4 weeks. A/J mice have narrow and highly mineralized femora and B6 mice have wide and less mineralized femora. Both strains averaged 2 to 9.5 km of running per day, with the average-distance run between strains not significantly different (p = 0.133). Exercised A/J femora showed an anabolic response to exercise with the diaphyses showing a 2.8% greater total area (Tt.Ar, p = 0.06) and 4.7% greater cortical area (Ct.Ar, p = 0.012) compared to controls. In contrast, exercised B6 femora showed a 6.2% (p < 0.001) decrease in Tt.Ar (p < 0.001) and a 6.7% decrease in Ct.Ar (p = 0.133) compared to controls, with the femora showing significant marrow infilling (p = 0.002). These divergent morphological responses to exercise, which did not depend on the daily distance run, translated to a 7.9% (p = 0.001) higher maximum load (ML) for exercised A/J femora but no change in ML for exercised B6 femora compared to controls. A consistent response was observed for the humeri but not the vertebral bodies. This differential outcome to exercise has not been previously observed in isolated loading or forced treadmill running regimes. Our findings suggest there are critical factors involved in the metabolic response to exercise during growth that require further consideration to understand how genotype, exercise, bone morphology, and whole-bone strength interact during growth. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

The biorhythm of human skeletal growth.

Evidence of a periodic biorhythm is retained in tooth enamel in the form of Retzius lines. The periodicity of Retzius lines (RP) correlates with body mass and the scheduling of life history events when compared between some mammalian species. The correlation has led to the development of the inter-specific Havers-Halberg oscillation (HHO) hypothesis, which holds great potential for studying aspects of a fossil species biology from teeth. Yet, our understanding of if, or how, the HHO relates to human skeletal growth is limited. The goal here is to explore associations between the biorhythm and two hard tissues that form at different times during human ontogeny, within the context of the HHO. First, we investigate the relationship of RP to permanent molar enamel thickness and the underlying daily rate that ameloblasts secrete enamel during childhood. Following this, we develop preliminary research conducted on small samples of adult human bone by testing associations between RP, adult femoral length (as a proxy for attained adult stature) and cortical osteocyte lacunae density (as a proxy for the rate of osteocyte proliferation). Results reveal RP is positively correlated with enamel thickness, negatively correlated with femoral length, but weakly associated with the rate of enamel secretion and osteocyte proliferation. These new data imply that a slower biorhythm predicts thicker enamel for children but shorter stature for adults. Our results develop the intra-specific HHO hypothesis suggesting that there is a common underlying systemic biorhythm that has a role in the final products of human enamel and bone growth.