Adolescent growth provides a unique opportunity for the growing body to adapt to external stimuli. A positive association between site-specific mechanical loading and increases in regional bone mineral content (BMC) during adolescence is established. Mechanical loads associated with middle-distance running expose the skeleton to a combination of compressive ground reaction forces and muscular contraction. Previous studies concerning musculoskeletal health in active adolescents are largely limited to planar, two-dimensional measures of bone mineral status, using Dual X-ray Absorptiometry (DXA). Intrinsic bone material properties are accurately measured using DXA. However, the interaction between bone material and structural properties that reflects the mechanical integrity of bone require a combination of imaging modalities. Magnetic Resonance Imaging (MRI) provides a three-dimensional geometric and biomechanical assessment of bone. When MRI is integrated with DXA technology, an effective non-invasive method of assessing in vivo bone strength is achieved. The impact of high training volumes on musculoskeletal development of male and female adolescent athletes engaged in repetitive, high magnitude mechanical loading has not been investigated. Specifically, differences in total body and regional bone mineral, bone and muscle geometry, bone biomechanical indices and bone strength at differentially-loaded skeletal sites have not been compared between adolescent middle-distance runners and age- and gender-matched non-athletic controls. Objectives: (i) to investigate the effects of intense sports participation involving mechanical loading patterns on bone mineral, bone and muscle geometry, biomechanical indices and estimated regional bone strength between elite adolescent male and female middle-distance runners and age- and gender-matched controls (ii) to examine factors predictive of total body BMC, distal tibial bone geometry, distal tibial bone strength, and Hip Strength Analysis (HSA)- derived indicators of bone strength at the femoral neck. Methods: Four groups of 20 adolescents were comprised of male (mean (SD) age 16.8 ± 0.6 yr, physical activity 14.1 ± 5.7 hr.wk-1) and female (age 16 ± 1.7 yr, physical activity 8.9 ± 2.1 hr.wk-1) middle-distance runners, and male (16.4 ± 0.7 yr, physical activity 2.2 ± 0.7 hr.wk-1) and female (age 16 ± 1.8 yr, physical activity 2.0 ± 0.07 hr.wk-1) controls. Total body and regional BMC were calculated using DXA. Distal tibial bone and muscle cross-sectional areas (CSA) were assessed using MRI. To calculate distal tibial bone strength index (BSI), a region of interest representing 10% of the mid distal tibia was analysed for DXA-derived bone mineral and was combined with bone geometry and biomechanical properties from MRI assessments. Calculations for femoral neck strength were acquired from DXA-derived HSA software. Results: No differences were found between male athletes and controls for unadjusted BMC at total body or regional sites. After covarying for fat mass (kg), male athletes displayed greater BMC at the lumbar spine (p = 0.001), dominant proximal femur (p = 0.001) and dominant leg (p = 0.03) than male controls. No differences were found in distal tibial bone geometry, bone strength at the distal tibia or HSA-derived indicators of bone strength at the femoral neck between male athletes and controls. Lean tissue mass and fat mass were the strongest predictors of total body BMC (R2 = 0.71), total muscle CSA explained 43.5% of variance in BSI at the distal tibia, and femur length and neck of femur CSA explained 33.4% of variance at the femoral neck. In females, athletes displayed greater unadjusted BMC at the proximal femur (+3.9 ±1.4 g, p = 0.01), dominant femoral neck (+0.5 ± 0.12 g, p = 0.01) and dominant tibia (+4.1 ± 2.1 g, p = 0.05) than female controls. After covarying for fat mass (kg), female athletes displayed greater (p = 0.001) total body, dominant proximal femur and dominant leg BMC than female controls. Female athletes also showed greater distal tibial cortical CSA (+30.9 ± 9.5 mm2, p = 0.003), total muscle (+240.2 ± 86.4 mm2, p = 0.03) and extensor muscle (+46.9 ±19.5 mm2, p = 0.02) CSA, smaller medullary cavity (-32.3 ± 14.7 mm2, p = 0.03) CSA and greater BSI at the distal tibia (+28037 ± 8214.7 g/cm3.mm4, p = 0.002) than female controls. Lean tissue mass and fat mass were the strongest predictors of total body BMC (R2 = 65), hours of physical weekly activity and total muscle CSA explained 58.3% of the variance of distal tibial BSI, and neck of femur CSA accounted for 64.6% of the variance in a marker of femoral neck HSA. Conclusion: High training loads are associated with positive musculoskeletal outcomes in adolescent middle-distance runners compared to non-athletic controls. Exposure to similar high training loads may advantage female adolescent athletes, more than male adolescent athletes compared with less active peers in bone strength at the distal tibia.

<p> Musculoskeletal injuries are one of the leading causes of human disability. Tissue engineers aim to restore damaged musculoskeletal tissues by creating scaffolds that promote cellular adhesion, proliferation, and eventual differentiation into functional tissue. It is known that the extracellular matrix (ECM) regulates cellular behavior and is often used as a basis for biological scaffolds; however, current scaffolds often mimic the ECM of adult, homeostatic tissue and frequently lead to poor tissue restoration. What is rarely taken into consideration is that the ECM undergoes extensive remodeling during development to facilitate growth.</p> <p>In the musculoskeletal system, myogenic progenitors (<em>Pax3</em>+) and connective tissue cells (<em>Prx1</em>+) proliferate and differentiate into muscle, tendon, cartilage, and conjoining interfaces (<em>e.g.</em> myotendinous junction), while depositing and remodeling the ECM. As tissues mature, cells continue to refine ECM networks to withstand the functional demands to facilitate movement. The ECM composition and architecture of adult musculoskeletal tissues have been studied individually and are thought to be distinct; however, there has yet to be a comprehensive comparative analysis of the ECM in adult muscle, tendon, and the myotendinous junction (MTJ) in a single study. Additionally, how the matrisome of adult musculoskeletal system compares to the ECM dynamics during forelimb development, remain largely unknown due to lack of techniques to analyze embryonic matrisome composition and synthesis. </p> <p>To address these research gaps, we (1) used quantitative proteomics to map the matrisome composition in the mature murine MTJ, relative to the tendon and muscle; (2) adapted tissue fractionation and biorthogonal non-canonical amino acid tagging techniques to embryonic tissues as a method to quantify the global and nascent embryonic matrisome; and (3) subsequently used these techniques to establish a baseline of ECM dynamics during forelimb morphogenesis (embryonic day, E11.5-E14.5) and growth (postnatal day, P3 and P35). We hypothesized that proteomic evaluation of ECM composition and synthesis in developing and adolescent limbs would resolve differences between embryonic and growing tissues. Indeed, we saw significant differences in global and nascent matrisome composition between embryonic and adolescent forelimbs. Notably, the relative abundance and ratios of collagens associated with type I fibrillogenesis (I, III, and V) were significantly different as a function of development embryogenesis and across the adult muscle, MTJ, and tendon.</p> <p>Type I collagen fibrils are critical for tissue architecture and function. Using genetic mouse models, the regulatory roles of COL5A1 in the initiation of type I collagen fibrillogenesis, and organization of subsequent fibrils, have been well characterized in tendons and ligaments; however, is it unknown which cell types contribute COL5A1 to the ECM in the forelimb. To identify the functional contribution of COL5A1 by myogenic or connective tissue cell populations, we generated conditional (cre-flox) knock-out mouse models to inactivate <em>Col5a1</em> using <em>Pax3</em>- or <em>Prx1</em>-drivers, respectively. Haploinsufficiency of <em>COL5A1</em> in humans is associated classical Ehlers-Danlos syndrome, characterized by skin fragility and join instability; similar, albeit more severe, phenotypes were present in <em>Prx1Cre/+;Col5a1fl/fl</em> mutants, but not in <em>Pax3Cre/+;Col5a1fl/fl</em> mutants or controls. Interestingly, THBS4+ and COL22A1+ networks at the MTJ were morphologically affected in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs. Additional work needs to be conducted to characterize the systematic phenotypes observed in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs.</p> <p>Together, our results indicate that there are distinct, complex ECM dynamics, originating from distinct cell-types, that drive musculoskeletal morphogenesis in the forelimb. Further, the tools developed here will serve as a foundation for quantitative proteomic analyses of the matrisome composition in embryonic tissues. Collectively, this work provides a baseline of ECM protein dynamics during musculoskeletal morphogenesis, a helpful guide for tissue engineers in designing scaffolds to promote restoration of damaged tissues, with enhanced integration into the host tissue.</p>