Prenatal murine skeletogenesis partially recovers from absent skeletal muscle as development progresses.

Skeletal muscle contractions are critical for normal skeletal growth and morphogenesis but it is unclear how the detrimental effects of absent muscle on the bones and joints change over time. Joint shape and cavitation as well as rudiment length and mineralisation were assessed in multiple rudiments at two developmental stages [Theiler stage (TS)24 and TS27] in the splotch-delayed "muscle-less limb" mouse model and littermate controls. Chondrocyte morphology was quantified in 3D in the distal humerus at the same stages. As development progressed, the effects of absent muscle on all parameters except for cavitation become less severe. All major joints in muscle-less limbs were abnormally shaped at TS24, while, by TS27, most muscle-less limb joint shapes were normal or nearly normal. In contrast, any joints that were fused at TS24 did not cavitate by TS27. At TS24, chondrocytes in the distal humerus were significantly smaller in the muscle-less limbs than in controls, while by TS27, chondrocyte volume was similar between the two groups, offering a cell-level mechanism for the partial recovery in shape of muscle-less limbs. Mineralisation showed the most pronounced changes over gestation. At TS24, all muscle-less rudiments studied had less mineralisation than the controls, while at TS27, muscle-less limb rudiments had mineralisation extents equivalent to controls. In conclusion, the effects of muscle absence on prenatal murine skeletogenesis reduced in severity over gestation. Understanding how mammalian bones and joints continue to develop in an environment with abnormal fetal movements provides insights into conditions including hip dysplasia and arthrogryposis.

Differential effect of frequency and duration of mechanical loading on fetal chick cartilage and bone development.

Developmental engineering strategies aim to recapitulate aspects of development in vitro as a means of forming functional engineered tissues, including cartilage and bone, for tissue repair and regeneration. Biophysical stimuli arising from fetal movements are critical for guiding skeletogenesis, but there have been few investigations of the biomechanical parameters which optimally promote cartilage and bone development events in in vitro explants. The effect of applied flexion-extension movement frequencies (0.33 and 0.67 Hz) and durations (2 h periods, 1, 2 or 3 × per day) on knee (stifle) joint cartilage shape, chondrogenesis and diaphyseal mineralisation of fetal chick hindlimbs, cultured in a mechanostimulation bioreactor, were assessed both quantitatively and qualitatively. It was hypothesised that increasing frequency and duration of movements would synergistically promote cartilage and bone formation in a dose-dependent manner. Increasing loading duration promoted cartilage growth, shape development and mineralisation of the femoral condyles and tibiotarsus. While increasing frequency had a significant positive effect on mineralisation, hyaline cartilage growth and joint shape were unaffected by frequency change within the ranges assessed, and there were limited statistical interactions between the effects of movement frequency and duration on cartilage or bone formation. Increased glycosaminoglycan deposition and cell proliferation may have contributed to the accelerated cartilage growth and shape change under increasing loading duration. The results demonstrated that frequencies and durations of applied biomechanical stimulation differentially promoted cartilage and bone formation, with implications for developmentally inspired tissue engineering strategies aiming to modulate tissue construct properties.

Prenatal muscle forces are necessary for vertebral segmentation and disc structure, but not for notochord involution in mice.

Embryonic muscle forces are necessary for normal vertebral development and spinal curvature, but their involvement in intervertebral disc (IVD) development remains unclear. The aim of the current study was to determine how muscle contractions affect (1) notochord involution and vertebral segmentation, and (2) IVD development including the mechanical properties and morphology, as well as collagen fibre alignment in the annulus fibrosus. Muscular dysgenesis (mdg) mice were harvested at three prenatal stages: at Theiler Stage (TS)22 when notochord involution starts, at TS24 when involution is complete, and at TS27 when the IVD is formed. Vertebral and IVD development were characterised using histology, immunofluorescence, and indentation testing. The results revealed that notochord involution and vertebral segmentation occurred independently of muscle contractions between TS22 and TS24. However, in the absence of muscle contractions, we found vertebral fusion in the cervical region at TS27, along with (i) a displacement of the nucleus pulposus towards the dorsal side, (ii) a disruption of the structural arrangement of collagen in the annulus fibrosus, and (iii) an increase in viscous behaviour of the annulus fibrosus. These findings emphasise the important role of mechanical forces during IVD development, and demonstrate a critical role of muscle loading during development to enable proper annulus fibrosus formation. They further suggest a need for mechanical loading in the creation of fibre-reinforced tissue engineering replacement IVDs as a therapy for IVD degeneration.

Initiation and emerging complexity of the collagen network during prenatal skeletal development.

The establishment of a complex collagen network is critical for the architecture and mechanical properties of cartilage and bone. However, when and how the key collagens in cartilage and bone develop has not been characterised in detail. The study provides a detailed qualitative characterisation of the spatial localisations of collagens I-III, V-VI and IX-XI in the mouse and their regional architecture variation over three developmentally significant time points: when the rudiment starts to form at E13.5 [Theiler stage (TS) 22], when mineralisation is present at E16.5 (TS25) and during the latest prenatal stage at E18.5 (TS27). Dynamic changes in collagen distribution between stages with the progression of the growth plate and mineralisation (particularly collagens I, II, V, X and XI) and dramatic changes in collagen structural organisation and complexity with maturation, especially for collagens II and XI, were observed. The future articular cartilage region was demarcated by pronounced collagens II and VI expression at TS27 and the emergence of collagens I, III, V, IX and XI in the tendon and its insertion site was observed. The present study revealed, for the first time, the emergence and maturation of key cartilage and bone collagens, in high resolution, at multiple locations across the entire rudiment, including the joint regions, at three of the most developmentally significant stages of skeletogenesis, furthering the understanding of disease and regeneration of skeletal tissues.

Short-term foetal immobility temporally and progressively affects chick spinal curvature and anatomy and rib development.

Congenital spine deformities may be influenced by movements in utero, but the effects of foetal immobility on spine and rib development remain unclear. The purpose of the present study was to determine (1) critical time-periods when rigid paralysis caused the most severe disruption in spine and rib development and (2) how the effects of an early, short-term immobilisation were propagated to the different features of spine and rib development. Chick embryos were immobilised once per single embryonic day (E) between E3 and E6 and harvested at E9. To assess the ontogenetic effects following single-day immobilisation, other embryos were immobilised at E4 and harvested daily between E5 and E9. Spinal curvature, vertebral shape and segmentation and rib development were analysed by optical projection tomography and histology. The results demonstrated that periods critical for movement varied for different aspects of spine and rib development. Single-day immobilisation at E3 or E4 resulted in the most pronounced spinal curvature abnormalities, multiple wedged vertebrae and segmentation defects, while single-day immobilisation at E5 led to the most severe rib abnormalities. Assessment of ontogenetic effects following single-day immobilisation at E4 revealed that vertebral segmentation defects were subsequent to earlier vertebral body shape and spinal curvature abnormalities, while rib formation (although delayed) was independent from thoracic vertebral shape or curvature changes. A day-long immobilisation in chicks severely affected spine and rib development, highlighting the importance of abnormal foetal movements at specific time-points and motivating targeted prenatal monitoring for early diagnosis of congenital scoliosis.

Biomechanics of foetal movement.

Foetal movements commence at seven weeks of gestation, with the foetal movement repertoire including twitches, whole body movements, stretches, isolated limb movements, breathing movements, head and neck movements, jaw movements (including yawning, sucking and swallowing) and hiccups by ten weeks of gestational age. There are two key biomechanical aspects to gross foetal movements; the first being that the foetus moves in a dynamically changing constrained physical environment in which the freedom to move becomes increasingly restricted with increasing foetal size and decreasing amniotic fluid. Therefore, the mechanical environment experienced by the foetus affects its ability to move freely. Secondly, the mechanical forces induced by foetal movements are crucial for normal skeletal development, as evidenced by a number of conditions and syndromes for which reduced or abnormal foetal movements are implicated, such as developmental dysplasia of the hip, arthrogryposis and foetal akinesia deformation sequence. This review examines both the biomechanical effects of the physical environment on foetal movements through discussion of intrauterine factors, such as space, foetal positioning and volume of amniotic fluid, and the biomechanical role of gross foetal movements in human skeletal development through investigation of the effects of abnormal movement on the bones and joints. This review also highlights computational simulations of foetal movements that attempt to determine the mechanical forces acting on the foetus as it moves. Finally, avenues for future research into foetal movement biomechanics are highlighted, which have potential impact for a diverse range of fields including foetal medicine, musculoskeletal disorders and tissue engineering.

Smaller, weaker, and less stiff bones evolve from changes in subsistence strategy.

UNLABELLED: We propose a computational model with which to examine the evolution of bone. Our results indicate that changes in subsistence strategy have influenced the evolution of bone growth and mechanoregulation, and predict that bone size, stiffness, and structural strength may decrease in future generations, bringing increased risk of fracture and prevalence of osteoporosis. INTRODUCTION: Archeological data suggest that bone size and strength have decreased over evolution. We hypothesize that changing evolutionary pressures and levels of physical activity, both arising from changes in subsistence strategy, have affected the evolution of bone. We propose a computational model with which to examine the evolution of bone growth and mechanoregulation due to the transitions from hunter-gatherer to agricultural to modern lifestyles. METHODS: The evolution of genes governing growth and mechanoregulation in a population of bones is simulated, where each individual is represented by a 2-D bone cross-section. Genetic variability is assumed to modulate growth through mechanoregulatory factors that direct periosteal expansion, endosteal expansion/infilling, and ash content accretion in response to strains incurred during walking. RESULTS: The model predicts decreases in cortical area and section modulus (a measure of structural strength) and increases in maximum compressive strain over the course of the simulation, meaning evolution of smaller, less strong, and less stiff bones is predicted for the population average. The model predicts small but continued decreases in size, strength, and stiffness in modern populations, despite the absence of a strong evolutionary advantage to efficient bones during this phase. CONCLUSION: In conclusion, our results show that changing loading regimes and evolutionary pressures may have influenced the evolution of bone growth and mechanoregulation, and predict that bone size and strength may continue to decrease in future generations, bringing increased risk of fracture and prevalence of osteoporosis.