Journal of Anatomy

Chondrichthyans (cartilaginous fishes) form the sister group to osteichthyans (bony fishes) and therefore occupy a key phylogenetic position for comparative studies of early vertebrate evolution. Despite their importance, chondrichthyan development remains understudied relative to established model systems such as mouse, chick, and zebrafish, in part because of limited embryo accessibility and the lack of standardized laboratory resources for rearing. Here, we present the epaulette shark Hemiscyllium ocellatum, a small, oviparous shark as a tractable laboratory system for studying shark development. We provide an overview of epaulette shark husbandry requirements and generate a comprehensive micro-computed tomography imaging series spanning embryonic development through hatching. This dataset provides a three-dimensional anatomical atlas of development for a representative chondrichthyan species. By preserving whole embryos in three dimensions, micro-CT imaging enables developmental morphologies to be visualized at high resolution and in near-native anatomical context. Together with the recently published epaulette shark genome, this developmental atlas helps establish the Epaulette shark for comparative anatomical, developmental, and genomic studies.

Although cartilage in tetrapod skeletons is typically said to lack blood vessels, this is only true for adult cartilage. In young bird and mammal cartilage, a dense network of vasculature-containing tunnels --cartilage canals-- perforate the growing skeleton, helping nourish the cartilage and develop the ossification centers that will later form the skeletons epiphyseal bone. As the canals and their rich vascular network typically recede as animals age, the healthy cartilage of adult animals is typically known to be avascular. Here, however, we use a range of tissue characterization and visualization techniques --including light/electron microscopy and microCT-- to show that the skeletons of rays and sharks (elasmobranch fishes) not only possess cartilage canals, but that these structures persist in the adult skeleton. The morphology and tissue composition of elasmobranch cartilage canals argues homology with mammalian cartilage canals and an ancient invasion of the vascular system into cartilage. However, the anatomical location of canals --extending away from mineralized tissue not toward it-- and the lack of endochondral ossification in ray and shark cartilage suggest that cartilage canals developed early in vertebrates as a transport system for nutrients and mesenchymal cells into the growing skeleton. We describe distinctive features and variation in elasmobranch cartilage canals, discuss their possible roles and their potential for tissue mineralization, and the biomedical implications for their presence in a clade of animals with continuously growing cartilaginous skeletons.

Idiopathic scoliosis is a common spinal disorder characterized by progressive three-dimensional curvature of unknown cause. Although biomechanical imbalance has long been proposed to contribute to scoliosis, the early physiological states that precede curvature onset remain poorly understood. Here, we investigated this problem using zebrafish uts2r3 mutants, which develop fully penetrant juvenile-onset spinal curvature following disruption of urotensin signaling. Transcriptomic analysis before curvature revealed altered expression of muscle-associated genes, suggesting that Uts2r3 influences axial muscle development or function. However, immunofluorescence, birefringence imaging, and quantitative analysis of myotome morphology showed that mutants lack overt muscle architectural defects or dystrophic pathology. By contrast, direct measurements of isolated larval trunks revealed pre-curvature biomechanical abnormalities: namely, uts2r3 mutants generated reduced active force following electrical stimulation while also exhibiting increased passive resistance to stretch. These findings identify urotensin signaling as a regulator of axial tissue biomechanics during growth and suggest that scoliosis-like curvature can arise from an early imbalance between active force generation and passive tissue stiffness. SignificanceSpinal curvature is common, but the biological events that cause the spine to bend during growth remain poorly understood. Animal models, especially zebrafish, make it possible to study these events before curvature begins. Zebrafish lacking urotensin signaling develop spinal curves that arise during juvenile growth, similar to idiopathic scoliosis in humans. Here, we demonstrate that zebrafish lacking the urotensin pathway receptor Uts2r3 develop an abnormal biomechanical state prior to curve onset. Their axial tissues generate less active force when contracting and, at the same time, show increased passive resistance to stretch--an unexpected combination that reveals a distinct pre-curvature biomechanical state. These findings suggest that spinal curvature can arise from an early imbalance in tissue mechanics during growth and identify urotensin signaling as a pathway that helps preserve spinal morphology through a biomechanical mechanism.

ObjectiveX-linked Hypophosphatemia is associated with dental complications, including spontaneous endodontic infections (abscesses) in non-carious teeth and severe periodontal loss. Previous studies have mainly focused on dentin Hypomineralization; however, the structural basis underlying periodontal tissue failure remains unclear. We aimed to investigate histoanatomical abnormalities in the dentin and periodontium of Hyp mice to clarify structural consequences of Phex deficiency in adult molars. MethodsWe performed detailed histological and scanning electron microscopy analyses on the molar regions of untreated adult Hyp mice and wild-type littermates, with particular attention to the structural integrity of the root and periodontal ligament. Additionally, odontoblast process morphology and periodontal attachment abnormalities were evaluated. ResultsHyp molars exhibited marked root abnormalities, including radicular shunt-like defects and disorganized odontoblast processes, particularly in furcation and radicular dentin. Periodontal attachment showed characteristic asymmetry: detachment from the cementum surface was frequently observed, whereas attachment to the alveolar bone surface was relatively preserved. These changes were accompanied by thinning and discontinuity of Sharpeys fibers and increased vascularity in the periodontal ligament. ConclusionsThese findings provide a histoanatomical framework for understanding refractory dental complications in X-linked hypophosphatemia and support the importance of intervention during root development.

Mammal tails have long been recognized for their diversity of morphological form and function, however, there remains a substantial gap between the motivation to understand and emulate the various performance functions of the tail and what is known about tail anatomy. In this study, we were motivated to discover the anatomical foundations of the fast, whipping motions of the tail of the lesser Egyptian jerboa (Jaculus jaculus), which may aid in the quick changes of direction as the animal escapes from predators using ricochetal bipedal hopping. We employed microCT scans, dissections, and museum data to describe the musculoskeletal anatomy of the jerboa in comparison with the laboratory mouse (Mus musculus) and rat (Rattus norvegicus). While many aspects of tail anatomy are conserved across these species, the jerboa does possess unique characteristics such as an extremely long tail arising from caudal vertebral elongation, development of extensive dorsal musculature differentiated into lateral and medial components to increase points of skeletal attachment, and a novel anatomical feature - the bi-lobed cranial transverse process - which serves as a supernumerary dorsal tendon attachment site and possible brace to protect the ventral tendons and intrinsic muscles for a section of caudal vertebrae which likely experiences high mechanical stress.

The diversity of body shapes is one of the most prominent features of phenotypic variation in mammals. Yet, mammalian body shapes are poorly quantified and the underlying components contributing to its diversity as well as its relationship to other components of the skeleton are rarely tested. Here, we use lagomorphs (hares, rabbits and pikas) as a model system to (1) investigate which components of the skeleton contributed the most to body shape diversity, (2) examine the relationships between body shape and relative limb lengths, and (3) test how body size, ecotype, burrowing behavior, and locomotor mode influenced variation in lagomorph body shape and appendicular morphology. We quantified the body shape and functional proxies of the appendicular skeleton in 40 lagomorph species from osteological specimens held at museum collections. Using phylogenetic comparative methods, we found the relative length of the ribs and elongation or shortening of the thoracic and lumbar regions contributed the most to body shape evolution across lagomorphs. Second, we found that only leporids (hares and rabbits) exhibited a significant relationship between limb length and body shape, where more elongate species exhibit relatively shorter forelimbs and hindlimbs. Lastly, we found that models incorporating body size were the best predictors of lagomorph body shape and the majority of the appendicular traits, whereas models incorporating burrowing behavior and locomotor mode were largely poor fits. Broadly, these results indicate that larger lagomorphs tend to exhibit more robust body shapes with longer, more gracile forelimbs, whereas smaller lagomorphs tend to exhibit more elongate body shapes with shorter, more robust forelimbs. Overall, this work contributes to the growing understanding of mammalian body shape evolution and demonstrates the importance of not omitting body size in ecomorphological analyses.

Abstract Introduction: Proximal humerus fractures account for approximately 5-6% of all adult fractures and are primarily managed nonoperatively. Healing is conventionally monitored with radiographs, with radiopaque callus formation indicating healing. Visible radiographic callus appears weeks after biological union begins. Ultrasound provides a dynamic, radiation-free, and cost-effective method that can detect early callus formation before x-ray visibility. Although ultrasound has demonstrated utility for fracture healing in the clavicle and humeral shaft, its role in proximal humerus fractures remains unclear. Methods: This single-centre prospective study will be conducted in two phases. The pilot phase will measure inter-rater reliability for ultrasound detection of early callus formation at 2 and 4 weeks post-injury. Ten patients with proximal humerus fractures treated nonoperatively will undergo standardized anterior and lateral scans. Each patient will generate four saved images (short- and long-axis views), producing forty anonymized images independently reviewed by two raters. The prospective cohort phase will recruit approximately thirty additional patients. Results: Reliability will be quantified using Cohens kappa. A power calculation will be performed after pilot analysis. Results from the prospective cohort phase will help determine the association and predictive value of early ultrasound-detected bridging callus for radiographic and clinical union at three and six months. Patient reported outcome measures will be assessed using the Quick Disabilities of Arm, Shoulder and Hand (QuickDASH) questionnaire. Discussion: This study will develop and validate a standardized ultrasound protocol for assessing early fracture healing in proximal humerus fractures. By establishing both inter-rater reliability and predictive value, the findings may support ultrasound as a reproducible, radiation-free adjunct to conventional imaging and enable earlier identification of union status.

Congenital heart defects affect females and males differently. Several congenital heart defects arise during the formation of the heart tube, suggesting that heart tube morphogenesis may differ between females and males. We investigated if the fruit fly Drosophila melanogaster displays sexual dimorphisms in the cellular mechanisms of heart tube formation. Quantitative microscopy revealed no differences between females and males in the migration of cardiac progenitors to form the heart tube. Our results suggest that Drosophila do not display sexual dimorphisms in early cardiac development, and support the omission of sex as an experimental variable when investigating Drosophila heart tube morphogenesis.

Craniofacial birth defects, such as cleft lip and palate, are among the most common congenital anomalies and often arise from disruptions in early facial patterning. Many of these defects are linked to environmental teratogens, yet such exposures cannot be directly tested in humans, making animal models essential for evaluating developmental risks. Xenopus laevis offers a powerful solution: its tadpoles develop externally, share deeply conserved craniofacial patterning mechanisms with humans, and provide an accessible platform for uncovering how environmental exposures reshape facial structures during development. Here, we present the XenCart Protocol, a reproducible workflow for Alcian Blue staining and quantitative morphometric analysis of Xenopus craniofacial cartilage. This method provides clear visualization of individual cartilage elements and can be readily applied to investigate genetic or environmental perturbations. The Xenopus craniofacial skeleton contains distinct cartilaginous structures that perform key biomechanical functions and share strong homology with regions of the human craniofacial skeleton. These similarities allow direct comparison of developmental outcomes across vertebrates. As part of a CURE-based undergraduate course, the XenCart Protocol was used to measure jaw cartilage dimensions in tadpoles exposed to an emerging teratogen, e-liquids used in vaping. E-liquid exposure caused consistent reductions across major craniofacial cartilages, including shorter Meckels cartilage, narrowed infrarostral width, decreased basihyobranchial and ceratohyal dimensions, and reduced suprarostral angles, reflecting an overall shift toward a smaller, more compact craniofacial morphology. These patterns suggest potential disruption of neural crest cell migration or signaling pathways for craniofacial cartilage development, mechanisms that, if similarly affected in humans, could contribute to midfacial narrowing, jaw underdevelopment, or increased vulnerability to conditions such as orofacial clefts. The ability to detect robust, structure-specific differences highlights the sensitivity of the protocol and its strong alignment with student-led research. These findings also pinpoint the precise regions of the jaw most affected by e-liquid exposure, providing a foundation for uncovering the developmental mechanisms driving these craniofacial changes. In summary, the XenCart Protocol provides a standardized, scalable method for quantifying craniofacial cartilage development and offers a powerful platform for both mechanistic research and undergraduate training in developmental biology and toxicology.

The peripheral auditory system of dolphins comprises specialised bony, fatty, vascular, and neural structures adapted for underwater hearing and diving physiology. These include the external ear canal, acoustic fat bodies, sinuses, and associated neurovascular networks, which together support sound conduction, protection, and possibly sensory functions. Despite advances in gross anatomical description, the detailed integration of these tissues, particularly the innervation, neurovascular organisation, and their functional implications, remains poorly understood. Previous studies have described the presence of sensory nerve formations and vascular plexuses, but their arrangement, connectivity, and relation to each other are unresolved. Here, we combine macroscopic dissection, DICE-{micro}CT, histology, and high-resolution confocal microscopy to characterise several neurovascular and sensory components of the dolphin peripheral auditory system in several delphinid species. Macroscopic dissection and DICE-{micro}CT revealed the traditional acoustic fat body distribution with detailed morphology of the posterolateral extension that is not well-known. The cranial nerve distribution, and specifically the mandibular nerve branching patterns, are described in detail. Confocal microscopy uncovered a stratified neurovascular plexus around the external ear canal with a complex sensory system comprising lamellar corpuscles, Merkel cell-neurite complexes, and intraepithelial nerve fibres. Notably, the lamellar corpuscles formed a continuous, three-dimensional neural network with frequent merging and splitting of axonal bundles, shared perineuria, and vascular integration, features not observed in previous studies. Our findings demonstrate that the dolphin external ear canal and surrounding structures form a sophisticated, multimodal somatosensory organ, integrating structural, vascular, and neural specialisations likely adapted for proprioceptive mechanosensation in the aquatic environment. This study provides insights into the integration of the various components of the peripheral hearing apparatus. Future studies integrating anatomical, electrophysiological, and biomechanical approaches are needed to fully elucidate these adaptations.

Sharks exhibit extraordinary morphological diversity across a wide range of ecological niches, yet large-scale, high-resolution digital datasets of their internal anatomy remain limited. Here we present an open-access 3D shark anatomical repository derived from published X-ray computed tomography (CT) data, featuring manually segmented and systematically annotated models of the chondrocranium, visceral arches, axial skeleton, musculature, and viscera in standard STL format. The dataset comprises 117 individuals, representing 72 species across 25 families and all nine extant shark orders, with 115 full-body reconstructions and two head-only models. This open-access dataset offers a comprehensive resource for comparative anatomy, biomechanical simulations, evolutionary developmental biology and biomimetics research of extant sharks.

Gorgonopsian therapsids represent a transitional condition in the evolution of synapsid locomotion and postcranial structure. Most descriptions of gorgonopsians have focused on cranial material, however, limiting their usefulness for informing patterns of postcranial evolution on the mammal stem. While some recent work has begun to focus on postcrania, especially the pectoral girdle and forelimbs, comparatively little data are available on the pelvic girdle, hind limbs and tail. We report a new specimen of the late Permian gorgonopsian Aelurognathus tigriceps comprising a partial skull and well-preserved postcranial skeleton, including the near-complete series of dorsal vertebrae and ribs, complete pelvic girdle, hind limbs, feet, and a nearly complete tail. The tail is longer than any other published gorgonopsian. The new material presented here provides an opportunity to better establish broader patterns of morphology in the gorgonopsian postcranial skeleton.

IntroductionHealthy and diseased placentae alike often display some degree of pathology. However, quantitative techniques to characterize common pathologies and their relationship to local maternal hemodynamics in healthy primate placentae are currently limited. MethodsPlacentae from seven rhesus macaques were imaged by MRI at three time points across mid-to late-gestation, to quantify placental blood volume, flow, and perfusion from maternal spiral arteries across pregnancy. Near term, we collected placental cotyledons, digitized hematoxylin/eosin-stained slides, then segmented and annotated sub-tissues and major pathologies (intervillous gaps, fibrin deposition, villous agglutination, inflammatory agglutination, and stromal mineralization) within each cotyledon. Individual pathologies were assessed in relation to each other and MRI perfusion metrics, in a cotyledon-specific manner. Parallel analyses were performed to investigate both basic (Spearman correlation) and animal variance-negated (dimensionality-reduction) relationships. ResultsCotyledons with increased stromal mineralization demonstrated low blood perfusion across pregnancy, alongside significant compensatory changes. Mineralization was further associated with decreased fetal weight, across all sub-tissues. Dimensionality reduction revealed maternal vascular malperfusion-associated pathologies as the largest contributor to dataset variance. Additionally, pathologies commonly associated with healthy placental function demonstrated low cotyledon blood flow and volume at all timepoints, with no evidence of compensatory changes across gestation. ConclusionsComprehensive digital annotation revealed several relationships connecting pathology and maternal blood perfusion in the healthy primate pregnancy, at the smallest functional unit of the placenta. This methodological framework embeds pathologist-refined morphological expertise into a quantitative, spatially resolved format that can ground, rather than be replaced by, unsupervised computational approaches to placental analysis.

Relapse of aligned mandibular anterior teeth and the progressive collapse of the mandibular anterior arch are historically striking problems for orthodontists. The etiology of this collapse, and the cause of mesial molar drift, are unknown. However, light continuous (quasi-continuous) intra-oral pressures and forces applied to the mandibular dentition have been implicated. To explore this further, we use three-dimensional finite element analysis to investigate the influence of these intra-oral loads (tongue pressure, lip-cheek pressure, and interdental force) on mandibular arch collapse and mesial molar drift. Dentitions of three-dimensional finite element mandibular models were subjected to a wide range of simulated tongue pressures, lip-cheek pressures, and transseptal fiber-mediated interdental forces reported in the literature. Resulting crown displacement measurements from these isolated loads were made along with measurements resulting from simultaneous combined application of literature-defined mean tongue pressure, lip-cheek pressure, and interdental force. Our results indicate that tongue pressure alone results in generalized arch expansion and tooth spacing while lip-cheek pressure and interdental force result in generalized arch collapse, anterior crowding, and mesial molar displacement. Simultaneous application of tongue pressure, lip-cheek pressure, and interdental force mean values, as would occur in vivo, results in incisor crowding, intercanine width reduction, and mesial molar displacement. Our results suggest mandibular anterior arch collapse (incisor crowding / intercanine width reduction), and mesial molar displacement result from simultaneous application of tongue pressure, lip-cheek pressure, and interdental force.

The morphogenesis of the optic cup provides a robust system for studying how two apposed epithelial monolayers with distinct properties fold and stretch in a coordinated manner to form the primordial eye. While much research has been conducted on the temporal dynamics of retinal neuroepithelium invagination, the spatial organization and stretching of the retinal pigment epithelium has received less attention. The fish species Astyanax mexicanus offers a unique model to examine the mechanisms of optic tissue morphogenesis through a comparative lens, as it exhibits natural variation in eye development between its river-dwelling and cave-adapted morphs. Using quantitative 3D imaging of optic cups from both morphs, we found that RPE morphogenesis involves transient, graded, and anisotropic cell stretching that patterns the epithelium during optic cup shaping. Analyses of RPE nuclear spacing and cell morphology showed that tissue stretching gradually increases along the proximo-distal axis, suggesting maximal tension in the elongated distal RPE cells aligned along the optic cup meridians. Furthermore, nuclear volumes and apical surface areas of RPE cells scaled spatially along the same axis, independently of endoreplication. In the cavefish natural mutant, RPE expansion was delayed by over six hours and proximal stretching exhibited altered isotropy, indicative of disrupted temporal coordination and suggesting modified mechanical constraints. These results demonstrate that RPE morphogenesis is a highly heterogeneous process from a spatiotemporal perspective, offering new insights into the study of the biomechanical principles of eye development in vertebrates. Summary statementThis study reveals the emergence of cell morphology gradients within the retinal pigment epithelium during morphogenesis of the eye in two distinct populations of the same species of fish.

Gene inactivation in model organisms has identified numerous genes and signaling pathways involved in mammalian cardiac OFT development. Human genetics data have implicated the VEGFR3 gene in OFT development but when and where it is required is unknown. In this study we determined the sensitivity of the developing murine cardiac OFT to reduced Vegfr3 gene dosage and we tested whether its requirement is dependent upon TBX1, a known regulator of Vegfr3 expression in cardiac and lymphatic endothelial cells. We found that in the mouse, a single copy if the Vegfr3 gene was sufficient for normal cardiac OFT development in most cases. Mutation of a single copy of the Tbx1 gene greatly enhanced the sensitivity of OFT development to Vegfr3 dosage reduction and led to the formation of severe OFT anomalies. In addition, deletion of Vegfr3 in the Tbx1 expression domain also led to OFT abnormalities. We used RNAscope to reveal the location of Vegfr3 and Tbx1 transcripts in midterm mouse embryos. This revealed co-localization of these transcripts that was restricted to the aortic sac endothelium, suggesting that the distal OFT is a potential site of genetic interaction between Vegfr3 and TBX1 that is critical for normal OFT development.

Extremely elongate necks have convergently evolved in several amniote lineages, including both aquatic and terrestrial forms (Fig. 1). The development of such a feature brings with it advantages in obtaining food items, but also biomechanical challenges, such as flexibility, stability, lift, and inertia. In Tanystropheus, a particularly long-necked Triassic archosauromorph, the neck is composed of only 13, mostly extraordinarily elongated and slender cervical vertebrae and accompanying rod-like, overlapping ribs, making it arguably the most extreme example of neck elongation in tetrapod evolution (Fig. 1;1-6). Understanding the function of this remarkable neck provides insights into the limits of neck elongation in amniotes and the evolution of morphological novelties in Triassic reptiles. Here we present the first quantitative biomechanical analysis of the Tanystropheus neck using a digital model based on three-dimensionally preserved bones. We assessed its range of motion (ROM) and performed finite element analysis (FEA) on the individual cervical ribs and the neck model in different configurations. Our results indicate that the neck of Tanystropheus was not extremely stiff, as previously postulated, and the ribs likely did not impair its movements. They transferred tensile forces towards the base of the neck, similar to what hypothesized for sauropods7. This study elucidates the bauplan of an extremely specialized animal and brings us closer to understanding the patterns of achieving neck elongation in vertebrates.

Flight feather vanes are the primary aerodynamic surface of the avian wing. Because loading varies across the wing, vane macrostructure should co-vary with local mechanical demands, yet comparative data on how barb and barbule traits change among remiges and between vane surfaces remain scarce. We quantified barb density, barbule density, barb angle, barb length, and vane width on both vanes at three measurement positions along the rachis of all remiges in four species with contrasting flight modes (white stork, common buzzard, house sparrow, pygmy cormorant), generating over 40,000 measurements across 15 response variables from 992 feathers of 41 individuals. Two complementary generalised additive models characterised variation along the spanwise, inter-vane, and longitudinal axes, and compared outer primaries, inner primaries, and secondaries as functional wing regions. Feather macrostructure varied along all three axes and outer primaries represent the most distinctive region, with lower leading-vane barb density, reduced barb angles, and vane width asymmetry two to three times higher than in inner primaries or secondaries. House sparrow exhibited the densest vane architecture and the highest vane width asymmetry, whereas the low wing-beat frequency species showed complex nonlinear spanwise patterns undetectable by single-feather sampling. Pygmy cormorant barbule density was 39-53% lower than in all other species, matching its wettable plumage strategy. Longitudinal gradients in barb density and barb angle (22-31% decline) were conserved across species. The avian wing is thus functionally regionalised at the macrostructural level, with vane architecture reflecting both aerodynamic and ecological pressures. Summary statementFine-scale vane measurements across all remiges in four species show macrostructural regionalisation of the avian wing, with outer primaries showing the most distinctive vane architecture.

BackgroundThe pivot shift (PS) test is the most specific clinical examination for anterolateral rotational instability in ACL-deficient knees, yet grading remains subjective, as evidenced by poor inter-observer reliability, particularly for Grade 2. Since low-grade (Grade 1) versus high-grade (Grades 2/3) PS is the threshold for recommending lateral extra-articular augmentation, performing the test in awake clinic patients limits grading reproducibility and introduces variability in surgical decision-making. Existing methods to quantify the pivot shift usually require examiner-performed testing under general anaesthesia. No prior approach has ascertained PS grading from a separate patient-performed functional movement. PurposeTo evaluate the feasibility of a machine learning (ML) classifier, trained on kinematic ultrasound bone-tracking signals acquired during a patients sit-stand-sit (SSS) knee movement, to predict their PS grade, and to clinically validate its ability to differentiate low versus high-grade PS. MethodsUltrasound bone-tracking kinematic data were collected during SSS manoeuvres in 23 ACL-injured patients using the GATOR device, and ground truth PS grades (0-3) were assigned under general anaesthesia by fellowship-trained orthopaedic sports surgeons. From the data collected, Leave-one-out cross-validation (LOOCV) was used to train the ML classifier. Clinical SSS data from 6 ACL-deficient patients was used for independent held-out validation of their low-grade (Grade 1) versus high-grade (Grade 2/3) PS. Multiple deep learning architectures (XceptionTime, InceptionTime, FCN, ResNet, ResCNN) and training strategies (including mixup augmentation and supervised contrastive learning) were tested. Performance was measured by one-versus-rest (OVR) AUC under LOOCV and by AUC (low vs high grade PS) from the held-out patient sessions. ResultsThe ML classifier achieved a maximum OVR AUC of 0.928 {+/-} 0.084 under LOOCV. Classifier performance increased with pivot-shift severity: Grade 3 was identified most reliably (AUC ~0.81; sensitivity 0.70-0.80), whereas Grade 2 remained the most challenging boundary (sensitivity 0.20-0.75 across configurations). For the clinically relevant binary classification of low-versus high-grade pivot shift, the classifier generalised well to a completely unseen patient cohort (AUC 0.889; accuracy 0.860; sensitivity 0.850; minimum-class sensitivity 0.767). ConclusionThe study demonstrates that kinematic ultrasound bone-tracking during sit-stand-sit contains transferable information about rotational instability severity in ACL-deficient patients, and represents the first reported approach to predict pivot shift grade from a patient-performed functional movement. The strong cross-validation performance confirms that the signals contain meaningful PS grade-discriminative information, but larger datasets targeting 50-100 sessions per grade will be required to achieve patient-level generalisation and advance this novel rotational instability assessment tool toward full clinical adoption. Level of EvidenceLevel IV, diagnostic feasibility study.

Large full-thickness (LFT) skin wounds remain a major clinical challenge, and progress in regenerative medicine has been limited by poor translation from animal models to humans. A key limitation is that commonly used species such as mice, rats, and rabbits are loose-skinned, whereas humans are tight-skinned with distinct skin architecture. Although pigs more closely resemble human skin, widely used breeds have lost secondary (vellus-like) hair follicles through artificial selection, restricting their utility for studying ectodermal organ regeneration. Here, we characterize the development, patterning, and molecular features of secondary hair follicles in the Lanyu pig (Sus scrofa taivanus), an indigenous breed that retains these structures. Whole-mount and histological analyses revealed two distinct follicle populations: primary follicles arranged in stable triplet clusters and smaller secondary follicles distributed interstitially. A developmental time course using alkaline phosphatase (ALP) staining identified sequential stages of secondary follicle morphogenesis--placode, hair germ, hair peg, and mature follicle--occurring after primary follicle establishment. Immunohistochemical analysis demonstrated conserved epithelial- mesenchymal interactions, progressive epithelial stratification, and dynamic {beta}-catenin signaling during secondary follicle development. Keratin expression patterns and follicular architecture closely resembled those of human vellus hair follicles, supporting the translational relevance of this model. Notably, secondary follicles were retained into adulthood, and genetic analyses of outcrossed animals suggest that this trait follows an autosomal dominant inheritance pattern. Together, these findings establish the Lanyu pig as a tight-skinned mammalian model that preserves vellus-like hair follicles, providing a platform for investigating hair follicle-mediated skin regeneration and improving translational relevance for human wound healing.