The Role of Gene Expression Changes in ceRNA Network Underlying Ossification of Ligamentum Flavum Development.

We aimed at exploring the role of gene expression changes regulated by non-coding RNAs in ossification of ligamentum flavum (OLF). Three microarray datasets, including long non-coding RNA (lncRNA)/mRNA expression profile (GSE106253), circular RNA (circRNA) expression profile (GSE106255), and microRNA (miRNA) expression profile (GSE106256), were downloaded from the public Gene Expression Omnibus repository. The differentially expressed (DE) mRNAs, lncRNAs, miRNAs, and circRNAs in OLF tissues were analyzed, compared with normal tissues. Two competing endogenous RNA (ceRNA) networks with lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA interactions were constructed, separately. Random walk with the restart model was applied to calculate the correlations of mRNAs with the published OLF-related genes. The top 50 mRNAs were subjected to function enrichment analysis and active small-molecule prediction. Total 2323 DE mRNAs, 1168 lncRNAs, 336 circRNAs, and 29 miRNAs were identified based on the microarray datasets. The LncRNA-related ceRNA network was constructed with 614 lncRNA-miRNA, 494 miRNA-mRNA, and 2099 lncRNA-mRNA interaction pairs; the circRNA-related ceRNA network was constructed with 153 circRNA-miRNA, 190 miRNA-mRNA, and 210 circRNA-mRNA interaction pairs. There were 17 OLF-related genes retrieved from previous literature, such as NPPS, COL6A1, and COL11A2, among which COL6A1 was the overlapped gene with mRNAs in the ceRNA network. Subsequently, top 50 mRNAs that closely correlated with COL6A1 in the ceRNA network were captured and these genes were closely related with the collagen catabolic process, regulation of cell growth, and neuronal action potential. DRD1 and COL6A1 were predicted to be the targets by small active molecule drugs. The collagen catabolic process may be implicated in OLF development. COL6A1 and DRD1 may be the candidate targets for OLF. However, further validations were needed.

Histological development and integration of the Zebrafish Weberian apparatus.

BACKGROUND: The Weberian apparatus enhances hearing in otophysan fishes, including Zebrafish (Danio rerio). Several studies have examined aspects of morphological development of the Weberian apparatus and hearing ability in Zebrafish. A comprehensive developmental description including both hard and soft tissues is lacking. This information is critical for both interpretation of genetic developmental analyses and to better understand the role of morphogenesis and integration on changes in hearing ability. RESULTS: Histological development of hard and soft tissues of the Weberian apparatus, including ossicles, ear, swim bladder, and ligaments are described from early larval stages (3.8 mm notochord length) through adult. Results show a strong relationship in developmental timing and maturation across all regions. All required auditory elements are present and morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and changes in shape continue throughout the examined developmental period. CONCLUSIONS: This study provides the first comprehensive histological description of Weberian apparatus development in Zebrafish. Morphological integration was found early, before increases in hearing ability were detected in functional studies (>10 mm total length), suggesting morphological integration precedes functional integration. Further research is needed to examine the nature of the functional delay, and how maturation of the Weberian apparatus influences functionality.

An overview of decellularisation techniques of native tissues and tissue engineered products for bone, ligament and tendon regeneration.

Decellularised tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications. Because of the complexity of each tissue (size, porosity, extracellular matrix (ECM) composition etc.), there is no standardised protocol and the decellularisation methods vary widely, thus leading to heterogeneous outcomes. Physical, chemical, and enzymatic methods have been developed and optimised for each specific application and this review describes the most common strategies utilised to achieve decellularisation of soft and hard tissues. While removal of the DNA is the primary goal of decellularisation, it is generally achieved at the expense of ECM preservation due to the harsh chemical or enzymatic processing conditions. As denaturation of the native ECM has been associated with undesired host responses, decellularisation conditions aimed at effectively achieving simultaneous DNA removal and minimal ECM damage will be highlighted. Additionally, the utilisation of decellularised matrices in regenerative medicine is explored, as are the most recent strategies implemented to circumvent challenges in this field. In summary, this review focusses on the latest advancements and future perspectives in the utilisation of natural ECM for the decoration of synthetic porous scaffolds.

Age-related changes in inguinal region anatomy from 0 to 19 years of age.

Knowledge of the age-related changes in inguinal region anatomy is essential in pediatric urological and abdominal surgery, yet little is published. This study aimed to determine the position of inguinal region structures and growth of the surrounding pelvis and inguinal ligament in subjects from 0 to 19 years of age. Anonymized contrast-enhanced CT DICOM datasets of 103 patients (63 male: 40 female) aged from 0 to 19 years had left and right sides analyzed by three independent observers. Exclusion criteria were applied. Growth of the pelvis and inguinal ligament were determined using fixed bony reference points. The position of the deep inguinal ring and femoral vasculature were determined as ratio of inguinal ligament length, measured from the anterior superior iliac spine. Growth of the pelvis in vertical and horizontal dimensions and of the inguinal ligament followed a positive polynomial relationship with increasing age, with no observed increase in growth rate during puberty. From 0 to 19 years, the deep inguinal ring moved superolaterally with respect to the inguinal ligament (from 0.74 to 0.60 of the distance along the inguinal ligament) and the femoral artery and vein moved medially (from 0.50 to 0.58, and 0.61 to 0.65 of the distance along the inguinal ligament, respectively). The position of the femoral artery, vein, and deep inguinal ring followed a logarithmic relationship with age. No significant left:right side or male:female differences were observed. From 0 to 19 years of age the femoral vasculature and deep inguinal ring change position as the pelvis grows around them. Clin. Anat. 32:794-802, 2019. © 2019 Wiley Periodicals, Inc.

Age-dependent variations in the expression of myosin isoforms and myogenic factors during the involution of the proximal sesamoidean ligament of sheep.

In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180 days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.

Electrospinning of highly porous yet mechanically functional microfibrillar scaffolds at the human scale for ligament and tendon tissue engineering.

Electrospun fibers offer tremendous potential for tendon and ligament tissue engineering, yet developing porous scaffolds mimicking the size, stiffness and strength of human tissues remains a challenge. Previous studies have rolled, braided, or stacked electrospun sheets to produce three-dimensional (3D) scaffolds with tailored sizes and mechanical properties. A common limitation with such approaches is the development of low porosity scaffolds that impede cellular infiltration into the body of the implant, thereby limiting their regenerative potential. Here, we demonstrate how varying the rotational speed of the collecting mandrel during the electrospinning of poly(ε-caprolactone) (PCL) can be used to limit inter-fiber fusion (or fiber welding). Increasing the fraction of unfused fibers reduced the flexural rigidity of the electrospun sheets, which in turn allowed us to bundle the fibers into 3D scaffolds with similar dimensions to the human anterior cruciate ligament (ACL). These unfused fibers allowed for higher levels of porosity (up to 95%) that facilitated the rapid migration of mesenchymal stem cells (MSCs) into the body of the scaffolds. Mechanical testing demonstrated that the fiber-bundles possessed a Young's modulus approaching that of the native human ACL. The scaffolds were also capable of supporting the differentiation of MSCs towards either the fibrocartilage or ligament/tendon lineage. This novel electrospinning strategy could be used to produce mechanically functional, yet porous, scaffolds for a wide range of biomedical applications.

The natural involution of the sheep proximal sesamoidean ligament is due to depletion of satellite cells and simultaneous proliferation of fibroblasts: Ultrastructural evidence.

The rapid involution that happens in some muscles of ungulate fetlock joints has never been investigated at an ultrastructural level. In this study, the proximal sesamoidean ligament (PSL) of sheep was chosen as a model to investigate, at the cellular level, the transition from muscle to connective structures that occurs during early development. In particular, we were interested in observing the presence of satellite cells and fibroblasts, detecting fluctuations in their numbers in the postnatal developing PSL, and evaluating putative apoptotic mechanisms. Interestingly, some features were shared by both PSL involution and muscle ageing; the most relevant being the significant and rapid decrease in the number of satellite cells together with a quick proliferation of fibroblasts in the muscle-connective transitional area (MCT-TA). Electron microscopy and immunohistochemical analyses revealed putative cellular mechanisms that led to a progressive involution of the muscle portion of the PSL during postnatal growth. Our findings showed a fast transition from muscle to connective tissue due to the depletion of satellite cells, apoptosis of some muscle fibres, and simultaneous proliferation of fibroblasts originating from mesenchymal progenitors or from differentiation of satellite cells typically located at the border between muscle and connective tissue of the PSL.

Ligament, hinge, and shell cross-sections of the Atlantic surfclam (Spisula solidissima): Promising marine environmental archives in NE North America.

The Atlantic surfclam (Spisula solidissima) is a commercially important species in North American waters, undergoing biological and ecological shifts. These are attributed, in part, to environmental modifications in its habitat and driven by climate change. Investigation of shell growth patterns, trace elements, and isotopic compositions require an examination of growth lines and increments preserved in biogenic carbonates. However, growth pattern analysis of S. solidissima is challenging due to multiple disturbance lines caused by environmental stress, erosion in umbonal shell regions, and constraints related to sample size and preparation techniques. The present study proposes an alternative method for describing chronology. First, we analyzed growth patterns using growth lines within the shell and hinge. To validate the assumption of annual periodicity of growth line formation, we analyzed the oxygen isotope composition of the outer shell layer of two specimens (46°54'20"N; 56°18'58"W). Maximum δ18Oshell values occurred at the exact same location as internal growth lines in both specimens, confirming that they are formed annually and that growth ceases during winter. Next, we used growth increment width data to build a standardized growth index (SGI) time-series (25-year chronology) for each of the three parts of the shell. Highly significant correlations were found between the three SGI chronologies (p < 0.001; 0.55 < τ < 0.68) of all specimens. Thus, ligament growth lines provide a new method of determining ontogenetic age and growth rate in S. solidissima. In a biogeographic approach, the shell growth performance of S. solidissima in Saint-Pierre and Miquelon was compared to those in other populations along its distribution range in order to place this population in a temporal and regional context.

[Cartilage/chondrocyte research and osteoarthritis. Current topics and future of ligament regeneration therapy.]

Ligaments of joint have an essential role of proper mobilization and stabilization between bone and bone. Damage to ligaments caused by ageing, injury, and arthritis induce a disability of musculoskeletal system and has a problem to reduce our quality of life. To aim for the regeneration of ligaments, we have researched from the point of view of the developmet, found out that the transcription factor Mohawk has been important for the development and homeostasis of tendons and ligaments, and analyzed its function. Furthermore, we have also attempted to induce stem cells to tendon and ligament cells to produce type Ⅰ collagen fibers. In this article, we outline the mechanism of the development that has been reported including our approaches.

The impact of artificial selection on morphological integration in the appendicular skeleton of domestic horses.

The relationships between the different component parts of organisms, such as the sharing of common development or function, produce a coordinated variation between the different traits. This morphological integration contributes to drive or constrain morphological variation and thus impacts phenotypic diversification. Artificial selection is known to contribute significantly to phenotypic diversification of domestic species. However, little attention has been paid to its potential impact on integration patterns. This study explores the patterns of integration in the limb bones of different horse breeds, using 3D geometric morphometrics. The domestic horse is known to have been strongly impacted by artificial selection, and was often selected for functional traits. Our results confirm that morphological integration among bones within the same limb is strong and apparently partly produced by functional factors. Most importantly, they reveal that artificial selection, which led to the diversification of domestic horses, impacts covariation patterns. The influence of selection on the patterns of covariation varies along the limbs and modulates bone shape, likely due to a differential ligament or muscle development. These results highlight that, in addition to not being constrained by a strong morphological integration, artificial selection has modulated the covariation patterns according to the locomotor specificities of the breeds. More broadly, it illustrates the interest in studying how micro-evolutionary processes impact covariation patterns and consequently contribute to morphological diversification of domestic species.

Biofabrication of soft tissue templates for engineering the bone-ligament interface.

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition, and function of the native enthesis. In the ligament-to-bone interface, this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous, or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression toward an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. Biotechnol. Bioeng. 2017;114: 2400-2411. © 2017 Wiley Periodicals, Inc.

Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system.

Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is expressed persistently in tendons/ligaments, but transiently in entheseal cartilage. In this study, we generated a novel ScxCre knock-in (KI) allele, by in-frame replacement of most of Scx exon 1 with Cre recombinase (Cre), to drive Cre expression using Scx promoter and to inactivate the endogenous Scx. Reflecting the intensity and duration of endogenous expression, Cre-mediated excision occurs in tendinous and ligamentous tissues persistently expressing Scx. Expression of tenomodulin, a marker of mature tenocytes and ligamentocytes, was almost absent in tendons and ligaments of ScxCre/Cre KI mice lacking Scx to indicate defective maturation. In homozygotes, the transiently Scx-expressing entheseal regions such as the rib cage, patella cartilage, and calcaneus were small and defective and cartilaginous tuberosity was missing. Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 were also observed in the developing entheseal cartilage, patella, and deltoid tuberosity of ScxCre/Cre KI mice. These results highlighted the functional importance of both transient and persistent expression domains of Scx for proper integration of the musculoskeletal components.

Restrained Differential Growth: The Initiating Event of Adolescent Idiopathic Scoliosis?

STUDY DESIGN: An experimental model study and a short review of literature. OBJECTIVE: The purpose of this study was to explore a new hypothesis suggesting that the curvatures seen in adolescent idiopathic scoliosis (AIS) originate from restrained differential growth between the vertebral column and the surrounding musculo-ligamentary structures. SUMMARY OF BACKGROUND DATA: Despite decades of research, there is no generally accepted theory on the physical origin of the severe spinal deformations seen in AIS. The prevailing theories tend to focus on left-right asymmetry, rotational instability, or the sagittal spinal profile in idiopathic scoliosis. METHODS: We test our hypothesis with a physical model of the spine that simulates growth, counteracted by ligaments and muscles, modeled by tethers and springs. Growth of the spine is further restrained by an anterior band representing the thorax, the linea alba, and abdominal musculature. We also explore literature in search of molecular mechanisms that may induce differential growth. RESULTS: Differential growth in the restrained spine model first induces hypokyphosis and mild lateral bending of the thoracic spine, but then suddenly escalates into a scoliotic deformity, consistent with clinical observations of AIS. The band simulating the ventral structures of the body had a pivotal effect on sagittal curvature and the initiation of lateral bending and rotation. In literature, several molecular mechanisms were found that may explain the occurrence of differential growth between the spine and the musculo-ligamentary structures. CONCLUSION: While AIS is a three-dimensional deformation of the spine, it appears that restrained differential growth in the sagittal plane can result in lateral bending and rotation without a pre-existing left-right asymmetry. This supports the concept that AIS may result from a growth imbalance rather than a local anatomical defect. LEVEL OF EVIDENCE: N/A.

Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.

Heightened phenotypic variation among mutant animals is a well-known, but poorly understood phenomenon. One hypothetical mechanism accounting for mutant phenotypic variation is progenitor cells variably choosing between two alternative fates during development. Zebrafish mef2cab1086 mutants develop tremendously variable ectopic bone in their hyoid craniofacial skeleton. Here, we report evidence that a key component of this phenotype is variable fate switching from ligament to bone. We discover that a 'track' of tissue prone to become bone cells is a previously undescribed ligament. Fate-switch variability is heritable, and comparing mutant strains selectively bred to high and low penetrance revealed differential mef2ca mutant transcript expression between high and low penetrance strains. Consistent with this, experimental manipulation of mef2ca mutant transcripts modifies the penetrance of the fate switch. Furthermore, we discovered a transposable element that resides immediately upstream of the mef2ca locus and is differentially DNA methylated in the two strains, correlating with differential mef2ca expression. We propose that variable transposon epigenetic silencing underlies the variable mef2ca mutant bone phenotype, and could be a widespread mechanism of phenotypic variability in animals.

Functional regeneration of ligament-bone interface using a triphasic silk-based graft.

The biodegradable silk-based scaffold with unique mechanical property and biocompatibility represents a favorable ligamentous graft for tissue-engineering anterior cruciate ligament (ACL) reconstruction. However, the low efficiency of ligament-bone interface restoration barriers the isotropic silk graft to common ACL therapeutics. To enhance the regeneration of the silk-mediated interface, we developed a specialized stratification approach implementing a sequential modification on isotropic silk to constitute a triphasic silk-based graft in which three regions respectively referring to ligament, cartilage and bone layers of interface were divided, followed by respective biomaterial coating. Furthermore, three types of cells including bone marrow mesenchymal stem cells (BMSCs), chondrocytes and osteoblasts were respectively seeded on the ligament, cartilage and bone region of the triphasic silk graft, and the cell/scaffold complex was rolled up as a multilayered graft mimicking the stratified structure of native ligament-bone interface. In vitro, the trilineage cells loaded on the triphasic silk scaffold revealed a high proliferative capacity as well as enhanced differentiation ability into their corresponding cell lineage. 24 weeks postoperatively after the construct was implanted to repair the ACL defect in rabbit model, the silk-based ligamentous graft exhibited the enhancement of osseointegration detected by a robust pullout force and formation of three-layered structure along with conspicuously corresponding matrix deposition via micro-CT and histological analysis. These findings potentially broaden the application of silk-based ligamentous graft for ACL reconstruction and further large animal study.

[Application of silk-based tissue engineering scaffold for tendon / ligament regeneration].

Tendon/ligament injury is one of the most common impairments in sports medicine. The traditional treatments of damaged tissue repair are unsatisfactory, especially for athletes, due to lack of donor and immune rejection. The strategy of tissue engineering may break through these limitations, and bring new hopes to tendon/ligament repair, even regeneration. Silk is a kind of natural biomaterials, which has good biocompatibility, wide range of mechanical properties and tunable physical structures; so it could be applied as tendon/ligament tissue engineering scaffolds. The silk-based scaffold has robust mechanical properties; combined with other biological ingredients, it could increase the surface area, promote more cell adhesion and improve the biocompatibility. The potential clinical application of silk-based scaffold has been confirmed by in vivo studies on tendon/ligament repairing, such as anterior cruciate ligament, medial collateral ligament, achilles tendon and rotator cuff. To develop novel biomechanically stable and host integrated tissue engineered tendon/ligament needs more further micro and macro studies, combined with product development and clinical application, which will give new hope to patients with tendon/ligament injury.

Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate.

PURPOSE: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer "neoligament" seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. METHODS: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell-cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). RESULTS: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts. SUMMARY: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration.

Controlled Bioactive Molecules Delivery Strategies for Tendon and Ligament Tissue Engineering using Polymeric Nanofibers.

The interest in polymeric nanofibers has escalated over the past decade given its promise as tissue engineering scaffolds that can mimic the nanoscale structure of the native extracellular matrix. With functionalization of the polymeric nanofibers using bioactive molecules, localized signaling moieties can be established for the attached cells, to stimulate desired biological effects and direct cellular or tissue response. The inherently high surface area per unit mass of polymeric nanofibers can enhance cell adhesion, bioactive molecules loading and release efficiencies, and mass transfer properties. In this review article, the application of polymeric nanofibers for controlled bioactive molecules delivery will be discussed, with a focus on tendon and ligament tissue engineering. Various polymeric materials of different mechanical and degradation properties will be presented along with the nanofiber fabrication techniques explored. The bioactive molecules of interest for tendon and ligament tissue engineering, including growth factors and small molecules, will also be reviewed and compared in terms of their nanofiber incorporation strategies and release profiles. This article will also highlight and compare various innovative strategies to control the release of bioactive molecules spatiotemporally and explore an emerging tissue engineering strategy involving controlled multiple bioactive molecules sequential release. Finally, the review article concludes with challenges and future trends in the innovation and development of bioactive molecules delivery using polymeric nanofibers for tendon and ligament tissue engineering.

The cross-sectional area changes in digital flexor tendons and suspensory ligament in foals by ultrasonographic examination.

REASONS FOR PERFORMING STUDY: Flexural deformities are common conditions of growing horses and are suggested to have a relationship with the contraction of musculotendinous units. However, limited studies have documented the changes in each tendon and ligament in the metacarpal region with age. OBJECTIVES: To investigate the changes in the cross-sectional area (CSA) of each tendon and ligament in the metacarpal region with age by ultrasonographic examination. STUDY DESIGN: Longitudinal study of foals from Day 1 to age 24 months. METHODS: The CSA of the superficial digital flexor tendon, deep digital flexor tendon, accessory ligament of the deep digital flexor tendon and suspensory ligament was measured by ultrasonographic examination at monthly intervals from Day 1 to age 24 months in 7 Thoroughbred foals. RESULTS: Changes in superficial digital flexor CSA in all regions were larger than those of other structures from 10 months to 15 months. The suspensory ligament CSA was significantly larger than those of other structures on Day 1 in both the region of suspensory origin (RSO) and region of suspensory body (RSBO). This condition continued until 2 months in the RSO and until 5 months in the RSBO. The changes in deep digital flexor CSA were larger than those of other structures from 2 to 5 months in both the RSO and RSBO. CONCLUSIONS: The rate of change in each structure varies with age. Thus, the functional adaptation with age that takes place may differ among structures because the primary function of each structure differs.