Fetal Tendon Research Articles

Mechanobiological Strategies to Enhance Ovine (Ovis aries) Adipose-Derived Stem Cells Tendon Plasticity for Regenerative Medicine and Tissue Engineering Applications.

Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their phenotypic properties pre-transplantation. Moreover, their tenogenic potential was assessed using two in vitro-validated approaches: (1) teno-inductive conditioned media (CM) derived from a co-culture between ovine amniotic stem cells and fetal tendon explants, and (2) short- (48 h) and long-term (14 days) seeding on highly aligned PLGA (ha-PLGA) electrospun scaffold. Our findings indicate that oADSCs can be expanded without senescence and can maintain the expression of stemness (Sox2, Oct4, Nanog) and mesenchymal (CD29, CD166, CD44, CD90) markers while remaining negative for hematopoietic (CD31, CD45) and MHC-II antigens. Of note, oADSCs' tendon differentiation potential greatly depended on the in vitro strategy. oADSCs exposed to CM significantly upregulated tendon-related genes (COL1, TNMD, THBS4) but failed to accumulate TNMD protein at 14 days of culture. Conversely, oADSCs seeded on ha-PLGA fleeces quickly upregulated the tendon-related genes (48 h) and in 14 days accumulated high levels of the TNMD protein into the cytoplasm of ADSCs, displaying a tenocyte-like morphology. This mechano-sensing cellular response involved a complete SOX9 downregulation accompanied by YAP activation, highlighting the efficacy of biophysical stimuli in promoting tenogenic differentiation. These findings underscore oADSCs' long-term self-renewal and tendon differentiative potential, thus opening their use in a preclinical setting to develop innovative stem cell-based and tissue engineering protocols for tendon regeneration, applied to the veterinary field.

From fetal tendon regeneration to adult therapeutic modalities: TGF-β3 in scarless healing.

Tendon injuries are common disorders that can significantly impact people's lives. Unfortunately, the limited regenerative ability of tendons results in tissue healing in a scar-mediated manner. The current therapeutic strategies fail to fully recover the functions of the injured tendons, and as such, the conception of 'scarless healing' has gained prominent attention in the field of regenerative medicine. Interestingly, injured fetal tendons possess the capability to heal through regeneration, which builds an ideal blueprint for adult tendon regeneration. Studies have shown that fetal biochemical cues have the potential to improve adult tendon healing. Here we review the biological factors that contribute to fetal tendon regeneration and how manipulation of these biochemical cues in the adult tendon healing process could achieve regeneration.

Establishing in vivo and ex vivo chick embryo models to investigate fetal tendon healing

Injured adult tendons heal fibrotically and possess high re-injury rates, whereas fetal tendons appear to heal scarlessly. However, knowledge of fetal tendon wound healing is limited due in part to the need for an accessible animal model. Here, we developed and characterized an in vivo and ex vivo chick embryo tendon model to study fetal tendon healing. In both models, injury sites filled rapidly with cells and extracellular matrix during healing, with wound closure occurring faster in vivo. Tendons injured at an earlier embryonic stage improved mechanical properties to levels similar to non-injured controls, whereas tendons injured at a later embryonic stage did not. Expression levels of tendon phenotype markers, collagens, collagen crosslinking regulators, matrix metalloproteinases, and pro-inflammatory mediators exhibited embryonic stage-dependent trends during healing. Apoptosis occurred during healing, but ex vivo tendons exhibited higher levels of apoptosis than tendons in vivo. Future studies will use these in vivo and ex vivo chick embryo tendon injury models to elucidate mechanisms of stage-specific fetal tendon healing to inform the development of therapeutic approaches to regeneratively heal adult tendons.

Histoarchitecture of the fibrillary matrix of human fetal posterior tibial tendons

Adult tendons are highly differentiated. In mature individuals, tendon healing after an injury occurs through fibrotic tissue formation. Understanding the intrinsic reparative properties of fetal tendons would help to understand the maturation tissue process and tendon tissue repair. The present study evaluated the evolution of histoarchitecture, cellularity and the distribution of collagens I, III and V in the posterior tibial tendon in human fetuses at different gestational ages. Morphological profiles were assessed in nine fresh spontaneously aborted fetuses (Group I: five fetuses aged between 22 and 28 weeks of gestation; Group II: four fetuses aged between 32 and 38 weeks of gestation), characterized by a combination of histology, fluorescence and immunohistochemistry. In Group I, the posterior tibial tendon showed statistically significant greater cellularity and presence of collagen III and V than in Group II tendon, which showed a predominance of collagenous I and a better organization of the extracellular matrix compared with Group I tendons. In addition, a statistically significant higher rate of CD90, a marker of mesenchymal cells, was found in Group I tendons. In fetuses with gestational age between 22 and 28 weeks, the posterior tibialis tendons showed a thin and disorganized fibrillar structure, with an increase in collagen III and V fibers and mesenchymal cells. In the posterior tibialis tendons of fetuses with gestational age between 32 and 38 weeks, the fibrillar structure was thicker with a statistically significant increase in type I collagen and decreased cellularity.

Classification by degree of twisted structure of the fetal Achilles tendon.

The purpose of this study was to classify the twisted structure of the fetal Achilles tendon. The study was conducted using 30 legs from 15 Japanese fetuses (mean weight, 1764.6 ± 616.9g; mean crown-rump length, 283.5 ± 38.7mm; 16 males, 14 females). According to attachment to the deep layer of the calcaneal tuberosity, cases showing only soleus attachment were classified as least twist (Type I), cases showing both lateral head of the gastrocnemius and soleus were classified as moderate twist (Type II), and cases with only lateral head of the gastrocnemius were classified as extreme twist (Type III). Viewing the Achilles tendon from cranially shows a structure twisted counterclockwise on the right side and clockwise on the left. The Achilles tendon was Type I in 4 legs (13%), Type II in 23 legs (77%), and Type III in 3 legs (10%). The twisted structure of the Achilles tendon can be classified as early as the second trimester and is similar to that seen in adults.

Molecular Mechanisms of Fetal Tendon Regeneration Versus Adult Fibrous Repair.

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.

The twisted structure of the fetal calcaneal tendon is already visible in the second trimester

IntroductionThe progress in morphological science results from the greater possibilities of intra-pubic diagnosis and treatment of congenital disabilities, including the motor system. However, the structure and macroscopic development of the calcaneal tendon have not been investigated in detail. Studies on the adult calcaneal tendon showed that the calcaneal tendon is composed of twisted subtendons. This study aimed to investigate the internal structure of the fetal calcaneal tendon in the second trimester.Materials and methodsThirty-six fetuses fixed in 10% formaldehyde were dissected using the layer-by-layer method and a surgical microscope.ResultsThe twisted structure of the calcaneal tendon was revealed in all specimens. The posterior layer of the calcaneal tendon is formed by the subtendon from the medial head of the gastrocnemius muscle. In contrast, the anterior layer is formed by the subtendon from the lateral head of the gastrocnemius muscle. The subtendon from the soleus muscle constitutes the anteromedial outline of the calcaneal tendon. The lateral outline of the calcaneal tendon is formed by the subtendon originating from the medial head of the gastrocnemius muscle. In contrast, the medial outline is formed by the subtendon from the soleus muscle. In most of the examined limbs, the plantaris tendon attached to the tuber calcanei was not directly connected to the calcaneal tendon.ConclusionsThe twisted structure of the subtendons of the fetal calcaneal tendon is already visible in the second trimester and is similar to that seen in adults.

Genome-wide transcriptome analysis reveals equine embryonic stem cell-derived tenocytes resemble fetal, not adult tenocytes

BackgroundTendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic.MethodsEquine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture were compared using RNA sequencing.ResultsNo qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome-wide transcriptomic analysis using RNA sequencing revealed that ESC-derived tenocytes’ transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this study adds to the growing evidence that monolayer cultured cells’ gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed.ConclusionThe information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.

Tendon Biomimetic Electrospun PLGA Fleeces Induce an Early Epithelial-Mesenchymal Transition and Tenogenic Differentiation on Amniotic Epithelial Stem Cells.

Background. The design of tendon biomimetic electrospun fleece with Amniotic Epithelial Stem Cells (AECs) that have shown a high tenogenic attitude may represent an alternative strategy to overcome the unsatisfactory results of conventional treatments in tendon regeneration. Methods. In this study, we evaluated AEC-engineered electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned fibers (ha-PLGA) that mimic tendon extracellular matrix, their biocompatibility, and differentiation towards the tenogenic lineage. PLGA fleeces with randomly distributed fibers (rd-PLGA) were generated as control. Results. Optimal cell infiltration and biocompatibility with both PLGA fleeces were shown. However, only ha-PLGA fleeces committed AECs towards an Epithelial-Mesenchymal Transition (EMT) after 48 h culture, inducing their cellular elongation along the fibers’ axis and the upregulation of mesenchymal markers. AECs further differentiated towards tenogenic lineage as confirmed by the up-regulation of tendon-related genes and Collagen Type 1 (COL1) protein expression that, after 28 days culture, appeared extracellularly distributed along the direction of ha-PLGA fibers. Moreover, long-term co-cultures of AEC-ha-PLGA bio-hybrids with fetal tendon explants significantly accelerated of half time AEC tenogenic differentiation compared to ha-PLGA fleeces cultured only with AECs. Conclusions. The fabricated tendon biomimetic ha-PLGA fleeces induce AEC tenogenesis through an early EMT, providing a potential tendon substitute for tendon engineering research.

Cellular and molecular maturation in fetal and adult ovine calcaneal tendons.

Processes of development during fetal life profoundly transform tendons from a plastic tissue into a highly differentiated structure, characterised by a very low ability to regenerate after injury in adulthood. Sheep tendon is frequently used as a translational model to investigate cell-based regenerative approaches. However, in contrast to other species, analytical and comparative baseline studies on the normal developmental maturation of sheep tendons from fetal through to adult life are not currently available. Thus, a detailed morphological and biochemical study was designed to characterise tissue maturation during mid- (2 months of pregnancy: 14 cm of length) and late fetal (4 months: 40 cm of length) life, through to adulthood. The results confirm that ovine tendon morphology undergoes profound transformations during this period. Endotenon was more developed in fetal tendons than in adult tissues, and its cell phenotype changed through tendon maturation. Indeed, groups of large rounded cells laying on smaller and more compacted ones expressing osteocalcin, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were identified exclusively in fetal mid-stage tissues, and not in late fetal or adult tendons. VEGF, NGF as well as blood vessels and nerve fibers showed decreased expression during tendon development. Moreover, the endotenon of mid- and late fetuses contained identifiable cells that expressed several pluripotent stem cell markers [Telomerase Reverse Transcriptase (TERT), SRY Determining Region Y Box-2 (SOX2), Nanog Homeobox (NANOG) and Octamer Binding Transcription Factor-4A (OCT-4A)]. These cells were not identifiable in adult specimens. Ovine tendon development was also accompanied by morphological modifications to cell nuclei, and a progressive decrease in cellularity, proliferation index and expression of connexins 43 and 32. Tendon maturation was similarly characterised by modulation of several other gene expression profiles, including Collagen type I, Collagen type III, Scleraxis B, Tenomodulin, Trombospondin 4 and Osteocalcin. These gene profiles underwent a dramatic reduction in adult tissues. Transforming growth factor-1 expression (involved in collagen synthesis) underwent a similar decrease. In conclusion, these morphological studies carried out on sheep tendons at different stages of development and aging offer normal structural and molecular baseline data to allow accurate evaluation of data from subsequent interventional studies investigating tendon healing and regeneration in ovine experimental models.

Indirect Co-Culture with Tendons or Tenocytes Can Program Amniotic Epithelial Cells towards Stepwise Tenogenic Differentiation

BackgroundAmniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue.Methodology/Principal FindingsFreshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43).Conclusions/SignificanceThe co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or explants. The co-cultural system can be proposed as a low cost and easy technique to engineer tendon for biological study and cell therapy approach.

Fetal Tendon Scar Formation is Associated With Increased Inflammation and Decreased MicroRNA-146a Production

Fetal Tendon Scar Formation is Associated With Increased Inflammation and Decreased MicroRNA-146a Production

Fetal tendon wound size modulates wound gene expression and subsequent wound phenotype

The fetal response to small tendon injury results in regenerative or scarless healing and is characterized by a markedly diminished cellular inflammatory response, lack of fibroplasia, and restoration of normal tissue architecture. We hypothesized that an increasing fetal tendon wound size would lead to increased wound inflammation and a change from regenerative to reparative healing and scar formation. We created small or large tendon wounds in early gestation fetal sheep and used histology to assess tissue architecture, immunohistochemistry to assess the cellular inflammatory response, ovine-specific gene microarrays, and real-time reverse transcription-polymerase chain reaction to measure the gene expression in response to injury. Small tendon wounds showed a regenerative healing phenotype with orderly deposition of collagen fibers while large tendon wounds showed disorderly collagen deposition consistent with scar formation. Small tendon wounds had few inflammatory cells at 7 and 28 days after injury, whereas the large wounds showed a significant inflammatory cell infiltrate at 7 days that resolved by 28 days. At 3 days, the differential expression of genes involved in the response to injury and inflammation were seen between large and small tendon wounds. By real-time polymerase chain reaction at 7 days, large tendon wounds also had significantly increased expression of interleukin-6, interleukin-8, transforming growth factor-β1, and transforming growth factor-β3, compared with the small wounds. Increasing the fetal tendon wound size results in increased proinflammatory gene expression, inflammatory cell infiltration, and a change from regenerative to reparative healing. This model allows the process of regenerative healing to be examined without the confounding variable of gestational age.

Increased fetal tendon wound size results in a transition from scarless regeneration to scar formation and is associated with upregulation of genes regulating inflammation and cell migration

Increased fetal tendon wound size results in a transition from scarless regeneration to scar formation and is associated with upregulation of genes regulating inflammation and cell migration

The Change from Scarless Regeneration to Scar Formation with Increased Fetal Tendon Wound Size Is Associated with Significant Upregulation of Inflammatory Genes

The Change from Scarless Regeneration to Scar Formation with Increased Fetal Tendon Wound Size Is Associated with Significant Upregulation of Inflammatory Genes

Stepwise Differentiation of Human Embryonic Stem Cells Promotes Tendon Regeneration by Secreting Fetal Tendon Matrix and Differentiation Factors

Human embryonic stem cells (hESCs) are ideal seed cells for tissue regeneration, but no research has yet been reported concerning their potential for tendon regeneration. This study investigated the strategy and efficacy of using hESCs for tendon regeneration as well as the mechanism involved. hESCs were first induced to differentiate into mesenchymal stem cells (MSCs), which had the potential to differentiate into the three mesenchymal lineages and were positive for MSC surface markers. hESC-derived MSCs (hESC-MSCs) regenerated tendon tissues in both an in vitro tissue engineering model and an in vivo ectopic tendon regeneration model, as confirmed by the expression of tendon-specific genes and structure. In in-situ rat patellar tendon repair, tendon treated with hESC-MSCs had much better structural and mechanical properties than did controls. Furthermore, hESC-MSCs remained viable at the tendon wound site for at least 4 weeks and secreted human fetal tendon-specific matrix components and differentiation factors, which then activated the endogenous regeneration process in tendon. Moreover, no teratoma was found in any samples. These findings demonstrate a safe and practical strategy of applying ESCs for tendon regeneration and may assist in future strategies to treat tendon diseases.

Fetal ACL Fibroblasts Exhibit Enhanced Cellular Properties Compared with Adults

Fetal tendons and skin heal regeneratively without scar formation. Cells isolated from these fetal tissues exhibit enhanced cellular migration and collagen production in comparison to cells from adult tissue. We determined whether fetal and adult fibroblasts isolated from the anterior cruciate ligament (ACL), a tissue that does not heal regeneratively, exhibit differences in cell migration rates and collagen elaboration. An in vitro migration assay showed fetal ACL fibroblasts migrated twice as fast as adult ACL fibroblasts at a rate of 38.90 +/- 7.69 microm per hour compared with 18.88 +/- 4.18 microm per hour, respectively. Quantification of Type I collagen elaboration by enzyme-linked immunosorbent assay showed fetal ACL fibroblasts produced four times the amount of Type I collagen compared with adult ACL fibroblasts after 7 days in culture. We observed no differences in Type III collagen with time for adult or fetal ACL fibroblasts. Our findings indicate fetal ACL fibroblasts are intrinsically different from adult ACL fibroblasts, suggesting the healing potential of the ACL may be age-dependent.

Regenerative properties of fetal sheep tendon are not adversely affected by transplantation into an adult environment

Tendon injuries account for a significant number of musculoskeletal afflictions each year. While new surgical techniques and rehabilitation protocols have led to improved clinical outcomes, postsurgical scarring remains the most problematic aspect of tendon repair. In contrast to this typical pattern of fibrosis, recent studies have shown that fetal tendon is capable of healing without scar. However, whether this regenerative healing pattern is intrinsic to the fetal tissue itself or the result of its environment is not known. Thus, the objective of this study is to examine the influence of an adult environment on healing in adult and fetal tendons. We hypothesized that injured fetal tendon tissue transplanted into an adult environment would retain a regenerative healing pattern after injury, demonstrating normal histological and mechanical properties. Our results support this hypothesis. Histological analyses revealed considerable alterations in adult tendon transplants after injury while fetal transplants showed no abnormalities. The injured adult tendons also demonstrated elevated levels of TGF-beta1, bFGF, and CD44 at the wound site, whereas the fetal specimens showed little or no such changes in response to injury. The data from our biomechanical studies further corroborate these observations, with significant decreases in the stiffness, modulus, and almost all viscoelastic properties in wounded versus unwounded adult tendons, and fetal specimens showing no differences in mechanical properties between the wounded and unwounded groups. Thus, the results of our investigation demonstrate that the adult environment is not an impediment to scarless repair and that this capability is intrinsic to the fetal tendon itself. Our study also begins to provide insight into the mechanisms controlling this regenerative response.

Serum‐dependent effects on adult and fetal tendon fibroblast migration and collagen expression

Cell migration and extracellular matrix synthesis play an important role in the wound-healing response to injury. Several studies have described differences in migratory behavior and collagen biosynthetic activity in adult vs. fetal skin fibroblasts. The objective of this study was to examine the serum- and age-dependent effects on cell migration and collagen expression in tendon fibroblasts. Medial tendon fibroblasts were isolated from pregnant ewes and their fetuses, and cultured with and without serum for up to 7 days. Cell migration was determined by quantitative image analysis, and collagen expression was assessed by reverse transcription-polymerase chain reaction and immunohistochemical staining. In serum-containing medium, tendon fibroblasts migrated significantly faster than cells in serum-free medium. Additionally, fetal tendon fibroblasts migrated significantly faster than adult tendon fibroblasts under both culture conditions. The expression of types I and III collagen mRNA was significantly up-regulated in tendon cell populations in serum-free medium compared with those in serum-containing medium. Quantitative assessment of collagen staining indicated that fetal tenocytes produced more type I collagen than adult tenocytes under both culture conditions. These findings suggest that there is an inherent difference between adult and fetal tendon fibroblasts, which may have implications in the wound-healing response in tendons.

Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in relation to collagen fibrils in the equine tendon

The structure and organisation of the extracellular matrix, and in particular the axial alignment of type I collagen fibrils, are essential for the tensile strength of tendons. The resident tenocytes synthesize and maintain the composition of the extracellular matrix, which changes with age and maturation. Other components of the extracellular matrix include less abundant collagen types II, III, V, VI, XII, proteoglycans and glycoproteins. Cartilage oligomeric matrix protein (COMP) is an abundant non-collagenous pentameric glycoprotein in the tendon, which can bind to collagen types I and II. The function of COMP in the tendon is not clear, but it may act as a catalyst in fibrillogenesis. Its concentration changes with age, maturation and load. The present study delineates the ultrastructural distribution of COMP and its correlation to collagen fibril thickness in different compartments in two flexor tendons from horses of different ages (foetus, 8 months, 3 years, 12 years). The immunolabeling for COMP was higher in the superficial digital flexor tendon compared with the deep digital flexor tendon and it increased with the age of the animal, with the highest concentration in the 3-year-olds. Fibril diameter differed between age groups and a more homogenous fibril population was found in the fetal tendons. A positive correlation between high COMP immunolabeling and the percentage of small fibrils (<60 nm) were present in the SDFT. COMP immunolabeling was enriched at the gap region of the collagen fibril. In situ hybridization revealed the strongest expression in tendons from the 3-year-old horses whereas there was no expression in foetal tendon.