Bioengineered 3D ovarian model for long-term multiple development of preantral follicle: bridging the gap for poly(ε-caprolactone) (PCL)-based scaffold reproductive applications.

BACKGROUND: Assisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development. METHODS: PA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level. RESULTS: The ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence. CONCLUSIONS: The study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.

Navigating the Intersection of Glycemic Control and Fertility: A Network Perspective.

The rising incidence of metabolic diseases is linked to elevated blood glucose levels, contributing to conditions such as diabetes and promoting the accumulation of advanced glycation end products (AGEs). AGEs, formed by non-enzymatic reactions between sugars and proteins, build up in tissues and are implicated in various diseases. This article explores the relationship between glycemic control and AGE accumulation, focusing on fertility implications. A computational model using network theory was developed, featuring a molecular database and a network with 145 nodes and 262 links, categorized as a Barabasi-Albert scale-free network. Three main subsets of nodes emerged, centered on glycemic control, fertility, and immunity, with AGEs playing a critical role. The transient receptor potential vanilloid 1 (TRPV1), a receptor expressed in several tissues including sperm, was identified as a key hub, suggesting that the modulation of TRPV1 in sperm by AGEs may influence fertility. Additionally, a novel link between glycemic control and immunity was found, indicating that immune cells may play a role in endocytosing specific AGEs. This discovery underscores the complex interplay between glycemic control and immune function, with significant implications for metabolic, immune health, and fertility.

The Molecular Link between Obesity and the Endometrial Environment: A Starting Point for Female Infertility.

Female infertility constitutes a growing health problem in developing countries and could be associated with several possible causes including reproductive disorders, congenital malformations, infections and hormonal dysfunction. Nonetheless, a series of additional factors can also negatively impact female fertility and are represented by chronic exposure to environmental pollutants, stress, unhealthy lifestyle choices such as cigarette smoking and, among others, obesity. Excess weight is associated with several chronic diseases, and growing evidence demonstrates that it can compromise reproductive physiology due to its influence on endometrial gene expression and receptivity. Thus, the current review of the literature mainly focused on how obesity can impair uterine receptivity, mostly from a molecular point of view throughout the window of implantation (WOI) period at an endometrial level. It was also highlighted that an obesity-related increase in adipose tissue may lead to a modulation in the expression of multiple pathways, which could cause a hostile endometrial environment with a consequent negative impact on the uterine receptivity and the establishment of pregnancy. Thanks to the use of the endometrial receptivity assay (ERA), a specific microarray that studies the expression of a series of genes, it is now possible to evaluate the endometrial status of patients with infertility problems in a more detailed manner. Moreover, female fertility and endometrial receptivity could be affected by endometriosis, a chronic benign gynecological disease, whose cause-and-effect relationship to obesity is still uncertain. Therefore, further investigations would be required to better elucidate these mechanisms that govern embryo implantation and could be potentially useful for the generation of new strategies to overcome implantation failure and improve the pregnancy rates in obese women.

IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture.

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

AEC and AFMSC Transplantation Preserves Fertility of Experimentally Induced Rat Varicocele by Expressing Differential Regenerative Mechanisms.

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.

Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability-An In Vitro Study in a Swine Model.

Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.

Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations.

Gonadotropin Cell Transduction Mechanisms.

The intention of this Special Edition was to collect review and original research articles that illustrate and stimulate the growing efforts to highlight the mechanisms of action of gonadotropins, as well as deepen our understanding of their biological roles in health and disease, aiming at revealing novel therapeutic opportunities in reproductive and regenerative medicine [...].

ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa.

Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin system. Recently, ACE2 has been in the spotlight due to its involvement in the SARS-CoV-2 entry into cells. There are no data available regarding the expression of ACE2 and its short-ACE2 isoform at the protein level on human spermatozoa. Here, protein expression was demonstrated by western blot and the percentage of sperm displaying surface ACE2 was assessed by flow cytometry. Immunocytochemistry assays showed that full-length ACE2 was mainly expressed in sperm midpiece, while short ACE2 was preferentially distributed on the equatorial and post-acrosomal region of the sperm head. To our knowledge, this is the first study demonstrating the expression of protein ACE2 on spermatozoa. Further studies are warranted to determine the role of ACE2 isoforms in male reproduction.

A Systematic Review of the Effects of High-Fat Diet Exposure on Oocyte and Follicular Quality: A Molecular Point of View.

Worldwide, infertility affects between 10 and 15% of reproductive-aged couples. Female infertility represents an increasing health issue, principally in developing countries, as the current inclinations of delaying pregnancy beyond 35 years of age significantly decrease fertility rates. Female infertility, commonly imputable to ovulation disorders, can be influenced by several factors, including congenital malformations, hormonal dysfunction, and individual lifestyle choices, such as smoking cigarettes, stress, drug use and physical activity. Moreover, diet-related elements play an important role in the regulation of ovulation. Modern types of diet that encourage a high fat intake exert a particularly negative effect on ovulation, affecting the safety of gametes and the implantation of a healthy embryo. Identifying and understanding the cellular and molecular mechanisms responsible for diet-associated infertility might help clarify the confounding multifaceted elements of infertility and uncover novel, potentially curative treatments. In this view, this systematic revision of literature will summarize the current body of knowledge of the potential effect of high-fat diet (HFD) exposure on oocyte and follicular quality and consequent female reproductive function, with particular reference to molecular mechanisms and pathways. Inflammation, oxidative stress, gene expression and epigenetics represent the main mechanisms associated with mammal folliculogenesis and oogenesis.

Evaluation of cartilage biomechanics and knee joint microenvironment after different cell-based treatments in a sheep model of early osteoarthritis.

PURPOSE: Aiming to prevent cartilage damage during early osteoarthritis (OA), the therapeutic challenge is to restore and maintain the physiological and functional properties of such a tissue with minimally invasive therapeutic strategies. METHODS: Accordingly, an in vivo model of early OA in sheep was here treated through three different cell therapies (culture expanded ADSCs, SVF, and culture expanded AECs) thus to preserve the joint surface from the progression of the pathology. Three months after the treatment injections, their performance was assessed through mechanical automated mapping (Young's modulus and cartilage thickness), gross evaluation of articular surfaces, and biochemical analysis of the synovial fluid. RESULTS: No severe degeneration was observed after three months from OA induction. Cartilage mechanical properties were crucial to identify early degeneration. All the treatments improved the macroscopic cartilage surface aspect and reduced pro-inflammatory cytokines in the synovial fluid. Among the three treatments, SVF highlighted the best performance while ADSCs the worst. CONCLUSION: Despite that the evaluated experimental time is an early follow-up and, thus, longer trial is mandatory to properly assess treatments effectiveness, the proposed multidisciplinary approach allowed to obtain preliminary, but also crucial, results concerning the reduction in OA signs on cartilage properties, in osteophyte development and in all the inflammatory markers.

Looking for In Vitro Models for Retinal Diseases.

Retina is a layered structure of the eye, composed of different cellular components working together to produce a complex visual output. Because of its important role in visual function, retinal pathologies commonly represent the main causes of visual injury and blindness in the industrialized world. It is important to develop in vitro models of retinal diseases to use them in first screenings before translating in in vivo experiments and clinics. For this reason, it is important to develop bidimensional (2D) models that are more suitable for drug screening and toxicological studies and tridimensional (3D) models, which can replicate physiological conditions, for investigating pathological mechanisms leading to visual loss. This review provides an overview of the most common retinal diseases, relating to in vivo models, with a specific focus on alternative 2D and 3D in vitro models that can replicate the different cellular and matrix components of retinal layers, as well as injury insults that induce retinal disease and loss of the visual function.

Role and Modulation of TRPV1 in Mammalian Spermatozoa: An Updated Review.

Based on the abundance of scientific publications, the polymodal sensor TRPV1 is known as one of the most studied proteins within the TRP channel family. This receptor has been found in numerous cell types from different species as well as in spermatozoa. The present review is focused on analyzing the role played by this important channel in the post-ejaculatory life of spermatozoa, where it has been described to be involved in events such as capacitation, acrosome reaction, calcium trafficking, sperm migration, and fertilization. By performing an exhaustive bibliographic search, this review gathers, for the first time, all the modulators of the TRPV1 function that, to our knowledge, were described to date in different species and cell types. Moreover, all those modulators with a relationship with the reproductive process, either found in the female tract, seminal plasma, or spermatozoa, are presented here. Since the sperm migration through the female reproductive tract is one of the most intriguing and less understood events of the fertilization process, in the present work, chemotaxis, thermotaxis, and rheotaxis guiding mechanisms and their relationship with TRPV1 receptor are deeply analyzed, hypothesizing its (in)direct participation during the sperm migration. Last, TRPV1 is presented as a pharmacological target, with a special focus on humans and some pathologies in mammals strictly related to the male reproductive system.

Equine Chorionic Gonadotropin as an Effective FSH Replacement for In Vitro Ovine Follicle and Oocyte Development.

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.

Effects of P4 Antagonist RU486 on VEGF and Its Receptors' Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles.

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors' signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles' microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.

In Vitro Innovation of Tendon Tissue Engineering Strategies.

Tendinopathy is the term used to refer to tendon disorders. Spontaneous adult tendon healing results in scar tissue formation and fibrosis with suboptimal biomechanical properties, often resulting in poor and painful mobility. The biomechanical properties of the tissue are negatively affected. Adult tendons have a limited natural healing capacity, and often respond poorly to current treatments that frequently are focused on exercise, drug delivery, and surgical procedures. Therefore, it is of great importance to identify key molecular and cellular processes involved in the progression of tendinopathies to develop effective therapeutic strategies and drive the tissue toward regeneration. To treat tendon diseases and support tendon regeneration, cell-based therapy as well as tissue engineering approaches are considered options, though none can yet be considered conclusive in their reproduction of a safe and successful long-term solution for full microarchitecture and biomechanical tissue recovery. In vitro differentiation techniques are not yet fully validated. This review aims to compare different available tendon in vitro differentiation strategies to clarify the state of art regarding the differentiation process.

Two-Player Game in a Complex Landscape: 26S Proteasome, PKA, and Intracellular Calcium Concentration Modulate Mammalian Sperm Capacitation by Creating an Integrated Dialogue-A Computational Analysis.

Recent experimental findings suggest the involvement of the 26S proteasome, the main protease active in eukaryotic cells, in the process that leads mammalian sperm to become fully fertile, so-called capacitation. Unfortunately, its role in male gametes signaling is still far from being completely understood. For this reason, here, we realized a computational model, based on network theory, with the aim of rebuilding and exploring its signaling cascade. As a result, we found that the 26S proteasome is part of a signal transduction system that recognizes the bicarbonate ion as an input terminal and two intermediate layers of information processing. The first is under the control of the 26S proteasome and protein kinase A (PKA), which are strongly interconnected, while the latter depends on intracellular calcium concentrations. Both are active in modulating sperm function by influencing the protein phosphorylation pattern and then controlling several key events in sperm capacitation, such as membrane and cytoskeleton remodeling. Then, we found different clusters of molecules possibly involved in this pathway and connecting it to the immune system. In conclusion, this work adds a piece to the puzzle of protease and kinase crosstalk involved in the physiology of sperm cells.

Fabrication and Plasma Surface Activation of Aligned Electrospun PLGA Fiber Fleeces with Improved Adhesion and Infiltration of Amniotic Epithelial Stem Cells Maintaining their Teno-inductive Potential.

Electrospun PLGA microfibers with adequate intrinsic physical features (fiber alignment and diameter) have been shown to boost teno-differentiation and may represent a promising solution for tendon tissue engineering. However, the hydrophobic properties of PLGA may be adjusted through specific treatments to improve cell biodisponibility. In this study, electrospun PLGA with highly aligned microfibers were cold atmospheric plasma (CAP)-treated by varying the treatment exposure time (30, 60, and 90 s) and the working distance (1.3 and 1.7 cm) and characterized by their physicochemical, mechanical and bioactive properties on ovine amniotic epithelial cells (oAECs). CAP improved the hydrophilic properties of the treated materials due to the incorporation of new oxygen polar functionalities on the microfibers' surface especially when increasing treatment exposure time and lowering working distance. The mechanical properties, though, were affected by the treatment exposure time where the optimum performance was obtained after 60 s. Furthermore, CAP treatment did not alter oAECs' biocompatibility and improved cell adhesion and infiltration onto the microfibers especially those treated from a distance of 1.3 cm. Moreover, teno-inductive potential of highly aligned PLGA electrospun microfibers was maintained. Indeed, cells cultured onto the untreated and CAP treated microfibers differentiated towards the tenogenic lineage expressing tenomodulin, a mature tendon marker, in their cytoplasm. In conclusion, CAP treatment on PLGA microfibers conducted at 1.3 cm working distance represent the optimum conditions to activate PLGA surface by improving their hydrophilicity and cell bio-responsiveness. Since for tendon tissue engineering purposes, both high cell adhesion and mechanical parameters are crucial, PLGA treated for 60 s at 1.3 cm was identified as the optimal construct.

Cyclin-CDK Complexes are Key Controllers of Capacitation-Dependent Actin Dynamics in Mammalian Spermatozoa.

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional maturation process to acquire the competence to fertilize the female egg. During this process, called capacitation, the actin cytoskeleton dramatically changes its organization. First, actin fibers polymerize, forming a network over the anterior part of the sperm cells head, and then it rapidly depolymerizes and disappears during the exocytosis of the acrosome content (the acrosome reaction (AR)). Here, we developed a computational model representing the actin dynamics (AD) process on mature spermatozoa. In particular, we represented all the molecular events known to be involved in AD as a network of nodes linked by edges (the interactions). After the network enrichment, using an online resource (STRING), we carried out the statistical analysis on its topology, identifying the controllers of the system and validating them in an experiment of targeted versus random attack to the network. Interestingly, among them, we found that cyclin-dependent kinase (cyclin-CDK) complexes are acting as stronger controllers. This finding is of great interest since it suggests the key role that cyclin-CDK complexes could play in controlling AD during sperm capacitation, leading us to propose a new and interesting non-genomic role for these molecules.

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-ß~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.