Comparative Ungual Drug Uptake Studies: Equine Hoof Membrane vs. Human Nail Plate.

Human nail diseases, mostly caused by fungal infections, are common and difficult to treat. The development and testing of new drugs and drug delivery systems for the treatment of nail diseases is often limited by the lack of human nail material for permeation studies. Animal material is frequently used, but there are only few comparative data on the human nail plate, and there is neither a standardized test design nor a nail bed analogue to study drug uptake into the nail. In this study, a new permeation device was developed for permeation studies, and the permeation behavior of three model substances on the human nail plate and a model membrane from the horse hoof was investigated. A linear correlation was found between drug uptake by the human nail plate and the uptake by the equine hoof. The developed and established permeation device is suitable for investigations of ungual drug transport and enables the use of different membrane diameters and the use of a gel-based nail bed analog. The hydrogel-based acceptor medium used ensures adequate stabilization and hydration of the nail membrane.

Hierarchical modeling of elastic moduli of equine hoof wall.

This study predicts analytically effective elastic moduli of substructures within an equine hoof wall. The hoof wall is represented as a composite material with a hierarchical structure comprised of a sequence of length scales. A bottom-up approach is employed. Thus, the outputs from a lower spatial scale serve as the inputs for the following scale. The models include the Halpin-Tsai model, composite cylinders model, a sutured interface model, and classical laminate theory. The length scales span macroscale, mesoscale, sub-mesoscale, microscale, sub-microscale, and nanoscale. The macroscale represents the hoof wall, consisting of tubules within a matrix at the mesoscale. At the sub-mesoscale, a single hollow tubule is reinforced by a tubule wall made of lamellae; the surrounding intertubular material also has a lamellar structure. The lamellae contain sutured and layered cells at the microscale. A single cell is made of crystalline macrofibrils arranged in an amorphous matrix at the sub-microscale. A macrofibril contains aligned crystalline rod-like intermediate filaments at the nanoscale. Experimentally obtained parameters are used in the modeling as inputs for geometry and nanoscale properties. The predicted properties of the hoof wall material agree with experimental measurements at the mesoscale and macroscale. We observe that the hierarchical structure of the hoof wall leads to a decrease in the elastic modulus with increasing scale, from the nanoscale to the macroscale. Such behavior is an intrinsic characteristic of hierarchical biological materials. This study can serve as a framework for designing impact-resistant hoof-inspired materials and structures.

Equine hoof wall: Structure, properties, and bioinspired designs.

The horse hoof wall exhibits exceptional impact resistance and fracture control due to its unique hierarchical structure which contains tubular, lamellar, and gradient configurations. In this study, structural characterization of the hoof wall was performed revealing features previously unknown. Prominent among them are tubule bridges, which are imaged and quantified. The hydration-dependent viscoelasticity of the hoof wall is described by a simplified Maxwell-Weichert model with two characteristic relaxation times corresponding to nanoscale and mesoscale features. Creep and relaxation tests reveal that the specific hydration gradient in the hoof keratin likely leads to reduced internal stresses that arise from spatial stiffness variations. To better understand realistic impact modes for the hoof wall in-vivo, drop tower tests were executed on hoof wall samples. Fractography revealed that the hoof wall's reinforced tubular structure dominates at lower impact energies, while the intertubular lamellae are dominant at higher impact energies. Broken fibers were observed on the surface of the tubules after failure, suggesting that the physically intertwined nature of the tubule reinforcement and intertubular matrix improves the toughness of this natural fiber reinforced composite. The augmented understanding of the structure-mechanical property relationship in dynamic loading led to the design of additively manufactured bioinspired structures, which were evaluated in quasistatic and dynamic loadings. The inclusion of gradient structures and lamellae significantly reduced the damage sustained in drop tower tests, while tubules increased the energy absorption of samples tested in compact tension. The samples most similar to the hoof wall displayed remarkably consistent fracture control properties. STATEMENT OF SIGNIFICANCE: The horse hoof wall, capable of withstanding large, repeated, dynamic loads, has been touted as a candidate for impact-resistant bioinspiration. However, our understanding of this biological material and its translation into engineered designs is incomplete. In this work, new features of the horse hoof wall are quantified and the hierarchical failure mechanisms of this remarkable material under near-natural loading conditions are uncovered. A model of the hoof wall's viscoelastic response, based on studies of other keratinous materials, was developed. The role of hydration, strain rate, and impact energy on the material's response were elucidated. Finally, multi-material 3D printed designs based on the hoof's meso/microstructure were fabricated and exhibited advantageous energy absorption and fracture control relative to control samples.

The Presence of Treponema spp. in Equine Hoof Canker Biopsies and Skin Samples from Bovine Digital Dermatitis Lesions.

Equine hoof canker and bovine digital dermatitis are infectious inflammatory diseases of the hooves with an unknown etiology. However, anaerobic spirochetes of the genus Treponema are considered to be potential etiological agents. The aim of this study was to find a suitable way to isolate DNA and to detect the presence of treponemal DNA in samples of equine hoof canker and bovine digital dermatitis. DNAzol®® Direct and column kits were used to isolate DNA from samples of equine hoof canker and bovine digital dermatitis. The presence of Treponema spp. was detected using PCR and Sanger sequencing. DNAzol®® Direct is suitable for isolating DNA from these types of samples. Treponemal DNA was detected in equine hoof samples as well as in bovine digital dermatitis skin samples. In equine hoof biopsies, the most frequently detected was Treponema pedis (8/13). Treponema brennaborense (2/13) and Treponema denticola (2/13) were also found. In the case of bovine digital dermatitis, Treponema medium ssp. bovis was confirmed in 14 of 36 skin samples. Treponema pedis (9/36), Treponema vincentii (1/36), Treponema phagedenis (1/36), and Treponema brennaborense (1/36) were detected as well. DNAzol®® Direct was more appropriate for isolation of treponemal DNA because the columns isolation method was more equipment and time-consuming. The presence of several Treponema spp. was determined in the samples. In horses, the most commonly detected species was a T. pedis, while in cattle it was T. medium ssp. bovis.

Linear elastic and hyperelastic studies of equine hoof mechanical response at different hydration levels.

Most simulation studies on equine hoof biomechanics employed linear elastic (LE) material models. However, the equine hoof wall's stress-strain relationship is nonlinear and varies with hydration level. Therefore, it is essential to investigate the accuracy of the LE model compared to more advanced material models, such as hyperelastic (HE) or viscoelastic models. The current research investigated performances of LE and three HE models (Mooney-Rivlin, Neo-Hookean, and Marlow) in describing equine hoof's mechanical behavior using finite element (FE) analysis. In the first attempt, a rectangular tissue specimen was simulated using the previously published experimental data. The Marlow HE model predicted the hoof wall stress-strain curve more accurately than the LE, Mooney-Rivlin, and Neo-Hookean models. The LE model accuracy, compared with the experimental results, varied within the reported range of the strain. However, the Marlow HE model perfectly matched the experimental data for a wide range of strains. In the second attempt, the entire hoof, including nine associated tissues, was modeled from computed tomography (CT) scans of an equine forelimb, and analyzed at trotting and standing modes of locomotion. The effect of environmental humidity on the hoof wall material properties was incorporated at four hydration levels; 0%, 53%, 75%, and 100%. The simulation results of the LE and HE models indicated that the minimum principal strain distribution on the hoof wall remained under 2% for various hydration levels and gait conditions. The numerical results of the Marlow HE model demonstrated better agreement with published experimental data compared to the LE, Mooney-Rivlin, and Neo-Hookean models. Higher hydration levels significantly increased the strains - a potential explanation could be the fact that the higher hydration levels decreased stiffness of the hoof wall tissues and ultimately increased strains. Higher ground reaction forces increased the von Mises stress at various points in the hoof wall, especially in the quarter regions and close to the coronet, where cracks and fractures are found more often in the physiological conditions.

EquiSim: An Open-Source Articulatable Statistical Model of the Equine Distal Limb.

Most digital models of the equine distal limb that are available in the community are static and/or subject specific; hence, they have limited applications in veterinary research. In this paper, we present an articulatable model of the entire equine distal limb based on statistical shape modeling. The model describes the inter-subject variability in bone geometry while maintaining proper jointspace distances to support model articulation toward different poses. Shape variation modes are explained in terms of common biometrics in order to ease model interpretation from a veterinary point of view. The model is publicly available through a graphical user interface (https://github.com/jvhoutte/equisim) in order to facilitate future digitalization in veterinary research, such as computer-aided designs, three-dimensional printing of bone implants, bone fracture risk assessment through finite element methods, and data registration and segmentation problems for clinical practices.

Preliminary Study on the Connection Between the Mineral Profile of Horse Hooves and Tensile Strength Based on Body Weight, Sex, Age, Sampling Location, and Riding Disciplines.

Hoof mineral profile is important as it could affect locomotion. Factors such as body weight, sex, age, and riding disciplines affect hoof mineral profile. In Mexico and globally, studies are needed on the characterization of mineral profile of hooves and tensile strength, as this could help to prevent overgrowth or microfractures. Therefore, in the present survey, 165 samples of equine hoof cuttings from different sex, ages, breeds, and riding disciplines from different regions of Mexico were analyzed for their mineral composition, and a universal testing machine was used to measure tensile strength. More than half of the samples were from males (63%) and aged 3 to 5 years (52%). Most samples were obtained from horses used for reproduction (36%) and working (29%) purposes. The most preponderant minerals were K (3,416 µg/g), Na (2,242 µg/g), and Ca (631 µg/g). Tensile strength ranged from 1.2 to 45 N. Females had higher (P < 0.05) amounts of Zn than males. Animals younger than 3 years old have lower (P < 0.05) levels of Na than those between 3 and 5 years old. Horses used for reproduction had lower (P < 0.05) Mg concentrations than animals used for running and working. Tensile strength was similar between demographic characteristics. Horses from Santa Gertrudis military ranch had higher (P < 0.05) levels of Ca, Se, and Na compared with horses from other sampling location. Copper was higher (P < 0.05) in horses from racecourse. Potassium was higher (P < 0.05) in horses from the Presidential General Staff. Overall, there was no evident connection between sex, ages, breeds, and riding disciplines from different regions of Mexico and the mineral composition of the hoof or its tensile strength. Further research should focus on the relation on specific feeding regimes, horse individual characteristic, hoof mineral contents, and tensile strength.

Equine Hoof Canker: Bovine Papillomavirus Infection Is Not Associated With Impaired Keratinocyte Differentiation.

Impaired keratinocyte differentiation has recently been suggested as a key event in equine hoof canker development. Koilocytotic appearance of keratinocytes, one of the most characteristic morphological alterations in hoof canker tissue, is also a common marker for papillomavirus (PV) infection, and bovine PV-1 and/or -2 (BPV-1/2) has previously been detected in equine canker patients. Therefore, the present study aimed to correlate the frequency and severity of koilocytotic keratinocytes with BPV detection in hoof canker samples. Hoof tissue of 5/18 canker-affected horses and 2/6 control horses tested positive for BPV-1/2 DNA using polymerase chain reaction. Thus, no association between the presence of BPV-1/2 papillomaviral DNA and koilocytotic appearance was found. Proteins associated with but not specific for PV infection were also investigated. Using immunohistochemistry, specific adhesion molecules (E-cadherin and ß-catenin) and intermediate filaments (keratins 6 and 14) important for intact epidermal barrier function and keratinocyte differentiation were documented in control samples (n = 6) and in hoof canker tissue samples (n = 19). Altered expression patterns of intermediate filaments and adhesion molecules were demonstrated in canker tissue, confirming the importance of incomplete keratinocyte differentiation, as well as the crucial role of keratinocyte differentiation in hoof canker.

A natural energy absorbent polymer composite: The equine hoof wall.

The equine hoof has been considered as an efficient energy absorption layer that protects the skeletal elements from impact when galloping. In the present study, the hierarchical structure of a fresh equine hoof wall and the energy absorption mechanisms are investigated. Tubules are found embedded in the intertubular matrix forming the hoof wall at the microscale. Both tubules and intertubular areas consist of keratin cells, in which keratin crystalline intermediate filaments (IFs) and amorphous keratin fill the cytoskeletons. Cell sizes, shapes and IF fractions are different between tubular and intertubular regions. The structural differences between tubular and intertubular areas are correlated to the mechanical behavior of this material tested in dry, fresh and fully hydrated conditions. The stiffness and hardness in the tubule areas are higher than that in the intertubular areas in the dry and fresh samples when loaded along the hoof wall; however, once the samples are fully hydrated, the intertubular areas become stiffer than the tubular areas due to higher water absorption in these regions. The compression behavior of hoof in different loading speed and directions are also examined, with the isotropy and strain-rate dependence of mechanical properties documented. In the hoof walls, mechanistically the tubules serve as a reinforcement, which act to support the entire wall and prevent catastrophic failure under compression and impact loading. Elastic buckling and cracking of the tubules are observed after compression along the hoof wall, and no shear-banding or severe cracks are found in the intertubular areas even after 60% compression, indicating the highly efficient energy absorption properties, without failure, of the hoof wall structure. STATEMENT OF SIGNIFICANCE: The equine hoof wall is found to be an efficient energy absorbent natural polymer composite. Previous studies showed the microstructure and mechanical properties of the hoof wall in some perspective. However, the hierarchical structure of equine hoof wall from nano- to macro-scale as well as the energy absorption mechanisms at different strain rates and loading orientations remains unclear. The current study provides a thorough characterization of the hierarchical structure as well as the correlation between structure and mechanical behaviors. Energy dissipation mechanisms are also identified. The findings in the current research could provide inspirations on the designs of impact resistant and energy absorbent materials.

Isolation, cultivation and immunofluorescence characterization of lamellar keratinocytes from equine hoof by using explants / Isolamento, cultivo e caracterização por imunofluorescência de queratinócitos lamelares de casco equino utilizando explantes

The importance of the hoof to the horse health is clear, and the current knowledge regarding the cellular aspects of hoof keratinocytes is poor. Studies on equine keratinocyte culture are scarce. Developing keratinocyte cultures in vitro is a condition for studies on molecular biology, cell growth and differentiation. Some methods have already been established, such as those for skin keratinocyte culture. However, few methodologies are found for lamellar keratinocytes. The objective of this study was to standardize the equine hoof keratinocyte isolation and cultivation, and then characterize the cell immunophenotype. For this, the primary culture method used was through explants obtained from three regions of the equine hoof (medial dorsal, dorsal, and lateral dorsal). After the cell isolation and cultivation, the cell culture and its explants were stained with anti-pan cytokeratin (pan-CK) (AE1/AE3), vimentin (V9), p63 (4A4), and Ki-67 (MIB-1) antibodies. Cells were grown to third passage, were positive for pan-CK, p63 and Ki-67, and few cells had vimentin positive expression. As for the explants, the epidermal laminae were not stained for vimentin or Ki-67. However, some cells presented positive pan-CK and p63 expression. This study demonstrated the viability of lamellar explants of equine hooves as a form of isolating keratinocytes in primary cultures, as well as characterized the proliferation ability of such keratinocytes in monolayers.(AU)

É notória a importância do casco na saúde dos equinos, mas o conhecimento em nível celular é pouco entendido. Estudos envolvendo o cultivo de queratinócitos equinos são escassos. Sabe-se que o desenvolvimento de cultivos de queratinócitos in vitro é uma condição para estudos sobre a biologia molecular, crescimento e diferenciação celular. Alguns métodos já estão estabelecidos, como para cultivo de queratinócitos de pele, mas poucas metodologias são encontradas para queratinócitos lamelares. O objetivo desse estudo foi padronizar o cultivo de queratinócitos provenientes de casco equino visando futuramente associar ao estudo da medicina regenerativa para assim estabelecer um modelo experimental in vitro e indicar o uso criterioso de terapias regenerativas para a laminite equina. Desta forma, o cultivo em monocamada e a caracterização de queratinócitos lamelares foram realizados. Para isso, o método de cultura primária utilizado foi através de explantes obtidos de três regiões do casco (dorso-medial, dorsal e dorso-lateral). As células foram caracterizadas para os marcadores anti pan-cytokeratin (AE1/AE3), vimentin (V9), p63 (4A4) e Ki-67 (MIB-1) nos cultivos e nos explantes. As células foram cultivadas até terceira passagem, tendo marcação positiva para pan-CK, p63 e Ki-67 e fraca marcação para vimentina. Já as lâminas epidermais não tiveram marcação de vimentin e Ki-67, porém marcaram acentuadamente para pan-CK e p63. Este estudo demonstrou a exiquibilidade do uso de explantes lamelares do casco de equinos, como forma de isolamento de queratinócitos em cultivos primários, bem como caracterizou a habilidade de proliferação desses queratinócitos em monocamada.(AU)