Adolescent females are at a higher risk of anterior cruciate ligament (ACL) injury than males. While prior studies have associated injury timing and menstrual cycle phase, these data are limited by indirect cycle tracking and lack of analysis on ACL structure, mechanics, or composition. Additionally, little is known about how female sex hormones influence the distinct ACL bundles. This exploratory study investigated associations between serum sex hormone concentrations and size, mechanics, and composition of the ACL and its bundles in a female adolescent pig model. Serum from nine adolescent female Yorkshire crossbreed pigs was collected pre-euthanasia and analyzed for levels of estradiol, progesterone, and testosterone. The ACL and its bundles were assessed for size via magnetic resonance imaging (MRI), mechanics via robotic testing, and composition via biochemical and histological analyses. While individual hormone levels and the estradiol-to-progesterone (E/P) ratio had no association with most metrics, the E/P ratio was significantly associated with ACL size and T2* relaxation time. Higher E/P ratios were negatively associated with anteromedial (AM) bundle cross-sectional area (CSA) (R2 = 0.44) and overall ACL volume (R2 = 0.49) and positively associated with posterolateral (PL) bundle T2* relaxation time (R2 = 0.69, p < 0.05). Serum E/P ratio was also positively associated with normalized ACL stiffness, but there were no associations observed for tissue composition. The results of this exploratory study indicate that the ACL may be responsive to exposure to the relative concentration of female sex hormone in a bundle-specific manner.

Objective:To describe the proposed mechanism of action of shock wave therapy and discuss treatment considerations and guidelines for use in equine and canine practice. Animals:Client-owned animals with owner consent. Methods:Shock wave therapy is proposed to stimulate healing by generating forces that cause cells to undergo microtrauma and release anti-inflammatory cytokines and growth factors into the treated tissues. While the 4 types of shock wave therapies are discussed, electrohydraulic and piezoelectric are described in detail, as they are most utilized in veterinary medicine for the treatment of tendon and ligament injuries, osteoarthritis, and nonunion fractures. The dose that is applied per patient is relative to the selected settings of depth, energy level, and number of pulses delivered. It is important to recognize that the highest energy deposition and greatest biological effects are seen at anatomic regions of differing tissue types such as bone and soft tissue interfaces; thus, shock wave is particularly useful at areas of enthesopathy. Results:Because of the mechanism of action, the use of anti-inflammatory medications and/or cryotherapy around shock wave treatment times should be avoided. Additionally, because of the potent analgesic effects of shock wave for the first 48 hours after treatment, rest is recommended to prevent any further damage to the tissues. Competition rules surrounding the use of shock wave must also be followed and discussed with owners. Clinical Relevance:Shock wave therapy is an accessible and useful modality for the treatment of tendon and ligament injuries, osteoarthritis, and nonunion fractures.

Equine ocular disease is common and often challenging to treat using traditional methods. This has led to the development of new therapies. Like human medicine, veterinary medicine is adopting cellular and gene therapy as innovative approaches. Equine ocular disease is a particularly promising area for these techniques. Notably, immune-mediated diseases (such as immune-mediated keratitis and equine recurrent uveitis), ulcerative keratitis, and infectious ocular diseases are of interest. Several ocular gene therapy products are approved for use in humans, and more are currently being researched in veterinary medicine. In veterinary practice, cell therapy mainly involves multipotent stromal cells or mesenchymal stem cells (MSCs), which are also widely studied in human medicine. This review aims to summarize the status of cell and gene therapy in equine ocular disease and provide background on the principles behind these treatments, as well as insights from human medicine. Although many in vitro studies and case series exist, a significant research gap remains. Despite growing clinical use, there are limited controlled in vivo studies assessing their safety, routes of administration, or effectiveness.

Objective:To examine regional mechanical and biochemical differences across the length of the equine superficial digital flexor tendon (SDFT). Methods:Equine SDFTs were collected postmortem from 9 donors and divided into proximal, middle, and distal regions based on the location of the musculotendinous and osteotendinous junctions. Tensile testing was performed on samples to obtain macroscale mechanical properties, and atomic force microscopy was done to obtain microscale mechanical properties. Biochemical assays and histological staining were done to assess glycosaminoglycan (GAG), collagen, and DNA content in each region. Results:Regional differences were present in macroscale mechanical properties and biochemical properties. Differences were primarily seen between the middle and distal region, with the distal region having a 3-fold increased GAG content (Δ14.15 µg GAG/mg sample; 95% CI, -0.32 to 28.62) and a 1.67-fold decreased macroscopic tensile modulus (Δ-68.2 MPa; 95% CI, -107.9 to -28.5). A more disorganized collagen fibril matrix was seen in the distal region through histological staining. Conclusions:Mechanical, biochemical, and histological regional differences exist with the equine SDFT. In particular, the distal region showed a disorganized matrix with higher GAG content and decreased modulus in comparison to the proximal and middle regions. Clinical Relevance:Our data provide information that helps better understand normal SDFT function and presentation of tendinopathy by providing regional structural and biochemical and mechanical information within the SDFT.

BACKGROUND:The prevalence of insulin dysregulation (ID) has not been investigated in sport horses. Stall-side insulin assays offer convenience but need further validation. OBJECTIVES:To determine the prevalence of and risk factors for ID in a sport horse population using an oral sugar test (OST) and assess agreement between stall-side and validated laboratory assays for insulin quantification. STUDY DESIGN:Prospective cross-sectional study. METHODS:One hundred eighty sport horses presenting for orthopaedic evaluation at a university hospital (N = 140) or local farms (N = 40) were tested for ID using OST (0.15 mL/kg light corn syrup). Morphometric data were collected, and baseline serum leptin and adiponectin concentrations were measured. Insulin concentrations were measured using the validated fluorescence enzyme immunoassay (FEIA) and a stall-side lateral flow assay (LFA). ID was diagnosed if FEIA insulin measured >20 μIU/mL at T0 or >30 μIU/mL at T60/T90. Factors associated with ID were assessed using a mixed effects binary logistic regression model. Assay agreement was assessed using Pearson's correlation coefficient and Bland-Altman plot. Receiver operator characteristic curve analysis was performed to determine diagnostic cut-offs for LFA. RESULTS:ID was diagnosed in 22.8% (41/180) of horses. Only 11/41 ID horses had increased fasted insulin. Increasing leptin/adiponectin ratio in males was the only significant predictor of ID (OR = 1.102, 95% CI 1.036-1.171). Correlation between FEIA and LFA insulin measures was good (r = 0.81, 95% CI 0.78-0.84), but the LFA read on average 13.62 μIU/mL higher than the FEIA and had greater variability at higher insulin concentrations. Area under the ROC curve showed the LFA was excellent at correctly classifying ID status (AUC = 0.95, 95% CI 0.93-0.97). MAIN LIMITATIONS:Single-centre study, voluntary enrolment may overestimate ID prevalence. CONCLUSIONS:ID is common in sport horses and often requires dynamic insulin testing to diagnose. Physical appearance does not predict ID risk. Agreement between stall-side and laboratory assays is good.

Objective:To determine the effect of limb position, exercise, and sedation on neovascularization detectability with the use of power Doppler ultrasonography (PDU) in superficial digital flexor tendon core lesions of the equine forelimb. Methods:9 Thoroughbred horses with superficial digital flexor tendon core tendinopathies were assessed with PDU before lesion induction (week 0) and 12 weeks after lesion induction, with 5 horses in the study period ranging from January to April 2024 and 4 horses from January to April 2025. During weeks 0 and 12, ultrasound examinations were performed 24 hours apart, with the limb weight-bearing and non-weight-bearing, before and after light exercise, and before and after IV detomidine sedation. The opposite forelimb served as a control. Results:Limb position was the only variable with statistical significance. No blood flow was visualized in the prelesion surgical or control limb under all conditions. After lesion induction, neovascularization was significantly increased under all conditions when the limb was imaged during non-weight-bearing. Conclusions:Practitioners may utilize the information from this study to acknowledge the importance of non-weight-bearing limb position for neovascularization detection with PDU and may continue the standard exercise performed during a lameness examination and detomidine sedation administration to facilitate a complete diagnostic evaluation. Clinical Relevance:Light exercise, such as a typical lameness examination, and the administration of IV detomidine sedation to facilitate an ultrasound evaluation will not impact the detectability or degree of neovascularization with the use of PDU. Imaging the limb during non-weight-bearing will significantly increase the detectability and is a critical component of a complete evaluation.

To determine the utility of a dorsolateral arthroscopic approach to the tarsocrural joint (TCJ) to examine and surgically access the medial malleolus (MM) and compare this to the standard dorsomedial approach to the MM. Experimental cadaver study. Six pelvic limbs from four adult horses. Arthroscopic examination of the dorsal aspect of the TCJ was performed followed by the dorsomedial and dorsolateral surgical approaches to the MM, in randomized order on cadaver limbs (n = 6). The dorsomedial approach involved placing the arthroscope and instrument in the dorsomedial pouch. The dorsolateral approach involved placing the arthroscope dorsolaterally and the instrument dorsomedially. Identification and surgical access grades for the MM were assessed and recorded. Using the dorsomedial approach, identification grades were excellent for the MM and surgical access to the MM was excellent or good in all limbs. Using the dorsolateral approach, identification and surgical access grades for the MM were excellent in all limbs. Interference between the arthroscope and instrument only occurred during the dorsomedial approach. The view of the axial aspect of the MM was improved with the dorsolateral approach. The dorsolateral approach allowed identification and surgical access to the MM and provided an improved view of the axial aspect of the MM. No interference between the instrument and arthroscope was encountered. The dorsolateral arthroscopic approach to the TCJ can be used for debridement of MM OCD lesions.

This review explores the use of biologics in the treatment of tendon and ligament injuries in horses, focusing on current and emerging therapies aimed at enhancing tissue repair and regeneration. It discusses the role of scaffold-based therapies, growth factors, blood-derived and tissue-derived biologics, and gene therapy as biologic regenerative therapies for improving healing outcomes. The review evaluates the mechanisms, efficacy, and challenges of biologics in clinical applications, highlighting recent advances and clinical studies. It provides an overview of current trends and future directions for biologic therapies in equine musculoskeletal injuries.

Musculoskeletal knee injuries are common and debilitating, with the most prevalent soft tissue injuries being anterior cruciate ligament (ACL) and meniscal tears. These tears do not heal well naturally, and biological therapies involving scaffolds are often unsuccessful, due in part to the synovial fluid environment of the joint. Viscous synovial fluid contains high concentrations of degradative enzymes, including plasmin, which prevents the stable formation of provisional fibrin scaffolds. Lack of provisional scaffold formation prevents bridging of torn tissue and subsequent remodeling for permanent tissue repair. Coagulation factors such as fibrinogen and thrombin, reinforced with synthetic platelet-like particles (PLPs), can be introduced to synovial fluid to promote fibrin scaffold formation. PLPs bind to and retract fibrin fibers to enhance stiffness, density, and stability of fibrin scaffolds. Therefore, the objective of this work is to investigate the role of PLPs in enhancing fibrin scaffold formation and degradation capabilities within synovial fluid and to characterize the resulting scaffold structure, density, and mechanics. We investigated effects in synovial fluid with high or low viscosity, as viscosity can change with injury and can vary between individuals. Following the addition of clotting factors and PLPs to synovial fluid, we found an increase in fibrin scaffold density, structure, and maximum mechanics for low viscosity, but not high viscosity, synovial fluid groups. Furthermore, by lowering the viscosity of synovial fluid with hyaluronidase, the increase in scaffold density following PLP addition was restored, indicating the strong role of synovial fluid viscosity on stable scaffold formation. This technology contributes to the development of a more robust fibrin-based therapy for intra-articular musculoskeletal injuries.

IntroductionNeutrophil extracellular traps (NETs) play a significant role in response to a variety of infectious and inflammatory stimuli in human and veterinary medicine. Although entrapment of bacteria can be an important function of NETs, the exuberant release of DNA and other intracellular molecules has also been negatively implicated in the pathogenesis of different diseases. Thus, NET formation must be tightly controlled and represents an opportunity for therapeutic interventions. Horses are particularly sensitive to bacterial stimuli that have previously been shown to cause NETs in other species, but the species-specific processes that control NET release have not been fully elucidated.MethodsThe purpose of this study was to compare the magnitude of response of equine neutrophils to different chemical and bacterial stimuli, including phorbol 12-myristate 13-acetate (PMA), a calcium ionophore (A23187), Staphylococcus aureus, and Escherichia coli. In addition, we investigated whether ex vivo equine NET formation is controlled by the NADPH-oxidase (NOX) pathway and by autophagy, both of which control NET formation in other species.ResultsWe demonstrated that equine neutrophils produce robust NETs in response to calcium ionophore and E. coli stimuli and produce fewer NETs in response to PMA and S. aureus. Both NOX-dependent and NOX-independent pathways of NET formation were identified in equine neutrophils. Autophagy inhibition altered the mechanics of NET release, by reducing the amount of extracellular DNA stranding.DiscussionThese results provide insight into equine-specific neutrophil biology, which could be key for managing equine diseases such as asthma and laminitis.

Orthobiologics rich in alpha-2-macroglobulin (A2M) are being used with increasing frequency to treat equine and human osteoarthritis (OA). The glycoprotein is concentrated from whole blood, typically prepared by a commercial device, and is administered IA with the goal of ameliorating inflammation and associated pain. In recent years, numerous investigations have elucidated A2M's mechanism of action and its effects on joint cells; however, none have used the horse as a model nor have they investigated the commercially available kit for equine orthobiologic preparation, Alpha2EQ. This narrative review presents the most pertinent work on A2M, which supports its current clinical use as an OA treatment. Alpha-2-macroglobulin has been studied in a variety of preclinical models, in vivo, and in clinical patients. As a naturally occurring and potent protease inhibitor, A2M acts to regulate key components of the OA inflammatory cascade, from cytokines and chemokines, which propagate synovitis, to disintegrins and metalloproteinases that degrade the cartilage extracellular matrix. Three main mechanisms of action contribute to A2M's modulation of joint inflammation and concomitant OA progression across species: the bait-and-trap mechanism, direct binding interactions, and regulation of gene expression. In vivo, A2M treatment results in improved histopathology scores, gait improvement in animals, and improved patient-reported outcomes in people. Though substantial evidence exists for A2M's anti-inflammatory effects and role in OA treatment, further studies will be required to elucidate the mechanisms of the equine orthobiologic.

Objective:To investigate the ability of the equine orthobiologic Alpha2EQ to control inflammation in cultured synovial fibroblasts. Methods:Equine synovial fibroblasts (n = 16) were cultured in a monolayer, and a targeted transcriptomic analysis (NanoString nCounter) was performed to screen for upregulated inflammatory gene expression. Cells were classified according to their IL-6 expression level. In the first experiment, high IL-6 expression (n = 4) and low IL-6 expression (4) cells were treated with Alpha2EQ, and in the second, cells with basal IL-6 expression were stimulated with 10 ng/mL IL-1β (4) before treatment. Allogeneic Alpha2EQ was pooled from sound healthy horses (n = 6) at a dose of 25% vol/vol of cell culture media. Twenty-four hours later, RNA was isolated for NanoString gene expression analysis. The t tests assessed differences in mean gene expression fold changes between baseline and treatment, while one-way ANOVA or Wilcoxon signed-rank tests were used for multiple comparisons (P < .05). Results:Alpha2EQ downregulated inflammatory genes in 2 cell culture models. Compared to baseline, Alpha2EQ treatment significantly reduced expression of IL-6, IL-15, and CCL2/MCP1 in IL-6HIGH synovial fibroblasts by 1.88- to 4.21-fold, as well as expression of IL-1β, CCL5/RANTES, and PPBP/CXCL7 by 2.14- to 4.07-fold in a 10 ng/mL IL-1β model. In addition, CXCL6/GCP-2 and TNF-α were significantly downregulated by Alpha2EQ in both models (2.64- to 5.38-fold). Conclusions:Alpha2EQ reduces inflammation by modulating the expression of cytokines and chemokines by synovial cells. Clinical Relevance:This study provides early insights into Alpha2EQ's anti-inflammatory mechanisms in vitro and evidence to support its clinical use in the treatment of equine osteoarthritis.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly ( N -isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Herein we report a new library of 2,3-pyrrolidinedione analogues that expands on our previous report on the antimicrobial studies of this heterocyclic scaffold. The novel 2,3-pyrrolidinediones reported herein have been evaluated against S. aureus and methicillin-resistant S. aureus (MRSA) biofilms, and this work constitutes our first report on the antibiofilm properties of this class of compounds. The antibiofilm activity of these 2,3-pyrrolidinediones has been assessed through minimum biofilm eradication concentration (MBEC) and minimum biofilm inhibition concentration (MBIC) assays. The compounds displayed antibiofilm properties and represent intriguing scaffolds for further optimization and development.

Staphylococcus aureus skin and soft tissue infection is a common ailment placing a large burden upon global healthcare infrastructure. These bacteria are growing increasingly recalcitrant to frontline antimicrobial therapeutics like vancomycin due to the prevalence of variant populations such as methicillin-resistant and vancomycin-resistant strains, and there is currently a dearth of novel antibiotics in production. Additionally, S. aureus has the capacity to hijack the host clotting machinery to generate fibrin-based biofilms that confer protection from host antimicrobial mechanisms and antibiotic-based therapies, enabling immune system evasion and significantly reducing antimicrobial efficacy. Emphasis is being placed on improving the effectiveness of therapeutics that are already commercially available through various means. Fibrin-based nanoparticles (FBNs) were developed and found to interact with S. aureus through the clumping factor A (ClfA) fibrinogen receptor and directly integrate into the biofilm matrix. FBNs loaded with antimicrobials such as vancomycin enabled a targeted and sustained release of antibiotic that increased drug contact time and reduced the therapeutic dose required for eradicating the bacteria, both in vitro and in vivo. Collectively, these findings suggest that FBN-antibiotic delivery may be a novel and potent therapeutic tool for the treatment of S. aureus biofilm infections.

Objectives: The number of anterior cruciate ligament (ACL) injuries are increasing in pediatric patients. These ACL injuries are commonly associated with meniscal tears or injuries. The lateral meniscus (LMEN) is more commonly torn at the time of injury, whereas the medial meniscus (MMEN) is more commonly injured over time in an ACL-deficient knee. Some clinicians favor non-surgical treatment of pediatric ACL injuries, though the risk of degeneration in the joint is increased. Studies have suggested that meniscal injuries may be isolated to the posterior region after ACL tears, suggesting that regional changes to the meniscus after ACL injury should be investigated. Our previous studies showed menisci volume increased after ACL transection (ACLT), however, regional volumes of the meniscus were not investigated. The objective of this study is to determine regional changes in magnetic resonance imaging (MRI)-based size and signal parameters, histology, and biochemistry of the menisci after an ACLT using a juvenile porcine model. Methods: All animal protocols were approved by the institutional animal care and use committee. Seven female juvenile (3 month) Yorkshire crossbreed pigs underwent a unilateral ACLT arthroscopically and a sham incision was made on the contralateral joint. After 12 weeks, both hind limbs were imaged using the 3.0-T Siemens MAGNETOM Skyra MRI system (T2 SWI sequence, voxel size 0.5 x 0.5 x 0.8 mm) (Figure 1A). Meniscal measurements were done for both the medial and lateral menisci. These measurements included middle height and width. The medial and lateral menisci were then segmented manually from the MRI scans. The segmented menisci were also separated by the posterior, middle and anterior regions respectively. All volumes (total, posterior, middle, and anterior) were recorded. Using the meniscal segmentations, T2* relaxation maps were created by fitting a monoexponential decay function on a voxel-by-voxel based on 6 echo times using MATLAB. Biochemical analysis was performed on the inner and outer areas of the middle region of both the medial and lateral menisci. A dimethylmethylene blue (DMMB) assay was performed to investigate the glycosaminoglycan (GAG) content of the meniscus before and after injury. Furthermore, compositional changes were histologically evaluated using a safranin O and fast green stain, where safranin O stains GAGs. Meniscus volume, height, meniscus T2* values and meniscus GAG content were all compared between the ACLT and sham operated joints using paired t-tests with Bonferroni-Dunn method as correction for multiple comparisons. Overall significance was set at α = 0.05. Results: At 12 weeks post-ACLT, total volume of both medial and lateral menisci increased by 27% (P=0.090) and 22% (P = 0.111) relative to contralateral sham-operated controls (Figure 1B), respectively. Specific meniscal regions also increased in volume. The middle region of both the MMEN (P = 0.048) and LMEN (P = 0.035) significantly increased along with the posterior region of the MMEN (P = 0.014) (Figure 1D-F). Similarly, the MMEN posterior horn height (P=0.024) significantly increased along with the MMEN middle width (P < 0.001) and LMEN middle height (P < 0.01) which both significantly increased (Figure 1G-I). T2* values significantly decreased compared to the sham operated controls in the entire lateral meniscus (P = 0.05) and regionally in the posterior lateral meniscus (P = 0.017) (Figure 2A-B). However, the T2* values in the medial posterior meniscus remained unchanged (Figure 2C). Via histology, GAG content is stained by the Saf-O (red). In normal tissue, the inner two thirds mainly consist of GAGs and the outer third is primarily fibrous tissue. Visually, the GAG content remains similar in LMEN, but decreases in MMEN after ACLT (Figure 3A). Overall, similar mean changes were observed via biochemistry, but with substantial variability (LMEN (P = 0.122); MMEN (P = 0.525) (Figure 3B). Conclusions: ACLT caused significant changes to the meniscus within 12 weeks of injury, specifically in the posterior and middle regions of the meniscus. Although meniscus remodeling has not been a primary metric for degeneration, studies have shown hypertrophy to be indicative of osteoarthritis. Our findings align with this. After an ACLT, the meniscus size increased. Furthermore, our study determined specific regions to better understand how the meniscus is impacted and where degeneration begins after an ACLT. Higher T2* values show greater disorganization in collagen fibers. Our results show a decrease in T2*, which could be a result of tissue remodeling in response to altered loading. Our results show greater differences when looking at size versus T2* values, which may be valuable since more complex imaging sequences would not be required. In future studies, T1rho mapping via MRI mapping can be used to associate changes in regional T1rho values with changes in GAGs due to degeneration. This could be an additional metric with histology to better quantify the compositional makeup of the meniscus after injury. Better understanding of where meniscal changes occur after ACL injury can lead to appropriate metrics for early detection of degeneration, which may aid in prevention of long-term changes, such as osteoarthritis.

Tendinopathy is a leading cause of mobility issues. Currently, the cell-matrix interactions involved in the development of tendinopathy are not fully understood. In vitro tendon models provide a unique tool for addressing this knowledge gap as they permit fine control over biochemical, micromechanical, and structural aspects of the local environment to explore cell-matrix interactions. In this study, direct-write, near-field electrospinning of gelatin solution was implemented to fabricate micron-scale fibrous scaffolds that mimic native collagen fiber size and orientation. The stiffness of these fibrous scaffolds was found to be controllable between 1 MPa and 8 MPa using different crosslinking methods (EDC, DHT, DHT+EDC) or through altering the duration of crosslinking with EDC (1 h to 24 h). EDC crosslinking provided the greatest fiber stability, surviving up to 3 weeks in vitro. Differences in stiffness resulted in phenotypic changes for equine tenocytes with low stiffness fibers (similar to 1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (similar to 8 MPa) were rounded. High stiffness fibers resulted in the upregulation of matrix metalloproteinase (MMPs) and proteoglycans (possible indicators for tendinopathy) relative to low stiffness fibers. These results demonstrate the feasibility of direct-written gelatin scaffolds as tendon in vitro models and provide evidence that matrix mechanical properties may be crucial factors in cell-matrix interactions during tendinopathy formation.

Introduction: One major obstacle in validating drugs for the treatment or prevention of hearing loss is the limited data available on the distribution and concentration of drugs in the human inner ear. Although small animal models offer some insights into inner ear pharmacokinetics, their smaller organ size and different barrier (round window membrane) permeabilities compared to humans can complicate study interpretation. Therefore, developing a reliable large animal model for inner ear drug delivery is crucial. The inner and middle ear anatomy of domestic pigs closely resembles that of humans, making them promising candidates for studying inner ear pharmacokinetics. However, unlike humans, the anatomical orientation and tortuosity of the porcine external ear canal frustrates local drug delivery to the inner ear.Methods: In this study, we developed a surgical technique to access the tympanic membrane of pigs. To assess hearing pre- and post-surgery, auditory brainstem responses to click and pure tones were measured. Additionally, we performed 3D segmentation of the porcine inner ear images and used this data to simulate the diffusion of dexamethasone within the inner ear through fluid simulation software (FluidSim).Results: We have successfully delivered dexamethasone and dexamethasone sodium phosphate to the porcine inner ear via the intratympanic injection. The recorded auditory brainstem measurements revealed no adverse effects on hearing thresholds attributable to the surgery. We have also simulated the diffusion rates for dexamethasone and dexamethasone sodium phosphate into the porcine inner ear and confirmed the accuracy of the simulations using in-vivo data.Discussion: We have developed and characterized a method for conducting pharmacokinetic studies of the inner ear using pigs. This animal model closely mirrors the size of the human cochlea and the thickness of its barriers. The diffusion time and drug concentrations we reported align closely with the limited data available from human studies. Therefore, we have demonstrated the potential of using pigs as a large animal model for studying inner ear pharmacokinetics.

The absence of novel antibiotic classes, coupled with the rising threat of antibiotic persistence and resistance, is pushing the world perilously close to a new pre-antibiotic era. Over 35 000 people die every year in the US as a consequence of antimicrobial-resistant infections. Bacterial biofilms further complicate this scenario, as they are inherently more resistant to antibiotic treatments. Currently, there are no approved single agent or adjuvant small molecules for treating biofilm-complicated infections. Herein, we report the synthesis and microbiological evaluation of a novel library of 25+ monomeric and dimeric pyrrolidine-2,3-dione scaffolds. These compounds have displayed improved aqueous solubility, potent anti-biofilm properties, a low MBEC-to-MIC ratio, and synergism with FDA-approved antimicrobials against biofilm infections, constituting a promising technology as antimicrobial adjuvants.

OBJECTIVE:The study objectives were to 1) determine the mesenchymal stem cell (MSC) surface expression of major histocompatibility complex (MHC) class I and transcriptome-wide gene expression changes following IL-1β + TGF-β2 dual licensing and 2) evaluate if IL-1β + TGF-β2 dual-licensed MSCs had a greater ability to positively modulate tenocyte function compared to naive MSCs. SAMPLE:Equine bone marrow-derived MSCs from 6 donors and equine superficial digital flexor tenocytes from 3 donors. METHODS:Experiments were performed in vitro. Flow cytometry and bulk RNA sequencing were utilized to determine naive and dual-licensed MSC phenotype and transcriptome-wide changes in gene expression. Conditioned media were generated from MSCs and utilized in tenocyte cell culture assays as a method to determine the effect of MSC paracrine factors on tenocyte function. RESULTS:Dual-licensed MSCs have a reduced expression of MHC class I and exhibit enrichment in functional pathways associated with the extracellular matrix, cell signaling, and tissue development. Additionally, dual-licensed MSC-conditioned media significantly improved in vitro tenocyte migration and metabolism to a greater degree than naive MSC-conditioned media. In tenocytes exposed to IL-1β, dual-licensed conditioned media also positively modulated tenocyte gene expression. CLINICAL RELEVANCE:Our data indicate that conditioned media containing paracrine factors secreted from dual-licensed MSCs significantly modulates in vitro tenocyte function, which may confer benefits in vivo to healing tendons following injury. Additionally, due to reduced MHC class I expression in dual-licensed MSCs, this technique may also provide an avenue to provide an effective "off-the-shelf" allogenic source of MSCs.