Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study.

OBJECTIVE: The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA). ANIMALS: EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA. METHODS: Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes. RESULTS: Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA. CLINICAL RELEVANCE: We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

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.

IL-1ß + TGF-ß2 dual-licensed mesenchymal stem cells have reduced major histocompatibility class I expression and positively modulate tenocyte migration, metabolism, and gene expression.

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-1ß + TGF-ß2 dual licensing and 2) evaluate if IL-1ß + TGF-ß2 dual-licensed MSCs had a greater ability to positively modulate tenocyte function compared to naive MSCs. SAMPLES: 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-1ß, 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.

Controlled Stiffness of Direct-Write, Near-Field Electrospun Gelatin Fibers Generates Differences in Tenocyte Morphology and Gene Expression.

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 (∼1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (∼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.

Mesenchymal stem cell licensing: enhancing MSC function as a translational approach for the treatment of tendon injury.

Tendon injuries are common in both veterinary and human clinical patients and result in morbidity, pain, and lost athletic performance. Consequently, utilizing naturally occurring injuries in veterinary patients as a comparative model could inform the development of novel therapies and increase translation for the treatment of human tendon injuries. Mesenchymal stem cells (MSCs) have shown considerable efficacy for the treatment of experimental and clinical superficial digital flexor tendon injury in the horse; however, the reinjury rate following treatment can remain high and MSC efficacy in treating other tendons is less well known. Additionally, the translation of MSC therapy to human tendon injury has remained poor. Recent evidence indicates that naïve MSC function can be enhanced through exogenous stimulation or manipulation of their environment. This stimulation or activation, herein termed MSC licensing, markedly alters MSC functions associated with immunomodulation, extracellular matrix remodeling, vascular development, bioactive factor production, and endogenous stromal/progenitor cell support. Additionally, a variety of licensing strategies has proven to influence MSC-secreted factors that have positively influenced outcome parameters in both in vitro and in vivo disease models separate from musculoskeletal tissues. Therefore, identifying the optimal licensing strategy for MSCs could ultimately provide an avenue for reliable and repeatable treatment of a broad range of tendon injuries of both veterinary and human clinical patients. This article details current evidence on the effects of licensed MSCs in both in vitro and in vivo disease models of different species and provides commentary on how those effector functions identified may be translated to the treatment of tendon injuries.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

Use of mesenchymal stem cells for tendon healing in veterinary and human medicine: getting to the "core" of the problem through a one health approach.

The purpose of this manuscript, which is part of the Currents in One Health series, is to take a comparative approach to stem cell treatment for tendon injury and consider how the horse might inform treatment in other veterinary species and humans. There is increasing experimental and clinical evidence for the use of bone marrow-derived mesenchymal stem cells to treat tendon injuries in the horse. The same evidence does not currently exist for other species. This manuscript will review why the equine superficial digital flexor tendon core lesion might be considered optimal for stem cell delivery and stem cell interaction with the injury environment and will also introduce the concept of stem cell licensing for future evaluation. The companion Currents in One Health by Koch and Schnabel, AJVR, October 2023, addresses in detail what is known about stem cell licensing for the treatment of other diseases using rodent models and how this information can potentially be applied to tendon healing.

Biomechanical evaluation of interlocking nail and locking compression plating for stabilization of ovine critical-sized segmental tibia defects.

Background: Segmental large volume bone loss resulting from fracture or osseous neoplasia is a major challenge to orthopedic surgeons and there is an ongoing quest to identify treatments that optimize healing. To advance treatment, large animal translational models-such as the ovine critical-sized tibia defect model-are instrumental for testing of novel scaffolds for bone regeneration. However, little standardization in the implants utilized for defect stabilization has been determined and current commercially available implants may be inadequate to replicate the strength of the native tibia. We hypothesize that a 10-mm interlocking nail (ILN) would be stiffer in axial, bending, and torsional loading than its 8-mm counterpart and would be stiffer in axial and torsional loading compared to a 4.5-mm broad locking compression plate (LCP). Methods: Tibias were harvested from 24 ovine hind limbs from skeletally mature ewes euthanized for reasons unrelated to this study and were randomized to treatment group. An ex vivo comparison of a novel 10-mm angle-stable non-tapered ILN was compared to a commercially available 8-mm angle-stable tapered ILN and a broad LCP in an ovine critical-sized (5-cm) tibia defect model. Axial stiffness, torsional stiffness, and bending stiffness were determined in control intact tibia and tibial constructs in the three treatment groups. Following implantation, radiography was performed in all limbs and tibia length and cortical and medullary cavity diameter were measured. Comparisons between groups were assessed with a one-way analysis of variance. Significance was set at P<0.05. Results: The 10-mm ILN in tibia containing a 5-cm ostectomy gap most closely replicated the structural properties of intact tibia compared with other constructs. The 10-mm ILN had significantly stronger torsional (P<0.001) and bending (P=0.002) stiffness than the 8-mm ILN, and was significantly stronger than the LCP in axial (P=0.04) and torsional (P=0.01) stiffness. Conclusions: A 10-mm ILN used to stabilize an ovine critically-sized tibia defect most closely mimicked the structural properties of the intact tibia when compared to a 8-mm ILN or broad LCP. Further in vivo testing will aid in determining which stabilization method is best suited for testing of novel tissue engineering and bone healing studies.

Pneumatic compression therapy using the EQ Press accelerates lymphatic flow in healthy equine forelimbs as determined by lymphoscintigraphy.

OBJECTIVE: Limb lymphedema in horses can be debilitating and painful. Pneumatic compression therapy has shown significant benefits for people suffering from lymphedema. The objective of this study was to determine the effect of a novel, equine-specific pneumatic compression device on the lymphatic flow of healthy horse forelimbs as determined by Tc-99m sulfur colloid lymphoscintigraphy. ANIMALS: 6 healthy Thoroughbreds. PROCEDURES: In a randomized crossover design, horses underwent bilateral forelimb lymphoscintigraphy following subcutaneous injection of Tc-99m sulfur colloid at the coronary band as untreated control or with pneumatic compression therapy using the EQ Press. Lateral, static images were obtained of the distal limb (time 0 to 60 minutes) and proximal limb (time 30 to 60 minutes) using a standard gamma camera. Lymphatic flow was determined by assigning a score to the time point at which Tc-99m sulfur colloid was first visualized at the level of the accessory carpal bone (1 to 7) in the distal limb and the cubital lymph node (1 to 4) in the proximal limb. RESULTS: EQ Press treatment led to a significantly faster lymphatic flow of Tc-99m sulfur colloid to the predetermined anatomic locations of the accessory carpal bone (P = .002) in the distal limb and the cubital lymph node (P = .001) in the proximal limb. CLINICAL RELEVANCE: Pneumatic compression therapy as provided by an equine-specific device encouraged lymphatic flow in healthy, nonedematous equine forelimbs. These data support further study of the EQ Press for pneumatic compression therapy in horses clinically affected by lymphedema and lymphatic drainage disorders.

TGF-ß2 enhances expression of equine bone marrow-derived mesenchymal stem cell paracrine factors with known associations to tendon healing.

BACKGROUND: Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix proteins that contribute to their ability to support tissue healing and regeneration. Both the transcriptome and the secretome of MSCs can be altered by treating the cells with cytokines, but neither have been thoroughly investigated following treatment with the specific cytokine transforming growth factor (TGF)-ß2. METHODS: RNA-sequencing and western blotting were used to compare gene and protein expression between untreated and TGF-ß2-treated equine bone marrow-derived MSCs (BM-MSCs). A co-culture system was utilized to compare equine tenocyte migration during co-culture with untreated and TGF-ß2-treated BM-MSCs. RESULTS: TGF-ß2 treatment significantly upregulated gene expression of collagens, extracellular matrix molecules, and growth factors. Protein expression of collagen type I and tenascin-C was also confirmed to be upregulated in TGF-ß2-treated BM-MSCs compared to untreated BM-MSCs. Both untreated and TGF-ß2-treated BM-MSCs increased tenocyte migration in vitro. CONCLUSIONS: Treating equine BM-MSCs with TGF-ß2 significantly increases production of paracrine factors and extracellular matrix molecules important for tendon healing and promotes the migration of tenocytes in vitro.

Interleukin-1ß in tendon injury enhances reparative gene and protein expression in mesenchymal stem cells.

Tendon injury in the horse carries a high morbidity and monetary burden. Despite appropriate therapy, reinjury is estimated to occur in 50-65% of cases. Although intralesional mesenchymal stem cell (MSC) therapy has improved tissue architecture and reinjury rates, the mechanisms by which they promote repair are still being investigated. Additionally, reevaluating our application of MSCs in tendon injury is necessary given recent evidence that suggests MSCs exposed to inflammation (deemed MSC licensing) have an enhanced reparative effect. However, applying MSC therapy in this context is limited by the inadequate quantification of the temporal cytokine profile in tendon injury, which hinders our ability to administer MSCs into an environment that could potentiate their effect. Therefore, the objectives of this study were to define the temporal cytokine microenvironment in a surgically induced model of equine tendon injury using ultrafiltration probes and subsequently evaluate changes in MSC gene and protein expression following in vitro inflammatory licensing with cytokines of similar concentration as identified in vivo. In our in vivo surgically induced tendon injury model, IL-1ß and IL-6 were the predominant pro-inflammatory cytokines present in tendon ultrafiltrate where a discrete peak in cytokine concentration occurred within 48 h following injury. Thereafter, MSCs were licensed in vitro with IL-1ß and IL-6 at a concentration identified from the in vivo study; however, only IL-1ß induced upregulation of multiple genes beneficial to tendon healing as identified by RNA-sequencing. Specifically, vascular development, ECM synthesis and remodeling, chemokine and growth factor function alteration, and immunomodulation and tissue reparative genes were significantly upregulated. A significant increase in the protein expression of IL-6, VEGF, and PGE2 was confirmed in IL-1ß-licensed MSCs compared to naïve MSCs. This study improves our knowledge of the temporal tendon cytokine microenvironment following injury, which could be beneficial for the development and determining optimal timing of administration of regenerative therapies. Furthermore, these data support the need to further study the benefit of MSCs administered within the inflamed tendon microenvironment or exogenously licensed with IL-1ß in vitro prior to treatment as licensed MSCs could enhance their therapeutic benefit in the healing tendon.

Clinical outcome of horses with guttural pouch infection following transpharyngeal fenestration.

OBJECTIVE: To report the clinical outcomes of horses with chronic guttural pouch infection characterized by accumulation of mucopurulent material following transpharyngeal diode laser fenestration. ANIMALS: 13 client-owned horses. PROCEDURES: Horses undergoing diode laser fenestration for chronic guttural pouch infection were identified by medical record search. Signalment, disease history, presence of mucopurulent empyema or chondroids, and pre- and postoperative therapy were recorded. Owners were contacted for follow-up information at a minimum of 6 months following surgery. RESULTS: 13 horses underwent laser fenestration for chronic guttural pouch infection. Thirteen had mucopurulent nasal discharge on presentation, and 3 were coughing. At follow-up, 12 horses treated with transpharyngeal diode laser fenestration had complete resolution of nasal discharge and coughing. One horse, despite resolution of guttural pouch infection on endoscopy, continued to have nasal discharge and coughing attributed to concurrent equine asthma syndrome. All owners expressed satisfaction with the surgical procedure and clinical resolution of guttural pouch infection. CLINICAL RELEVANCE: This surgical technique for transpharyngeal diode laser fenestration of the guttural pouch was uncomplicated to perform and well tolerated in sedated horses and attributed to resolution of clinical signs associated with guttural pouch infection, and owners reported a high satisfaction with the clinical outcome. Implementing this surgical technique could be considered to hasten resolution of chronic guttural pouch disease in horses with few technique-related complications.

Polyaxial pedicle screw external fixation to stabilize oblique mandibular fractures in three standing, sedated horses.

OBJECTIVE: To report the radiographic, surgical and postoperative features in horses with unstable oblique mandibular fractures secured with polyaxial pedicle screws (PPS) external fixation construct and intraoral wiring. ANIMALS: Three client-owned horses. STUDY DESIGN: Short case series. METHODS: Two horses each had a unilateral fracture, which did not improve after conservative management, and one horse had bilateral fractures. Clinical and radiographic features were documented. Polyaxial pedicle screw external fixators and intraoral tension band wiring were applied in standing horses after combining sedation and regional nerve anesthesia. Intraoral wires were implanted through a lateral buccotomy between teeth (two horses) or burred through exposed crown (one horse) and then secured around the incisors. The PPS were inserted under radiographic guidance to avoid tooth roots. Healing was assessed with radiographic examination. The PPS external fixator rod and intraoral wires were removed first. The mandible was manipulated, and, if it was stable, the PPS were removed. RESULTS: Implants were removed at 6, 8, or 10 weeks after the mandibles were palpably stable. Complications included broken wires in one horse, bone sequestration in one horse, and infection in one horse. Follow-up communication with the owners 12 to 18 months later confirmed complete healing without further complications of the fractured mandibles or teeth. CONCLUSION: Polyaxial pedicle screw external fixation led to fracture healing and a return to function in all three horses. The complications encountered did not preclude a successful outcome. CLINICAL SIGNIFICANCE: Polyaxial pedicle screw external fixation coupled with intraoral wiring provides an alternative to treat unstable equine mandibular fractures without general anesthesia.

Laparoscopic Ovariectomy in a Domestic Yak.

Owners of a juvenile domestic yak elected bilateral ovariectomy to prevent future reproduction. The yak was noted to be healthy at presentation. Both ovaries were removed using a laparoscopic approach as follows: after induction and maintenance of general inhalant anesthesia, 15 degrees Trendelenburg positioning was required to view the ovaries. Ovariectomy was conducted within a surgical time of 50 minutes. Due to the small ovarian size, portal enlargement was not necessary for removal. Mild hemorrhage from the left ovarian pedicle was controlled with application of a vessel-sealing device. Postoperative complications were not encountered during hospitalization. At 12 months following surgery, the yak was healthy, and the owner was highly satisfied with the procedure. The described approach was successful for performing laparoscopic ovariectomy in a juvenile yak. Positioning for surgery was similar to other small ruminant species. Further case enrollment is needed to optimize the surgical approach and better describe clinical outcomes.

Prospective randomized investigation of topical anesthesia during unilateral laparoscopic ovariectomy in horses.

OBJECTIVE: To compare pain-related responses in mares receiving topical or injected anesthesia of the ovarian pedicle prior to standing unilateral laparoscopic ovariectomy. STUDY DESIGN: Prospective randomized, blinded, placebo-controlled study. ANIMALS: Fifteen healthy research mares. METHODS: Mares were restrained in stocks and administered sedation. A right or left paralumbar ovariectomy was performed by using a laparoscopic portal and two instrument portals. Mares were divided into two treatment groups, and equal volumes of mepivacaine anesthesia were administered either topically (n = 8) or by injection into the ovarian pedicle (n = 7). Saline controls were simultaneously administered topically (n = 7) or by injection (n = 8), and surgeons were blinded to the treatment group. Ovarian removal was performed with traumatic forceps and a blunt tip vessel sealer and divider. Pain responses were measured by operative visual analog scale (VAS) scoring and perioperative serum cortisol response. Visual analog scale and serum cortisol were compared between groups by using Mann-Whitney testing. Serum cortisol concentrations were evaluated using repeated-measures one-way analysis of variance. RESULTS: Ovaries were removed in all mares by using the described technique without operative complications. Quantity of sedation required to complete the procedure, operative VAS scores, and perioperative cortisol concentrations did not differ between treatment groups. CONCLUSION: Application of topical mepivacaine to the ovary provided intraoperative analgesia similar to injection of the ovarian pedicle when performing unilateral standing laparoscopic ovariectomy in mares. CLINICAL SIGNIFICANCE: Topical anesthesia application to the ovary could provide an alternative to laparoscopic needle use, reducing the risk of inadvertent trauma to the pedicle or other visceral organs during laparoscopic ovariectomy.

Comparison of two techniques for transpharyngeal endoscopic auditory tube diverticulotomy in the horse.

Auditory tube diverticula, also known as guttural pouches, are naturally occurring dilations of the auditory tube in horses that communicate with the nasopharynx through a small ostium. Infection and select other conditions can result in inflammation and narrowing of the nasopharyngeal ostium, which prevents drainage of fluid or egress of air and can lead to persistent infection or guttural pouch tympany. Auditory tube diverticulotomy allows continuous egress from the auditory tube diverticula and is a feature of disease treatment in horses, in which medical treatment alone is not successful. Transpharyngeal endoscopic auditory tube diverticulotomy was performed using a diode laser either at a single dorsal pharyngeal recess location or bilaterally caudal to the nasopharyngeal ostium in 10 horse head specimens. Both methods resulted in clear communication between the nasopharynx and auditory tube diverticula. Diverticulotomy performed in the dorsal pharyngeal recess required less laser energy and activation time and had a shorter surgical duration than diverticulotomy performed caudal to the nasopharyngeal ostium. Further study related to the clinical application of both techniques is warranted.