Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

Proximal sesamoid bone microdamage is localized to articular subchondral regions in Thoroughbred racehorses, with similar fracture toughness between fracture and controls.

OBJECTIVE: To determine whether proximal sesamoid bone (PSB) microdamage and fracture toughness differ between Thoroughbred racehorses sustaining PSB fracture and controls. STUDY DESIGN: Cadaveric case-control. ANIMALS: Twenty-four Thoroughbred racehorses (n = 12 PSB fracture, n = 12 control). METHODS: Proximal sesamoid bones were dissected, and gross pathological changes and morphological measurements were documented. High-speed exercise history data were evaluated. Microdamage was assessed in fracture, fracture-contralateral limb (FXCL) and control PSBs using whole bone lead uranyl acetate (LUA) staining with micro-CT imaging or basic fuchsin histological analysis. Fracture toughness mechanical testing was carried out in 3-point-bending of microbeams created from PSB flexor cortices. Data were analyzed using ordinal logistic and linear regression models. RESULTS: Microdamage was detected most commonly in the articular subchondral region of PSBs via LUA micro-CT and basic fuchsin histology. There were no differences in microdamage between FXCL and control PSBs. Fracture toughness values were similar for FXCL (1.31 MPa√m) and control (1.35 MPa√m) PSBs. Exercise histories were similar except that horses sustaining fracture spent a greater percentage of their careers in rest weeks. CONCLUSION: Microdamage was detected in the articular region of PSBs but was not greater in horses sustaining catastrophic PSB fracture. Fracture toughness of PSB flexor cortices did not differ between FXCL and control PSBs. CLINICAL SIGNIFICANCE: Although uncommon, microdamage is localized to the articular region of Thoroughbred racehorse PSBs. Catastrophic PSB failure is not associated with lower PSB flexor cortex fracture toughness.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models.

BACKGROUND: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1ß-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1ß synovitis and tarsal intra-articular lavage. RESULTS: Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17-20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands - 2, - 3, - 5 and - 11) as compared to lavage. CONCLUSIONS: Synovial fluid lubricin increased in response to IL-1ß synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Surgical treatment of uterine neoplasia in 13 production size pigs with a comparison to pot-bellied pigs.

OBJECTIVES: The primary objective of this study was to describe the clinical management and surgical treatment of production size pigs (PrdP) with uterine neoplasia. A secondary objective was to compare tumor diagnoses as well as short- and long-term survival between PrdP and a published report of pot-bellied pigs (PBP) following surgical intervention. STUDY DESIGN: Retrospective clinical study. ANIMALS OR SAMPLE POPULATION: Client-owned PrdP (n = 13) treated with exploratory celiotomy ±ovariohysterectomy for uterine neoplasia. METHODS: Medical records from a university hospital were reviewed for historical treatment, presenting complaint, clinical signs, diagnostics, surgical intervention, pathology, and outcome. An online owner survey was performed for follow-up. The novel PrdP cohort was compared to a previously published PBP cohort for differences in tumor diagnoses, surgical complications, and survival. Descriptive statistics, Fischer's exact tests and odds ratios were reported. RESULTS: PrdP were affected by uterine leiomyoma (4/11), leiomyosarcoma (2/11), adenoma (1/11), adenocarcinoma (3/11), and carcinosarcoma (1/11) with no difference in tumor types between PrdP and PBP. PrdP surviving to hospital discharge (6/13) survived at least 1 year postoperatively, with median follow-up of 16 months (14-60 months). PrdP were less likely than PBP to survive in the short-term despite similar frequencies of marked intraoperative hemorrhage. PrdP and PBP had comparable rates of long-term survival following hospital discharge. CONCLUSION: PrdP are afflicted by similar uterine neoplasia diagnoses as PBP, but they have lower rates of short-term survival to hospital discharge with surgical treatment. CLINICAL SIGNIFICANCE/IMPACT: PrdP have a guarded prognosis for survival to hospital discharge when operated for uterine neoplasia.

Stable recombinant production of codon-scrambled lubricin and mucin in human cells.

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or "synDNAs," for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems.

Selective and Tunable Galectin Binding of Glycopolymers Synthesized by a Generalizable Conjugation Method.

Glycopolymers, conjugates of synthetic polymers with pendant carbohydrates, are becoming increasingly important to probe the role of carbohydrates in cellular processes and for applications like biosensors and drug delivery. A library of glycopolymers bearing different sugar moieties was synthesized by grafting amino-functionalized sugars to poly(acrylic acid) via DMTMM coupling. Primary amines were introduced at the anomeric (C-1) position to a number of unprotected mono-, di-, and trisaccharides using ammonium carbamate and conjugated to poly(acrylic acid) of different molecular weights, synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. This approach provides a simple and efficient route for the preparation of glycopolymers that differ only in the identity or degree of substitution of the sugar moiety on the polymer. The binding parameters (ka, kd, and KD) of these new glycopolymers to galectins-1 and -3 were quantified using surface plasmon resonance. The galectins selectively bound only to lactose-containing polymers, and the binding affinity was dependent on the galectin type, degree of sugar substitution and the molecular weight of polymer chains. Binding to both galectin-1 and -3 increased with a higher degree of sugar substitution, and higher molecular weight of the polymer backbone, reaching KD values on the order of 10-11 M.

Return to racing after surgical management of third carpal bone slab fractures in thoroughbred and standardbred racehorses.

OBJECTIVE: To determine the prognosis for racing of horses surgically treated for slab fractures of the third carpal bone (C3). STUDY DESIGN: Retrospective case study. ANIMALS: Horses (n = 125) surgically treated for C3 slab fractures. METHODS: Medical records of horses surgically treated for dorsal or sagittal C3 fractures were reviewed for age, sex, breed, limb, fracture type, degree of cartilage damage, and surgical treatment. Radiographs were evaluated to determine fracture depth, width, and displacement. Osteophytes, C3 lysis, and fragmentation were scored. Racing performance was obtained from online databases. Univariable and multivariable analyses were used to determine associations between independent variables and outcomes. RESULTS: Fifty-four (43%) horses raced postoperatively. Among thoroughbreds, 35% (30/86) with dorsal fractures and 63% (17/27) with sagittal fractures raced postoperatively. Among standardbreds, 77% (10/13) with dorsal fractures and 0% (0/2) with sagittal fractures raced postoperatively. Fracture displacement, C3 lysis, and cartilage damage affected the likelihood of racing postoperatively. Placement of 3.5-mm screws vs 4.5-mm screws and the placement of fewer screws were associated with improved likelihood of racing. CONCLUSION: The prognosis for postoperative racing of thoroughbreds with dorsal C3 fractures was less favorable than that previously reported. Concurrent joint pathology, such as cartilage damage at time of surgery, affected the ability of the horse to race postoperatively. CLINICAL SIGNIFICANCE: Although internal fixation of C3 slab fractures is required to restore joint congruity, return to racing should be expected in only 42% of thoroughbreds and 67% of standardbreds.

Foal Fractures: Osteochondral Fragmentation, Proximal Sesamoid Bone Fractures/Sesamoiditis, and Distal Phalanx Fractures.

Foals are susceptible to many of the same types of fractures as adult horses, often secondary to external sources of trauma. In addition, some types of fractures are specific to foals and occur routinely in horses under 1 year of age. These foal-specific fractures may be due to the unique musculoskeletal properties of the developing animal and may present with distinct clinical signs. Treatment plans and prognoses are tailored specifically to young animals. Common fractures not affecting the long bones in foals are discussed in this article, including osteochondral fragmentation, proximal sesamoid bone fractures/sesamoiditis, and distal phalanx fractures.

Laparoscopic-guided compared to skilled instructor support for student rectal examination training using live horses in the veterinary curriculum.

OBJECTIVES: To evaluate the veterinary student learning outcome of 2 methods of equine rectal examination training. STUDY DESIGN: Randomized prospective study. SAMPLE POPULATION: Veterinary students (3rd and 4th year; n = 40) and practicing equine veterinarians (n = 10). METHODS: Year 1: Group 1 (n = 11) and Group 2 students (n = 10) received skilled instructor (SI) and laparoscopic-guidance (LG), respectively, during rectal exam instruction. All students were tested on rectal identification of 4 abdominal organs. Year 2: One group of students (n = 19) was trained and subsequently tested using each technique, first SI, followed by LG. Subjective evaluation of laparoscopy as a teaching tool was achieved with veterinary students and equine practitioners. RESULTS: A significantly greater percentage of students having LG compared to SI were able to correctly identify the left kidney (Year 1) and the spleen, cecum, and right ovary (Year 2). A significantly greater proportion of LG trained students in years 1 and 2 (100% and 95%, respectively) were also able to identify 75% of organs compared with SI (27% and 21%, respectively). Both students and veterinarians uniformly provided favorable feedback for LG in teaching rectal palpation skills. CONCLUSION: The LG method of equine rectal examination instruction resulted in improved learning for identification of several key abdominal organs compared with SI.

Loss of effective lubricating viscosity is the primary mechanical marker of joint inflammation in equine synovitis.

Inflammation of the synovium, known as synovitis, plays an important role in the pathogenesis of osteoarthritis (OA). Synovitis involves the release of a wide variety of pro-inflammatory mediators in synovial fluid (SF) that damage the articular cartilage extracellular matrix and induce death and apoptosis in chondrocytes. The composition of synovial fluid is dramatically altered by inflammation in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. However, the relationship between key biochemical markers of joint inflammation and mechanical function of SF is not well understood. Here, we demonstrate the application of a novel analytical framework to measure the effective viscosity for SF lubrication of cartilage, which is distinct from conventional rheological viscosity. Notably, in a well-established equine model of synovitis, this effective lubricating viscosity decreased by up to 10,000-fold for synovitis SF compared to a ~4 fold change in conventional viscosity measurements. Further, the effective lubricating viscosity was strongly inversely correlated (r = -0.6 to -0.8) to multiple established biochemical markers of SF inflammation, including white blood cell count, prostaglandin E2 (PGE2), and chemokine ligand (CCLs) concentrations, while conventional measurements of viscosity were poorly correlated to these markers. These findings demonstrate the importance of experimental and analytical approaches to characterize functional lubricating properties of synovial fluid and their relationships to soluble biomarkers to better understand the progression of OA.

Recombinant manufacturing of multispecies biolubricants.

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O-glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

Equine non-septic tenosynovitis: A systematic literature review of site-specific pathological lesions, outcomes and surgical complications.

BACKGROUND: Non-septic tenosynovitis is a clinically relevant and often performance limiting musculoskeletal injury in the horse. OBJECTIVES: To review the published literature to determine which tendon sheaths are commonly affected by non-septic tenosynovitis and to describe the most frequently reported pathological lesions, outcomes, and surgical complications in equine non-septic tenosynovitis. STUDY DESIGN: Systematic review. METHODS: Literature searches were conducted in July 2021 from the online search engines PubMed, Scopus, Web of Science Core, VetMed Resource and ProQuest Theses & Dissertations. The inclusion criteria followed a participants, interventions, comparisons, outcome and study design (PICOS) approach. For inclusion, studies had to include live equids with non-septic tenosynovitis of any tendon sheath. Studies were excluded if they only described non-equine species, if they included data only on non-tendon sheath structures, or if they included data exclusively on cases of septic or contaminated tendon sheaths. Determination of non-sepsis relied on the diagnosis of the original authors; however, if non-sepsis was not explicitly specified, then cases that had a history of contamination of the sheath, a wound near the sheath or a positive bacterial culture were excluded from analysis. Data analysed included the distribution of structures affected by non-septic tenosynovitis, the most common pathological lesions identified within each sheath, and the most frequently reported surgical complications of non-septic tenosynovitis. The quality of each study was assessed using a methodological quality analysis. RESULTS: A total of 85 studies describing non-septic tenosynovitis in the horse were included. Across all 85 studies, there were a total of 2449 tendon sheaths in 2101 horses reported to be affected by non-septic tenosynovitis. The digital flexor sheath was the most reported structure to be diagnosed with non-septic tenosynovitis: 41/85 (48%) studies examined the digital flexor sheath exclusively, followed by the carpal flexor sheath, tarsal flexor sheath, carpal extensor sheaths, tarsal extensor sheaths, and one case of biceps brachii non-septic tenosynovitis. For most tendon sheaths, the most common pathological lesion was an intrathecal tear of a soft tissue structure, including tears of the deep digital flexor tendon and tears of the manica flexoria. Bilateral disease was most common in the carpal flexor sheath, where distal radial physeal exostoses were the most common pathological lesions. Less common causes of non-septic tenosynovitis included neoplasia, fracture of a bone adjacent to a tendon sheath, and mineralisation of an intrathecal tendon. The likelihood of return to previous level of athletic function following non-septic tenosynovitis of most structures was approximately 50%, and the most common complication was persistent effusion following tenoscopy. While iatrogenic infection following surgery was uncommon, it was most likely following tenoscopy of the digital flexor sheath. CONCLUSION: Non-septic tenosynovitis is commonly reported in equine athletes, with intra-thecal tears of the deep digital flexor tendon, superficial digital flexor tendon and manica flexoria frequently reported. Directions for future research include more thorough assessment of and reporting of complications following non-septic tenosynovitis and correlation of characteristics of intrathecal pathological lesions with clinical outcomes.

Synovial sepsis diagnostics and antimicrobial resistance: a one-health perspective.

This article, as part of the Currents in One Health series, reviews the current state of diagnostics for synovial sepsis. Synovial sepsis is a condition that affects veterinary and human medicine and requires coordinated efforts from both parties, as well as environmental considerations to accurately diagnose and preserve effective treatments. The article discusses best practices to identify the causative agent in septic synovitis, trends in bacterial identification and antimicrobial resistance patterns across common bacterial species, and a one-health perspective to optimize diagnostics across species. Antimicrobial resistance is a challenge facing both human and veterinary medicine and requires mindful and attentive prescribing to reduce the development of antimicrobial resistance and preserve antimicrobials for future application. The current standard of care for bacterial identification in veterinary practice is culture and antimicrobial susceptibility; however, positive culture rates from synovial sepsis cases often remain < 50%. Recent developments in advanced bacterial identification present opportunities for improved bacterial identification in synovial sepsis. Increased bacterial isolation will also help guide empirical antimicrobial therapy. Utilizing information and recommendations from both the human and veterinary literature will improve timely and accurate bacterial identification and therefore rapid and effective treatment of synovial sepsis across species and limit the development of antimicrobial resistance.

Recombinant mucin biotechnology and engineering.

Mucins represent a largely untapped class of polymeric building block for biomaterials, therapeutics, and other biotechnology. Because the mucin polymer backbone is genetically encoded, sequence-specific mucins with defined physical and biochemical properties can be fabricated using recombinant technologies. The pendent O-glycans of mucins are increasingly implicated in immunomodulation, suppression of pathogen virulence, and other biochemical activities. Recent advances in engineered cell production systems are enabling the scalable synthesis of recombinant mucins with precisely tuned glycan side chains, offering exciting possibilities to tune the biological functionality of mucin-based products. New metabolic and chemoenzymatic strategies enable further tuning and functionalization of mucin O-glycans, opening new possibilities to expand the chemical diversity and functionality of mucin building blocks. In this review, we discuss these advances, and the opportunities for engineered mucins in biomedical applications ranging from in vitro models to therapeutics.

Equine synovial sepsis laboratory submissions yield a low rate of positive bacterial culture and a high prevalence of antimicrobial resistance.

OBJECTIVE: To investigate (1) variables associated with the likelihood of obtaining a positive culture, (2) commonly isolated microorganisms, and (3) antimicrobial resistance patterns of isolates from horses with presumptive synovial sepsis. SAMPLES: Synovial fluid, synovium, and bone samples from equine cases with presumptive synovial sepsis submitted to the Cornell University Animal Health Diagnostic Center from 2000 to 2020 for microbial culture and antimicrobial sensitivity testing. PROCEDURES: Univariable and multivariable analyses were performed to determine the effect of variables on the likelihood of positive culture. Frequency distributions for isolated organisms and antimicrobial resistance were generated. Multidrug resistance patterns and associations were assessed with association rule mining. RESULTS: The positive culture rate for all samples was 37.4%, while the positive culture rate among samples confirmed to be septic by a combination of clinical pathological variables and case details was 43%. Blood culture vial submissions were 1.7 times more likely to yield a positive culture compared to samples submitted in a serum tube. Structure sampled, tissue submitted, and horse age were associated with a positive culture. Staphylococcus spp (23.7%), Streptococcus spp (22.4%), and Enterococcus spp (9.67%) were commonly isolated. Multidrug resistance prevalence decreased from 92% (2000 to 2009) to 76% (2010 to 2020) of gram-negative isolates and 60% (2000 to 2009) to 52% (2010 to 2020) of gram-positive isolates. CLINICAL RELEVANCE: The positive culture rate from synovial fluid submissions with traditional sampling and culture methods remains low and may be optimized by submitting samples in blood culture vials. Overall, antimicrobial resistance was frequently observed but did not increase from the first to second decade for most genera.

Metacarpophalangeal Joint Pathology and Bone Mineral Density Increase with Exercise but Not with Incidence of Proximal Sesamoid Bone Fracture in Thoroughbred Racehorses.

Proximal sesamoid bone (PSB) fracture is the leading cause of fatal musculoskeletal injury in Thoroughbred racehorses in Hong Kong and the US. Efforts are underway to investigate diagnostic modalities that could help identify racehorses at increased risk of fracture; however, features associated with PSB fracture risk are still poorly understood. The objectives of this study were to (1) investigate third metacarpal (MC3) and PSB density and mineral content using dual-energy X-ray absorptiometry (DXA), computed tomography (CT), Raman spectroscopy, and ash fraction measurements, and (2) investigate PSB quality and metacarpophalangeal joint (MCPJ) pathology using Raman spectroscopy and CT. Forelimbs were collected from 29 Thoroughbred racehorse cadavers (n = 14 PSB fracture, n = 15 control) for DXA and CT imaging, and PSBs were sectioned for Raman spectroscopy and ash fraction measurements. Bone mineral density (BMD) was greater in MC3 condyles and PSBs of horses with more high-speed furlongs. MCPJ pathology, including palmar osteochondral disease (POD), MC3 condylar sclerosis, and MC3 subchondral lysis were greater in horses with more high-speed furlongs. There were no differences in BMD or Raman parameters between fracture and control groups; however, Raman spectroscopy and ash fraction measurements revealed regional differences in PSB BMD and tissue composition. Many parameters, including MC3 and PSB bone mineral density, were strongly correlated with total high-speed furlongs.

Radiomics Modeling of Catastrophic Proximal Sesamoid Bone Fractures in Thoroughbred Racehorses Using µCT.

Proximal sesamoid bone (PSB) fractures are the most common musculoskeletal injury in race-horses. X-ray CT imaging can detect expressed radiological features in horses that experienced catastrophic fractures. Our objective was to assess whether expressed radiomic features in the PSBs of 50 horses can be used to develop machine learning models for predicting PSB fractures. The µCTs of intact contralateral PSBs from 50 horses, 30 of which suffered catastrophic fractures, and 20 controls were studied. From the 129 intact µCT images of PSBs, 102 radiomic features were computed using a variety of voxel resampling dimensions. Decision Trees and Wrapper methods were used to identify the 20 top expressed features, and six machine learning algorithms were developed to model the risk of fracture. The accuracy of all machine learning models ranged from 0.643 to 0.903 with an average of 0.754. On average, Support Vector Machine, Random Forest (RUS Boost), and Log-regression models had higher performance than K-means Nearest Neighbor, Neural Network, and Random Forest (Bagged Trees) models. Model accuracy peaked at 0.5 mm and decreased substantially when the resampling resolution was greater than or equal to 1 mm. We find that, for this in vitro dataset, it is possible to differentiate between unfractured PSBs from case and control horses using µCT images. It may be possible to extend these findings to the assessment of fracture risk in standing horses.

Metabolism and global protein glycosylation are differentially expressed in healthy and osteoarthritic equine carpal synovial fluid.

BACKGROUND: Carpal osteochondral fragmentation and subsequent post-traumatic osteoarthritis (PTOA) are leading causes of wastage in the equine athlete. Identification of synovial fluid biomarkers could contribute to the diagnosis and understanding of osteoarthritis (OA) pathophysiology. OBJECTIVE: The aim of this study was to identify differentially expressed metabolic and glycosylation pathways in synovial fluid from healthy horses and horses with naturally occurring carpal OA. STUDY DESIGN: Cross-sectional, in vivo metabolomics and glycomics study. METHODS: In cohort 1, carpal synovial fluid (n = 12 horses; n = 6 healthy, n = 6 OA) was analysed using high-resolution liquid chromatography mass spectrometry (LC-MS). In cohort 2 (n = 40 horses; n = 20 healthy, n = 20 OA), carpal synovial fluid was analysed using lectin microarrays and a lubricin sandwich ELISA. RESULTS: Metabolomic analysis identified >4900 LC-MS features of which 84 identifiable metabolites were differentially expressed (P < .05) between healthy and OA joints, including key pathways related to inflammation (histidine and tryptophan metabolism), oxidative stress (arginine biosynthesis) and collagen metabolism (lysine metabolism). Principle Component Analysis and Partial Least Squares Discriminant Analysis demonstrated separation between healthy and OA synovial fluid. Lectin microarrays identified distinct glycosylation patterns between healthy and OA synovial fluid, including increased Core 1/Core 3 O-glycosylation, increased α-2,3 sialylation and decreased α-1,2 fucosylation in OA. O-glycans predominated over N-glycans in all synovial fluid samples, and synovial fluid lubricin was increased in OA joints as compared to controls. MAIN LIMITATIONS: The sample size in cohort 1 was limited, and there is inherent variation in severity and duration of joint injury in naturally occurring OA. However, LC-MS identified up to 5000 unique features. CONCLUSIONS: These data suggest new potential diagnostic and therapeutic targets for equine OA. Future targeted metabolomic and glycomic studies should be performed to verify these results. Lectin microarrays could be investigated as a potential screening tool for the diagnosis and therapeutic monitoring of equine OA.