Department of Clinical Sciences

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These digital collections include theses, dissertations, student publications, faculty publications, and datasets from the Department of Clinical Sciences.

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Open Access
Cartilage repair using trypsin enzymatic pretreatment combined with growth-factor functionalized self-assembling peptide hydrogel
(Colorado State University. Libraries, 2019) Zanotto, Gustavo Miranda, author; Frisbie, David D., advisor; Grodzinsky, Alan, committee member; McIlwraith, C. Wayne, committee member; Barrett, Myra F., committee member; Puttlitz, Christian, committee member
Treatment of cartilage defects remains challenging in the orthopedic field. Several techniques are currently available to treat cartilage defects, with subchondral bone microfracture being the most commonly used marrow stimulation technique. However, despite satisfactory results in the short-term, clinical and functional outcomes of microfracture treated patients tend to decline over time. Improving microfracture technique using tissue engineering principles may be a more attractive way to treat cartilage defects compared to other more complex and expensive alternatives. Self-assembling peptide hydrogel has been extensively studied as a scaffold for cartilage repair. This hydrogel is biocompatible within the joint environment and has been shown to increase cartilage healing and improve clinical and functional outcomes in both rabbit and equine models of cartilage repair. Recently, a clinically applicable technique was described using trypsin enzymatic pretreatment of the surrounding cartilage combined with local delivery of heparin binding insulin growth factor-1 (HB-IGF-1). The results of this study demonstrated improved cartilage integration in vitro when this technique is utilized. Thus, in the present study we evaluated the combination of trypsin enzymatic pretreatment with a self-assembling peptide hydrogel functionalized with growth factors to improve cartilage repair. First, the effect of trypsin enzymatic pretreatment alone or combined with self-assembling peptide hydrogel functionalized with HB-IGF-1 and/or platelet-derived growth factor- BB (PDGF-BB) was tested using a rabbit model (48 rabbits). Subsequently, trypsin enzymatic pretreatment combined with self-assembling peptide hydrogel functionalized with HB-IGF-1 and PDGF-BB was used to augment microfracture augmentation in an equine model of cartilage defects (8 horses). In the small animal model, trypsin enzymatic pre-treatment resulted in an overall increase in defect filling, as well as improvements in subchondral bone reconstitution, surface regularity, cartilage firmness, reparative tissue color, cell morphology and chondrocyte clustering. The presence of PDGF-BB alone improved subchondral bone reconstitution and basal integration, while the combination of HB-IGF-1 and PDGF-BB resulted in an overall improvement in tissue and cell morphology. In the equine model, microfracture augmentation using trypsin enzymatic pretreatment combine with self-assembling peptide hydrogel functionalized with growth factors (HB-IGF-1 and PDGF-BB) resulted in better functional outcomes, better defect healing on second look arthroscopy at 12 months, as well as improved reparative tissue histology and increased biomechanical proprieties of the adjacent cartilage compared to defects treated with microfracture only. In conclusion, trypsin enzymatic pretreatment combined with self-assembling peptide hydrogel functionalized with growth factors (HB-IGF-1 and PDGF-BB) resulted in successful microfracture augmentation. These therapeutic approaches can result in a more cost effective way to improve cartilage healing in patients undergoing subchondral bone microfracture.
Open Access
Equine cervical pain and dysfunction
(Colorado State University. Libraries, 2021) Story, Melinda R., author; Kawcak, Christopher E., advisor; Haussler, Kevin K., advisor; McIlwraith, C. Wayne, committee member; Nout-Lomas, Yvette S., committee member; Barrett, Myra F., committee member; Frisbie, David D., committee member
Cervical pain and dysfunction in horses has become more recognized in recent years. However, a horse may present with a long list of different clinical syndromes and the examination findings can be confusing, resulting in difficulty effectively treating the horse. This frequently leads to frustration by the owner, as well as the veterinarian charged with helping the horse. This body of work aims to enlighten the reader of the dearth of understanding of cervical pain and dysfunction, to highlight how dangerous behavior may be related to cervical pain, and describe the course and development of future research. There is a paucity of peer-reviewed equine literature available describing cervical pain and dysfunction in the horse. The first chapter is designed to provide a synopsis of the current state of understanding of the disease processes, diagnostic capabilities, and possible treatment strategies available to manage cervical pain and dysfunction in horses. The second chapter describes a series of horses displaying unwanted behavior that became dangerous to the rider and often times to the horse itself. The included horses all had moderate to severe ganglionitis at multiple vertebral levels. Ganglionitis has been associated with neuropathic pain in other species, and is believed to be causing a state of neuropathic pain in this series of horses. This study highlights the need for deeper understanding of pain behavior in horses. Chapter 3 describes a prospective evaluation of cervical pain and dysfunction in 12 horses. Recombinant equine interleukin-1β (reIL-1β) has been used as an acute synovitis model within the appendicular skeleton and was utilized in this study to create transient synovitis at the cervical articulation of C5-C6. This study evaluated the clinical, biomechanical and ultrasonographic features in horses with a known source of neck pain. Acute synovitis of the articular process joint (APJ) induced clinical signs of myofascial pain and neck stiffness with variable degrees of forelimb lameness. Ultrasonographic evidence of the presence and severity of APJ effusion could be readily identified and tracked over time. Utilizing this model in the future could further add to our understanding of the clinical presentations in horses experiencing cervical pain and dysfunction. Through this collection of work, we have developed collaborations to investigate many unanswered questions that have been raised. We will look to define pathways related to neuropathic pain mechanisms in order to ultimately improve the quality of life, not only for our equine patients, but potentially of other veterinary species and even the human population experiencing chronic pain.
Open Access
Investigation of cationic contrast-enhanced computed tomography for the evaluation of equine articular cartilage
(Colorado State University. Libraries, 2017) Nelson, Bradley B., author; Kawcak, Christopher E., advisor; Goodrich, Laurie R., advisor; McIlwraith, C. Wayne, committee member; Grinstaff, Mark W., committee member; Barrett, Myra F., committee member
Osteoarthritis and articular cartilage injury are substantial problems in horses causing joint pain, lameness and decreased athleticism resonant of the afflictions that occur in humans. This debilitating joint disease causes progressive articular cartilage degeneration and coupled with a poor capacity to heal necessitates that articular cartilage injury is detected early before irreparable damage ensues. The use of diagnostic imaging is critical to identify and characterize articular cartilage injury, though currently available methods are unable to identify these early degenerative changes. Cationic contrast-enhanced computed tomography (CECT) uses a cationic contrast media (CA4+) to detect the early molecular changes that occur in the extracellular matrix. Glycosaminoglycans (GAGs) within the extracellular matrix are important for the providing the compressive stiffness of articular cartilage and their degradation is an early event in the development of osteoarthritis. Cationic CECT imaging capitalizes on the electrostatic attraction between CA4+ and GAGs; exposing the proportional relationship between the amount of GAGs present within and the amount of CA4+ that diffuses into the tissue. The amount of CA4+ that resides in the tissue is then quantified through CECT imaging and estimates tissue integrity through nondestructive assessment. Despite the emergence of this promising technology to capture quantitative information on articular cartilage quality, cationic CECT has not been thoroughly investigated in equine tissues in vitro or in vivo, nor has it been investigated in in vivo in any large animal or human subjects. This compilation of studies was designed to critically examine the capacity of cationic CECT to provide quantitative information on articular cartilage quality across a continuum of disease states in horses while dually serving as a translational model to showcase its potential application in humans. The first experiments successfully characterized the diffusion properties of CA4+ into equine articular cartilage in vitro and in vivo, and also established the femoropatellar joint as the optimum joint for use in the subsequent in vivo experimental models. These initial studies also established preliminary evidence of the safety of CA4+ on articular tissues. The experimental in vivo equine impact model delivered a contusive force to articular cartilage that successfully instituted degeneration of the extracellular matrix components and decreased tissue stiffness. This subtle degradation was discernable from healthy articular cartilage using cationic CECT imaging (microCT). In vivo, the clinical cationic CECT attenuation demonstrated fair correlations with the biochemical, mechanical and histologic staining measures, though there was not sufficient delineation among groups to detect significant differences between disease states. Further investigations are warranted to determine how focal sites of injury can be identified from the resolution limited by current CT technology. The second in vivo experimental study generated reparative and degenerative articular cartilage to provide comparisons with healthy tissue. The cationic CECT method, using microCT and clinical scanners, demonstrated an ability to distinguish between these groups of varying articular cartilage quality. Though the results were promising, this study revealed the complexity of detecting subtle articular cartilage injury in a solitary imaging examination owing to the inherent biochemical and biomechanical variation that exists across articular surfaces. However, this study confirmed the successful monitoring of articular cartilage through longitudinal examinations. Overall, this collection of studies established that cationic CECT imaging in vitro and in vivo is capable of distinguishing articular cartilage across a spectrum of disease states exemplifying its utility in the comprehensive evaluation of equine articular cartilage. Further work of how this technique compares to other quantitative diagnostic imaging techniques and optimization strategies for routine use are required. Nonetheless, the results of this work reveal the excellent potential of cationic CECT imaging to be incorporated into research and clinical investigations in horses and highlight the feasible translation of this work into human tissues.
Open Access
Presale diagnostic imaging in Thoroughbred horses: the prevalence and progression of radiological and ultrasonographical findings and their associations with racing performance
(Colorado State University. Libraries, 2024) Peat, Frances J., author; Kawcak, Christopher E., advisor; McIlwraith, C. Wayne, advisor; King, Melissa R., committee member; Selberg, Kurt T., committee member; Barrett, Myra F., committee member
Background: The sale of young horses at bloodstock auctions plays an important role in the success of the Thoroughbred industry worldwide. The Keeneland September Yearling Sale in Lexington, Kentucky, is the largest sale of Thoroughbred yearlings in the world. Conducted over 12 days and presenting between 2500 and 3000 horses through the auction ring, the sale now records gross receipts exceeding 400 million United States Dollars (USD) per year. Veterinarians at the sale perform presale inspections on future racing prospects and consult with prospective purchasers regarding a horse's suitability for its intended use. Radiography and ultrasonography are used in presale examinations to identify orthopaedic issues that may affect soundness during athletic training and racing. Modern diagnostic imaging technology produces high quality images that have enabled the detection of a number of presale findings of unknown significance in young horses. Variable interpretation of these findings and conflicting assignments of potential clinical importance have become a source of contention in the Thoroughbred industry and this requires resolution. Specific imaging findings for which further scientific evidence is needed include radiological changes in the equine medial femoral condyles (MFCs) and proximal sesamoid bones (sesamoids) and ultrasonographic findings in the medial and lateral branches of the suspensory ligament (branches). Changes in the sesamoid and the insertional region of the adjacent suspensory branch are of particular importance in young Thoroughbreds, due to the potential for catastrophic injury to the suspensory apparatus in which they are an integral structure. Little is known about the prevalence of concurrent ultrasonographic branch change relative to the various grades of radiological sesamoid appearance seen in horses. Objective scientific data would enable prepurchase and training management decisions to be made that are in the best interests of the horse and its connections and the wider industry. Objectives: In the equine stifle (femorotibial joint) and fetlock (metacarpo/metatarsophalangeal joint), the objectives of this doctoral research were firstly to identify the prevalence of subchondral lucencies (SCLs) in the distal aspect of the MFC and the prevalence of various sesamoid changes on sales repository radiographs in yearling and 2-year-old Thoroughbreds, and to identify the prevalence of ultrasonographic suspensory branch changes in the same population of horses. Secondly, the studies aimed to monitor changes in MFC, sesamoid and suspensory branch grades between yearling and 2-year-old sales in horses that presented for sale at both ages. Thirdly, the research aimed to determine any associations between grade of MFC, sesamoid or suspensory branch findings and future racing performance. Lastly, the research was designed to examine the existence of concurrent radiological and ultrasonographic findings in individual sesamoid-branch units in sales horses; to determine whether there are any radiological findings that are consistently accompanied by a particular degree of insertional branch change and to provide practical recommendations as to when suspensory branch ultrasonography may be warranted in the sales environment. The overriding objective was to provide an evidence-based determination of which presale imaging findings should be regarded as an acceptable appearance at a given age in sales horses and which findings constitute a risk to future performance. Methods: The research was performed via prospective cohort studies using enrolled samples. Sales repository radiographs were obtained with consignor permission from the 2016 Keeneland September Yearling Sale and the five major North American 2-year-old sales in 2017 run by Fasig-Tipton Company and Ocala Breeders' Sales Company. Ultrasonography was performed immediately prior to the sales on the forelimb suspensory branches of horses with consignor permission. Stifle and fetlock radiographs were evaluated for MFC and sesamoid changes, respectively. MFC SCL were graded on a scale of 0-3 according to radiological size and axial MFC lucencies were recorded separately. Sesamoid findings relating to vascular channel appearance (0-3), abaxial contour changes and apical and abaxial fragments were graded according to a grading system established for the purposes of this study. Ultrasonographic findings relating to suspensory branch size, fibrillar pattern, the presence of hyperechoic foci, periligamentar tissue thickness and the adjacent proximal sesamoid bone surface were recorded during post-sale image evaluation. Racing performance was assessed for all study horses until the end of their 4-year-old racing season and performance data was obtained from Equibase Company LLC. Racing performance was measured via eight outcome variables: whether the horse started at least one race by the end of their 4-year-old year, age at first race start, total number of race starts, total prizemoney earned, earnings per start, class of career best start achieved, weighted Listed and Group race starts and Class Performance Index. Clinical follow-up was sought to ascertain why horses that did not race never started. Distributions of imaging findings were examined using descriptive statistics at the individual bone and branch level and at the horse level. Associations between imaging findings and racing performance from 2 to 4 years of age were examined using multivariate regression analyses, controlling for horse sex. Analysis was via logistic, negative binomial or linear regression as appropriate, with the threshold for significance set at a=0.05. Results: Sales radiographs from 2,508 yearlings and 436 2-year-olds were included. This sample represented 11% of the annual US Thoroughbred foal crop. It comprised 36% of all yearlings sold at auction in North America in 2016 and 20% of all 2-year-olds sold at auction in North America in 2017. Radiographs of 5,016 yearling stifles and 872 2-year-old stifles were evaluated. MFC SCLs of Grades 1-3 were observed in 242 yearlings (9.7%) and 49 2-year-olds (11.2%). Bilateral MFC SCLs of Grades 1-3 were observed in 54 yearlings (2.2%) and 12 2-year-olds (2.8%). Yearling Grade 1 MFC SCLs had either resolved (11/31), remained unchanged (14/31) or progressed to a Grade 2 (6/31) by 2-year-old sales. Yearling Grade 2 MFC SCLs had either improved to a Grade 1 (2/10), remained unchanged (6/10) or progressed to a Grade 3 (2/10) by 2-year-old sales. Yearlings with a Grade 3 MFC SCL had a 78% probability of starting a race (95% Confidence Interval (CI): 58.2-89.6%), compared to 84% for MFC Grade 0 yearlings (95% CI: 82.7-85.8%). Six of the seven yearlings with axial MFC lucencies raced. Radiographs of 20,064 yearling sesamoids and 3,488 2-year-old sesamoids were evaluated. Interobserver agreement using the new radiological grading system was substantial. Yearling findings associated with a significantly reduced probability of starting a race were: Grade 3 vascular channels in forelimb sesamoids (0.52, P<0.001, 95% CI: 0.37-0.67), abaxial new bone in forelimb sesamoids (0.62, P=0.01, 95% CI: 0.49-0.73), apical or abaxial fragments in forelimb sesamoids (0.55, P=0.005, 95% CI: 0.37-0.72). For affected horses that did race, Grade 3 vascular channels in forelimb sesamoids were associated with fewer race starts (9.9 starts, P=0.03, 95% CI: 8.0-12.2) and Grade 3 vascular channels in hindlimb sesamoids were associated with a delayed start to racing careers (54 days, P=0.01, 95% CI: 20-89). Abaxial new bone in forelimb sesamoids was associated with a 54% reduction in total earnings (P=0.003, 95% CI: 24-72) and a 46% reduction in earnings per start (P=0.002, 95% CI: 21-64). Abaxial concavity occurred predominantly in yearling medial forelimb sesamoids, had no impact on racing performance and mostly resolved by two-year-old sale. A total of 593 sales yearlings and 367 2-year-olds had ultrasonography performed on all four forelimb suspensory branches per horse. Grade ≥2 fibrillar branch change was present in 8.9% of yearlings and 14.4% of 2-year-olds. A 0.25cm increase in branch width was associated with a 49-day delayed start to racing careers (P<0.001, 95% CI: 21-77 days). The presence of Grade 2 hyperechoic foci was associated with significantly lower total earnings (P=0.01, 95% CI: $2,000-$16,022) and lower earnings per start (P=0.003, 95% CI: $349-$1,718) in USD. Grade 3 fibrillar branch change had clinically important reductions in the probability of racing, calibre of racing performance and earnings. Grade 1 fibrillar pattern was associated with significantly higher earnings per start (P=0.004, 95% CI: $2,641-$5,759). A total of 2,204 yearling forelimb sesamoid-branch units and 1,336 2-year-old forelimb sesamoid-branch units were available for evaluation of concurrent imaging findings. The proportion of yearling sesamoids with Grade ≤1 vascular channels that had adjacent Grade ≥2 fibrillar branch change was 1.2%. The same proportion for 2-year-olds was 3.8%, with medial forelimb sesamoids with Grade 1 vascular channels overrepresented in 2-year-olds. In yearlings, 31% of sesamoids with Grade 2 vascular channels had adjacent Grade ≥2 fibrillar branch change and 59% of sesamoids with Grade 3 vascular channels had adjacent Grade ≥2 fibrillar branch change. In 2-year-olds, 47% of sesamoids with Grade 2 vascular channels had adjacent Grade ≥2 fibrillar branch change and 67% of sesamoids with Grade 3 vascular channels had the same. Only 1 yearling and 1 2-year-old sesamoid with radiological abaxial concavity had Grade 2 fibrillar branch change. Limitations: The samples used in this research are representative of the population of interest at Thoroughbred sales but may underestimate the prevalence of severe lesions in non-sale horses. The study design could not address exclusions prior to sale. The findings are applicable to horses prepared for public auction and deemed fit to be entered for sale by consignors and their veterinarians. Clinical examinations were not performed for the purposes of this research. Conclusions: Regarding stifle lucencies, Grade 1 MFC SCLs were the most common type seen in yearling and 2-year-old sales horses. The majority of yearling Grade 1 MFC SCLs resolved or remained unchanged by 2-year-old sales. It was also possible for Grade 2 and 3 MFC SCLs to improve one grade between sales. Fewer sales yearlings with a Grade 3 MFC SCL raced, but in those that did race there was no evidence of worse performance compared to unaffected peers. Axial MFC lucencies did not affect racing performance. For sesamoids, Grade 3 vascular channels, forelimb sesamoid abaxial new bone and forelimb sesamoid fragments are important findings in sales repository radiology. The new grading scale assigns a numerical grade for vascular channel appearance that matches the number of enlarged vascular channels evident in a given sesamoid. Abaxial contour changes, when present in sesamoids that are Grade 0 for vascular channels, are noted separately as either abaxial new bone or abaxial concavity. Fragments are also noted and interpreted separately. Reference values specific to young Thoroughbreds have been established for suspensory branch ultrasonography. Grade 1 fibrillar suspensory branch change should be regarded as an acceptable appearance in sales yearlings and 2-year-olds. Approximately one third of Grade 2 yearling branches progressed to a Grade 3 lesion. Evidence of enlarged branch width and Grade 2 hyperechoic foci at 2-year-old sales constitute a risk to racing performance. The existence and prevalence of concurrent radiological and ultrasonographic findings in the proximal sesamoid bones and adjacent suspensory ligament branches has been established in yearling and 2-year-old Thoroughbred sales horses. General recommendations have been made for selective branch ultrasonography on the basis of sesamoid radiological appearance. The results support a separate aetiology for radiological sesamoid abaxial concavity that does not primarily involve the suspensory branch insertion. This research provides veterinarians and the wider Thoroughbred industry with evidence-based determinations of the importance or otherwise of the various presale imaging findings seen in the MFCs, sesamoids and suspensory branches of yearlings and 2-year-olds. Many findings can be regarded as an acceptable appearance in yearlings and 2-year-olds. For those findings that are associated with reduced performance, sale and management decisions can be made based on quantitative evaluations of risk that are in the best interests of the horse.

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