Lauren Schnabel

The National Research Council has documented a dire national need for veterinary specialists trained in biomedical research. Furthermore, veterinary researchers play a key role in comparative and translational research activities since they naturally bridge basic and clinical research. To address this training need, we request continued NIH funding for 7 fellows per year for 3 years. NC State University will provide funding for up to 2 fellows per year and 2 pre-T32 positions. Trainees will be degree-seeking fellows in the Comparative Medicine and Translational Research training program established by the faculty in the College of Veterinary Medicine (CVM) and the Comparative Medicine Institute (CMI) at North Carolina State University. This training program specifically targets individuals with the DVM degree who have completed specialty training and is designed to prepare trainees to compete for an early career development award and a rapid transition to independence as a principal investigator or in another research-intensive career. Trainees complete requirements leading to the PhD degree in Comparative Biomedical Sciences (CBS) in one of 7 areas of concentration: 1) Immunology 2) Cell Biology, 3) Pharmacology, 4) Neurosciences, 5) Infectious Diseases, 6) Population Medicine and Global Health, and 7) Pathology. Training faculty are well-funded productive scientists that have a strong training track-record and diverse research expertise. Training faculty are all members of the CBS graduate program and the CMI and represent 5 departments from 3 Colleges. Research projects emphasize comparative and translational themes fostered by the CVM and CMI in functional tissue engineering, translational pharmacology and physiology, and emerging and infectious diseases. Program requirements include: (1) a capstone comparative animal models course; (2) professional development courses and workshops; (3) courses in research ethics and research rigor and reproducibility; (4) a grant writing course and (5) annual research symposia. These requirements are in addition to those associated with the graduate program. Twenty four fellows have completed training. Twenty hold faculty positions in academia, one is a research pathologist, one is a clinical pathologist in industry, and one is a postdoctoral fellow. Fellows were awarded 18 NIH or other federal grants as PI and more than 50 extramural research grants and published 125 papers (84 first author) arising from their research while in training.

Meniscal tears are the most commonly reported knee injuries, and approximately 1 million surgeries involving the meniscus are performed annually in the US. Tissue engineering and regenerative medicine approaches are being actively pursued as potential alternatives to overcome limitations of current clinical treatments. Yet, the translation of these approaches to clinical application has been hampered by their limited ability to efficiently and reproducibly create physiologic-sized, patient-specific scaffolds featuring anisotropic structural and mechanical properties on the order of native meniscus. 3D printing can create scaffolds that replicate physiologic size and patient-specific geometry in a highly repeatable manner and with good handling characteristics for surgical implantation, yet, these fiber sizes are typically on the order of hundreds of microns, several orders of magnitude higher than the native tissue. On the other hand, nonwoven textiles approaches allow the fabrication of fibers on the scale of native collagen fibrils, but it is infeasible to create complex anatomical 3D geometries, such as that of the knee meniscus. The overall goal of this proposal is to investigate the ability of a new 3D nonwoven scaffold fabrication approach that synergistically integrates attributes of traditional nonwoven melt blowing and 3D printing to overcome these limitations and recapitulate complex anisotropic structural characteristics of the meniscus at multiple scales as a means to provide superior outcomes, in-vitro and in-vivo. We hypothesize that this 3D Melt Blowing (3DMB) approach can allow physiological fiber morphology (similar to other nonwovens such as electrospinning), while also enabling the creation of patient-specific meniscus 3D geometry via the customized rotating mandrel (similar to 3D printing). We further hypothesize that the resulting scaffold features will permit superior in-vivo outcomes, particularly, cell infiltration, new matrix production, and the prevention of cartilage degeneration via control of porosity and fiber size. Aim 1 is to determine how primary 3DMB process variables influence the structural architecture and biomechanical properties of anatomically-sized meniscus scaffolds made of selected biopolymers and decellularized meniscus ECM (dECM) hydrogels. Aim 2 is to determine whether the incorporation of zone-specific meniscus decellularized ECM (dECM) improves infiltration and tissue formation by cells as well as integration with the surrounding meniscus tissue. Aim 3 is to determine whether cartilage degeneration following meniscectomy is reduced through the addition of an appropriate dECM within scaffolds with meniscus-matched mechanics. On completion, this project will provide fundamental knowledge about the micro- and macro-level process-structure-function relationships in meniscus-relevant scaffolds fabricated using our new 3DMB nonwovens approach, and will serve as a base technology of great significance allowing advances in the treatment of orthopaedic fibrous soft tissue injuries.

The overall goal of this current proposal is to evaluate the effects of licensed MSCs on tendon healing, first in vitro and then in vivo. Our broad hypothesis is that licensed MSCs will further enhance tendon healing compared to na��������ve MSCs. Aim 1 studies will focus on injured tenocyte repair in vitro when co-cultured with MSCs in order to optimize MSC licensing conditions with IL-1B, TGF-B2, or a combination of IL-1B and TGF-B2. Aim 2 studies will determine the effect of licensed MSC treatment (as optimized in Aim 1) on SDFT healing in vivo when treatment is initiated after the acute inflammatory phase has subsided at 14 days post injury to mimic the clinical setting. We expect these studies to markedly advance MSC therapy for the treatment of tendon injuries in horses by demonstrating that licensing is critical to produce an MSC secretome for enhanced tendon repair regardless of timing of administration.

Over the past decade, equine research at North Carolina State University College of Veterinary Medicine (NCSU CVM) has grown exponentially under the leadership of Dr. Paul Lunn (Dean 2012-2022) and Dr. Kate Meurs (Associate Dean for Research and Graduate Studies date 2011- 2022; Dean 2022 - present). At present, our Equine Group is comprised of 16 faculty members with 1 additional search underway and 3 clinical veterinarians (See table below). Each member of our group has an active research program with group funding to date totaling over 45 million dollars. In addition, we are responsible for the training and specialty board certification of 5-6 equine residents/year, each of which must complete a research project of their own, as well as for the training of equine graduate (PhD and MS) students. While our group has been very successful at obtaining external funding (both federal and foundation) for our studies, we have struggled to maintain consistent funding for permanent NCSU CVM equine research herds, which are vital to all facets of our research and clinical work. Full funding for such herds is extremely difficult to fit within the limited budgets of most equine foundation grants. Additionally, funding for these herds is not directly applicable to the budget of many federal grants, whose proposed experiments rely on terminal studies or the use of clinical cases. However, the pilot data for these federal grants, which is absolutely essential for a successful application, does usually come from research herds. Lastly, horses in these herds, and particularly that of Dr. Schnabel���s research herd, are being used to make biologic products that have been highly successful for the treatment of equine hospital patients suffering from infectious arthritis and osteoarthritis as well as other ailments. While hospital revenue from such treatments helps to cover the costs of producing the biologic products themselves, it is not enough to cover the overall care and mandatory screening of these research herd horses for transmissible diseases. In addition to benefiting equine health and translational research, NCSU CVM equine research Herds also provide an important educational opportunity for NCSU students at all levels of training. Between them, Drs. Schnabel, Sheats and the Theriogenology group have trained over 40 undergraduate, 35 veterinary and 12 PhD students in research projects that rely on equine research herds. When appropriate, some of the research horses (Sheats) are also available for use in non-invasive equine science and veterinary teaching labs.

Recent data indicates that the fastest rising rates of anterior cruciate ligament (ACL) injuries of the knee are reported in children and adolescents with significant growth remaining. In the skeletally immature patient population, surgical reconstruction is increasingly suggested for complete ACL tears. However, the choice of non-surgical treatment or immediate surgical reconstruction of ACL tears remains a subject of debate in young patients with significant growth remaining or in the case of partial tears involving one ACL bundle. Sex appears to be a major risk factor for ACL injury during adolescence, but not in childhood, adding another layer of complexity. For both complete and partial ACL injuries, treatment algorithms have been developed without considering the potential sex- and age-dependent function of the ACL, due to the paucity of available data. Thus, the objective of this proposal is to determine how age and sex impact ACL maturation and joint function during skeletal growth and to assess if this knowledge can be applied to improve treatment after ACL injury. Aim 1 will determine how sex impacts the maturation of the ACL as well as that of its individual AM and PL bundles during skeletal growth. Aim 2 will determine how age and sex impact the immediate loading of secondary tissues in-vitro and the remodeling response of the joint in-vivo following loss of function of the AM bundle, PL bundle, or the entire ACL. Aim 3 will determine if a replacement graft positioned to more accurately reflect age- and sex-specific ACL function improves graft viability and function and reduces the risk of secondary injury and long-term degenerative changes. Successful completion of these aims will provide a basic science foundation for the development of age- and sex-specific algorithms for the treatment of ACL injuries.

The proposed Mentored Research Scientist Development Award (K01) will provide the candidate, Dr. Alix Berglund, DVM, PhD, with the necessary knowledge, training, and experience to become an independent translational biomedical researcher in the fields of immunology and mesenchymal stem cell (MSC) biology. MSCs are a promising cell source for treating inflammatory and immune-mediated diseases. Allogeneic therapy would provide cost-effective and efficient treatment, but is currently hindered by recipient immune rejection of donor MSCs expressing mismatched-major histocompatibility complex (MHC) molecules. MSCs are strongly immunomodulatory, however, and manipulation of MHC expression may be sufficient to allow donor MSCs to evade recipient immune responses. The central hypothesis of this proposal, which is supported by strong preliminary research, is that treating MSCs with transforming growth factor-��������2 (TGF-��������2) downregulates MHC I and MHC II gene transcription thereby reducing the in vivo immunogenicity of MHC-mismatched MSCs. The aims of this project are to 1) Identify how TGF-��������2 downregulates MHC expression in MSCs and 2) Determine how TGF-��������2 treatment affects MSC immunogenicity in vivo. Both murine and equine MSCs will be utilized to elucidate TGF-��������2 signaling pathways to increase translational potential to humans (Aim 1) and non-inflammatory and inflammatory murine models will be used to analyze how the immune system responds to TGF-��������2-treated MSCs in vivo (Aim 2). Completion of the proposed research is a first step towards improving the efficacy and safety of allogeneic MSCs for clinical use. To accomplish these aims, Dr. Berglund will build on her background in mesenchymal stem cell biology, immunology, and large animal models by developing expertise in molecular immunology techniques, in vivo alloimmune response analysis, and the utilization of murine models. Dr. Berglund������������������s mentoring team has combined expertise in immunology, mesenchymal stem cells, and genetics to facilitate her transition to an independent translational biomedical researcher. This work will be completed primarily at the North Carolina State University College of Veterinary Medicine, which is eminently qualified to train translational clinician scientists, with additional work at the University of North Carolina-Chapel Hill.

Laminitis is a devastating cause of morbidity and mortality in horses, yet many aspects of its etiology and management remain poorly understood. Corticosteroids have many applications in horses, but their use is sometimes limited by the perceived risk of corticosteroid-induced laminitis. Research has established that corticosteroid administration alters several metabolic parameters, but a direct association between corticosteroid administration and increased laminitis risk has not been proven. Nevertheless, it is a clinical finding that one-time administration of corticosteroids at appropriate doses can induce acute laminitis. In the case of joint therapy, horses receiving sacroiliac injections may be at increased risk compared to horses receiving injections at standard intra-synovial sites. In this study, we will compare absorption of triamcinolone following injection at a standard intra-synovial site (antebrachiocarpal joint) to an extra-synovial site (sacroiliac joint), and evaluate glucose, insulin, and cortisol levels following injection. We hypothesize that there will be increased systemic absorption from the extra-synovial site as compared to the intra-synovial site, and that triamcinolone administration will result in cortisol suppression, hyperglycemia, and hyperinsulinemia. This information is critical for guiding future research and the clinical use of triamcinolone in order to mitigate the risk of laminitis.

The objectives of this project are to determine how licensed MSCs affect tenocyte function including migration, proliferation, and gene and protein expression of factors associated with tenocyte development and ECM production/remodeling. Our hypothesis is that when grown in coculture with SDFT tenocytes, licensed MSCs will enhance parameters of tenocyte function beneficial to the healing tendon compared to na��ve MSCs.

Articular cartilage damage and subsequent degenerative joint disease is a common cause of lameness in the horse (Kawack 2016). Although magnetic resonance imaging (MRI) is commonly used for evaluation of lameness localized to the foot in the horse, studies have shown that standard MRI sequences have limited sensitivity in detecting cartilage lesions (Olive et al. 2010; Nelson et al. 2018; van Zadelhoff et al. 2020). Due to this limited sensitivity, advanced sequences are increasingly used in human medicine for improved detection of articular cartilage lesions. These sequences include dual-echo steady state (DESS), ultrashort TE (UTE), and high-resolution volume interpolated breath-hold examination (VIBE) sequences. Susceptibility[MBF1] weighted imaging (SWI) sequences show promise in the evaluation of juvenile cartilage in humans and foals (Martel et al. 2016; Kolb et al. 2018). Use of these sequences to improve detection of articular cartilage lesion in the horse would allow for better direction of therapy and determination of prognosis. DESS is a high-resolution 3D gradient echo MR sequence that has high spatial resolution and cartilage signal-to-noise ratio, increasing sensitivity for detection of cartilage lesions. UTE sequences employ ultrashort TE value (0.008 to 0.50 ms) (Robson and Bydder, 2006), allowing improved evaluation of tissues with a short relaxation time such as the deeper layers of the articular cartilage. VIBE is a T1-weighted 3D gradient echo MR sequence that can provide isotropic or near-isotropic resolution of cartilage (0.5 mm or smaller isotropic voxels), but these higher resolution scans for good cartilage detail require a longer scan time. At a lower resolution (>2 mm slices and larger voxel sizes), VIBE scan times are shorter but smaller cartilage lesions may be less apparent due to volumetric averaging. SWI is a 3D high-spatial-resolution fully velocity corrected gradient-echo MRI sequence. While it is more commonly used in the detection of blood products and calcium within tissues, it has been used in a research setting in the evaluation of relaxation times of the vascular architecture of juvenile cartilage (Martel et al. 2016; Kolb et al. 2018), and was proven useful in evaluation of the articular cartilage in our initial use. By employing multiple different TE������������������s within one image acquisition it can help detect early inflammation/changes in cartilage that would not be apparent in standard sequences. The purpose of this study is to evaluate the ability of advanced (DESS, UTE, SWI, hi-res VIBE) MRI sequences and standard sequences used clinically for MRI of the equine foot (PD, low-res VIBE) to detect cartilage lesions of the distal interphalangeal (DIP) joint. Gross inspection and histopathology will be used as the gold standard. A scoring system will be used for gross evaluation. As a preliminary study, the DIP joint in six cadaver limbs were imaged using these advanced sequences. Four of the limbs had naturally occurring cartilage lesions detected on gross examination. At least one of the advanced sequences successfully detected cartilage lesions present in these four limbs. We will evaluate the ability of these advanced sequences to detect cartilage lesions of the DIP joint in equine cadaver limbs, by comparing the sensitivity and specificity of each advanced sequence and the standard MRI protocol against gross inspection and histopathology as the gold standard. We hypothesise that the advanced sequences will each individually have higher sensitivity/specificity for cartilage lesions than our current clinical imaging protocol.

Dr. Schnabel along with Mrs. Julie Long of the Schnabel Laboratory will be responsible for the in vivo screening of synthetic platelet like particles provided by Selsym Biotech, Inc. Maximum tolerated dose studies following intravenous injection of PLPs will be evaluated in male and female mice. Mortality, morbidity, and weight of mice will be monitored for 5 days after injection; organs will then be collected, fixed, and provided to Selsym Biotech, Inc. for evaluation of signs of thrombosis and/or organ damage. Dr. Schnabel will additionally contribute to study design, data analysis, manuscript writing, and project reporting.