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Background Currently there are no standard of care treatment strategies for IH prevention (IHP). Dehydrated human amnion-chorion (dHACM) is a healing adjunct that elutes growth factors including several that have reduced IH in animal models. We therefore performed a double-blinded, prospective randomized controlled trial (RCT) to test the hypothesis that dHACM significantly reduces IH formation in a well-studied animal model of acute IH. Material and Methods Forty 16-week-old male Sprague-Dawley rats were randomized to one of four groups: No Treatment vs. dHACM Sheet (Group A), and Saline vs. dHACM Injection (Group B). Each animal underwent a 5-cm midline laparotomy which was incompletely closed with 5-0 plain gut sutures; this was performed by a surgeon blinded to treatment group (first blind). After 28 days, the primary endpoints of IH formation and hernia size were determined by study staff blinded to treatment (second blind). Secondary endpoints included healed fascia tensile strength as determined by tensiometry, systemic and local inflammatory markers as measured by ELISA, and fascial scar collagen I/III ratios per Western blotting. Results In Group A, No Treatment developed IH at 87.5% vs. 62.5% for Sheet (P = 0.28). Hernias that formed in the Sheet group were significantly smaller (P = 0.036). In Group B, Injection and Saline yielded identical IH rates of 77.8%. Molecular characterization of fascial scar demonstrated non-inferior tensile strength, collagen I/III ratios, and inflammatory markers in dHACM-treated animals. Conclusions dHACM sheets significantly reduced the size of IH following laparotomy when compared to no treatment.

Acute spinal cord injury (ASCI) is a devastating event that can have severe hemodynamic consequences, depending on location and severity of the lesion. Knowledge of hyperacute hemodynamic changes is important for researchers using porcine models of thoracic ASCI. The goal of this study was to determine the hyperacute hemodynamic changes observed after ASCI when using pigs as their own controls. Five Yucatan gilts were anesthetized, and a dorsal laminectomy performed at T10-T12. Standardized blunt trauma was applied for 5 consecutive min, and hemodynamic variables were collected 5 min before ASCI, and at 2, 4, 6, 8, 10, 20, 30, 60, 80 and 120 min after ASCI. Arterial blood gas samples were collected at 60 min and 10 min before, and at 30 min and between 120 and 240 min after ASCI. Parametric data were analyzed using a mixed effects model with time point as the fixed factor and subject as the random factor. We found no effect on heart rate, pulse pressure, SpO2, EtCO2, and respiratory rate between baseline and timepoints after ASCI. Diastolic arterial pressure, mean arterial pressure, and systolic arterial pressure fell significantly by 18%, 16%, and 15%, respectively, at 2 min after ASCI. However, none of the decrements in arterial pressures resulted in hypotension at any time point. Heart rate did not change significantly after ASCI. Blood glucose progressively increased to 50% above baseline between 120 and 240 minutes after ASCI. Low thoracic ASCI caused a consistent and statistically significant but clinically minor hyperacute decrease in arterial pressures (-15%) that did not produce hypotension or metabolic changes suggestive of tissue hypoperfusion. Our findings using this model suggest that mean arterial pressures should be maintained above 85 mm Hg prior to spinal trauma in order to avoid hypotensive states after ASCI.

OBJECTIVE To evaluate differentiation of canine adipose–derived multipotent stromal cells (ASCs) into ligamentoblasts on tensioned collagen type I (Col1) templates in a perfusion culture system. SAMPLES Infrapatellar fat pad ASCs from healthy stifle joints of 6 female mixed-breed dogs. PROCEDURES Third-passage ASCs (6 × 106 cells/template) were loaded onto suture-augmented Col1 templates under 15% static strain in perfusion bioreactors. Forty-eight ASC-Col1 constructs were incubated with ligamentogenic (ligamentogenic constructs; n = 24) or stromal medium (stromal constructs; 24) for up to 21 days. Specimens were collected from each construct after 2 hours (day 0) and 7, 14, and 21 days of culture. Cell number, viability, distribution, and morphology; construct collagen content; culture medium procollagen-I-N-terminal peptide concentration; and gene expression were compared between ligamentogenic and stromal constructs. RESULTS ASCs adhered to collagen fibers. Cell numbers increased from days 0 to 7 and days 14 to 21 for both construct types. Relative to stromal constructs, cell morphology and extracellular matrix were more mature and collagen content on day 21 and procollagen-I-N-terminal peptide concentration on days 7 and 21 were greater for ligamentogenic constructs. Ligamentogenic constructs had increased expression of the genes biglycan on day 7, decorin throughout the culture period, and Col1, tenomodulin, fibronectin, and tenascin-c on day 21; expression of Col1, tenomodulin, and tenascin-c increased between days 7 and 21. CONCLUSIONS AND CLINICAL RELEVANCE Ligamentogenic medium was superior to stromal medium for differentiation of ASCs to ligamentoblasts on suture-augmented Col1 scaffolds. Customized ligament neotissue may augment treatment options for dogs with cranial cruciate ligament rupture.

Osteoarthritis (OA) accounts for up to 60% of equine lameness. Agmatine, a decarboxylated arginine, may be a viable option for OA management, based on reports of its analgesic properties. Six adult thoroughbred horses, with lameness attributable to thoracic limb OA, received either daily oral phenylbutazone (6.6 mg/kg), agmatine sulfate (25 mg/kg) or a control for 30 days, with 21-day washout periods between treatments. Subjective lameness, thoracic limb ground reaction forces (GRF), plasma agmatine and agmatine metabolite levels were evaluated using an established rubric, a force platform, and mass spectrometry, respectively, before, during and after each treatment period. Gastric ulceration and plasma chemistries were evaluated before and after treatments. Braking GRFs were greater after 14 and 29 days of agmatine compared to phenylbutazone administration. After 14 days of phenylbutazone administration, vertical GRFs were greater than for agmatine or the control. Glandular mucosal ulcer scores were lower after agmatine than phenylbutazone administration. Agmatine plasma levels peaked between 30 and 60 min and were largely undetectable by 24 h after oral administration. In contrast, plasma citric acid levels increased throughout agmatine administration, representing a shift in the metabolomic profile. Agmatine may be a viable option to improve thoracic limb GRFs while reducing the risk of glandular gastric ulceration in horses with OA.

Karanvir S Aulakh,1 Mandi J Lopez,1 Caleb Hudson,2 Lorrie Gaschen,1 Michelle Fabiani,2 Brian Beale,2 Frank M Andrews,1 Chin-Chi Liu,1 Jimmy Lattimer3 1School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA; 2Gulf Coast Veterinary Specialists, Houston, TX, USA; 3College of Veterinary Medicine, University of Missouri, Columbia, MO, USACorrespondence: Karanvir S AulakhSchool of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, USATel +1 217-722-2937Email kaulakh@lsu.eduPurpose: To evaluate the clinical effects of an intra-articular injection of 117mSn-colloid for management of canine grade 1 or 2 elbow osteoarthritis (OA).Patients and Methods: This was a prospective study in 23 dogs with grade 1 or 2 elbow OA. An orthopedic examination and elbow radiographs were performed to confirm the presence of OA. Dogs were randomly assigned to receive unilateral intra-articular (IA) injection of low-dose (LD: 1.0mCi, n =8), medium-dose (MD: 1.75mCi, n =6), or high-dose (HD: 2.5mCi, n =9) of 117mSn-colloid. The primary outcome measure was peak vertical force (PVF) from force-plate gait analysis and secondary outcome measures included the Canine Brief Pain Inventory score (CBPI) and elbow goniometry. The CBPI was evaluated at pretreatment and then monthly post treatment for 1 year, and goniometry and PVF were evaluated at pretreatment, and at 1, 3, 6, 9 and 12 months post treatment.Results: PVF improved at 3, and 9 months compared to pretreatment values in the HD group. CBPI scores improved at most of the time points in all dose groups. There was no significant difference in elbow goniometry between treated and untreated elbows. There were no self-reports of any adverse effects of the injection by the owners and none were noted by the examining veterinarian at the time of regularly scheduled re-evaluations.Conclusion: 117mSn IA injection was free of any obvious adverse effects, improved CBPI scores, and increased weight bearing in limbs with elbow OA providing preliminary evidence that 117mSn may be beneficial in the management of elbow OA in dogs. Although 17mSn appeared to be effective for management of elbow OA in these dogs, this pilot study has inherent limitations; therefore, future studies with larger numbers and with placebo group are needed.Keywords: osteoarthritis, Radiosynoviorthesis, Tin-117m (117mSn), CBPI, goniometry, peak vertical force

Equine shoes provide hoof protection and support weakened or damaged hoof tissues. Two hypotheses were tested in this study: 1) motion of the third phalanx (P3) and hoof wall deformation are greater in laminitic versus unaffected hooves regardless of shoe type; 2) P3 displacement and hoof wall deformation are greatest while unshod (US), less with open-heel (OH), then egg-bar (EB) shoes, and least with heart-bar (HB) shoes for both hoof conditions. Distal forelimbs (8/condition) were subjected to compressive forces (1.0x102–5.5x103 N) while a real-time motion detection system recorded markers on P3 and the hoof wall coronary band, vertical midpoint, and solar margin. Magnitude and direction of P3 displacement and changes in proximal and distal hemi-circumference, quarter and heel height and proximal and distal heel width were quantified. Hoof condition and shoe effects were assessed with 2-way ANOVA (p

This paper summarizes the results of a study that was conducted to identify all possible changes that could be made to horseshoes used by the Amish community to mitigate their damage to local roads. Several alternative horseshoes were identified, including: horseshoes with a new calk design, horseshoes coated with select tungsten carbide coating design, and a composite horseshoe. Laboratory tests were performed to quantify the reduction in damage to local roads from using the alternative horseshoes. Experiments were also conducted to evaluate the effects of the new horseshoes on horses’ hooves and legs. Finally, comprehensive cost analyses were conducted to estimate the costs to users as well as the life-cycle costs of Amish buggy routes when the different horseshoes considered in this paper are used. The results of laboratory tests indicated that the use of alternative horseshoes can significantly reduce the road damage. The experiments performed to evaluate the effect of alternative horseshoes on horses indicated that the alternative horseshoes reduced stress on the horse hoof by at least 45%, while maintaining traction. The results of life-cycle cost analyses indicated that using the alternative horseshoes with new calk design can result in reducing the annual repair costs of Amish buggy routes by at least 40%. The results also suggested that using the alternative horseshoes could reduce the annual cost of horse keeping, considering the effects of the shoes on the service life of a horse.

Some of the earliest success in de novo tissue generation was in bone tissue, and advances, facilitated by the use of endogenous and exogenous progenitor cells, continue unabated. The concept of one health promotes shared discoveries among medical disciplines to overcome health challenges that afflict numerous species. Carefully selected animal models are vital to development and translation of targeted therapies that improve the health and well‐being of humans and animals alike. While inherent differences among species limit direct translation of scientific knowledge between them, rapid progress in ex vivo and in vivo de novo tissue generation is propelling revolutionary innovation to reality among all musculoskeletal specialties. This review contains a comparison of bone deposition among species and descriptions of animal models of bone restoration designed to replicate a multitude of bone injuries and pathology, including impaired osteogenic capacity.

Epidural puncture in swine is technically challenging. Several combinations of limb and body positions have been suggested to increase lumbosacral interlaminar space (LSS) and lumbosacral angle (LSA). This study investigated whether cranial hyperflexion of pelvic limbs increased LSS and LSA in laterally and sternally recumbent juvenile Duroc and adult Yucatan pigs and assessed which position produced the largest LSS. Juvenile Duroc (n = 7) and adult Yucatan (n = 7) pigs were euthanized and randomly placed in 4 positions: sternal with neutral limbs, sternal with cranially hyperflexed limbs, lateral with neutral limbs, and lateral with hyperflexed limbs. LSS and LSA were measured on transverse axial CT images of the spine and compared by using multivariate ANOVA and the Student t test. In both age groups, LSS was greater in lateral flexed (juvenile, 7.0 ± 0.7 mm; adult, 15.9 ± 1.1 mm) and sternal flexed (juvenile, 7.5 ± 1 mm; adult, 17.1 ± 1.1 mm) positions than in lateral neutral (juvenile, 5.4 ± 0.9 mm; adult, 9.6 ± 1.6 mm) position. In addition, in both age groups, LSS and LSA in lateral neutral position were smaller than lateral flexed, sternal neutral, and sternal flexed positions. In adults, LSS was greater in lateral flexed and sternal flexed than in sternal neutral position. Hyperflexion of pelvic limbs increases LSS and LSA in sternally recumbent adult Yucatan pigs and laterally recumbent adult Yucatan and juvenile Duroc swine. Increased LSS from positioning pigs with pelvic limbs flexed in sternal or lateral recumbence may facilitate epidural puncture compared with neutral limb positioning.

The equine hoof capsule, composed of modified epidermis and dermis, is vital for protecting the third phalanx from forces of locomotion. There are descriptions of laminitis, defined as inflammation of sensitive hoof tissues but recognized as pathologic changes with or without inflammatory mediators, in the earliest records of domesticated horses. Laminitis can range from mild to serious, and signs can be acute, chronic, or transition from acute, severe inflammation to permanently abnormal tissue. Damage within the intricate dermal and epidermal connections of the primary and secondary lamellae is often associated with lifelong changes in hoof growth, repair, and conformation. Decades of research contribute to contemporary standards of care that include systemic and local therapies as well as mechanical hoof support. Despite this, consistent mechanisms to restore healthy tissue formation following a laminitic insult are lacking. Endogenous and exogenous progenitor cell contributions to healthy tissue formation is established for most tissues. There is comparably little information about equine hoof progenitor cells. Equine hoof anatomy, laminitis, and progenitor cells are covered in this review. The potential of progenitor cells to advance in vitro equine hoof tissue models and translate to clinical therapies may significantly improve prevention and treatment of a devastating condition that has afflicted equine companions throughout history.

The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning electron microscopy, immunocytochemistry, and CD105+K14+ cell trilineage plasticity. Cells from normal tissue had typical progenitor cell characteristics. Those from inflamed tissue had organelles and morphology consistent with catabolic activities including lysosomes, irregular rough endoplasmic reticulum, and fewer vacuoles and early endosomes than those from normal tissue. Cryopreserved tissue cells appeared apoptotic with an irregular cell membrane covered by cytoplasmic protrusions closely associated with endocytic and exocytic vesicles, chromatin aggregated on the nuclear envelop, abundant, poorly organized rough endoplasmic reticulum, and plentiful lysosomes. Cells that were CD105+K14+ were distinguishable from heterogenous cells by infrequent microvilli on the cell surface, sparse endosomes and vesicles, and desmosomes between cells. Cells expressed ectodermal (K15) and mesodermal (CD105) proteins in 2D and 3D cultures. Inflamed and cryopreserved tissue isolates attached poorly to tissue scaffold while normal tissue cells attached well, but only CD105+K14+ cells produced extracellular matrix after 4 d. The CD105+K14+ cells exhibited osteoblastic, adipocytic, and neurocytic differentiation. Ultrastructural information provided by this study contributes to understanding of equine hoof progenitor cells to predict their potential contributions to tissue maintenance, healing, and damage as well post-implantation behavior.

OBJECTIVE To assess the effect of horseshoes with and without traction adaptations on the gait kinetics of nonlame horses during a trot on a concrete runway. ANIMALS 5 nonlame adult light-breed horses. PROCEDURES Kinetic data were obtained for each horse when it was trotted across a force platform within a concrete runway unshod (control) and shod with standard horseshoes; standard horseshoes with high profile–low surface area calks, with low profile–high surface area calks, and coated with a thin layer of tungsten carbide (TLTC); and plastic-steel composite (PSC) horseshoes. Kinetic data were obtained for the control treatment first, then for each of the 5 shoe types, which were applied to each horse in a random order. Kinetic variables were compared among the 6 treatments. RESULTS Body weight distribution did not differ among the 6 treatments. Compared with the control, the greatest increase in forelimb peak vertical force was observed when horses were shod with PSC shoes. In the hind limbs, the greatest increase in peak braking force was observed when horses were shod with PSC shoes, followed by the TLTC and low profile–high surface area calked shoes. The PSC shoes yielded the greatest coefficient of friction in both the forelimbs and hind limbs. Stance time was longest when horses were shod with standard shoes. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that PSC and TLTC shoes provided the best hoof protection and traction and might be good options for horses that spend a large amount of time traversing paved surfaces.

Background: Chronic myelogenous leukemia (CML) is a clonal proliferative disorder of the myeloid, megakaryocyte, and erythroid lineages. The onset and subsequent progression of CML is well-described in humans. There is comparably little information surrounding CML progression in veterinary species, including Yucatan miniature swine that are common for preclinical pharmaceutical and device testing. In humans, more than 90% of CML cases are associated with a chromosomal translocation that results in the Philadelphia gene (BCR/ABL mutation). In this report, the presence of the Philadelphia gene in a Yucatan burrow was confirmed in white blood cells collected prior to onset of clinical signs with primers designed from the human BCR/ABL sequence. Case Presentation: A 24 month old, 70 kg, Yucatan barrow received a prefabricated bovine cortical bone xenograft following a unilateral zygomatic ostectomy for a preclinical study. Complete blood count and serum chemistries were performed prior to and 28, 53, 106, and 129 days after facial surgery. Fifty three days after surgery, a bone marrow biopsy was performed due to anorexia, severe basophilia, and mild anemia. A finding of a moderate increase in basophilic precursors in bone marrow cytology was followed by lymphocyte immunophenotyping via flow cytometry and RT-PCR amplification of the Philadelphia gene in white blood cell samples from the affected barrow and an unaffected barrow in the same treatment group. Bone marrow, lymph node, liver, spleen, lung, kidney, and adrenal gland lesions of mostly myeloblasts were identified after the affected barrow died 146 days after surgery. Flow cytometry confirmed lymphopenia and suggested basophilia, and RT-PCR established the presence of the BCR/ABL gene. Conclusions: The information in this report confirms the presence of the BCR/ABL mutation and documents progression of chronic myelogenous (basophilic) leukemia from a chronic phase to a terminal blast crisis in an adult Yucatan barrow. The natural occurrence and progression of CML associated with the BCR/ABL mutation in miniature swine establishes potential for future porcine models of human CML. The information also establishes a genetic test to confirm porcine CML to prevent inadvertent attribution of clinical signs to treatment complications during preclinical testing.

Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host.

Introduction Autologous reconstruction of segmental craniomaxillofacial bone defects is limited by insufficient graft material, donor site morbidity, and need for microsurgery. Reconstruction is challenging due to the complex three-dimensional (3D) structure of craniofacial skeleton. Customized 3D-printed patient-specific biologic scaffolds hold promise for reconstruction of the craniofacial skeleton without donor site morbidity. The authors report a porcine craniofacial defect model suitable for further evaluation of custom 3D-printed engineered bone scaffolds. Methods The authors created a 6 cm critical load-bearing defect in the left mandibular angle and a 1.5 cm noncritical, nonload bearing defect in the contralateral right zygomatic arch in 4 Yucatan minipigs. Defects were plated with patient-specific titanium hardware based on preoperative CT scans. Serial CT imaging was done immediately postoperatively, and at 3 and 6 months. Animals were clinically assessed for masticatory function, ambulation, and growth. At the 6-month study endpoint, animals were euthanized, and bony regeneration was evaluated through histological staining and micro-CT scanning compared to contralateral controls. Results All 4 animals reached study endpoint. Two mandibular plates fractured, but did not preclude study completion due to loss of masticatory function. One zygoma plate loosened while the site of another underwent heterotopic ossification. Gross examination of site defects revealed heterotopic ossification, confirmed by histological and micro-CT evaluation. Biomechanical testing was unavailable due to insufficient bony repair. Conclusions The presented porcine zygoma and mandibular defect models are incapable of repair in the absence of bone scaffolds. Based on the authors' results, this model is appropriate for further study of custom 3D-printed engineered bone scaffolds.

Background Use of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration. Methods This study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC) and adipose tissue-derived MSCs (ASC) were culture expanded to P3 and immunophenotyped with flow cytometry. They were then loaded by spinner flask on to scaffolds composed of tricalcium phosphate (TCP)/hydroxyapatite (HA) (40:60; HT), polyethylene glycol (PEG)/poly-l-lactic acid (PLLA) (60:40; GA), or PEG/PLLA/TCP/HA (36:24:24:16; GT). Scaffolds with and without cells were maintained in static culture for up to 21 days or implanted subcutaneously in athymic mice that were radiographed every 3 weeks up to 9 weeks. In vitro cell viability and proliferation were determined. Explant composition (double-stranded (ds)DNA, collagen, sulfated glycosaminoglycan (sGAG), protein), equine and murine osteogenic target gene expression, microcomputed tomography (μCT) mineralization, and light microscopic structure were assessed. Results The ASC and BMSC number increased significantly in HT constructs between 7 and 21 days of culture, and BMSCs increased similarly in GT constructs. Radiographic opacity increased with time in GT-BMSC constructs. Extracellular matrix (ECM) components and dsDNA increased significantly in GT compared to HT constructs. Equine and murine osteogenic gene expression was highest in BMSC constructs with mineral-containing scaffolds. The HT constructs with either cell type had the highest mineral deposition based on μCT. Regardless of composition, scaffolds with cells had more ECM than those without, and osteoid was apparent in all BMSC constructs. Conclusions In this study, both exogenous and host MSCs appear to contribute to in vivo osteogenesis. Addition of mineral to polymer scaffolds enhances equine MSC osteogenesis over polymer alone, but pure mineral scaffold provides superior osteogenic support. These results emphasize the need for bioscaffolds that provide customized osteogenic direction of both exo- and endogenous MSCs for the best regenerative potential.

Developmental dysplasia of the hip (DDH) in humans is a common condition that is associated with hip pain, functional limitations, and secondary osteoarthritis (OA). Surgical treatment of DDH has improved in the last decade, allowing excellent outcomes at short- and mid-term follow-up. Still, the etiology, mechanobiology, and pathology underlying this disease are not well understood. A pre-clinical animal model of DDH could help advance the field with a deeper understanding of specific pathways that initiate hip joint degeneration secondary to abnormal biomechanics. An animal model would also facilitate different interventional treatments that could be tested in a rigorous and controlled environment. The dog model exhibits several important characteristics that make it valuable as a pre-clinical animal model for human DDH. Dogs are naturally prone to develop canine hip dysplasia (CHD), which is treated in a similar manner as in humans. Comparable to human DDH, CHD is considered a pre-OA disease; if left untreated it will progress to OA. However, progression to OA is significantly faster in dogs than humans, with progression to OA within 1-2 years of age, associated with their shorter life span compared to humans. Animal studies could potentially reveal the underlying biochemical pathway(s), which can inform refined treatment modalities and provide opportunities for new treatment and prevention targets. Herein, we review the similarities and differences between the two species and outline the argument supporting CHD as an appropriate pre-clinical model of human DDH. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1807-1817, 2018.

Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive improvements in veterinary regenerative medicine. Inherent limitations to long‐term culture preclude use for storage. Until cell line creation from primary isolates becomes routine, MSC stasis at cryogenic temperatures is required for this purpose. Many protocols and reagents, including cryoprotectants, used for veterinary MSCs are derived from those for human and rodent cells. Dissimilarities in cryopreservation strategies play a role in variable MSC behaviors. Familiarity with contemporary cryopreservation reagents and processes is essential to an appreciation of their impact on MSC survival and post‐cryopreservation behavior. In addition to these points, this review includes a brief history and description of current veterinary stem cell regulation.

Objective To quantify stability in cranial cruciate ligament (CrCL) deficient canine stifles with hamstring grafts affixed at 3 femoral locations. Study Design Canine stifle motion study using a multi-cohort, repeated measures design. Sample Population 27 canine cadaver stifles. Methods Hamstring grafts (HG) were affixed at the gracilis-semitendinosus insertion and on the lateral femur (1) proximal trochlear ridge (TR), (2) craniodistal to fabella (F), or (3) condyle center (CC). Total, cranial, and caudal tibial translation and total, medial, and lateral angular displacement, with and without translational load, were quantified with the CrCL intact, transected, and reconstructed. Angular displacement was quantified from points on the distal femur and proximal tibia. Graft strain was calculated from tissue displacement measured at joint angles of 30°, 60°, 90°, and 120°. Results Tibial translation was lowest in F constructs, which also achieved the least difference in tibial translation from intact stifles. Tibial translation was lower in intact stifles than in CrCL transected or reconstructed stifles. Less angular displacement of the proximal tibia was detected in the medial than in the lateral direction, and tibial displacement was lower in the cranial than the caudal direction. Angular displacement was lowest in the F treatment group. F constructs had the lowest graft strain at joint angles greater than 30°. Conclusions Femoral fixation of a canine hamstring graft craniodistal to the lateral fabella conferred the best joint stability and lowest graft strain in vitro. No fixation method restored joint stability of the intact CrCL.

Damage to an ectodermal-mesodermal interface like that in the equine hoof and human finger nail bed can permanently alter tissue structure and associated function. The purpose of this study was to establish and validate in vitro culture of primary progenitor cell isolates from the ectodermal-mesodermal tissue junction in equine hooves, the stratum internum, with and without chronic inflammation known to contribute to lifelong tissue defects. The following were evaluated in hoof stratum internum cell isolates up to 5 cell passages (P): expansion capacity by cell doublings and doubling time; plasticity with multi-lineage differentiation and colony-forming unit (CFU) frequency percentage; immunophenotype with immunocytochemistry and flow cytometry; gene expression with RT-PCR; and ultrastructure with transmission electron microscopy. The presence of keratin (K)14, 15 and K19 as well as cluster of differentiation (CD)44 and CD29 was determined in situ with immunohistochemistry. To confirm in vivo extracellular matrix (ECM) formation, cell-scaffold (polyethylene glycol/poly-L-lactic acid and tricalcium phosphate/hydroxyapatite) constructs were evaluated with scanning electron microscopy 9 weeks after implantation in athymic mice. Cultured cells had characteristic progenitor cell morphology, expansion, CFU frequency percentage and adipocytic, osteoblastic, and neurocytic differentiation capacity. CD44, CD29, K14, K15 and K19 proteins were present in native hoof stratum internum. Cultured cells also expressed K15, K19 and desmogleins 1 and 3. Gene expression of CD105, CD44, K14, K15, sex determining region Y-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) was confirmed in vitro. Cultured cells had large, eccentric nuclei, elongated mitochondria, and intracellular vacuoles. Scaffold implants with cells contained fibrous ECM 9 weeks after implantation compared to little or none on acellular scaffolds. In vitro expansion and plasticity and in vivo ECM deposition of heterogeneous, immature cell isolates from the ectodermal-mesodermal tissue interface of normal and chronically inflamed hooves are typical of primary cell isolates from other adult tissues, and they appear to have both mesodermal and ectodermal qualities in vitro. These results establish a unique cell culture model to target preventative and restorative therapies for ectodermal-mesodermal tissue junctions.

Background: Pain from coxofemoral joint (CFJ) osteoarthritis (OA) characteristic of canine hip dysplasia (CHD) afflicts many dogs. Intervertebral disc (IVD) degeneration is a common CFJ OA comorbidity. Non-steroidal anti-inflammatory drug (NSAID) administration is standard for treatment of pain from degenerative joint disease. Potential side effects and tolerance from prolonged administration drive efforts to identify compounds that may be alternatives to or combined with NSAIDs. Agmatine, decarboxylated arginine, reportedly alleviates neuropathic pain, a likely component of OA pain. The objective of this study was to compare treatment response to agmatine and carprofen in dogs with varying degrees of CFJ OA with or without IVD degeneration and to test the hypothesis that agmatine improves hindlimb use comparably to carprofen and more than placebo. Methods: Nine hound-type dogs received oral carprofen (4.4 mg/kg, sid) for 7 days. Six months later, oral agmatine sulfate (25 mg/kg, bid) or placebo (hydroxypropyl methylcellulose, bid) was administered to the same dogs for 28 days with a 2 week washout period between treatments. Validated pain assessment scores were measured before treatment and every seven days throughout the treatment periods. Serum chemistry levels and ground reaction forces (GRF) were quantified before and after each treatment period. A board-certified radiologist quantified radiographic CFJ OA based on Orthopedic Foundation for Animals criteria and IVD degeneration on magnetic resonance images. GRFs were compared among treatments at each time point and among time points for each treatment. Results: There were no detectable adverse effects with any treatment. Significant results included improved GRFs in dogs with mild CFJ OA (N = 3) following agmatine administration compared to carprofen or placebo and a trend for improved GRFs in dogs with moderate CFJ OA (N = 2) following carprofen vs. agmatine or placebo. Neither agmatine nor carprofen improved GRFs in dogs with severe CFJ OA (N = 4). The GRFs improved in dogs with IVD degeneration (N = 3) following carprofen treatment compared to agmatine or placebo regardless of CFJ OA score, but no effect was observed in dogs with normal lumbar spines (N = 6). Conclusions: Results support agmatine over carprofen treatment to improve limb use in dogs with early or mild CFJ OA, while carprofen may be the better choice for dogs with moderate CFJ OA or IVD degeneration regardless of CFJ OA severity.

OBJECTIVE: To determine the influence of postoperative extracorporeal shock wave therapy (ESWT) on hind limb use after tibial plateau leveling osteotomy (TPLO). STUDY DESIGN: Randomized, prospective clinical trial. ANIMALS: Sixteen client‐owned dogs, 2 to 10 years old weighing 18 to 75 kg. METHODS: Dogs were randomly assigned to treatment cohorts, TPLO with ESWT (ESWT, n = 9) or TPLO without ESWT (control, n = 7). Treatment consisted of 1000 pulses at 0.15 mJ/mm² immediately and 2 weeks after surgery. Subjective pain, stifle goniometry, stifle circumference, peak vertical force (PVF) and vertical impulse (VI) were measured before surgery, prior to ESWT, and 2 and 8 weeks after surgery. Measures were compared between treatments at each time point and among time points for each treatment (P

OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44+, CD105+, and major histocompatability complex class II (MHCII; CD44+-CD105+-MHCII+ cells and CD44+-CD105+-MHCII− cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44+-CD105+-MHCII− CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.

The cat, as a species, is somewhat new to the field of adult multipotent stromal cells. Despite the relative phylogenetic distance between the domestic cat, Felis silvestris catus, and humans, they share some similar health challenges like diabetes, kidney disease and asthma. There is a plethora of current investigative efforts focused on adult adipose tissue-derived multipotent stromal cell (ASC) therapies to address these and other conditions. Given the small size of domestic cats, particular attention to optimize cell isolation from relatively little tissue is a necessary condition of feline ASC studies and therapies. Additionally, there are some unique features of culture conditions to test and confirm feline ASC plasticity. This chapter contains a few of the novel aspects of feline ASC isolation and culture.

Adult multipotent stromal cells (MSCs) are increasingly popular for direct therapeutic applications and bioengineering. Canine patients constitute a major component of veterinary practice, and the dog is an established preclinical animal model for numerous traumatic, degenerative, and disease conditions. Current information supports the presence and relative abundance of adipose tissue-derived multipotent stromal cells (ASCs) in various canine adipose tissue depots. Refined isolation and characterization techniques contribute to collective knowledge of ASC phenotypes and subpopulations for customized, targeted applications. Continued efforts to augment understanding of canine ASCs is critical to progressive treatment advances and high-impact study outcomes. This chapter contains a description of techniques to isolate and characterize canine ASCs.

Adult multipotent stromal cells (MSCs) are increasingly popular for direct therapeutic applications and bioengineering. Canine patients constitute a major component of veterinary practice, and the dog is an established preclinical animal model for numerous traumatic, degenerative, and disease conditions. Current information supports the presence and relative abundance of adipose tissue-derived multipotent stromal cells (ASCs) in various canine adipose tissue depots. Refined isolation and characterization techniques contribute to collective knowledge of ASC phenotypes and subpopulations for customized, targeted applications. Continued efforts to augment understanding of canine ASCs is critical to progressive treatment advances and high-impact study outcomes. This chapter contains a description of techniques to isolate and characterize canine ASCs.

The cat, as a species, is somewhat new to the field of adult multipotent stromal cells. Despite the relative phylogenetic distance between the domestic cat, Felis silvestris catus, and humans, they share some similar health challenges like diabetes, kidney disease and asthma. There is a plethora of current investigative efforts focused on adult adipose tissue-derived multipotent stromal cell (ASC) therapies to address these and other conditions. Given the small size of domestic cats, particular attention to optimize cell isolation from relatively little tissue is a necessary condition of feline ASC studies and therapies. Additionally, there are some unique features of culture conditions to test and confirm feline ASC plasticity. This chapter contains a few of the novel aspects of feline ASC isolation and culture.

Canine hip dysplasia (CHD) is a polygenic and multifactorial developmental disorder characterized by coxofemoral (hip) joint laxity, degeneration, and osteoarthritis (OA). Current diagnostic techniques are largely subjective measures of joint conformation performed at different stages of development. Recently, measures on three-dimensional images generated from computed tomography scans predicted the development of OA associated with CHD. Continued refinement of similar imaging methods may improve diagnostic imaging techniques to identify dogs predisposed to degenerative hip joint changes. By current consensus, joint changes consistent with CHD are influenced by genetic predisposition as well as environmental and biomechanical factors; however, despite decades of work, the relative contributions of each to the development and extent of CHD signs remain elusive. Similarly, despite considerable effort to decipher the genetic underpinnings of CHD for selective breeding programs, relevant genetic loci remain equivocal. As such, prevention of CHD within domestic canine populations is marginally successful. Conservative management is often employed to manage signs of CHD, with lifelong maintenance of body mass as one of the most promising methods. Surgical intervention is often employed to prevent joint changes or restore joint function, but there are no gold standards for either goal. To date, all CHD phenotypes are considered as a single entity in spite of recognized differences in expression and response to environmental conditions and treatment. Identification of distinct CHD phenotypes and targeting evidence-based conservative and invasive treatments for each may significantly advance prevention and management of a prevalent, debilitating condition in canine companions.

OBJECTIVE To evaluate associations of measures assessed by radiography, 2-D CT, and 3-D CT of the hip joints of immature dogs with osteoarthritis in the same joints at maturity. ANIMALS 46 hound-type dogs from a colony predisposed to osteoarthritis. PROCEDURES Images of hip joints (1/dog) were obtained at 16, 32, and 104 weeks of age. Radiographic measures included Norberg angle, distraction index, and osteoarthritis score. Two-dimensional CT measures included acetabular index, percentage of femoral head coverage, and center edge, horizontal toit externe, acetabular anteversion, and ventral, dorsal, and horizontal acetabular sector angles. Three-dimensional CT measures were femoral head and neck volume, femoral neck angle, and femoral head and neck radius. Differences among measures at 16 and 32 weeks in dogs with different osteoarthritis scores at later time points, relationships among variables at each time point, and relationships of single and combined measures with the presence of osteoarthritis at 104 weeks were evaluated. RESULTS The 16- and 32-week distraction index, center edge angle, dorsal acetabular sector angle, horizontal acetabular sector angle, percentage of femoral head coverage, acetabular index, and Norberg angle and the 32-week femoral neck angle varied significantly with osteoarthritis severity at 104 weeks. Presence of osteoarthritis in mature dogs was most strongly associated with 16-week combined measures of distraction index and center edge angle and 32-week combined measures of dorsal acetabular sector angle and Norberg angle. CONCLUSIONS AND CLINICAL RELEVANCE Changes in hip joint morphology associated with radiographic signs of osteoarthritis were detectable as early as 16 weeks of age and varied with osteoarthritis severity in adult dogs. The use of combined hip joint measures may improve early identification of dogs predisposed to hip joint osteoarthritis.

Developmental dysplasia of the hip (DDH) in humans is a common condition that is associated with hip pain, functional limitations, and secondary osteoarthritis (OA). Surgical treatment of DDH has improved in the last decade, allowing excellent outcomes at short- and mid-term follow-up. Still, the etiology, mechanobiology, and pathology underlying this disease are not well understood. A pre-clinical animal model of DDH could help advance the field with a deeper understanding of specific pathways that initiate hip joint degeneration secondary to abnormal biomechanics. An animal model would also facilitate different interventional treatments that could be tested in a rigorous and controlled environment. The dog model exhibits several important characteristics that make it valuable as a pre-clinical animal model for human DDH. Dogs are naturally prone to develop canine hip dysplasia (CHD), which is treated in a similar manner as in humans. Comparable to human DDH, CHD is considered a pre-OA disease; if left untreated it will progress to OA. However, progression to OA is significantly faster in dogs than humans, with progression to OA within 1-2 years of age, associated with their shorter life span compared to humans. Animal studies could potentially reveal the underlying biochemical pathway(s), which can inform refined treatment modalities and provide opportunities for new treatment and prevention targets. Herein, we review the similarities and differences between the two species and outline the argument supporting CHD as an appropriate pre-clinical model of human DDH. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1807-1817, 2018.

Summary According to Greek mythology, Prometheus' liver grew back nightly after it was removed each day by an eagle as punishment for giving mankind fire. Hence, contrary to popular belief, the concept of tissue and organ regeneration is not new. In the early 20th century, cell culture and ex vivo organ preservation studies by Alexis Carrel, some with famed aviator Charles Lindbergh, established a foundation for much of modern regenerative medicine. While early beliefs and discoveries foreshadowed significant accomplishments in regenerative medicine, advances in knowledge within numerous scientific disciplines, as well as nano- and micromolecular level imaging and detection technologies, have contributed to explosive advances over the last 20 years. Virtually limitless preparations, combinations and applications of the 3 major components of regenerative medicine, namely cells, biomaterials and bioactive molecules, have created a new paradigm of future therapeutic options for most species. It is increasingly clear, however, that despite significant parallels among and within species, there is no ‘one-size-fits-all’ regenerative therapy. Likewise, a panacea has yet to be discovered that completely reverses the consequences of time, trauma and disease. Nonetheless, there is no question that the promise and potential of regenerative medicine have forever altered medical practices. The horse is a relative newcomer to regenerative medicine applications, yet there is already a large body of work to incorporate novel regenerative therapies into standard care. This review focuses on the current state and potential future of stem cells in equine regenerative medicine.

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

Objective To identify the optimum intra-articular multipotent stromal cell (MSC) tissue source in the canine stifle. Study Design Experimental. Sample Population Infrapatellar adipose tissue, synovium lining the joint capsule, and synovium surrounding the cranial cruciate ligament (CrCL) from normal stifles of 6 dogs. Methods Nucleated cell density for each tissue was determined, and cell doublings (CD) and doubling times (DT) were quantified for expansion rates. Adipogenic, osteogenic, and chondrogenic differentiation was confirmed with light microscopy. Fibroblastic, adipogenic, and osteogenic colony forming unit frequencies were determined for multipotentiality. Tissue-specific target gene expression was assessed, and percentages of CD29+, CD34+, CD44+, CD45+, and CD90+ cells quantified. Results Adipose tissue had the highest MSC density (ASC). The CD decreased with increasing passages for all cell types, and ASC values tended to be higher. Multipotentiality decreased with passage, but remained highest in ASC. Tissue-specific target gene expression was higher in induced versus noninduced cells, and ASCs had the highest upregulation across passages. Most cells were CD29+, CD44+, CD90+, and percentages decreased with passage. Within cell types, there were more CD29+ ASC in early passages and more CD44+ and CD90+ ASC in later passages. Conclusions ASC had the highest in vitro expansion rates, CFU frequencies, tissue-specific target gene expression, and percentages of MSC immunophenotypes.

Since the discovery of bone marrow derived stromal cell osteogenesis in the 1960s, tissue engineering with adult multipotent stromal cells (MSCs) has evolved as a promising approach to restore structure and function of bone compromised by injury or disease. To date, accelerated bone formation with MSCs has been demonstrated with a variety of tissue engineering strategies. Though MSC bone tissue engineering has advanced over the last few decades, limitations to clinical translation remain. A current review of this promising field is presented with a specific focus on equine investigations.

Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care—the use of bone harvested from another region in the body—has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, without bone morphogenic proteins, using native bovine bone matrix and a perfusion bioreactor for the growth and transport of living grafts. The ramus-condyle unit (RCU), the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatan minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material, and crafted it into an anatomically correct shape using image-guided micromilling, to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either non-seeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering.