C. Wayne McIlwraith

Evaluation of Adipose-Derived Stromal Vascular Fraction or Bone Marrow-Derived Mesenchymal Stem Cells for Treatment of Osteoarthritis

The purpose of this study was the assessment of clinical, biochemical, and histologic effects of intraarticular administered adipose‐derived stromal vascular fraction or bone marrow‐derived mesenchymal stem cells for treatment of osteoarthritis. Osteoarthritis was induced arthroscopically in the middle carpal joint of all horses, the contralateral joint being sham‐operated. All horses received treatment on Day 14. Eight horses received placebo treatment and eight horses received adipose‐derived stromal vascular fraction in their osteoarthritis‐affected joint. The final eight horses were treated the in osteoarthritis‐affected joint with bone marrow‐derived mesenchymal stem cells. Evaluations included clinical, radiographic, synovial fluid analysis, gross, histologic, histochemical, and biochemical evaluations. No adverse treatment‐related events were observed. The model induced a significant change in all but two parameters, no significant treatment effects were demonstrated, with the exception of improvement in synovial fluid effusion PGE2 levels with bone marrow‐derived mesenchymal stem cells when compared to placebo. A greater improvement was seen with bone marrow‐derived mesenchymal stem cells when compared to adipose‐derived stromal vascular fraction and placebo treatment. Overall, the findings of this study were not significant enough to recommend the use of stem cells for the treatment of osteoarthritis represented in this model.

Evaluation of intra-articular mesenchymal stem cells to augment healing of microfractured chondral defects.

PURPOSE This study evaluated intra-articular injection of bone marrow-derived mesenchymal stem cells (BMSCs) to augment healing with microfracture compared with microfracture alone. METHODS Ten horses (aged 2.5 to 5 years) had 1-cm2 defects arthroscopically created on both medial femoral condyles of the stifle joint (analogous to the human knee). Defects were debrided to subchondral bone followed by microfracture. One month later, 1 randomly selected medial femorotibial joint in each horse received an intra-articular injection of either 20 × 10(6) BMSCs with 22 mg of hyaluronan or 22 mg of hyaluronan alone. Horses were confined for 4 months, with hand walking commencing at 2 weeks and then increasing in duration and intensity. At 4 months, horses were subjected to strenuous treadmill exercise simulating race training until completion of the study at 12 months. Horses underwent musculoskeletal and radiographic examinations bimonthly and second-look arthroscopy at 6 months. Horses were euthanized 12 months after the defects were made, and the affected joints underwent magnetic resonance imaging and gross, histologic, histomorphometric, immunohistochemical, and biochemical examinations. RESULTS Although there was no evidence of any clinically significant improvement in the joints injected with BMSCs, arthroscopic and gross evaluation confirmed a significant increase in repair tissue firmness and a trend for better overall repair tissue quality (cumulative score of all arthroscopic and gross grading criteria) in BMSC-treated joints. Immunohistochemical analysis showed significantly greater levels of aggrecan in repair tissue treated with BMSC injection. There were no other significant treatment effects. CONCLUSIONS Although there was no significant difference clinically or histologically in the 2 groups, this study confirms that intra-articular BMSCs enhance cartilage repair quality with increased aggrecan content and tissue firmness. CLINICAL RELEVANCE Clinical use of BMSCs in conjunction with microfracture of cartilage defects may be potentially beneficial.

In Vivo Evaluation of Autologous Cartilage Fragment-Loaded Scaffolds Implanted Into Equine Articular Defects and Compared With Autologous Chondrocyte Implantation

Background Current autologous chondrocyte implantation (ACI) techniques require 2 surgical procedures: 1 for cell harvest and 1 for reimplantation of cultured cells. A 1-step procedure is more desirable. Purpose A 1-step surgical procedure using autologous cartilage fragments on a polydioxanone scaffold, or CAIS (cartilage autograft implantation system), in a clinically relevant defect (15-mm diameter) within equine femoral trochlea was compared with a 2-step ACI technique as well as with empty defects and defects with polydioxanone foam scaffolds alone. Study Design Controlled laboratory study. Methods Ten skeletally mature horses were used. Articular cartilage from the lateral trochlea of the femur was harvested arthro-scopically (n 5 5), and chondrocytes were cultured on small intestinal submucosa to produce ACI constructs. The CAIS procedure had cartilage harvested during defect creation to prepare minced cartilage on polydioxanone-reinforced foam. The ACI and CAIS constructs were placed in defects using polydioxanone/polyglycolic acid staples. Defects were examined arthroscopically at 4, 8, and 12 months and with gross, histological, and immunohistochemical examination at 12 months. Results Arthroscopic, histologic, and immunohistochemistry results show superiority of both implantation techniques (ACI and CAIS) compared with empty defects and defects with polydioxanone foam alone, with CAIS having the highest score. Conclusion This is the first demonstration of long-term healing with strenuous exercise using ACI and CAIS in a critically sized defect. Clinical Relevance Given these results with the CAIS procedure, testing in human patients is the next logical step (a phase 1 human clinical study has proceeded from this work).

Proposed Definitions and Criteria for Reporting Time Frame, Outcome, and Complications For Clinical Orthopedic Studies in Veterinary Medicine

Outcome, and Complications For Clinical Orthopedic Studies in Veterinary Medicine James L. Cook, DVM, PhD, Diplomate ACVS, Richard Evans, PhD, Michael G. Conzemius, DVM, PhD, Diplomate ACVS, B. Duncan X. Lascelles, BVSc, PhD, Diplomate ECVS, Diplomate ACVS, C. Wayne McIlwraith, BVSc, PhD, Diplomate ACVS, Antonio Pozzi, DMV, MS, Diplomate ACVS, Peter Clegg, MA, VetMB, PhD, Diplomate ECVS, MRCVS, John Innes, BVSc, PhD, DSAS (Orth), MRCVS, Kurt Schulz, DVM, Diplomate ACVS, John Houlton, MA, VetMB, DVR, DSAO, MRCVS, Diplomate ECVS, Lisa Fortier, DVM, PhD, Diplomate ACVS, Alan R. Cross, DVM, Diplomate ACVS, Kei Hayashi, DVM, PhD, Diplomate ACVS, Amy Kapatkin, DVM, MS, Diplomate ACVS, Dorothy Cimino Brown, DVM, MSCE, Diplomate ACVS, and Allison Stewart, DVM, MS, Diplomate ACVS Comparative Orthopaedic Laboratory, University of Missouri, Columbia, MO, College of Veterinary Medicine, University of Illinois, Urbana, IL, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, Comparative Pain Research Laboratory, North Carolina State University,

Autologous Conditioned Serum in the Treatment of Orthopedic Diseases The Orthokine ® Therapy

The common strategies for the treatment of patients with orthopedic diseases do not address the underlying pathogenesis. Several biologically based, local therapies aiming to influence the cytokine imbalance are either in development or in the initial stages of clinical use. A method based on exposure of blood leukocytes to pyrogenfree surfaces (e.g. glass spheres) elicits an accumulation of anti-inflammatory cytokines, including interleukin-1 receptor antagonist, and several growth factors, including insulin-like growth factor-1, platelet-derived growth factor, and transforming growth factor-β1, in the liquid blood phase. Based on these observations, a new therapy using cell-free, autologous conditioned serum (ACS) from the incubation of whole blood with glass spheres was developed. The injection of ACS into affected tissue (s) has shown clinical effectiveness and safety in animal models and studies, as well as in human clinical studies, for the treatment of osteoarthritis, lumbar stenosis, disc prolapse, and muscle injuries.

IL-1ra/IGF-1 gene therapy modulates repair of microfractured chondral defects.

Repair of cartilage defects involves sequential participation of specific hormones and growth factors with potential impairment by inflammatory cytokines. We explored an in vivo gene therapy treatment to supply adenoviral vectors carrying the genes of interleukin-1 receptor antagonist protein (IL-1ra) and insulinlike growth factor-1 (IGF-1), hoping to enhance repair of full-thickness equine chondral defects treated with microfracture. We asked whether our treatment could (1) increase proteoglycan and Type II collagen content in the repair tissue, (2) improve the macroscopic and histomorphometric aspect of the repair tissue, and (3) induce prolonged and increased IL-1ra and IGF-1 production in treated joints. Twelve horses had full-thickness chondral defects created in their carpus and stifle followed by microfracture. Joints were injected with either equine IL-1ra/IGF-1 adenoviral preparation or Geys balanced salt solution. Sixteen weeks later, defect healing was evaluated macroscopically, histologically, histochemically, and biochemically. Production of IL-1ra and IGF-1 was measured by enzyme-linked immunosorbent assay and radioimmunoassay. We found increased proteoglycan content in treated defects along with augmented Type II collagen associated with substantial transgene expression of IL-1ra during the first 3 weeks. These data suggest in vivo gene therapy can improve biologic processes associated with chondral defect repair.

Evaluation of Ad-BMP-2 for enhancing fracture healing in an infected defect fracture rabbit model

The objective of this study was to evaluate the use of adenoviral transfer of the BMP‐2 gene (Ad‐BMP‐2) for enhancing healing in an infected defect fracture model. A femoral defect stabilized with plates and screws was surgically created in sixty‐four skeletally mature New Zealand white rabbits. Experimental groups were: (1) non‐infected Ad‐luciferase (Ad‐LUC, NONLUC), (2) non‐infected Ad‐BMP‐2 (NONBMP), (3) infected Ad‐LUC (INFLUC), and (4) infected Ad‐BMP‐2 (INFBMP). A sclerosing agent was applied to the ends of the bone at surgery to facilitate the development of osteomyelitis. Fracture healing was evaluated radio‐graphically and histologically. Data were analyzed using an ANOVA, with statistical significance set as p < 0.05.

Preclinical Studies for Cartilage Repair Recommendations from the International Cartilage Repair Society

Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic chondral defects can be used but the later are more like the lesions found in patients and may be more predictive. Quantitative and qualitative outcome measures such as macroscopic appearance, histology, biochemistry, functional imaging, and biomechanical testing of cartilage, provide reliable data to support investment decisions and subsequent applications to regulatory bodies for clinical trials. No one model or species can be considered ideal for pivotal studies, but the larger animal species are recommended for pivotal studies. Larger species such as the horse, goat and pig also allow arthroscopic delivery, and press-fit or sutured implant fixation in thick cartilage as well as second look arthroscopies and biopsy procedures.

Clinical Outcome After Intra-Articular Administration of Bone Marrow Derived Mesenchymal Stem Cells in 33 Horses With Stifle Injury

OBJECTIVE To report outcome of horses with femorotibial lesions (meniscal, cartilage or ligamentous) treated with surgery and intra-articular administration of autologous bone marrow derived mesenchymal stem cells (BMSCs). STUDY DESIGN Prospective case series. ANIMALS Horses (n = 33). METHODS Inclusion criteria included horses that had lameness localized to the stifle by diagnostic anesthesia, exploratory stifle arthroscopy and subsequent intra-articular administration of autologous BMSCs. Case details and follow-up were gathered from medical records, owner, trainer or veterinarian. Outcome was defined as returned to previous level of work, returned to work, or failed to return to work. RESULTS Follow-up (mean, 24 months) was obtained; 43% of horses returned to previous level of work, 33% returned to work, and 24% failed to return to work. In horses with meniscal damage (n = 24) a higher percentage in the current study (75%) returned to some level of work compared to those in previous reports (60-63%) that were treated with arthroscopy alone, which resulted in a statistically significant difference between studies (P = .038). Joint flare post injection was reported in 3 horses (9.0%); however, no long-term effects were noted. CONCLUSIONS Intra-articular administration of BMSC postoperatively for stifle lesions appeared to be safe, with morbidity being similar to that of other biologic agents. Improvement in ability to return to work may be realized with BMSC treatment compared to surgery alone in horses with stifle injury.

Evaluation of the effect of extracorporeal shock wave treatment on experimentally induced osteoarthritis in middle carpal joints of horses.

OBJECTIVE-To assess the clinical, biochemical, and histologic effects of extracorporeal shock wave therapy (ESWT) in the treatment of horses with experimentally induced osteoarthritis (OA). ANIMALS-Twenty-four 2- to 3-year-old horses without evidence of lameness. PROCEDURES-OA was induced arthroscopically in 1 middle carpal joint of each horse. Fourteen days after induction of OA, horses were treated with a sham ESWT probe (placebo; n = 8), polysulfated glycosaminoglycan (PSGAG) administered IM every 4 days for 28 days as a positive control treatment (8), or ESWT administered on days 14 and 28 with a focused shock wave unit (8). Evaluations included clinical assessments of degree of lameness every 2 weeks and weekly synovial fluid analyses. Horses were euthanized 70 days after induction of OA, and gross pathologic and histologic examinations of cartilage and synovial membrane specimens were performed at necropsy. A generalized linear mixed model was used to compare outcomes among treatment groups. RESULTS-No adverse treatment-related events were detected in any horse. The degree of lameness in horses treated with ESWT improved significantly, compared with the degree of lameness in placebo- or PSGAG-treated horses. No disease-modifying effects were evident in results for synovial fluid, synovial membranes, or cartilage from the ESWT- or PSGAG-treated horses. CONCLUSIONS AND CLINICAL RELEVANCE-Although a disease-modifying effect of ESWT was not detected, the significant clinical effect of ESWT suggested that this modality should be considered for treatment of horses with OA in combination with another modality that does affect the disease process.