David D. Frisbie

Early events in cartilage repair after subchondral bone microfracture.

The current study investigated healing of large full-thickness articular cartilage defects during the first 8 weeks with and without penetration of the subchondral bone using microfracture in an established equine model of cartilage healing. Chondral defects in the weightbearing portion of the medial femoral condyle were made bilaterally; one defect in each horse was microfractured whereas the contralateral leg served as the control. The expression of cartilage extracellular matrix components (Types I and II collagen and aggrecan) was evaluated using histologic techniques, reverse transcription coupled polymerase chain reaction, in situ hybridization, and immunohistochemistry. This study confirms an increase in Type II collagen mRNA expression in repair tissue as early as 6 weeks after microfracture. Although other matrix mRNA and protein levels changed in concentration and tissue location over the course of the study, no significant differences were seen in microfractured defects. Although the microfracture techniques appear to improve clinical functionality, volume of repair tissue, and augment Type II collagen content, aggrecan content is less than ideal. Therefore, methods to enhance key matrix components such as aggrecan after microfracture may additionally improve repair tissue observed after the procedure.

Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene

Osteoarthritis in horses and in humans is a significant social and economic problem and continued research and improvements in therapy are needed. Because horses have naturally occurring osteoarthritis, which is similar to that of humans, the horse was chosen as a species with which to investigate gene transfer as a potential therapeutic modality for the clinical treatment of osteoarthritis. Using an established model of equine osteoarthritis that mimics clinical osteoarthritis, the therapeutic effects resulting from intra-articular overexpression of the equine interleukin-1 receptor antagonist gene through adenoviral-mediated gene transfer were investigated. In vivo delivery of the equine IL-IRa gene led to elevated intra-articular expression of interleukin-1 receptor antagonist for approximately 28 days, resulting in significant improvement in clinical parameters of pain and disease activity, preservation of articular cartilage, and beneficial effects on the histologic parameters of synovial membrane and articular cartilage. Based on these findings, gene transfer of interleukin-1 receptor antagonist is an attractive treatment modality for the equine patient and also offers future promise for human patients with osteoarthritis.

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.

Effects of Calcified Cartilage on Healing of Chondral Defects Treated With Microfracture in Horses

Background Microfracture of full-thickness articular defects has been shown to significantly enhance the amount of repair tissue. However, there is a suggestion that leaving calcified cartilage inhibits this repair response. Hypothesis Removal of the calcified cartilage with retention of subchondral bone enhances the amount of attachment of the repair tissue compared with retention of the calcified cartilage layer. Study Design Controlled laboratory study. Methods There were 1-cm2 articular cartilage defects made in 12 skeletally mature horses on the axial weightbearing portion of both medial femoral condyles. Using a custom measuring device and direct arthroscopic observation of the subchondral bone beneath the calcified cartilage layer, the authors removed the calcified cartilage from 1 defect of each horse. The repair was assessed with arthroscopy, clinical examination, radiographic and magnetic resonance imaging examinations, biopsy at 4 months, gross and histopathologic examinations at 12 months, as well as mRNA and immunohistochemical evaluations. Results Removal of calcified cartilage with retention of the subchondral bone plate increased the overall repair tissue as assessed by arthroscopic (4 months) and gross evaluation (12 months). An increase in the level of the subchondral bone was also observed with removal of the calcified cartilage layer. The clinical pain, radiographic examinations, magnetic resonance imaging evaluations, histologic character, matrix proteins, or mRNA expression do not appear to differ based on level of defect debridement. Clinical Relevance Removal of the calcified cartilage layer appears to provide optimal amount and attachment of repair tissue. Therefore, close arthroscopic visualization is recommended for debridement of clinical lesions to ensure removal of the calcified cartilage layer.

Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses

OBJECTIVE To discriminate between changes in biomarkers with exercise compared to changes in biomarkers with osteoarthritis (OA) in exercising horses. METHOD Sixteen, 2-year-old horses were randomly assigned either to an exercise-alone (n=8) or OA-affected (also exercised) (n=8) group. All horses had both mid-carpal joints arthroscoped and OA induced in one mid-carpal joint in the OA-affected joints of OA-affected horses. Two weeks after surgery all horses commenced a strenuous exercise program on a high-speed treadmill. Clinical outcomes and synovial fluid and serum biomarkers, were evaluated weekly. Synovial and serum biomarkers evaluated were epitope CS846 (CS846), epitope CPII (CPII), glycosaminoglycans (GAGs), epitope Col CEQ (Col CEQ) (a marker of type II collagen degradation), type I and II collagen degradation fragments (C1,2C), osteocalcin, C-terminal of bone type I collagen (CTX1), type I collagen (Col I) and (synovial fluid only of cartilage) prostaglandin E2 (PGE2) levels. Horses were euthanized at day 91 and their joints assessed grossly, histopathologically, and histochemically. RESULTS Exercise induced a significant increase in synovial fluid CS846, CPII, GAG, Col CEQ, C1,2C, osteocalcin and Col I concentrations. There was a significant increase in synovial fluid CS846, CPII, Col CEQ, C1,2C, osteocalcin, Col I and PGE2 concentrations in OA-affected joints compared to exercise-alone joints. The concentration of serum CS846, CPII, GAG, osteocalcin, C1,2C and Col I increased with exercise. For each of these biomarkers there was also a statistically significant increase in serum biomarker levels in OA-affected horses compared to exercise-alone horses. CONCLUSIONS Six synovial fluid and serum biomarkers were useful in separating early experimental OA from exercise alone but synovial fluid CTX1 and serum Col CEQ and CTX1 were not.

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).

Gene-mediated restoration of cartilage matrix by combination insulin-like growth factor-I/interleukin-1 receptor antagonist therapy.

Combination of growth factor gene-enhanced cartilage matrix synthesis with interleukin-1 receptor antagonist protein (IL-1Ra) abrogation of cartilage matrix degradation may reduce and possibly reverse cartilage loss in synovitis and osteoarthritis. The feasibility of cotransduction of synovial membrane with two such genes that may act on cartilage homeostasis was investigated in an in vitro coculture system. Cultured synoviocytes in monolayer were cotransduced with E1-deleted adenoviral vectors, one containing IGF-I coding sequence under cytomegalovirus (CMV) promoter control (200 multiplicities of infection (moi)), and the second containing IL-1Ra sequence under CMV promoter control (100 moi). Adenovirus-IGF-I (AdIGF-I) transduction and AdIGF-I/AdIL-1Ra cotransduction of synovial monolayer cultures resulted in increased IGF-I mRNA and ligand expression, and similarly AdIL-1Ra and AdIGF-I/AdIL-1Ra-transduced cultures expressed high levels of IL-1Ra. Northern analysis confirmed a single mRNA transcript of the appropriate size for both IGF-I and IL-1Ra transgene expression. Synovial cell monolayer and cartilage explant coculture experiments were used to examine the effects of IGF-I and IL-1Ra protein expressed by transduced synoviocytes on normal and IL-1-depleted cartilage. Transduced monolayer cultures produced peak medium IGF-I content of 114±20.2 ng/ml and IL-1Ra levels of 241.8±10.5 ng/ml at 48 h after transduction. These IGF-I concentrations were sufficient to produce significantly increased proteoglycan (PG) content of normal cartilage cultured in medium conditioned by AdIGF-I and AdIGF-I/AdIL-1Ra-transduced synoviocytes. Interleukin-1-exposed cartilage was markedly depleted of PG, and this catabolic state was partially reversed in AdIGF-I-transduced cultures and fully reversed by AdIGF-I/AdIL-1Ra-transduced synovial cocultures. These data indicate that cultured synoviocytes are readily cotransduced by two recombinant adenoviral vectors containing transgenes active in restoring joint health. The AdIL-1Ra and AdIGF-I transgenes were abundantly expressed and the secreted products achieved therapeutic concentrations by day 2. The resulting increase in matrix biosynthesis returned cartilage PG content to normal levels. These data suggest that there may be significant value in cotransduction of synovial membrane to attenuate cartilage malacia associated with synovitis, injury, or early arthritis.

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.