Laurie R. Goodrich

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

Gene therapy in musculoskeletal repair.

Abstract:  Local and regional gene therapy has improved healing in preclinical trials of articular and other muculoskeletal conditions. Combinations of cell supplementation and cells overexpressing growth factor genes have shown promising results for improving cartilage repair, enhancing delayed union of fractures, and driving organized tendon repair. Proof of concept has been developed using viral vectors, predominantly adenovirus, to deliver growth factor genes, such as BMP‐2, TGF‐β1, and IGF‐I. Integrating vectors, such as retrovirus and lentivirus, have improved the duration of gene‐induced repair, however, increased risk factors have limited broad application. Cartilage repair can be improved using chondrocyte or stem cell transplantation with cells expressing IGF‐I, BMP‐2, or FGF‐2. In cartilage injury and secondary osteoarthritis models, a combination of IL‐1 knockdown and growth factor supplementation has restored cartilage matrix and stabilized the osteoarthritic process. Ultimately, nonviral vectors may provide similar control of catabolic activity in cartilage and synovial structures, which may further improve outcome after chondrocyte or mesenchymal stem cell (MSC) implantation. MSCs derived from bone marrow, fat, or other connective tissues provide a multipotent cell source that may be privileged vectors for skeletal gene therapy. MSCs expressing BMP‐2, TGF‐β1, LMP‐1, IGF‐I, or GDF‐5 have enhanced cartilage, bone, and tendon repair. Overall, the field of orthopedic gene therapy for enhanced tissue repair has made significant preclinical advances. Combining existing cell transplant technology to deliver differentiated cells in a minimally invasive way, with genes that improve matrix formation, provides a manageable protocol for a persisting anabolic impact.

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.

Arthroscopic injection of corticosteroids into the fibrous tissue of subchondral cystic lesions of the medial femoral condyle in horses: A retrospective study of 52 cases (2001–2006)

REASONS FOR PERFORMING STUDY There are no published results of subchondral cystic lesions (SCLs) in the medial femoral condyle (MFC) treated with arthroscopic injection of corticosteroids into the lining of the cyst. OBJECTIVES 1) To determine the success rate for treatment of SCLs in the MFC with arthroscopic injection of the fibrous tissue of the cyst with corticosteroids. 2) To identify any factors that may predict outcome. HYPOTHESES Injection of the fibrous tissue of SCLs of the MFC with corticosteroids utilising arthroscopic guidance yields a similar or higher chance for intended performance than does arthroscopic debridement as previously reported; this technique will be effective for treating SCLs in older horses. METHODS Horses with clinical and radiographic evidence of a SCL in the MFC were injected with corticosteroids under arthroscopic guidance, and case records and radiographs were reviewed retrospectively. A telephone survey of referring veterinarians, owners and trainers was conducted. RESULTS Thirty-five of 52 (67%) cases were classified as successful involving 73 SCLs of which 56 (77%) were classified as successful. There was no significant association between age group (age<or=3 years vs. >3 years) and outcome, or cyst configuration and outcome. Significantly more unilateral SCLs (28/31 [90%] SCLs) were classified as successful than bilateral (28/42: 67%). There were significant differences in outcome based on the surgeon operating the case and an association between pre-existing radiographic findings of osteophytes and negative outcome. CONCLUSIONS Injection of SCLs utilising arthroscopic guidance is an effective alternative method of surgical treatment of SCL. POTENTIAL RELEVANCE This technique offers a similar chance of success as has been reported with debridement and may allow for a shorter period of convalescence. If unsuccessful, the option remains to debride the cyst in a second surgery.

Osteoblastic differentiation of human and equine adult bone marrow-derived mesenchymal stem cells when BMP-2 or BMP-7 homodimer genetic modification is compared to BMP-2/7 heterodimer genetic modification in the presence and absence of dexamethasone.

Bone marrow‐derived mesenchymal stem cells (BMDMSCs) have been targeted for use in enhancement of bone healing; and their osteogenic potential may be further augmented by genes encoding bone morphogenetic proteins (BMPs). The purpose of this study was to compare the effect of genetic modification of human and equine BMDMSCs with BMP‐2 or ‐7 or BMP‐2 and ‐7 on their osteoblastogenic differentiation in the presence or absence of dexamethasone. The BMDMSCs were harvested from the iliac crest of three human donors and tuber coxae of three equine donors. Monolayer cells were genetically modified using adenovirus vectors encoding BMP‐2, ‐7 or both and cultured in the presence or absence of dexamethasone. Expression of BMPs was confirmed by enzyme linked immunosorbent assay (ELISA). To evaluate osteoblastic differentiation, cellular morphology was assessed every other day and expression and secretion of alkaline phosphatase (ALP), as well as expression levels of osteonectin (OSTN), osteocalcin (OCN), and runt‐related transcription factor‐2 (Runx2) were measured for up to 14 days. Human and equine BMDMSCs showed a capacity for osteogenic differentiation regardless of genetic modification or dexamethasone supplementation. Dexamethasone supplementation was more important for osteoblastogenic differentiation of equine BMDMSCs than human BMDMSCs. Genetic modification of BMDMSCs increased ALP secretion with AdBMP‐2 homodimer having the greatest effect in both human and equine cells compared to AdBMP 7 or AdBMP 2/7. BMP protein elution rates reached their maximal concentration between day 4 and 8 and remained relatively stable thereafter, suggesting that genetically modified BMDMSCs could be useful for cell‐based delivery of BMPs to a site of bone formation. Published by Wiley Periodicals, Inc. J Orthop Res 28:1330–1337, 2010

Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis

Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200–800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA.

Effect of scaffold dilution on migration of mesenchymal stem cells from fibrin hydrogels

OBJECTIVE To evaluate the effect of fibrin concentrations on mesenchymal stem cell (MSC) migration out of autologous and commercial fibrin hydrogels. SAMPLE Blood and bone marrow from six 2- to 4-year-old horses. PROCEDURES Autologous fibrinogen was precipitated from plasma and solubilized into a concentrated solution. Mesenchymal stem cells were resuspended in fibrinogen solutions containing 100%, 75%, 50%, and 25% of the fibrinogen precipitate solution. Fibrin hydrogels were created by mixing the fibrinogen solutions with MSCs and thrombin on tissue culture plates. After incubation for 24 hours in cell culture medium, the MSCs that had migrated onto the tissue culture surface and beyond the boundary of the hydrogels were counted. This procedure was repeated with a commercial fibrin sealant. RESULTS Hydrogel-to-surface MSC migration was detected for all fibrin hydrogels. Migration from the 25% autologous hydrogels was 7.3-, 5.2-, and 4.6-fold higher than migration from 100%, 75%, and 50% autologous hydrogels, respectively. The number of migrating cells from 100%, 75%, and 50% autologous hydrogels did not differ significantly. With commercial fibrin sealant, the highest magnitude of migration was from the 25% hydrogels, and it was 26-fold higher than migration from 100% hydrogels. The 75% and 50% hydrogels resulted in migration that was 9.5- and 4.2-fold higher than migration from the 100% hydrogels, respectively. CONCLUSIONS AND CLINICAL RELEVANCE MSC migration from fibrin hydrogels increased with dilution of the fibrinogen component for both autologous and commercial sources. These data supported the feasibility of using diluted fibrin hydrogels for rapid delivery of MSCs to the surface of damaged tissues.

Ex Vivo Serotype-Specific Transduction of Equine Joint Tissue by Self-Complementary Adeno-Associated Viral Vectors

Cell transplantation for the treatment of joint disease is an important clinical tool. Genetic modification of cells before transplantation has shown enhanced healing. Ex vivo genetic modification of joint tissue cells with various adeno-associated virus (AAV) serotypes has not been investigated. The transduction efficiencies of self-complementary AAV serotypes (1-6 and 8) were determined in joint tissue containing chondrocytes and synoviocytes isolated from equine models. When comparing scAAV serotypes for efficient transduction ex vivo, in chondrocytes versus synoviocytes, serotypes 6 and 2, and serotypes 3 and 2, respectively, appeared superior for gene expression. Unlike adenoviral vectors, no upregulation of inflammatory markers, such as matrix metalloproteinases and aggrecanase, was seen on treatment of joint tissue with AAV vectors ex vivo. Our findings also corroborate that ex vivo transduction of joint tissue can result in high transgene protein levels over time, and transplantation modalities might be feasible using AAV vectors in the treatment of joint-related diseases.

Incidence of support limb laminitis in horses treated with half limb, full limb or transfixation pin casts: a retrospective study of 113 horses (2000-2009).

REASONS FOR PERFORMING STUDY To determine the incidence of support limb laminitis among horses treated with half limb, full limb or transfixation pin casts and determine potential risk factors. METHODS Medical records of 113 horses treated with half limb, full limb or transfixation pin casts at an equine referral hospital from 2000 to 2009 were reviewed. Associations between potential risk factors and development of support limb laminitis were evaluated by bivariable and multivariable logistic regression analyses. RESULTS Of the 113 horses that received casts, 14 (12%) developed confirmed support limb laminitis. The bodyweight of the horse and duration of casting in weeks were significantly associated with support limb laminitis. Horses requiring full limb casts or transfixation pin casts were more likely to develop this complication than horses requiring half limb casts. There were no significant associations between developing support limb laminitis and weightbearing capacity on presentation to the hospital, the limb affected (fore- or hind), whether there was a fracture present or breed of horse. CONCLUSIONS Support limb laminitis is a relatively common complication among horses treated with half limb, full limb and transfixation pin casts. Greater durations of casting and higher bodyweights increase the likelihood of developing this complication. POTENTIAL RELEVANCE Support limb laminitis may occur secondary to any painful unilateral lameness and is not necessarily more likely to develop in horses with severe orthopaedic conditions such as fractures. However, heavier horses, those requiring casts for longer periods of time and those that require a full limb or transfixation pin cast as opposed to a half limb cast should be considered to have an increased risk for developing support limb laminitis post operatively.

Concurrent or sequential development of medial meniscal and subchondral cystic lesions within the medial femorotibial joint in horses (1996–2006)

UNLABELLED Summary Reasons for performing study: Medial meniscal injuries and subchondral cystic lesions (SCL) are known to occur independently within the medial femorotibial (MFT) joint in horses. However, there are no reports of a potential clinical relationship between these 2 types of lesions. OBJECTIVES To: 1) document the concurrent presence or sequential development of medial meniscal and SCL of the medial femoral condyle within the MFT joint; and 2) determine the prognosis with both types of lesions. METHODS Retrospective case series of horses with both a medial meniscal and SCL of the medial femoral condyle identified concurrently or sequentially by radiography, arthroscopy or post mortem examination. Case records and radiographs were reviewed, and a telephone survey of referring veterinarians, owners and trainers was conducted. RESULTS Twenty-one horses (9.1% of all horses undergoing MFT joint arthroscopy) were identified to have both a medial meniscal injury and SCL of the medial femoral condyle. Thirteen horses had both abnormalities identified concurrently, 6 developed a meniscal lesion subsequent to SCL debridement, and 2 developed a SCL subsequent to a medial meniscal injury. Only 4/19 horses were classified as successful and returned to their intended use. The severity of the meniscal injury was significantly associated with the severity of lameness but not with outcome. CONCLUSIONS A low percentage of horses may develop both a meniscal injury and SCL of the medial femoral condyle within the MFT joint and have a poor prognosis. POTENTIAL RELEVANCE Trauma to the MFT joint may lead to both meniscal and subchondral bone damage of the medial femoral condyle that may be recognised concurrently or sequentially.