Correlation of Arthroscopic Grading and Optical Coherence Tomography as Markers of Early Repair and Predictors of Later Healing Evident on MRI and Histomorphometric Assessment of Cartilage Defects Implanted with Chondrocytes Overexpressing IGF-I.

OBJECTIVE: Injury of articular cartilage is common, and due to the poor intrinsic capabilities of chondrocytes, it can precipitate joint degradation and osteoarthritis (OA). Implantation of autologous chondrocytes into cartilaginous defects has been used to bolster repair. Accurate assessment of the quality of repair tissue remains challenging. This study aimed to investigate the utility of noninvasive imaging modalities, including arthroscopic grading and optical coherence tomography (OCT) for assessment of early cartilage repair (8 weeks), and MRI to determine long-term healing (8 months). DESIGN: Large (15 mm diameter), full-thickness chondral defects were created on both lateral trochlear ridges of the femur in 24 horses. Defects were implanted with autologous chondrocytes transduced with rAAV5-IGF-I, autologous chondrocytes transduced with rAAV5-GFP, naïve autologous chondrocytes, or autologous fibrin. Healing was evaluated at 8 weeks post-implantation using arthroscopy and OCT, and at 8 months post-implantation using MRI, gross pathology, and histopathology. RESULTS: OCT and arthroscopic scoring of short-term repair tissue were significantly correlated. Arthroscopy was also correlated with later gross pathology and histopathology of repair tissue at 8 months post-implantation, while OCT was not correlated. MRI was not correlated with any other assessment variable. CONCLUSIONS: This study indicated that arthroscopic inspection and manual probing to develop an early repair score may be a better predictor of long-term cartilage repair quality following autologous chondrocyte implantation. Furthermore, qualitative MRI may not provide additional discriminatory information when assessing mature repair tissue, at least in this equine model of cartilage repair.

Synovial fluid lubricin and hyaluronan are altered in equine osteochondral fragmentation, cartilage impact injury, and full-thickness cartilage defect models.

The objectives of this study were to evaluate temporal changes in lubricin, hyaluronan (HA), and HA molecular weight (MW) distributions in three distinct models of equine joint injury affecting the carpal (wrist), tarsal (ankle), and femoropatellar (knee) joints. To establish ranges for lubricin, HA, and HA MW distributions across multiple joints, we first evaluated clinically healthy, high-motion equine joints. Synovial fluid was collected from high-motion joints in horses without clinical signs of joint disease (n = 11 horses, 102 joints) and from research horses undergoing carpal osteochondral fragmentation (n = 8), talar cartilage impact injury (n = 7), and femoral trochlear ridge full-thickness cartilage injury (n = 22) prior to and following arthroscopically induced joint injury. Lubricin and HA concentrations were measured via enzyme-linked immunosorbent assays, and gel electrophoresis was performed to evaluate HA MW distributions. Synovial fluid parameters were analyzed via linear regression models, revealing that lubricin and HA concentrations were conserved across healthy, high-motion joints. Lubricin concentrations increased post-injury in all osteoarthritis models (carpal fragmentation P = .001; talar impact P < .001; femoral trochlear ridge cartilage defect P = .03). Sustained loss of HA was noted post-arthroscopy following carpal osteochondral fragmentation (P < .0001) and talar impact injury (P < .001). Lubricin may be elevated to compensate for the loss of HA and to protect cartilage post-injury. Further investigation into the mechanisms regulating lubricin and HA following joint injury and their effects on joint homeostasis is warranted, including whether lubricin has value as a biomarker for post-traumatic osteoarthritis.

Return to racing after surgical management of third carpal bone slab fractures in thoroughbred and standardbred racehorses.

OBJECTIVE: To determine the prognosis for racing of horses surgically treated for slab fractures of the third carpal bone (C3). STUDY DESIGN: Retrospective case study. ANIMALS: Horses (n = 125) surgically treated for C3 slab fractures. METHODS: Medical records of horses surgically treated for dorsal or sagittal C3 fractures were reviewed for age, sex, breed, limb, fracture type, degree of cartilage damage, and surgical treatment. Radiographs were evaluated to determine fracture depth, width, and displacement. Osteophytes, C3 lysis, and fragmentation were scored. Racing performance was obtained from online databases. Univariable and multivariable analyses were used to determine associations between independent variables and outcomes. RESULTS: Fifty-four (43%) horses raced postoperatively. Among thoroughbreds, 35% (30/86) with dorsal fractures and 63% (17/27) with sagittal fractures raced postoperatively. Among standardbreds, 77% (10/13) with dorsal fractures and 0% (0/2) with sagittal fractures raced postoperatively. Fracture displacement, C3 lysis, and cartilage damage affected the likelihood of racing postoperatively. Placement of 3.5-mm screws vs 4.5-mm screws and the placement of fewer screws were associated with improved likelihood of racing. CONCLUSION: The prognosis for postoperative racing of thoroughbreds with dorsal C3 fractures was less favorable than that previously reported. Concurrent joint pathology, such as cartilage damage at time of surgery, affected the ability of the horse to race postoperatively. CLINICAL SIGNIFICANCE: Although internal fixation of C3 slab fractures is required to restore joint congruity, return to racing should be expected in only 42% of thoroughbreds and 67% of standardbreds.

Temporal changes in synovial fluid composition and elastoviscous lubrication in the equine carpal fracture model.

The objective of this study was to examine temporal variations in synovial fluid composition and lubrication following articular fracture. Post-traumatic osteoarthritis (PTOA) was induced by creating an osteochondral fracture in the middle carpal joint of four horses while the contralateral limb served as a sham-operated control. Horses were exercised on a high-speed treadmill, and synovial fluid was collected pre-operatively and at serial timepoints until 75 days post-operatively. Lubricin and hyaluronic acid (HA) concentrations were measured using sandwich ELISAs, and the molecular weight distribution of HA was analyzed via gel electrophoresis. Synovial fluid viscosity and cartilage friction coefficients across all modes of lubrication were measured on days 0, 19, 33, and 61 using a commercial rheometer and a custom tribometer, respectively. HA concentrations were significantly decreased post-operatively, and high molecular weight HA (>6.1MDa) did not recover to pre-operative values by the study termination at day 75. Lubricin concentrations increased after surgery to a greater extent in the OA as compared to sham-operated limbs. Viscosity was significantly reduced after surgery. While boundary and elastoviscous mode friction coefficients did not vary, the transition number, representing the shift between these modes, was lower. Although more pronounced in the OA limbs, similar derangements in HA, HA molecular weight distribution, viscosity, and transition number were observed in the sham-operated limbs, which may be explained by synovial fluid washout during arthroscopy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Persistence of fluorescent nanoparticle-labelled bone marrow mesenchymal stem cells in vitro and after intra-articular injection.

Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra-articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD-labelled MSCs [QD-MSCs]) in vivo after intra-articular injection into normal and osteoarthritic joints. One week after injection of QD-MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD-MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiation. In joints injected with QD-MSCs, labelled cells were identified on the synovial membrane and significantly less often on articular cartilage, without differences between normal and osteoarthritic joints. Joints injected with QD-MSCs and MSCs had increased synovial total nucleated cell count and protein compared with vehicle-injected joints. In vitro, QDs persisted in nonproliferating cells for up to 8 weeks (length of the study), but QD fluorescence was essentially absent from proliferating cells within two passages (approximately 3 to 5 days). QD labelling did not affect MSC differentiation into chondrocytes, adipocytes, and osteocytes. QD-MSCs had slightly different immunophenotype from control cells, but whether this was due to an effect of the QDs or to drift during culture is unknown. QD-MSCs can be visualized in histological sections 1 week after intra-articular injection and are more frequently found in the synovial membrane versus cartilage in both normal and osteoarthritic joints. QDs do not alter MSC viability and differentiation potential in vitro. However, QDs are not optimal markers for long-term tracking of MSCs, especially under proliferative conditions.

Galectins-1 and-3 Increase in Equine Post-traumatic Osteoarthritis.

Galectins are potent regulators of cell adhesion, growth and apoptosis in diverse cell types, including chondrocytes and synovial fibroblasts. Elevations in synovial fluid galectin-3 have been observed in rheumatoid arthritis, juvenile idiopathic arthritis and experimental inflammatory arthritis in animal models, whereas galectin-1 is thought to be protective. Less is known about galectins-1 and-3 in osteoarthritis (OA). Therefore, the purpose of this study was: (1) to determine whether galectin-1 and-3 synovial fluid concentrations and synovial membrane and cartilage histochemical staining were altered following osteochondral injury in an experimental equine osteoarthritis (OA) model and (2) to measure galectin-1 and-3 mRNA expression and synovial fluid concentrations in naturally occurring equine carpal OA. Synovial fluid galectin-1 and-3 concentrations were quantified using custom ELISAs in two research horse cohorts undergoing experimental OA induction (n = 5 and 4) and in a cohort of horses with naturally occurring carpal OA (n = 57). Galectin mRNA expression in synovial membrane and cartilage tissue obtained from carpal joints of horses with naturally occurring OA was measured using RT-qPCR, and galectin immunostaining was assessed in synovial membrane and osteochondral tissues in the experimental model (n = 5). Synovial fluid galectin-1 and-3 concentrations increased following experimental carpal osteochondral fragmentation. Cartilage galectin-1 mRNA expression increased with OA severity in naturally occurring disease. The superficial zone of healthy articular cartilage stained intensely for galectin-3 in sham-operated joints, whereas galectin-1 staining was nearly absent. Chondrocyte galectin-1 and-3 immunoreactivity increased following cartilage injury, particularly in galectin-1 positive chondrones. Galectins-1 and-3 are present in healthy equine synovial fluid and increase following post-traumatic OA. Healthy superficial zone chondrocytes express galectin-3, whereas galectin-1 chondrocyte staining is limited predominantly to chondrones and injured cartilage. Further work is needed to clarify the functions of galectins-1 and-3 in healthy and OA joints.

Disease-Modifying Osteoarthritis Treatment With Interleukin-1 Receptor Antagonist Gene Therapy in Small and Large Animal Models.

OBJECTIVE: Gene therapy holds great promise for the treatment of osteoarthritis (OA) because a single intraarticular injection can lead to long-term expression of therapeutic proteins within the joint. This study was undertaken to investigate the use of a helper-dependent adenovirus (HDAd)-mediated intraarticular gene therapy approach for long-term expression of interleukin-1 receptor antagonist (IL-1Ra) as sustained symptomatic and disease-modifying therapy for OA. METHODS: In mouse models of OA, efficacy of HDAd-IL-1Ra was evaluated by histologic analysis, micro-computed tomography (micro-CT), and hot plate analysis. In a horse OA model, safety and efficacy of HDAd-IL-1Ra were evaluated by blood chemistry, analyses of synovial fluid, synovial membrane, and cartilage, and gross pathology and lameness assessments. RESULTS: In skeletally immature mice, HDAd-IL-1Ra prevented development of cartilage damage, osteophytes, and synovitis. In skeletally immature and mature mice, treatment with HDAd-interleukin-1 receptor antagonist post-OA induction resulted in improved-albeit not significantly-cartilage status assessed histologically and significantly increased cartilage volume, cartilage surface, and bone surface covered by cartilage as assessed by micro-CT. Fewer osteophytes were observed in HDAd-IL-1Ra-treated skeletally immature mice. Synovitis was not affected in skeletally immature or mature mice. HDAd-IL-1Ra protected against disease-induced thermal hyperalgesia in skeletally mature mice. In the horse OA model, HDAd-IL-1Ra therapy significantly improved lameness parameters, indicating efficient symptomatic treatment. Moreover, macroscopically and histologically assessed cartilage and synovial membrane parameters were significantly improved, suggesting disease-modifying efficacy. CONCLUSION: These data from OA models in small and large animals demonstrated safe symptomatic and disease-modifying treatment with an HDAd-expressing IL-1Ra. Furthermore, this study establishes HDAd as a vector for joint gene therapy.

AAV-mediated Overexpression of IL-10 Mitigates the Inflammatory Cascade in Stimulated Equine Chondrocyte Pellets.

BACKGROUND: Following joint trauma, a posttraumatic inflammatory cascade drives degeneration of the joint. We aimed to assess whether transduction of chondrocytes with AAV5 overexpressing the immunomodulatory cytokine IL-10 would have protective effects in pellet cultures stimulated with IL-1ß. METHODS: Chondrocytes were isolated from 3 healthy horses and were transduced with AAV5-IL-10 at a dose of 1 x 105vg/cell. Chondrocyte pellets were formed by centrifugation and were stimulated with IL-1ß starting 48 hours following transduction. After 2, 6 and 14 days in culture, supernatants were collected for cytokine analysis and RNA was isolated from cells for gene expression analysis. Pellets were also collected for biochemical analysis. RESULTS: Transduction of chondrocytes led to significant increases in IL-10 expression. IL-10 expression was further enhanced by IL-1ß stimulation. IL-10 overexpression led to significantly decreased expression of IL-1ß and ADAMTS4. PGE2 synthesis was also significantly decreased. IL-1ß mediated suppression of GAG synthesis was not rescued by IL-10. CONCLUSIONS: Overexpression of IL-10 modulates the inflammatory response in chondrocytes, which may mitigate some of the deleterious effects of pro-inflammatory cascades in the posttraumatic joint. AAV5-IL-10 led to efficient and sustained overexpression of IL-10 in chondrocytes and could represent a viable treatment option for preventing osteoarthritis.

Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor.

Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art.

Matrix-Induced Autologous Chondrocyte Implantation (MACI) Using a Cell-Seeded Collagen Membrane Improves Cartilage Healing in the Equine Model.

BACKGROUND: Autologous chondrocyte implantation (ACI) using a collagen scaffold (matrix-induced ACI; MACI) is a next-generation approach to traditional ACI that provides the benefit of autologous cells and guided tissue regeneration using a biocompatible collagen scaffold. The MACI implant also has inherent advantages including surgical implantation via arthroscopy or miniarthrotomy, the elimination of periosteal harvest, and the use of tissue adhesive in lieu of sutures. This study evaluated the efficacy of the MACI implant in an equine full-thickness cartilage defect model at 1 year. METHODS: Autologous chondrocytes were seeded onto a collagen type-I/III membrane and implanted into one of two 15-mm defects in the femoral trochlear ridge of 24 horses. Control defects either were implanted with cell-free collagen type-I/III membrane (12 horses) or were left ungrafted as empty defects (12 horses). An additional 3 horses had both 15-mm defects remain empty as nonimplanted joints. The repair was scored by second-look arthroscopy (12 weeks), and necropsy examination (53 weeks). Healing was assessed by arthroscopic scoring, gross assessment, histology and immunohistology, cartilage matrix component assay, and gene expression determination. Toxicity was examined by prostaglandin E2 formation in joint fluid, and lymph node morphology combined with histologic screening of organs. RESULTS: MACI-implanted defects had improved gross healing and composite histologic scores, as well as increases in chondrocyte predominance, toluidine blue-stained matrix, and collagen type-II content compared with scaffold-only implanted or empty defects. There was minimal evidence of reaction to the implant in the synovial membrane (minor perivascular cuffing), subchondral bone, or cartilage. There were no adverse clinical effects, signs of organ toxicity, or evidence of chondrocytes or collagen type-I/III membrane in draining lymph nodes. CONCLUSIONS: The MACI implant appeared to improve cartilage healing in a critical-sized defect in the equine model compared with collagen matrix alone. CLINICAL RELEVANCE: These results indicate that the MACI implant is quick to insert, provides chondrocyte security in the defect, and improves cartilage healing compared with ACI.

Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs), synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility.

BACKGROUND: Mesenchymal stromal cells (MSCs) can be used intra-articularly to quell inflammation and promote cartilage healing; however, mechanisms by which MSCs mitigate joint disease remain poorly understood. Galectins, a family of ß-galactoside binding proteins, regulate inflammation, adhesion and cell migration in diverse cell types. Galectin-1 and galectin-3 are proposed to be important intra-articular modulators of inflammation in both osteoarthritis and rheumatoid arthritis. Here, we asked whether equine bone marrow-derived MSCs (BMSCs) express higher levels of galectin-1 and -3 relative to synovial fibroblasts and chondrocytes and if an inflammatory environment affects BMSC galectin expression and motility. METHODS: Equine galectin-1 and -3 gene expression was quantified using qRT-PCR in cultured BMSCs, synoviocytes and articular chondrocytes, in addition to synovial membrane and articular cartilage tissues. Galectin gene expression, protein expression, and protein secretion were measured in equine BMSCs following exposure to inflammatory cytokines (IL-1ß 5 and 10 ng/mL, TNF-α 25 and 50 ng/mL, or LPS 0.1, 1, 10 and 50 µg/mL). BMSC focal adhesion formation was assessed using confocal microscopy, and BMSC motility was quantified in the presence of inflammatory cytokines (IL-1ß or TNF-α) and the pan-galectin inhibitor ß-lactose (100 and 200 mM). RESULTS: Equine BMSCs expressed 3-fold higher galectin-1 mRNA levels as compared to cultured synovial fibroblasts (p = 0.0005) and 30-fold higher galectin-1 (p < 0.0001) relative to cultured chondrocytes. BMSC galectin-1 mRNA expression was significantly increased as compared to carpal synovial membrane and articular cartilage tissues (p < 0.0001). IL-1ß and TNF-α treatments decreased BMSC galectin gene expression and impaired BMSC motility in dose-dependent fashion but did not alter galectin protein expression. ß-lactose abrogated BMSC focal adhesion formation and inhibited BMSC motility. CONCLUSIONS: Equine BMSCs constitutively express high levels of galectin-1 mRNA relative to other articular cell types, suggesting a possible mechanism for their intra-articular immunomodulatory properties. BMSC galectin expression and motility are impaired in an inflammatory environment, which may limit tissue repair properties following intra-articular administration. ß-lactose-mediated galectin inhibition also impaired BMSC adhesion and motility. Further investigation into the effects of joint inflammation on BMSC function and the potential therapeutic effects of BMSC galectin expression in OA is warranted.

Galectin-3 Binds to Lubricin and Reinforces the Lubricating Boundary Layer of Articular Cartilage.

Lubricin is a mucinous, synovial fluid glycoprotein that enables near frictionless joint motion via adsorption to the surface of articular cartilage and its lubricating properties in solution. Extensive O-linked glycosylation within lubricin's mucin-rich domain is critical for its boundary lubricating function; however, it is unknown exactly how glycosylation facilitates cartilage lubrication. Here, we find that the lubricin glycome is enriched with terminal ß-galactosides, known binding partners for a family of multivalent lectins called galectins. Of the galectin family members present in synovial fluid, we find that galectin-3 is a specific, high-affinity binding partner for lubricin. Considering the known ability of galectin-3 to crosslink glycoproteins, we hypothesized that galectins could augment lubrication via biomechanical stabilization of the lubricin boundary layer. We find that competitive inhibition of galectin binding results in lubricin loss from the cartilage surface, and addition of multimeric galectin-3 enhances cartilage lubrication. We also find that galectin-3 has low affinity for the surface layer of osteoarthritic cartilage and has reduced affinity for sialylated O-glycans, a glycophenotype associated with inflammatory conditions. Together, our results suggest that galectin-3 reinforces the lubricin boundary layer; which, in turn, enhances cartilage lubrication and may delay the onset and progression of arthritis.

RNA Interference Mediated Interleukin-1ß Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses.

Posttraumatic activation of the catabolic cascade plays a major role in degradation of cartilage. Interleukin-1ß (IL-1ß), a primary instigator in the catabolic axis, is upregulated in chondrocytes following injury. IL-1ß activates key degradative enzymes, including MMPs and aggrecanases, and other proinflammatory mediators such as PGE2 which contribute to ECM breakdown. Posttranscriptional silencing of IL-1ß by RNA interference (RNAi) may drive a reduction in IL-1ß. We hypothesized that transduction of chondrocytes using rAAV2 expressing a short hairpin RNAi motif targeting IL-1ß (shIL-1ß) would significantly decrease IL-1ß expression and, in turn, decrease expression of other catabolic enzymes. Chondrocyte cultures were transduced with rAAV2-tdT-shIL-1ß in serum-free media. The fluorescent protein, tdTomato, was used to determine transduction efficiency via flow cytometry and fluorescent microscopy. Cells were stimulated with lipopolysaccharide (LPS) 48 hours following transduction. After 24-hour stimulation, supernatants were collected for cytokine analysis, and cells lysed for gene expression analysis. IL-1ß knockdown led to significantly decreased expression of IL-1ß, TNF-α, and ADAMTS5. PGE2 synthesis was also significantly downregulated. Overall, effective silencing of IL-1ß using rAAV2 vector expressing a short hairpin IL-1ß knockdown sequence was shown. Additionally, significant downstream effects were evident, including decreased expression of TNF-α and ADAMTS5. Targeted silencing of catabolic cytokines may provide a promising treatment avenue for osteoarthritic (OA) joints.

Cell-based cartilage repair strategies in the horse.

Damage to the articular cartilage surface is common in the equine athlete and, due to the poor intrinsic healing capabilities of cartilage, can lead to osteoarthritis (OA). Joint disease and OA are the leading cause of retirement in equine athletes and currently there are no effective treatments to stop the progression of OA. Several different cell-based strategies have been investigated to bolster the weak regenerative response of chondrocytes. Such techniques aim to restore the articular surface and prevent further joint degradation. Cell-based cartilage repair strategies include enhancement of endogenous repair mechanisms by recruitment of stem cells from the bone marrow following perforation of the subchondral bone plate; osteochondral implantation; implantation of chondrocytes that are maintained in defects by either a membrane cover or scaffold, and transplantation of mesenchymal stem cells into cartilage lesions. More recently, bioengineered cartilage and scaffoldless cartilage have been investigated for enhancing repair. This review article focuses on the multitude of cell-based repair techniques for cartilage repair across several species, with special attention paid to the horse.

Mechanical properties and structure-function relationships in articular cartilage repaired using IGF-I gene-enhanced chondrocytes.

Several studies have demonstrated the benefits of IGF-I gene therapy in enhancing the histologic and biochemical content of cartilage repaired by chondrocyte transplantation. However, there is little to no data on the mechanical performance of IGF-I augmented cartilage grafts. This study evaluated the compressive properties of full-thickness chondral defects in the equine femur repaired with and without IGF-I gene therapy. Animals were randomly assigned to one of three study cohorts based on chondrocyte treatment provided in each defect: (i) IGF-I gene delivered by recombinant adeno-associated virus (rAAV)-5; (ii) AAV-5 delivering GFP as a reporter; (iii) naïve cells without virus. In each case, the opposite limb was implanted with a fibrin carrier without cells. Samples were prepared for confined compression testing to measure the aggregate modulus and hydraulic permeability. All treatment groups, regardless of cell content or transduction, had mechanical properties inferior to native cartilage. Overexpression of IGF-I increased modulus and lowered permeability relative to other treatments. Investigation of structure-property relationships revealed that Ha and k were linearly correlated with GAG content but logarithmically correlated with collagen content. This provides evidence that IGF-I gene therapy can improve healing of articular cartilage and can greatly increase the mechanical properties of repaired grafts.

Mechanical characterization of matrix-induced autologous chondrocyte implantation (MACI®) grafts in an equine model at 53 weeks.

There has been much interest in using autologous chondrocytes in combination with scaffold materials to aid in cartilage repair. In the present study, a total of 27 animals were used to compare the performance of matrix-assisted chondrocyte implantation (MACI®) using a collagen sponge as a chondrocyte delivery vehicle, the sponge membrane alone, and empty controls. A total of three distinct types of mechanical analyses were performed on repaired cartilage harvested from horses after 53 weeks of implantation: (1) compressive behavior of samples to measure aggregate modulus (HA) and hydraulic permeability (k) in confined compression; (2) local and global shear modulus using confocal strain mapping; and (3) boundary friction coefficient using a custom-built tribometer. Cartilage defects receiving MACI® implants had equilibrium modulus values that were 70% of normal cartilage, and were not statistically different than normal tissue. Defects filled with Maix™ membrane alone or left empty were only 46% and 51-63% of control, respectively. The shear modulus of tissue from all groups of cartilage defects were between 4 and 10 times lower than control tissue, and range from 0.2 to 0.4 MPa. The average values of boundary mode friction coefficients of control tissue from all groups ranged from 0.42 to 0.52. This study represents an extensive characterization of the mechanical performance of the MACI® grafts implant in a large animal model at 53 weeks. Collectively, these data demonstrate a range of implant performance, revealing similar compressive and frictional properties to native tissue, with inferior shear properties.

Arthroscopic removal of discrete palmar carpal osteochondral fragments in horses: 25 cases (1999-2013).

OBJECTIVE: To characterize discrete palmar carpal osteochondral fragmentation in horses and to document the effect of osteoarthritis and surgical removal of these fragments on functional outcome. DESIGN: Retrospective case series. ANIMALS: 25 horses. PROCEDURES: Medical records and radiographic views were reviewed to identify horses that had radiographic evidence of palmar carpal fragmentation, which was subsequently treated by arthroscopic removal. Information collected included cause of fracture, initial and long-term clinical and radiographic findings, and functional outcome. RESULTS: Palmar carpal fragmentation of 30 carpal bones was identified in 25 unilaterally affected horses. A known traumatic event was reported to cause the fragmentation in 17 of the 25 (68%) horses. Of the 25 horses, 17 (68%) had fragmentation involving the antebrachiocarpal joint, 7 (28%) had fragmentation involving the middle carpal joint, and 1 (4%) had fragmentation involving the carpometacarpal joint. The proximal aspect of the radial carpal bone was the most commonly affected site (12/30 fragments), followed by the accessory carpal bone (6/30). Of the 25 horses, 19 (76%) were not lame (sound) after surgery and returned to their intended use, 4 (16%) were considered pasture sound, and 2 were euthanized (because of severe postoperative osteoarthritis or long bone fracture during recovery from anesthesia). Eight of the 14 horses with preoperative evidence of osteoarthritis returned to function after surgery. Twelve of 17 horses with antebrachiocarpal joint fragments and 6 of 7 horses with middle carpal joint fragments returned to their previous use. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the prognosis for horses after arthroscopic removal of palmar carpal osteochondral fragments is good. Early intervention, before the development of osteoarthritis, is recommended.

Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model.

Cartilage injury often precipitates osteoarthritis which has driven research to bolster repair in cartilage impact damage. Autologous chondrocytes transduced with rAAV5-IGF-I were evaluated in chondral defects in a well-established large animal model. Cartilage was harvested from the talus of 24 horses; chondrocytes were isolated and stored frozen. Twenty million cells were cultured and transduced with 10(5) AAV vg/cell prior to implantation. Chondrocytes from eight horses were transduced with rAAV5-IGF-I, chondrocytes from eight horses with rAAV5-GFP, and chondrocytes from eight horses were not transduced. A 15 mm full-thickness chondral defect was created arthroscopically in the lateral trochlear ridge of the femur in both femoropatellar joints. Treated defects were filled with naive or gene-enhanced chondrocytes, in fibrin vehicle. Control defects in the opposite limb received fibrin alone. rAAV5-IGF-I transduced chondrocytes resulted in significantly better healing at 8 week arthroscopy and 8 month necropsy examination when compared to controls. At 8 months, defects implanted with cells expressing IGF-I had better histological scores compared to control defects and defects repaired with naive chondrocytes. This included increased chondrocyte predominance and collagen type II, both features of hyaline-like repair tissue. The equine model closely approximates human cartilage healing, indicating AAV-mediated genetic modification of chondrocytes may be clinically beneficial to humans.

A comparison of three-dimensional culture systems to evaluate in vitro chondrogenesis of equine bone marrow-derived mesenchymal stem cells.

OBJECTIVE: To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). METHODS: MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-ß1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Toluidine Blue) and immunohistochemistry (collagen type II and X). RESULTS: alginate, fibrin alginate (FA), and both pellet culture systems resulted in chondrogenic transformation. Adequate RNA was not obtained from agarose cultures at any time point. There was increased COL2B, ACAN, and SOX9 expression on day 14 from both pellet culture systems. On day 28, increased expression of COL2B was maintained in 5×10(5) cell pellets and there was no difference in ACAN and SOX9 between FA and both pellet cultures. COL10 expression was significantly lower in FA cultures on day 28. Collagen type II was abundantly formed in all culture systems except alginate and collagen type X was least in FA hydrogels. CONCLUSION: equine MSCs respond to 3D culture in FA blended hydrogel and both pellet culture systems with chondrogenic induction. For prevention of terminal differentiation and hypertrophy, FA culture may be superior to pellet culture systems.

Cell- and gene-based approaches to tendon regeneration.

Repair of rotator cuff tears in experimental models has been significantly improved by the use of enhanced biologic approaches, including platelet-rich plasma, bone marrow aspirate, growth factor supplements, and cell- and gene-modified cell therapy. Despite added complexity, cell-based therapies form an important part of enhanced repair, and combinations of carrier vehicles, growth factors, and implanted cells provide the best opportunity for robust repair. Bone marrow-derived mesenchymal stem cells provide a stimulus for repair in flexor tendons, but application in rotator cuff repair has not shown universally positive results. The use of scaffolds such as platelet-rich plasma, fibrin, and synthetic vehicles and the use of gene priming for stem cell differentiation and local anabolic and anti-inflammatory impact have both provided essential components for enhanced tendon and tendon-to-bone repair in rotator cuff disruption. Application of these research techniques in human rotator cuff injury has generally been limited to autologous platelet-rich plasma, bone marrow concentrate, or bone marrow aspirates combined with scaffold materials. Cultured mesenchymal progenitor therapy and gene-enhanced function have not yet reached clinical trials in humans. Research in several animal species indicates that the concept of gene-primed stem cells, particularly embryonic stem cells, combined with effective culture conditions, transduction with long-term integrating vectors carrying anabolic growth factors, and development of cells conditioned by use of RNA interference gene therapy to resist matrix metalloproteinase degradation, may constitute potential advances in rotator cuff repair. This review summarizes cell- and gene-enhanced cell research for tendon repair and provides future directions for rotator cuff repair using biologic composites.