Intra-articular anaesthesia of the equine stifle improves foot lameness.

BACKGROUND: Equine diagnostic anaesthesia can be a useful tool in challenging lameness examinations. However, anaesthetics diffuse over time leading to nonspecific desensitisation of periarticular structures. Nerves that convey sensation from the distal limb to the central nervous system pass in close proximity to the caudal stifle joint capsule. Therefore, diffusion of intra-articular (IA) anaesthetics could cause inadvertent desensitisation of the distal limb resulting in a false diagnosis of stifle lameness. OBJECTIVES: To determine if IA stifle anaesthesia can alleviate lameness originating in the distal limb. STUDY DESIGN: Crossover experiment. METHODS: Nine horses were fitted with a circumferential hoof clamp to induce a moderate unilateral hindlimb lameness. Intra-articular stifle anaesthesia was performed and gait was evaluated every 10 min during the 90-min trial using an inertial sensor system. Push-off and landing components of the lameness were assessed by measuring the mean inter-stride difference between the maximum and minimum heights of the pelvis respectively. Differences were compared using a Wilcoxon signed-rank test. RESULTS: Overall, horses with hoof clamp-induced foot pain had a reduction in push-off lameness after IA stifle anaesthesia. The mean change in diffmax at 90 min was -4.3 mm (P = 0.005) for the experimental group vs. -2.3 mm (P = 0.2) for the control group. Lameness decreased over time, with an average improvement of 23% at 30 min, 33% at 60 min and 38% at 90 min. There was high inter-horse variability; 3/9 horses improved by ~50% within 30 min, while 2/9 improved by ~30% and 4/9 had minimal (<10%) or no improvement in lameness. Improvement after IA stifle anaesthesia was not related to the severity of baseline lameness (P = 0.3-0.7). MAIN LIMITATIONS: Limited clinical applicability of our lameness induction model. CONCLUSIONS: Intra-articular stifle anaesthesia reduces foot lameness in a third of horses by up to 50% within 30 min. Clinically, the results of IA stifle anaesthesia should be considered in the light of these findings before treatment recommendations are made, as additional diagnostics may be required to rule out pain originating in the distal limb.

Plasma firocoxib concentrations after intra-articular injection of autologous conditioned serum prepared from firocoxib positive horses.

Orthobiologics such as autologous conditioned serum (ACS) are often used to treat joint disease in horses. Because ACS is generated from the horse's own blood, any medication administered at the time of preparation would likely be present in stored ACS, which could lead to an inadvertent positive drug test following intra-articular (IA) injection. The main objective of this study was to determine if ACS prepared from firocoxib positive horses could result in detectable plasma concentrations of the drug following IA injection. Firocoxib was administered to six horses at 0.1mg/kg PO twice at a 24h interval. Blood was obtained at 4h following the second dose and transferred to a separate syringe (Arthrex IRAP II) for ACS preparation. Plasma and ACS concentrations of firocoxib were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). When horses were confirmed firocoxib negative, 7.5mL of ACS was injected into both tarsocrural joints. Blood samples were collected at 0, 4, 8, 12, 24, and 48h, and firocoxib concentration was measured. Mean (±standard error of the mean, SEM) plasma concentration of firocoxib 4h following the second dose was 33.3±4.72ng/mL. Mean (±SEM) firocoxib concentration in ACS was 35.4±4.47ng/mL. Fourteen days following the second and last dose of firocoxib, mean plasma concentration was below the lower limit of detection (LOD=1ng/mL) in all horses. Following IA injection of ACS, plasma concentrations of firocoxib remained below LOD at all times in all horses. ACS generated from horses with therapeutic plasma concentrations of firocoxib did not contain sufficient firocoxib to lead to a positive plasma drug test following IA administration.

Magnetic resonance imaging of an equine fracture model containing stainless steel metal implants.

REASONS FOR PERFORMING STUDY: Post operative imaging in subjects with orthopaedic implants is challenging across all modalities. Magnetic resonance imaging (MRI) is preferred to assess human post operative musculoskeletal complications, as soft tissue and bones are evaluated without using ionising radiation. However, with conventional MRI pulse sequences, metal creates susceptibility artefact that distorts anatomy. Assessment of the post operative equine patient is arguably more challenging due to the volume of metal present, and MRI is often not performed in horses with implants. Novel pulse sequences such as multiacquisition variable resonance image combination (MAVRIC) now provide improved visibility in the vicinity of surgical-grade implants and offer an option for imaging horses with metal implants. OBJECTIVES: To compare conspicuity of regional anatomy in an equine fracture-repair model using MAVRIC, narrow receiver bandwidth (NBW) fast spin echo (FSE), and wide receiver bandwidth (WBW) FSE sequences. STUDY DESIGN: Nonrandomised in vitro experiment. METHODS: MAVRIC, NBW FSE and WBW FSE were performed on 9 cadaveric distal limbs with fractures and stainless steel implants in the third metacarpal bone and proximal phalanx. Objective measures of artefact reduction were performed by calculating the total artefact area in each transverse image as a percentage of the total anatomic area. The number of transverse images in which fracture lines were visible was tabulated for each sequence. Regional soft tissue conspicuity was assessed subjectively. RESULTS: Overall anatomic delineation was improved using MAVRIC compared with NBW FSE; delineation of structures closest to the metal implants was improved using MAVRIC compared with WBW FSE and NBW FSE. Total artefact area was the highest for NBW FSE and lowest for MAVRIC; the total number of transverse slices with a visible fracture line was highest in MAVRIC and lowest in NBW FSE. CONCLUSION: MAVRIC and WBW FSE are feasible additions to minimise artefact around implants.

Joint-dependent response to impact and implications for post-traumatic osteoarthritis.

OBJECTIVE: The prevalence of osteoarthritis (OA) varies between joints. Cartilage in eight different joints was evaluated to elucidate the disparate susceptibilities between joints to post-traumatic OA (PTOA) and provide evidence for joint-specific clinical treatments. The hypothesis was that cartilage in different joints would have varying cell death and anabolic gene expression profiles after injury. METHODS: Adult equine cartilage explants were harvested from shoulder (SH), elbow (EL), carpal (CA), metacarpophalangeal (MC), patellofemoral (FP), tarsal (TA), metatarsophalangeal (MT), and proximal interphalangeal (PP) joints, and injured by loading with 30 MPa within 1 s. Fractional dissipated energy, cell density, cell death, and gene expression were quantified. RESULTS: PP had the highest fractional dissipated energy (94%, 95% confidence interval [CI] 88 to 101%). Cell density was highest in the superficial zone in all samples, with MC and MT having the highest peak density. Injured samples had significantly increased cell death (13.5%, 95% CI 9.1 to 17.9%) than non-injured samples (6.8%, 95% CI 2.5 to 11.1%, P = 0.016); however, cell death after injury was not significantly different between joints. Gene expression was significantly different between joints. CD-RAP expression in normal cartilage was lowest in FP (Cp = 21, 95% CI -80 to 122). After injury, the change in CD-RAP expression increased and was highest in FP (147% relative increase after injury, 95% CI 64 to 213). CONCLUSION: Different joints have different baseline characteristics, including cell density and gene expression, and responses to injury, including energy dissipation and gene expression. These unique characteristics may explain differences in OA prevalence and suggest differences in susceptibility to PTOA. CLINICAL RELEVANCE: Understanding differences in the response to injury and potential susceptibility to OA can lead to the development of preventative or treatment strategies. KEY TERMS: Gene expression, cartilage injury, chondrocyte, multiphoton microscopy, cartilage biomechanical properties, PTOA. WHAT IS KNOWN ABOUT THE SUBJECT: The prevalence of OA is variable among joints; however, most laboratory studies are performed on a single joint - most commonly the knee, and extrapolated to other joints such as the ankle or shoulder. A small number of studies have compared knee and ankle cartilage and reported differences in mechanical properties and gene expression. WHAT THIS STUDY ADDS TO EXISTING KNOWLEDGE: There are differences in baseline cell density and gene expression, and differences in response to injury, including gene expression and cell death. This suggests that there are inherent differences leading to varying susceptibilities in OA prevalence among joints. Joint-specific treatments may improve OA therapies.

Identification of cartilage injury using quantitative multiphoton microscopy.

OBJECTIVE: Cartilage injury can lead to post-traumatic osteoarthritis (PTOA). Immediate post-trauma cellular and structural changes are not widely understood. Furthermore, current cellular-resolution cartilage imaging techniques require sectioning of cartilage and/or use of dyes not suitable for patient imaging. In this study, we used multiphoton microscopy (MPM) data with FDA-approved sodium fluorescein to identify and evaluate the pattern of chondrocyte death after traumatic injury. METHOD: Mature equine distal metacarpal or metatarsal osteochondral blocks (OCBs) were injured by 30 MPa compressive loading delivered over 1 s. Injured and control sites were imaged unfixed and in situ 1 h post-injury with sodium fluorescein using rasterized z-scanning. MPM data was quantified in MATLAB, reconstructed in 3-D, and projected in 2-D to determine the damage pattern. RESULTS: MPM images (600 per sample) were reconstructed and analyzed for cell death. The overall distribution of cell death appeared to cluster into circular (n = 7) or elliptical (n = 4) patterns (p = 0.006). Dead cells were prevalent near cracks in the matrix, with only 26.3% (SE = 5.0%, p < 0.0001) of chondrocytes near cracks being viable. CONCLUSION: This study demonstrates the first application of MPM for evaluating cellular-scale cartilage injury in situ in live tissue, with clinical potential for detecting early cartilage damage. With this technique, we were able to uniquely observe two death patterns resulting from the same compressive loading, which may be related to local variability in matrix structure. These results also demonstrate proof-of-concept MPM diagnostic use in detecting subtle and early cartilage damage not detectable in any other way.

Plasma and synovial fluid concentration of doxycycline following low-dose, low-frequency administration, and resultant inhibition of matrix metalloproteinase-13 from interleukin-stimulated equine synoviocytes.

REASONS FOR STUDY: To determine whether low-dose, low-frequency doxycycline administration is capable of achieving chondroprotective concentrations within synovial fluid (SF) while remaining below minimum inhibitory concentration 90 (MIC90 ) of most equine pathogens and would be an option in the management of osteoarthritis. OBJECTIVES: To determine whether low-dose, low-frequency oral administration of doxycycline can attain in vivo SF concentrations capable of chondroprotective effects through reduction of matrix metalloproteinase (MMP)-13 activity, while remaining below MIC90 of most equine pathogens. STUDY DESIGN: Descriptive pharmacokinetic study with crossover design. METHODS: Two groups of 6 horses received oral doxycycline. Plasma and SF doxycycline concentrations were measured using high performance liquid chromatography. Group 1 received 5 mg/kg bwt q. 24 h with 21 blood and 8 SF samples collected over 120 h; Group 2 received 5 mg/kg bwt q. 48 h with 27 blood and 11 SF samples collected over 192 h. Cultured synoviocytes were treated with interleukin-1α (1 ng/ml) for 24 h to stimulate MMP synthesis, and then SF was added to the culture medium for 96 h. MMP-13 protein and mRNA were measured in synoviocyte culture medium and synoviocytes, respectively. RESULTS: Mean doxycycline concentration ≥0.043 µg/ml (previously demonstrated to inhibit MMP-13) was achieved in plasma by t = 0.25 h and SF by t = 48 h in Group 1, and in plasma by t = 0.17 h and SF by t = 1 h in Group 2. Synoviocyte culture medium containing doxycycline from Groups 1 and 2 had significantly decreased active MMP-13 protein concentration, and synoviocytes cultured in this medium had significantly decreased MMP-13 gene expression compared to controls. Plasma doxycycline concentration in both groups and SF doxycycline concentration in Group 2 demonstrated a cumulative effect. CONCLUSIONS: Low-dose orally administered doxycycline achieves SF concentrations in vivo capable of diminishing MMP-13 expression. This study supports the use of doxycycline as a disease modifying osteoarthritic drug.

Correlation of meniscal T2* with multiphoton microscopy, and change of articular cartilage T2 in an ovine model of meniscal repair.

OBJECTIVE: To correlate meniscal T2* relaxation times using ultra-short echo time (UTE) magnetic resonance imaging (MRI) with quantitative microscopic methods, and to determine the effect of meniscal repair on post-operative cartilage T2 values. DESIGN: A medial meniscal tear was created and repaired in the anterior horn of one limb of 28 crossbred mature ewes. MR scans for morphological evaluation, meniscal T2* values, and cartilage T2 values were acquired at 0, 4 and 8 months post-operatively for the Tear and Non-Op limb. Samples of menisci from both limbs were analyzed using multiphoton microscopy (MPM) analysis and biomechanical testing. RESULTS: Significantly prolonged meniscal T2* values were found in repaired limbs than in control limbs, P < 0.0001. No regional differences of T2* were detected for either the repaired or control limbs in the anterior horn. Repaired limbs had prolonged cartilage T2 values, primarily anteriorly, and tended to have lower biomechanical force to failure at 8 months than Non-Op limbs. MPM autofluorescence and second harmonic generation data correlated with T2* values at 8 months (ρ = -0.48, P = 0.06). CONCLUSIONS: T2* mapping is sensitive to detecting temporal and zonal differences of meniscal structure and composition. Meniscal MPM and cartilage T2 values indicate changes in tissue integrity in the presence of meniscal repair.

Treatment of subchondral cystic lesions of the medial femoral condyle of mature horses with growth factor enhanced chondrocyte grafts: a retrospective study of 49 cases.

REASONS FOR PERFORMING STUDY: To evaluate the long-term clinical outcome after allogeneic chondrocyte and insulin-like growth factor-I (IGF-I) grafting of subchondral cystic lesions (SCLs) of the femoral condyle in horses. OBJECTIVE: To test the hypothesis that chondrocyte and IGF-I grafts will improve the long-term clinical outcome in arthroscopically debrided SCLs. METHODS: Medical records of 49 horses with SCLs of the femoral condyle treated by debridement and implantation of chondrocytes and IGF-I were reviewed. Preoperative radiographs were obtained, and caudocranial radiographic projections were used to establish a ratio between cyst and femoral condyle size. Arthroscopic cyst debridement followed by filling of the bone void with autologous cancellous bone (45 horses) or tricalcium phosphate granules (4 horses) was performed. A paired syringe containing a fibrinogen and chondrocyte mixture in one syringe and calcium-activated bovine thrombin with IGF-I in the other was used to cover the surface. A successful outcome was defined as a horse that performed to its intended use without lameness. RESULTS: A successful outcome was achieved in 36 of 49 horses (74%). Preoperative radiography was performed in all horses, with 33 horses having unilateral SCLs of the medial femoral condyle, 15 horses having bilateral SCLs of the medial femoral condyle, and one horse having bilateral SCLs of the lateral femoral condyle. Median age of the horses was 3.3 years. Fifteen horses had preoperative radiographic and arthroscopic evidence of osteoarthritis (OA). A successful outcome was not influenced by age of horse, presence of pre-existing osteoarthritis or preoperative size of the subchondral cyst. Grafting resulted in success for 80% of horses >3 years old, and in 80% of horses with OA. CONCLUSIONS: Implantation of allogeneic chondrocytes supplemented with IGF-I is an effective treatment for horses with SCLs of the femoral condyle, and particularly for older horses and horses with pre-existing osteoarthritis. POTENTIAL RELEVANCE: Chondrocyte implantation may offer a greater chance of long-term success in older horses and horses with osteoarthritis than has been previously reported with cyst debridement alone.

Pharmacokinetics and distribution of minocycline in mature horses after oral administration of multiple doses and comparison with minimum inhibitory concentrations.

REASONS FOR PERFORMING STUDY: Minocycline holds great potential for use in horses not only for its antimicrobial effects but also for its anti-inflammatory and neuroprotective properties. However, there are no pharmacokinetic or safety data available regarding the use of oral minocycline in horses. OBJECTIVES: To determine pharmacokinetics, safety and penetration into plasma, synovial fluid, aqueous humour (AH) and cerebral spinal fluid (CSF) of minocycline after oral administration of multiple doses in horses and to determine the minimum inhibitory concentrations (MIC) of minocycline for equine pathogenic bacteria. METHODS: Six horses received minocycline (4 mg/kg bwt q. 12 h for 5 doses). Thirty-three blood and 9 synovial fluid samples were collected over 96 h. Aqueous humour and CSF samples were collected 1 h after the final dose. Minocycline concentrations were measured using high pressure liquid chromatography. The MIC values of minocycline for equine bacterial isolates were determined. RESULTS: At steady state, the mean ± s.d. peak concentration of minocycline in the plasma was 0.67 ± 0.26 µg/ml and the mean half-life was 11.48 ± 3.23 h. The highest trough synovial fluid minocycline concentration was 0.33 ± 0.12 µg/ml. The AH concentration of minocycline was 0.09 ± 0.03 µg/ml in normal eyes and 0.11 ± 0.04 µg/ml in blood aqueous barrier-disrupted eyes. The mean CSF concentration of minocycline was 0.38 ± 0.09 µg/ml. The MIC values were determined for 301 isolates. Minocycline concentrations were above the MIC(50) and MIC(90) for many gram-positive equine pathogens. POTENTIAL RELEVANCE: This study supports the use of orally administered minocycline at a dose of 4 mg/kg bwt every 12 h for the treatment of nonocular infections caused by susceptible (MIC ≤ 0.25 µg/ml) organisms in horses. Further studies are required to determine the dose that would be effective for the treatment of ocular infections.

Arthroscopic reattachment of osteochondritis dissecans cartilage flaps of the femoropatellar joint: long-term results.

REASONS FOR PERFORMING STUDY: Long-term efficacy of arthroscopic cartilage reattachment for the treatment of osteochondritis dissecans (OCD) lesions in the equine femoropatellar joint is unknown. OBJECTIVE: To evaluate radiographic outcome and long-term performance of horses undergoing OCD reattachment. HYPOTHESIS: Separated OCD cartilage flaps may be reincorporated into the joint surface by reattachment rather than flap removal. METHODS: Polydioxanone pins were utilised arthroscopically to reattach OCD lesions in 40 of 44 joints from 27 horses. Cartilage was reattached when it had persisting perimeter continuity, the surface was not deeply fissured or irregular, and the cartilage was not protuberant or extensively mineralised. Bone marrow aspirate concentrate was grafted to additional areas denuded of cartilage or alongside reattached cartilage. RESULTS: Breeds included Thoroughbred (n = 18), Quarter Horse (n = 4), Warmblood (n = 3), Standardbred (n = 1) and Arabian (n = 1). Mean age was 9.7 months. Radiographic lesion length was 1.5-6.3 cm. Reattachment alone was used in 32 of 44 affected joints, a combination of debridement and reattachment in 8 joints and debridement alone in 4 joints. One horse was destroyed due to tendon laceration. Of the remaining 26 horses, mean duration of follow-up was 15.6 months (range 2 months-12 years). Radiographic resolution of OCD lesions treated with reattachment was significantly improved at 6 months. Twenty horses had long-term performance data, of which 19 were sound and had reached intended athletic potential. One horse remained lame, and an additional 6 were sound but remained unbroken or were convalescing. Thus, an overall success rate based upon continued soundness in performing horses was 95% (19/20). CONCLUSION: Cartilage flap reattachment can salvage OCD cartilage by integration with the underlying bone. POTENTIAL RELEVANCE: Extensive OCD cartilage flaps may be salvaged by reattachment which can result in normal radiographic subchondral bone contour and long-term athletic performance.

Relationships between race earnings and horse age, sex, gait, track surface and number of race starts for Thoroughbred and Standardbred racehorses in North America.

REASONS FOR PERFORMING STUDY: There is no consensus on objective outcome measures that can be used to determine if a medical or surgical treatment affects race performance. OBJECTIVE: To determine the association between 2 commonly used outcome measures (total starts and total earnings) and age, sex, gait and race surface. METHODS: A cross-sectional study was performed using the race performance data for all Thoroughbred horses age 2, 3, 4 and 5 years racing in the United States, and Standardbred horses of the same ages racing in the United States and Canada during the year 2006. Median earnings and starts were determined for each combination of age, sex and track surface (for Thoroughbred) or gait (for Standardbred). The effect these variables had on starts on race earnings ($) was determined using linear regression. RESULTS: Race records for 68,649 Thoroughbreds and 25,830 Standardbreds were obtained. All independent variables (age, breed, sex, gait, track surface and total number of starts) had a significant impact on total earnings (P<0.0001). CONCLUSIONS: The data show considerable variation across age groups and track surfaces for Thoroughbreds and across age groups for Standardbreds. They also show that the decision to use earnings or starts as outcome measures could have a marked effect on reported success for a particular treatment. POTENTIAL RELEVANCE: Both earning and start data should be reported in studies evaluating outcome following surgery or other intervention. Considerations of age, breed, sex, track surface and gait should be included in the design of these studies.

Orally administered doxycycline accumulates in synovial fluid compared to plasma.

REASONS FOR PERFORMING STUDY: Tetracycline compounds have been used to slow the progression of osteoarthritis (OA) and rheumatoid arthritis but the concentration of doxycycline attained in synovial fluid following oral, low-dose administration has yet to be determined. OBJECTIVE: To determine the concentration of doxycycline in synovial fluid following oral, low-dose administration. METHODS: Six mature horses received doxycycline (5 mg/kg bwt q. 12 h for 5 doses). Venous blood and synovial fluid samples were collected at t=0, 0.25, 0.5, 1, 12, 24, 48 and 72 h. Doxycycline concentrations were measured using reverse phase high pressure liquid chromatography with ultraviolet detection. RESULTS: Doxycycline concentrations at all time points after t=0 were above the lower limit of quantification for the assay. Plasma concentrations of doxycycline were above 0.21 microg/ml at t=0.5 h. The mean+/-s.d. peak concentration (Cmax) of doxycycline in plasma was 0.37+/-0.22 microg/ml and time to peak concentration was 0.54+/-0.19 h. Synovial fluid concentrations of doxycycline were above 0.12 microg/ml 1 h after drug administration. The mean Cmax of doxycycline in the synovial fluid was 0.27+/-0.10 microg/ml. The penetration factor of doxycycline from plasma into synovial fluid, as determined by a ratio of the area-under-the-curve for synovial fluid:plasma during the sampling period, was 4.6. POTENTIAL RELEVANCE: Orally administered doxycycline distributes easily into synovial fluid with a penetration factor of 4.6. Terminal half-life of the drug in synovial fluid was longer than in the plasma, indicating possible accumulation in this compartment. Further in vivo studies are warranted to define a medication protocol prior to routine clinical use of doxycycline for the treatment of OA.

Effects of platelet rich plasma and acellular bone marrow on gene expression patterns and DNA content of equine suspensory ligament explant cultures.

REASONS FOR PERFORMING STUDY: Suspensory ligament (SL) desmitis is a common source of lameness. The results of this study will determine if blood-derived products stimulate SL matrix synthesis and have potential as regenerative therapies for SL desmitis OBJECTIVES: To determine if various blood-based biological products including plasma, blood, PRP, platelet poor plasma (PPP) and ABM aspirate stimulates anabolic and/or catabolic pathways in suspensory ligaments (SL). METHODS: The body of the SL was harvested from 6 horses and used to establish explant cultures. Explants were cultured in plasma, blood, PRP, PPP or ABM at concentrations of 10, 50 or 100%. Anabolic responses were assessed by use of quantitative PCR for collagens type I and III, cartilage oligomeric matrix protein (COMP) and decorin. Total DNA and collagen protein content were also measured. Catabolic reactions were measured by quantitative PCR for matrix metalloproteinases 3 and 13 (MMP-3, MMP-13). RESULTS: Acellular bone marrow aspirate at 100% stimulated decorin and COMP mRNA synthesis more than all other treatments at all concentrations. No treatment at any concentration stimulated the catabolic gene MMP-13; only 50% ABM stimulated MMP-13 mRNA expression. CONCLUSIONS: Acellular bone marrow is indicated, and might be preferred to plasma, blood or PPP, as a blood-based biological source for SL tissue regenerative therapy. Long-term, placebo controlled case studies are indicated to determine if ABM aids in recovery from SL desmitis. POTENTIAL RELEVANCE: Bone marrow aspirate is an autogenous, readily available biological source for SL regenerative therapy where the aim is to stimulate matrix synthesis.

Age-related changes in serum insulin-like growth factor-I, insulin-like growth factor-I binding protein-3 and articular cartilage structure in Thoroughbred horses.

REASONS FOR PERFORMING STUDY: Structural changes in articular cartilage associated with the ageing process require definition for investigators performing developmental and age-related studies, for which information is lacking. OBJECTIVES: To 1) determine the onset and end of puberty as defined by serum insulin like growth factor (IGF-I) and IGF-binding protein-3 (IGFBP-3) concentrations and 2) correlate articular-epiphyseal cartilage complex structural changes with the onset and end of puberty. METHODS: IGF-I and IGFBP-3 were measured in serum samples from normal female and male horses age 9-715 days to determine peak and steady-state values for horses transitioning through puberty. Osteochondral tissue sections were obtained from horses age 120-840 days (4-28 months) and examined histologically for cartilage canals and tidemark formation. RESULTS: In male and female horses, serum IGF-I/IGFBP-3 concentrations peaked at approximately 225 days, defining the onset of puberty. Cartilage canals were absent from articular cartilage just prior to this time point. IGF-I/IGFBP-3 concentrations declined to steady-state levels at approximately age 450 days, signalling exit from puberty and therefore the beginning of ageing. This time point correlated to initial formation of a tidemark in the osteochondral tissue sections. CONCLUSIONS: Horses may be considered pubescent at age 225-450 days, and post pubescent and ageing after age 450 days. POTENTIAL RELEVANCE: Defining the normal post natal to post pubescent concentrations for serum IGF-I and serum IGFBP-3 establishes subsets of animals for age-related studies and may be used to monitor horses for abnormally high IGF-I concentrations due to natural disease or subsequent to systemic growth hormone administration.

Arthroscopic reattachment of osteochondritis dissecans lesions using resorbable polydioxanone pins.

REASONS FOR PERFORMING STUDY: Debridement of osteochondritis dissecans (OCD) cartilage lesions results in fibrocartilage and imperfect hyaline repair tissue, and forms a permanent irregularity to the subchondral bone plate. OBJECTIVE: To evaluate the clinical, radiographic and outcome effects of OCD cartilage flap reattachment for select lesions as an alternative to OCD debridement. HYPOTHESIS: Separated cartilage flaps resulting from OCD lesions may be re-incorporated into the hyaline cartilage surface by reattachment rather than debridement and removal. METHODS: Resorbable polydioxanone pins were used to reattach OCD flap lesions in 16 joints of 12 horses. Criteria for attachment, rather than removal, included an unmineralised cartilage flap on preoperative radiographs and a relatively smooth surface with some residual perimeter attachment at surgery. RESULTS: There were 12 subjects, 6 males and 6 females, 7 Thoroughbred or Standardbred weanlings, 3 Warmbloods, 1 Arabian and 1 Quarter Horse, mean age at surgery 6.8 months. All horses had effusion of the affected femoropatellar joint (n = 9), tarsocrural joint (n = 1) or fetlock (n = 2). Radiographic lesions varied in length between 1.8-6.3 cm; reattachment was used in 16 of 18 affected joints and the OCD cartilage was not satisfactory for salvage in 2 stifles. Number of pins required was 2-10. One horse was subjected to euthanasia due to a tendon laceration 8 weeks after surgery; of the remaining 11 horses, mean duration of follow-up was 3.9 years (range 4 months-8 years). Nine of these were sound and had entered work, while 2 were sound but remained unbroken 4 and 6 months post operatively, respectively. Radiographic resolution of the OCD lesion occurred in 14 of 16 pinned joints in the 9 horses with long-term follow-up. The 2 remaining joints had a 3 and a 5 mm mineralised flap in the original defect sites. CONCLUSIONS: This study indicated cartilage flap reattachment was an alternative to removal in selected OCD lesions. POTENTIAL RELEVANCE: Relatively smooth OCD cartilage flaps may be salvaged by reattachment and can result in normal radiographic subchondral contour and a high likelihood of athletic performance. Further case numbers are required to determine which lesions are too irregular or contain too much mineral for effective incorporation after reattachment.

Insulin-like growth factor-I enhances cell-based repair of articular cartilage.

Composites of chondrocytes and polymerised fibrin were supplemented with insulin-like growth factor-I (IGF-I) during the arthroscopic repair of full-thickness cartilage defects in a model of extensive loss of cartilage in horses. Repairs facilitated with IGF-I and chondrocyte-fibrin composites, or control defects treated with chondrocyte-fibrin composites alone, were compared before death by the clinical appearance and repeated analysis of synovial fluid, and at termination eight months after surgery by tissue morphology, collagen typing, and biochemical assays. The structure of cartilage was evaluated histologically by Toluidine Blue reaction and collagen type-I and type-II in situ hybridisation and immunohistochemistry. Repair tissue was biochemically evaluated by DNA assay, proteoglycan quantitation and characterisation, assessment of collagen by reverse-phase high-performance liquid chromatography, and collagen typing using cyanogen bromide digestion and peptide separation by polyacrylamide gel electrophoresis. The results at eight months showed that the addition of IGF-I to chondrocyte grafts enhanced chondrogenesis in cartilage defects, including incorporation into surrounding cartilage. Gross filling of defects was improved, and the tissue contained a higher proportion of cells producing type-II collagen. Measurements of collagen type II showed improved levels in IGF-I-treated defects, supporting in situ hybridisation and immunohistochemical assessments of the defects. IGF-I improves the repair capabilities of chondrocyte-fibrin grafts in large full-thickness repair models.

Insulin-like growth factor-I gene expression patterns during spontaneous repair of acute articular cartilage injury.

This study evaluated the constitutive insulin-like growth factor-I (IGF-I) gene expression pattern in spontaneously healing cartilage defects over the course of 16 weeks, and correlated the tissue morphology and matrix gene expression with IGF-I mRNA levels. Full-thickness 15 mm cartilage defects were debrided in the femoral trochlea of both femoropatellar joints of 8 horses and the healing defects examined 2, 4, 8, or 16 weeks after surgery. Samples were harvested for histologic assessment of tissue healing using H&E staining, toluidine blue histochemical reaction for proteoglycan deposition, and in situ hybridization and immunohistochemistry procedures to demonstrate collagen type II mRNA and protein expression. Total RNA was isolated for Northern analysis to measure cartilage matrix molecule expression, and for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) to determine IGF-I gene expression patterns in healing cartilage defects. Full-thickness cartilage defects in horses were slow to heal compared to smaller lesions in similar locations in other animals. However, a progressive decline in tissue cellularity and vascularity, and increased tissue organization were observed on H&E stained specimens over the 16-week experiment. Evidence of early chondrogenic repair was detected through collagen type II in situ hybridization and immunohistochemistry. However, levels of collagen type II and aggrecan mRNA in lesions were not abundant on Northern analysis indicating incomplete chondrogenesis. IGF-I message expression followed a cyclic pattern with low levels at 2 weeks, followed by an increase at 4 and 8 weeks, and a subsequent decline at 16 weeks. There was no direct correlation between the stage of healing and cartilage matrix message expression, and the abundance of IGF-I mRNA in the healing lesions. In conclusion, this study demonstrated that the spontaneous healing of articular defects was accompanied by a temporal fluctuation in IGF-I gene expression which was discoordinate to the steady rise in expression of cartilage matrix molecules such as procollagen type II.

Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix.

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [35S]-sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF-beta1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two-fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF-beta1 followed by exposure of cells to IGF-I in three-dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSC's remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF-beta1 without sequential IGF-I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF-I supplemented cultures, particularly when used in concert with TGF-beta1 pretreatment.

Tenoscopic examination and proximal annular ligament desmotomy for treatment of equine "complex" digital sheath tenosynovitis.

OBJECTIVE: To determine the outcome of horses with "complex" digital tenosynovitis treated by tenoscopic proximal annular ligament desmotomy and resection of synovial masses or adhesions, or both, within the digital sheath. STUDY DESIGN: Retrospective evaluation. ANIMALS OR SAMPLE POPULATION: Twenty-five horses with a clinical and ultrasonographic diagnosis of palmar or plantar proximal annular ligament constriction and ultrasonographic evidence of synovial masses or adhesions within the digital tendon sheath. METHODS: Each horse had tenoscopic surgery for annular ligament desmotomy combined with adhesiolysis and/or synovial mass resection. Mean follow-up time was 3.4 years. Spearman's rank correlation was used to assess the relationship between functional outcome or cosmetic results and preoperative variables including duration of clinical signs, digital sheath synovial fluid total protein concentration and nucleated cell count, thickness of the palmar or plantar proximal annular ligament (PAL), severity of adhesions, severity of synovial masses, degree of synovial distention, or limb affected. RESULTS: A total of 18 (72%) horses returned to athletic soundness, 4 were improved but not sound, and 3 were not improved. Cosmetic outcome was normal in 10 horses, improved but not normal in 12, and not improved in 3 horses. Cosmetic and functional outcome were significantly adversely affected by the duration of clinical signs and the severity of synovial masses. CONCLUSIONS: With appropriate tenoscopic surgical attention, horses with complex tenosynovitis syndrome characterized by synovial masses, adhesions, or both adhesions and masses, and PAL constriction, have a good prognosis for return to athletic soundness. CLINICAL RELEVANCE: Horses with PAL constriction and additional digital tendon sheath pathology such as adhesions and synovial masses have a 72% chance of returning to sound athletic performance, however 60% of horses retain some degree of cosmetic blemish in the affected limb. There is an inverse relationship between the duration of clinical signs and outcome, and therefore, prompt surgical attention is advised.