Zonal characterization and differential trilineage potentials of equine intrasynovial deep digital flexor tendon-derived cells.

BACKGROUND: Intrasynovial deep digital flexor tendon (DDFT) injuries occur frequently and are often implicated in cases of navicular disease with poor outcomes and reinjuries. Cell-based approaches to tendon healing are gaining traction in veterinary medicine and ultimately may contribute to improved DDFT healing in horses. However, a better understanding of the innate cellular characteristics of equine DDFT is necessary for developing improved therapeutic strategies. Additionally, fibrocartilaginous, intrasynovial tendons like the DDFT are common sites of injury and share a poor prognosis across species, offering translational applications of this research. The objective of this study is to isolate and characterize tendon-derived cells (TDC) from intrasynovial DDFT harvested from within the equine forelimb podotrochlear bursa. TDC from the fibrocartilaginous and tendinous zones are separately isolated and assessed. Flow cytometry is performed for mesenchymal stem cell (MSC) surface markers (CD 29, CD 44, CD 90). Basal tenogenic, osteogenic and chondrogenic markers are assessed via quantitative real time-PCR, and standard trilineage differentiation is performed with third passage TDC from the fibrocartilaginous (fTDC) and tendinous (tTDC) zones of DDFT. RESULTS: Low-density plating isolated homogenous TDC populations from both zones. During monolayer passage, both TDC subpopulations exhibited clonogenicity, high in vitro proliferation rate, and fibroblast-like morphology. fTDC and tTDC were positive for MSC surface markers CD90 and CD29 and negative for CD44. There were no significant differences in basal tenogenic, osteogenic or chondrogenic marker expression between zones. While fTDC were largely restricted to chondrogenic differentiation, tTDC underwent osteogenic and chondrogenic differentiation. Both TDC subpopulations displayed weak adipogenic differentiation potentials. CONCLUSIONS: TDC at the level of the podotrochlear bursa, that potentially could be targeted for enhancing DDFT injury healing in horses were identified and characterized. Pending further investigation, promoting chondrogenic properties in cells administered exogenously into the intrasynovial space may be beneficial for intrasynovial tendon regeneration.

Subchondral lucencies of the proximal tibia in 17 horses.

OBJECTIVE: To describe subchondral lucencies (SCL) in the equine proximal tibia, several treatment options, and clinical outcomes. STUDY DESIGN: Retrospective study. ANIMALS: Seventeen horses with proximal tibial SCL. METHODS: Medical record and radiograph review. Follow-up was obtained via examination and radiography when possible and by telephone and race records when required. The median duration of follow-up was 20 months (range, 0-48). RESULTS: Proximal tibial SCL were associated with lameness in 14 of 17 horses. Subchondral lucencies were primary in 11 horses and secondary to an ipsilateral medial femoral condyle SCL in six horses. One foal with a primary SCL was euthanized because of osteomyelitis. Six horses ≤1 year old with primary SCL were managed with exercise restrictions only; SCL in three horses without lameness decreased in size, whereas three horses with lameness did not improve. One young horse treated with surgical debridement failed to improve and was euthanized. Lameness resolved in three horses with primary tibial SCL treated with screw fixation. Screw fixation of secondary SCL in five horses led to a reduction in SCL size and degree of lameness. CONCLUSION: Primary tibial SCL healed with rest in 3 non-lame young horses with small SCL, but was not successful in lame horses with larger SC. Radiographic size and associated lameness improved or resolved with screw fixation in primary and secondary proximal tibial SCL. CLINICAL SIGNIFICANCE: Primary tibial SCL that did not cause lameness healed with conservative management, but persistent primary and secondary tibial SCL required screw fixation to reduce lameness.

Evaluation of a New Surgical Treatment for Equine Hind Limb Proximal Suspensory Desmitis.

OBJECTIVE: To evaluate the effects of a new microfracture and ligament splitting procedure on ligament healing and to examine the usefulness of magnetic resonance (MR) imaging for monitoring ligament healing over time using a collagenase model of hind limb proximal suspensory desmitis. STUDY DESIGN: Experimental in vivo study. ANIMALS: Healthy adult horses (n=6). METHODS: Horses were free of lameness with normal hind limb proximal suspensory ligaments (PSL). The origin of both hind limb PSL was injected with collagenase and underwent MR imaging 2 weeks later, followed by the microfracture and ligament splitting procedure on 1 limb, with the opposite limb serving as the sham-operated control. Serial lameness and MR examinations were performed. Horses were euthanatized 210 days after surgery, the PSL harvested, and histology, biochemistry, and gene expression performed on both PSL. RESULTS: Collagenase lesions viewed on MR images appeared similar to those seen clinically. Serial MR images demonstrated resolution of abnormal signal intensity and tissue formation in the microfracture sites within the third metatarsal bone. Treated limbs had histologic evidence of connective tissue appearing to originate from the small perforations and blending into the ligament but no statistical differences were identified. Gene expression for cartilage oligomeric matrix protein and decorin were significantly increased in treated compared to control limbs. CONCLUSION: The microfracture and ligament splitting procedure did incite a tissue response but further clinical investigation is necessary to determine if this tissue remodeling at the bone-ligament interface translates to improved clinical outcome. MR imaging may be useful to follow healing in horses with hind limb proximal suspensory desmitis.

Ex vivo effects of corticosteroids on equine deep digital flexor and navicular fibrocartilage explant cell viability.

OBJECTIVE: To investigate the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on the viability of resident cells within the fibrocartilage on the dorsal surface of the deep digital flexor tendon (FC-DDFT) and fibrocartilage on the flexor surface of the navicular bone (FC-NB) of horses. SAMPLE: 12 to 14 explants of FC-DDFT and of FC-NB from grossly normal forelimbs of 5 cadavers of horses aged 9 to 15 years without evidence of musculoskeletal disease. PROCEDURES: Explants were incubated with culture medium (control) or TA-supplemented (0.6 or 6 mg/mL) or MPA-supplemented (0.5 or 5 mg/mL) medium for 6 or 24 hours. Explant metabolic activity and percentage of dead cells were assessed with a resazurin-based assay and live-dead cell staining, respectively, at each time point. Drug effects were assessed relative to findings for the respective control group. RESULTS: Application of TA (at both concentrations) did not significantly change the cell viability of FC-DDFT explants. For FC-NB explants, TA at 6 mg/mL significantly reduced the metabolic activity and increased the percentage of dead cells at both time points. With either MPA concentration, FC-DDFT and FC-NB explants had reduced metabolic activity and an increased percentage of dead cells at 24 hours, whereas only MPA at 5 mg/mL was cytotoxic at the 6-hour time point. CONCLUSIONS AND CLINICAL RELEVANCE: In ex vivo explants, TA was less cytotoxic to equine FC-DDFT and FC-NB cells, compared with MPA. Further work is warranted to characterize the drugs' transcriptional and translational effects as well as investigate their cytotoxicity at lower concentrations.

Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.

Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-µm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4',6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastin-collagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease; p < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease; p < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy. Impact statement This work demonstrated that second harmonic generation microscopy in conjunction with elastin immunofluorescence provided a comprehensive assessment of multiscale structural re-organization in healing tendon than when restricted to longitudinal collagen fiber alignment alone. Utilizing this approach for tendon histomorphometry is ideal not only to improve our understanding of hierarchical structural changes that occur after tendon injury and during remodeling but also to monitor the efficacy of therapeutic approaches.

In vitro Effects of Methylprednisolone Acetate on Equine Deep Digital Flexor Tendon-Derived Cells.

Primary deep digital flexor tendon (DDFT) pathologies and those accompanying degenerative changes of navicular bone fibrocartilage are major causes of lameness associated with navicular disease. Intrasynovial corticosteroids are mainstay in the treatment due to the anti-inflammatory effects, but their effect on DDFT cell biosynthesis are unknown. The objective of this in-vitro study was to investigate the effects of methylprednisolone acetate (MPA) on cells isolated from the dorsal fibrocartilaginous region of forelimb DDFTs (DDFT-derived cells) of 5 horses (aged 11-17 years). Non-adherent aggregate cultures were established from third passage cells over a 72 to 96-h duration prior to treating with medium containing 0 (control), 0.05 and 0.5 mg/mL MPA for 24 h. Tendon and cartilage extracellular matrix (ECM) related gene expression, cell aggregate and culture medium GAG contents, culture medium collagen and MMP-3 and-13 concentrations were measured. After 24 h of treatment, only the higher MPA concentration (0.5 mg/mL) significantly down-regulated tendon ECM related genes; whereas, both MPA doses significantly down-regulated cartilage ECM related genes. MPA treatment did not affect the total GAG content of DDFT-derived cells or total GAG, soluble collagen and MMP-3 and-13 contents in culture medium compared to untreated controls. Future studies to determine the response of DDFT-derived cells with longer exposure times to corticosteroids and in the presence of inflammatory cytokines are necessary. These results are a first step in assessing the effects of intrasynovial medications on equine DDFT, for which currently no information exists.

Evaluation of a Novel Bone Substitute Injection Technique for Potential Treatment of Impact Injury to the Equine Palmar Metacarpal Condyle.

OBJECTIVE: The aim of this study was to evaluate the injection of a bone substitute material (BSM) into an impact lesion in the palmar condyle of the third metacarpal bone. STUDY DESIGN: This was an in vivo controlled study performed on six horses. MATERIALS AND METHODS: Medial metacarpal condyles were exposed via arthrotomy and a compressive lesion created in anaesthetized horses using 80 psi (27.6 MPa) onto the articular surface (n = 12). Paired limbs were randomly selected as a control or for extra-articular injection of BSM towards the subchondral bone near the compressive lesion. Parameters of the surgical techniques and BSM distribution outcomes were evaluated using magnetic resonance imaging analysis, histology and histomorphometry. RESULTS: Injection of the BSM required significant pressure, as well as the use of a pilot hole. The BSM was visible in all magnetic resonance imagings in treatment limbs. Post-impact treatment limbs had greater average grey scale values than controls (p = 0.041), and greater average grey scale values than pre-impact treatment limbs (p = 0.004). Histology demonstrated haemorrhage and microfractures at the site of compression with no evidence of bone disruption from BSM injection. CONCLUSION: Injection of BSM into the dense subchondral bone of the equine palmar condyle could be targeted to a site of injury, distributed subchondrally and without further injury to bone or cartilage. CLINICAL SIGNIFICANCE: This procedure has potential for the treatment of clinical impact injury or osteoarthritis in horses, and long-term studies are warranted.

Surgical approach for removal of nonarticular base sesamoid fragments of the proximal sesamoid bones in horses.

OBJECTIVES: To describe a surgical approach for the removal of nonarticular base sesamoid fragments in performance horses and to report the outcome. STUDY DESIGN: Retrospective study. ANIMALS: Horses (n=11), 7 months to 10 years of age, with lameness because of nonarticular base sesamoid fragments. METHODS: Lameness was localized to the metacarpophalangeal/metatarsophalangeal region by clinical examination or response to diagnostic local anesthesia. Radiographs confirmed the diagnosis of a nonarticular base sesamoid fragment. Surgical removal was performed with an incision over the base of the affected sesamoid through the digital flexor tendon sheath. After identifying the fragment with the aid of needles, a small vertical incision was made in the straight distal sesamoidean ligament (SDSL) and the fragment was freed from its attachments and removed. Six months convalescence and rehabilitation was prescribed for all horses. RESULTS: Eleven horses had 16 nonarticular fragments of the base of the proximal sesamoid bones. Eleven (69%) fragments occurred in the forelimbs with the right front (82%) and medial sesamoid (73%) more commonly affected. Horses were treated by surgical fragment removal. In horses with follow-up, 9 of 10 returned to their intended use. CONCLUSIONS: Surgical removal of nonarticular base sesamoid fragments can be accomplished through a palmar/plantar approach through the digital flexor tendon sheath and SDSL. This "keyhole" approach minimizes damage to the distal sesamoidean ligament attachments to the sesamoid base and allows some horses to return to their intended use. CLINICAL RELEVANCE: Surgical removal of nonarticular base sesamoid fragments should be considered in horses with performance-limiting lameness as a result of the fragment.

Equine Dental Pulp Connective Tissue Particles Reduced Lameness in Horses in a Controlled Clinical Trial.

OBJECTIVE: To assess if injection of allogeneic dental pulp tissue particles would improve lameness in horses with naturally occurring osteoarthritis (OA) or soft tissue (ST) injury. DESIGN: Prospective, randomized, blinded, and controlled clinical trial and client survey assessment. ANIMALS: Forty lame client-owned horses. PROCEDURES: Sterile dental pulp, recovered from otherwise healthy foals that perish during dystocia, was processed under good manufacturing processing to produce mechanically manipulated, unexpanded pulp tissue particles containing viable cells surrounded in extracellular matrix. Forty lame client-owned horses with confirmed OA (n = 20), or ST injury (desmitis or tendonitis) received a 2 mL intra-articular (n = 20 OA) or intra-lesional (n = 20) injection of control transport vehicle (n = 20) or 10 × 106 dental pulp tissue particles (n = 20). Acclimatized horses had baseline measurements performed and were then injected on day 0. Horses were treadmill exercised for 2 weeks, evaluated by clinical parameters, lameness score, edema (score and circumference), pain on flexion (OA) or pressure (ST), and clients' scores for pain and discomfort before and through 45 days after pulp injection. Twenty horses were available for >2.5-year follow-up. RESULTS: Pulp-treated horses showed decrease in lameness compared to baseline (P < 0.009) or placebo controls (P < 0.013) for at least 2 weeks. Client assessments of comfort were improved between before and 45 days after pulp injection (P < 0.001). Clinical improvement with ST injury was significantly greater than OA (P < 0.001). At >2.5-year follow-up, at least 10 horses were in work. CONCLUSION AND CLINICAL RELEVANCE: Dental pulp tissue particles can be considered as a treatment option for equine lameness due to OA, desmitis, or tendonitis.

Concurrent or sequential tibial subchondral cystic lesions in 4 horses with medial femoral condyle subchondral cystic lesions.

CASE DESCRIPTION 4 horses were examined because of signs of chronic hind limb lameness. CLINICAL FINDINGS 3 horses had a history of lameness for > 6 months; specific duration was unknown for 1 horse. On initial evaluation, grade 3 to 4 (on a scale from 1 to 5) hind limb lameness was present in all 4 horses. Radiography of the stifle joint of the affected limb revealed medial femoral condyle subchondral lucencies or subchondral cystic lesions (SCLs) in all 4 horses, medial femorotibial osteoarthritis in 3 horses, and medial tibial condyle SCLs in 3 horses. TREATMENT AND OUTCOME 2 horses were treated medically (stall rest and oral NSAID administration), and 2 horses were treated surgically by means of medial femoral transcondylar lag screw placement through the medial femoral condyle SCLs. The 2 horses treated medically did not improve and were euthanized. Necropsy confirmed the presence of medial femoral condyle and medial tibial condyle SCLs. Surgical treatment did not resolve the lameness in 1 horse with SCLs in the medial tibial condyle and medial femoral condyle, and euthanasia was performed 150 days after surgery. In the second horse, a medial tibial condyle SCL was evident on radiographs obtained 3 months after surgery; however, this was not addressed surgically, and signs of lameness resolved 11 months after surgery. CLINICAL RELEVANCE Results of this small case series suggested that SCLs in the medial tibial condyle can occur in association with SCLs of the medial femoral condyle, with a poor prognosis for return to athletic function in affected horses. Further investigation is indicated.

Magnetic resonance imaging findings in horses with septic arthritis.

Fourteen horses with septic arthritis underwent high-field (1.5 T) magnetic resonance imaging (MRI). Septic arthritis was diagnosed based on results from historical and clinical findings, synovial fluid analyses and culture, and radiographic, ultrasonographic, arthroscopic, and histopathologic findings. MR findings included diffuse hyperintensity within bone and extracapsular tissue on fat-suppressed images in 14/14 horses (100%), joint effusion, synovial proliferation, and capsular thickening in 13/14 horses (93%), bone sclerosis in 11/14 horses (79%), and evidence of cartilage and subchondral bone damage in 8/14 horses (57%). Intravenous gadolinium was administered to five of the 14 horses and fibrin deposition was noted in all horses. Other findings after gadolinium administration included synovial enhancement in 4/5 (80%) horses, and bone enhancement in 1/5 (20%) horses. The MR findings of septic arthritis in horses were consistent with those reported in people. MRI may allow earlier and more accurate diagnosis of septic arthritis in horses as compared with other imaging modalities, especially when the clinical diagnosis is challenging. It also provides additional information not afforded by other methods that may influence and enhance treatment.

Magnetic resonance imaging features of proximal metacarpal and metatarsal injuries in the horse.

Magnetic resonance (MR) imaging abnormalities in horses with lameness localized to the proximal metacarpal or metatarsal region have not been described. To accomplish that, the medical records of 45 horses evaluated with MR imaging that had lameness localized to either the proximal metacarpal or metatarsal region were reviewed. Abnormalities observed in the proximal suspensory ligament or the accessory ligament of the deep digital flexor tendon included abnormal high signal, enlargement, or alteration in shape. Twenty-three horses had proximal suspensory ligament desmitis (13 hindlimb, 10 forelimb). Sixteen horses had desmitis of the accessory ligament of the deep digital flexor tendon. One horse had desmitis of the proximal suspensory ligament and the accessory ligament of the deep digital flexor tendon on the same limb and one horse had desmitis of the proximal suspensory ligament on one forelimb and desmitis of the accessory ligament of the deep digital flexor tendon on the other forelimb. Four horses did not have abnormalities in the proximal suspensory ligament or accessory ligament of the deep digital flexor tendon. Eighty percent of horses with forelimb proximal suspensory ligament desmitis and 69% of horses with hindlimb proximal suspensory ligament desmitis returned to their intended use. Sixty-three percent of horses with desmitis of the accessory ligament of the deep digital flexor tendon were able to return to their intended use. MR imaging is a valuable diagnostic modality that allows diagnosis of injury in horses with lameness localized to the proximal metacarpal and metatarsal regions. The ability to accurately diagnose the source of lameness is important in selecting treatment that will maximize the chance to return to performance.