Laparoscopic resection of an exostosis of the os pubis in a horse.

OBJECTIVE: To report the diagnostic findings and laparoscopic removal of an exostosis of the os pubis in a horse. STUDY DESIGN: Case report. ANIMAL: One 12-year-old Black Forest draught gelding. METHODS: History included recurrent colic before and during urination and poor performance. Findings at rectal examination included a pointed osseous prominence adjacent to the symphysis of the pecten ossis pubis. Cystoscopy revealed that this prominence caused a protrusion of the bladder wall into the lumen. Standing laparoscopy and laparoscopy under general anesthesia were performed. RESULTS: After a failed attempt at standing laparoscopy, the horse was anesthetized, and the exostosis of the os pubis was removed laparoscopically without complications. No recurrence of clinical signs associated with the exostosis was detected 12 months postoperatively. CONCLUSION: Minimally invasive surgical resection of an exostosis of the os pubis was achieved under general anesthesia with appropriately designed instruments. This treatment alleviated symptoms associated with the exostosis, including potential injury of the urinary bladder wall.

The Equine Gingiva: A Gross Anatomical Evaluation.

Equine periodontal disease (ePD) usually starts with food impaction, formation of diastemata, gingival inflammation and formation of periodontal pockets. This process proceeds toward the dentoalveolar space, causing detachment of tooth supporting periodontal fibers. Although several therapeutical procedures have been proposed, ePD is often only diagnosed in advanced stages, requiring dental extraction. A similar dilemma has been observed in small animal medicine, but has been overcome by the introduction of reliable examination protocols for the early diagnosis of periodontal diseases (PD). These protocols are based on detailed anatomical descriptions of healthy gingiva, allowing for the determination of the pathognomonic signs of the onset of PD and providing a basis for grading systems and treatment plans. Consequently, proposals have also been made for periodontal examination protocols in horses. However, these protocols were widely adopted from small animal medicine assuming a similar anatomy of the equine and canine gingiva. To provide a solid anatomical basis for equine specific periodontal examinations, 20 equine heads were examined macroscopically, with special attention to the gingival sulcus, the gingival margin and the interdental papillae. Constant morphological patterns of the gingival margin and the interdental papillae were found for the vestibular and lingual/palatal aspects of the upper and lower cheek teeth arcades, as well as for the incisor arcades. A gingival sulcus measuring greater than 1 mm was present in only 6% of the investigated specimens. The inspection of the gingival margin and the interdental papillae, as well as the recognition of a gingival sulcus, may serve as criteria to establish equine specific periodontal investigation protocols.

Identification of Novel Equine (Equus caballus) Tendon Markers Using RNA Sequencing.

Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 (EYA2) and a G-protein regulated inducer of neurite outgrowth 3 (GPRIN3) as specific tendon markers using RT-qPCR. Equine tendon cells cultured as three-dimensional spheroids expressed significantly greater levels of EYA2 than GPRIN3, and stained positively for EYA2 using immunohistochemistry. EYA2 was also found in fibroblast-like cells within the tendon tissue matrix and in cells localized to the vascular endothelium. In summary, we have identified EYA2 and GPRIN3 as specific molecular markers of equine tendon as compared to bone, cartilage and ligament, and provide evidence for the use of EYA2 as an additional marker for tendon cells in vitro.

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro.

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-ß1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.

Therapeutic potential of adipose-derived stromal cells in age-related osteoporosis.

Adipose-derived stromal cells (ASCs) are increasingly being used for orthopedic-based tissue engineering approaches due to their ability to readily undergo osteogenic differentiation. In the present study, we used in vitro and in vivo approaches to evaluate the use of ASCs as a treatment strategy for age-related osteoporosis. Molecular, histological and micro-computed tomography (micro-CT) based approaches confirmed that ASCs isolated from 18-week-old osteoporotic senescence-accelerated mice (SAMP6) were capable of undergoing osteogenesis when cultured in either silk fibroin (SF) scaffolds or scaffold-free microtissues (ASC-MT). A single intratibial injection of CM-Dil-labeled isogeneic ASCs or ASC-MT into SAMP6 recipients significantly improved trabecular bone quality after 6 weeks in comparison to untreated contralateral bones, as determined by micro-CT. Injected ASCs could be observed in paraffin wax bone sections at 24 h and 6 weeks post treatment and induced a significant increase in several molecular markers of bone turnover. Furthermore, a significant improvement in the osteogenic potential of osteoporotic patient-derived human bone marrow stromal cells (BMSCs) was observed when differentiated in conditioned culture media harvested from osteoporotic patient-derived human ASCs. These findings therefore support the use of ASCs as an autologous cell-based approach for the treatment of osteoporosis.

Evaluation of a motorized morcellator for laparoscopic removal of granulosa-theca cell tumors in standing mares.

OBJECTIVE: To describe a motorized morcellator technique for laparoscopic removal of granulosa-theca cell tumors (GCT) in standing mares and to evaluate long-term outcome. STUDY DESIGN: Case series. ANIMALS: Mares (n=7) aged 4-15 years, with unilateral GCT. METHODS: Tumor size was determined by transrectal palpation and ultrasonography. Standing sedated mares had 3 laparoscopic portal sites in the paralumbar fossa. After laparoscopic observation of the GCT, the mesovarium was desensitized, the ovarian pedicle transected with a LigaSure device, and the ovary grasped with forceps and cut in cylindrical tissue blocks using a motorized morcellator. Tissue blocks were removed through the laparoscopic sleeve. Outcome was determined by telephone interview of owners 6-40 months after surgery. RESULTS: Estimated ultrasonographic median GCT diameter was 17 cm (range, 10-22 cm). Surgical time was 2-4.5 hours. There were no surgical complications. Two mares had mild subcutaneous emphysema at the portals after surgery. Convalescence was short, owners were satisfied with cosmetic outcome, and clinical signs associated with GCT did not recur. CONCLUSION: The motorized morcellator allows piecemeal removal of large GCT through a relatively small laparoscopic portal. Surgical complications were rare and the cosmetic outcome is favorable. CLINICAL RELEVANCE: A motorized morcellator is a safe and minimally invasive technique for laparoscopic removal of GCT in mares.

Biocompatibility and resorption of a brushite calcium phosphate cement.

A hydraulic calcium phosphate cement with beta-tricalcium phosphate (TCP) granules embedded in a matrix of dicalcium phosphate dihydrate (DCPD) was implanted in experimentally created defects in sheep. One type of defect consisted of a drill hole in the medial femoral condyle. The other, partial metaphyseal defect was located in the proximal aspect of the tibia plateau and was stabilized using a 3.5 mm T-plate. The bone samples of 2 animals each per group were harvested after 2, 4, 6 and 8 weeks. Samples were evaluated for cement resorption and signs of immediate reaction, such as inflammation, caused by the cement setting in situ. Differences regarding these aspects were assessed for both types of defects using macroscopical, radiological, histological and histomorphometrical evaluations. In both defects the brushite matrix was resorbed faster than the beta-TCP granules. The resorption front was followed directly by a front of new bone formation, in which residual beta-TCP granules were embedded. Cement resorption occurred through (i) extracellular liquid dissolution with cement disintegration and particle formation, and (ii) phagocytosis of the cement particles through macrophages. Signs of inflammation or immunologic response leading to delayed new bone formation were not noticed at any time. Cement degradation and new bone formation occurred slightly faster in the femur defects.