Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.     

Transendoscopic injection of poly-L-lactic acid into the soft palate in horses: A new therapy for dorsal displacement of the soft palate?

The purpose of this study was to establish a minimal invasive, transendoscopic technique for injection of poly-L-lactic acid into the free edge of the soft palate that can be performed on the standing sedated horse. Furthermore, whether the implant material induces an increase in fibrous tissue at the injection site, resulting in a greater rigidity of the soft palate, was determined.In 4 horses, the injection was performed bilaterally along the caudal margin of the soft palate, and the influence of the treatment on the soft palate function was determined. Clinical, radiographic and endoscopic examinations at rest were performed before and up to 168 days after injection according to a standardized examination protocol.In 6 horses, the implant material was injected unilaterally into the right half of the caudal margin of the soft palate. After clinical and endoscopic follow-up examinations, 2 horses each were killed 28, 84, and 168 days after the implantation procedure. At necropsy, macroscopic comparison of the treated and the untreated halves of the soft palate showed the influence on the size of the ostium intrapharyngeum and on the soft palate thickness. The localization of the implant material in the soft palate and the tissue response to the poly-L-lactic acid were evaluated histologically.Transendoscopic injection into the soft palate requires neither general anesthesia nor wound management, and it is noted for minimal tissue trauma. After injection, no alteration of the soft palate function was observed in horses with normal upper airway mechanics. One horse with intermittent dorsal displacement of the soft palate showed an improvement of the soft palate function for 112 days after implantation. No significant alteration in the size of the ostium intrapharyngeum was evident in any horse. Histologic assessment indicated the presence of implant material during the entire observation period of 168 days. The implant was accompanied by a granulomatous inflammation, and it was encapsulated by fibrous tissue. This may result in an increase in stiffness, and subsequent a greater rigidity of the soft palate, preventing it from displacement. The injection may therefore be considered an alternative to established therapies in horses showing dorsal displacement of the soft palate because of its unstable free edge.     

Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.     

Soft palate hypoplasia in a horse

Several imaging techniques were used to diagnose hypoplasia of the soft palate in a horse. The absence of the caudal soft palate, hypertrophied lymphoid tissue and the formation of a pseudouvula were observed endoscopically. Plain and contrast radiography were used to demonstrate a soft palate remnant and to identify structures rostral to the epiglottis. Retrograde endoscopy of the pharynx via a tracheotomy incision is described.     

Epiglottic augmentation by use of polytetrafluoroethylene to correct dorsal displacement of the soft palate in a standardbred horse.

A 2-year-old 400-kg Standardbred colt with a history of exercise intolerance was determined to have dorsal displacement of the soft palate (DDSP). Standing endoscopy revealed a soft, flaccid, and hypoplastic epiglottis and arytenoid cartilages that moved symmetrically and abducted fully. Lateral laryngeal radiography was used to calculate a corrected thyroepiglottic length of 6.48 cm. Videoendoscopy during exercise on a high-speed treadmill at standardized treadmill speeds was performed weekly for 3 consecutive weeks. The soft palate dorsally displaced at each examination. A sternohyoideus/sternothyroideus myectomy was followed by 2 weeks of stall rest and 2 weeks of treadmill training. Because endoscopy during exercise at standardized treadmill speeds caused the horse to dorsally displace the soft palate once a week for 3 weeks after the myectomy, this surgery was deemed ineffective in this horse. Epiglottic augmentation was performed by injecting 7 ml of polytetrafluoroethylene into the ventral submucosal space of the epiglottis. After 21 days of stall rest and 5 weeks of pasture rest, the horse was placed on a 2-month treadmill conditioning program. Using endoscopy at standardized treadmill speeds, exercise efforts to encourage DDSP were unsuccessful, which suggested that epiglottic augmentation by use of polytetrafluoroethylene was an effective surgical treatment to correct dorsal displacement of the soft palate in this horse. It is believed that polytetrafluoroethylene increases the rigidity of the epiglottis, allowing it to maintain the ventral position of the soft palate.     

Free tissue transfer of the rectus abdominis myoperitoneal flap for oral reconstruction in a dog

A five-month-old intact/male Boxer dog was presented 5-days following bite wound trauma to the maxillary region resulting in an oronasal fistula extending from the maxillary canine teeth to the soft palate. Multiple surgical procedures using local, buccal mucosal flaps failed to repair the oronasal fistula. Free tissue transfer of the rectus abdominis myoperitoneal flap using microvascular surgical techniques was successful in providing soft tissue reconstruction of the hard palate area. Complications of these surgical techniques included muscle contraction and subsequent muzzle distortion. Small, refractory oronasal fistulae at the perimeter of the myoperitoneal flap were repaired by primary wound closure.     

Repair of an open radial fracture in an adult horse

An open radial fracture in an adult horse (450 kg) was repaired by internal fixation, using two 18-hole 4.5-mm broad dynamic compression plates and 5.5- and 4.5-mm bone screws. The fracture healed completely, but when evaluated 9 months after surgery, the horse was lame on the fractured limb at a trot. Local infiltration of anesthesia along the distal half of the bone plates greatly ameliorated the lameness, suggesting that the plates were irritating the soft tissues and extensor tendons along the cranial and lateral aspects of the antebrachium. Both bone plates were removed simultaneously with no complications, and the horse became sound.     

Scintigraphic evaluation of intra-arterial and intravenous regional limb perfusion of allogeneic bone marrow-derived mesenchymal stem cells in the normal equine distal limb using (99m) Tc-HMPAO

Reasons for performing study: Mesenchymal stem cells (MSCs) are commonly injected intralesionally for treatment of soft tissue injuries in the horse. Alternative routes of administration would be beneficial for treatment of lesions that cannot be accessed directly or to limit needle‐induced iatrogenic damage to the surrounding tissue. Objectives: The purpose of our study was to evaluate MSC distribution after intra‐arterial (IA) and intravenous (IV) regional limb perfusions (RLP) using scintigraphy. We hypothesised that MSCs would persist in the distal limb after tourniquet removal and that both techniques would lead to diffuse MSC distribution. Methods: Six horses were used in the study. MSCs were labelled with hexamethyl propylene amine oxime (HMPAO) and technetium‐99m. RLP was performed through the median artery of one forelimb and the cephalic vein of the opposite limb under general anaesthesia. The tourniquet was left in place for 45 min. Scintigraphic images were obtained at 0, 45, 75 min, 6 h and 24 h post injection. Results: Distribution of labelled MSCs through the entire distal limb was achieved with all 6 IA RLP, but 3 out of 6 IV RLP showed poor or absent uptake distal to the metacarpus. Mesenchymal stem cell persistence was 39% (30–60%) and 28% (14–50%) (median [minimum–maximum]) at 6 h for IA and IV RLP, respectively. Severe arterial thrombosis occurred in one horse after IA RLP. Conclusions: Both IA and IV RLP of the distal limb result in MSC persistence in perfused tissues. The IA perfusion resulted in more reliable cell distribution to the pastern and foot area. Potential relevance: Regional limb perfusion of MSCs might be used in cases where intralesional injection is not possible or in order to avoid iatrogenic needle damage. Further work is needed to assess the safety of IA RLP before its clinical use.     

The use of porcine urinary bladder matrix (UBM) to repair a perforated corneal ulcer with iris prolapse in a horse

This report describes the use of porcine urinary bladder matrix (UBM) to repair a full thickness defect in the cornea of a Thoroughbred racehorse. The horse presented with acute keratomalacia of the right eye of 12 h duration that had rapidly progressed to perforation of the cornea with partial iris prolapse. The cornea was surgically debrided with the horse under general anaesthesia and the defect repaired with ACell multilayer wound matrix. A temporary tarsorrhaphy was performed. The defect in the cornea was successfully repaired and the horse regained vision in the eye. Four months after surgery the defect was still undergoing remodelling and the area of opacity in the cornea was progressively reducing.     

Generation of neuronal-like cells from umbilical cord blood-derived mesenchymal stem cells of a RFP-transgenic cloned cat

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells, which can differentiation into cells of connective tissue and neural lineages. This study investigated the potential for neuronal differentiation of red fluorescent protein (RFP)-transgenic cat UCB-derived MSCs. The cells were cultured in pre-induction medium for 24 hr and in neuronal-induction medium for 72 hr. Immunofluorescent staining showed that 6.85% of the total cells were beta III-tubulin-positive, 3.37% were neurofilament light (NF-L)-positive and 7.04% were neurofilament medium (NF-M)-positive. A beta III-tubulin band was detected by western blot analysis. Our results demonstrate that RFP-transgenic UCB-derived MSCs can be differentiated into neuronal cells in vitro. Thus, RFP-transgenic MSCs could provide alternative tracing material for stem cell transplantation.     

Osteogenic potential of sorted equine mesenchymal stem cell subpopulations

The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 10³ MSCs from each tissue source were sorted into 5 fractions using non-equilibrium GrFFF (GrFFF proprietary system). Pooled fractions were cultured and expanded for use in osteogenic assays, including flow cytometry, histochemistry, bone nodule assays, and real-time quantitative polymerase chain reaction (qPCR) for gene expression of osteocalcin (OCN), RUNX2, and osterix. Equine MMSCs and BMSCs were consistently sorted into 5 fractions that remained viable for use in further osteogenic assays. Statistical analysis confirmed strongly significant upregulation of OCN, RUNX2, and osterix for the BMSC fraction 4 with P < 0.00001. Flow cytometry revealed different cell size and granularity for BMSC fraction 4 and MMSC fraction 2 compared to unsorted controls and other fractions. Histochemisty and bone nodule assays revealed positive staining nodules without differences in average nodule area, perimeter, or stain intensity between tissues or fractions. As there are different subpopulations of MSCs with different osteogenic capacities within equine muscle- and bone marrow-derived sources, these differences must be taken into account when using equine stem cell therapy to induce bone healing in veterinary medicine.     

Use of Buccal Mucosal Flaps for the Correction of Congenital Soft Palate Defects in Three Dogs

Objective —To present an alternative surgical technique for the repair of soft palate defects in dogs.
Animal Population —Three Foxhound cross dogs.
Methods —Bilateral buccal mucosal flaps, based at the palatoglossal arches, were raised. One flap was rotated so that the mucosal side created the floor of the nasopharynx. The second flap was rotated so that the mucosal side formed the roof of the oropharynx. The mucosa of the remnant of the soft palate and the pharyngeal walls was incised and the flaps sutured to these free mucosal edges. The caudal ends of the two flaps were sutured to each other.
Results —Complete closure of the soft palate defect was obtained in all three dogs. The dogs were monitored for between 1.5 and 3 years postoperatively, and no long-term problems were encountered.
Conclusions —This surgical technique resulted in an excellent functional separation of the oropharynx and nasopharynx and allowed reconstruction of a large bilateral soft palate defect.
Clinical Relevance —By using the buccal mucosal flaps, as described in this report, a tension-free closure of large soft palate defects can be obtained, thereby eliminating a major cause of failure of this type of reconstructive surgery.     

Use of a Cortico-Cancellous Bone Graft in the Repair of a Cleft Palate in a Dog

We report the successful use of a cortico-cancellous bone graft to repair a cleft of the secondary palate in a 6-month-old Akita dog. The cleft extended from the incisal papilla to the posterior border of the soft palate. It was 10 mm wide in the hard palate and 16 mm in the soft palate. The cleft was repaired using the "push-back" method with single-pedicled mucoperiosteal flaps to close the cleft in the soft tissues, and a free bone graft from the tibia (cortex with cancellous bone attached) to fill the defect in the hard palate. Two and one-half years after the operation the graft was indistinguishable from surrounding bone on radiographs. Maxillary growth appeared to have progressed normally, resulting in normal occlusion.     

Soft palate hypoplasia and concurrent middle ear pathology in six dogs

This retrospective clinical study describes six consecutive cases of bilateral hypoplasia/malformation of the soft palate in dogs in which associated middle ear disease was investigated and the palatine defects were surgical repaired. Radiographic abnormalities of the tympanic bullae were seen in both ears of all six dogs (12 of 12). Negative tympanocentesis findings were recorded in 11 of 12 ears. A purulent otitis media was confirmed in one ear of one dog, and loss of hearing was also demonstrated in this ear on brainstem auditory evoked response hearing assessment. There was no evidence of hearing loss on brainstem auditory evoked response in any of the remaining ears. Surgical repair of the soft palate defect was undertaken in all six dogs. Long-term assessment of the clinical outcome was considered excellent in five dogs and reasonable in one dog (mean 18 months, range seven to 27 months). It would appear that surgical intervention for the treatment of bilateral palatine malformation/hypoplasia may be associated with a better prognosis than reported previously. The lack of middle ear effusion and associated hearing impairment suggests that the underlying aetiology of middle ear pathology in dogs suffering from congenital palatine defects may be different from that observed in human beings. The true nature of the radiographic bullae changes seen in dogs with soft palate defects remains unclear.     

Effects of atelocollagen gel containing bone marrow-derived stromal cells on repair of osteochondral defect in a dog

To clarify the contribution of autologous transplantation of mesenchymal stromal cells (MSCs), an atelocollagen gel containing or not containing fluorescently-labeled canine MSCs was transplanted into an osteochondral defect which did not repair spontaneously and the histological repair of the defect was compared. Although an early repair of the cartilage was not observed in either defect, the reproduction of subchondral bone was remarkable in the MSCs-implanted defect. Moreover, in 2 weeks after operation, the implanted MSCs were located in the deeper regions of the defect, suggesting the differentiation of osteoblasts. There was a possibility that the movement of the implanted MSCs was due to an increase in intra-articular pressure from postoperative inflammation.     

Repair of a diaphragmatic hernia in a horse

One month after falling heavily a four-year-old horse developed acute colic while being exercised. A tentative diagnosis of diaphragmatic hernia was reached on the basis of the clinical findings and confirmed at laparotomy. The defect was repaired and the horse made an uneventful recovery.     

Bilateral hypoplasia of the soft palate and aryepiglottic entrapment in a horse

Endoscopic examination of a 7-year-old gelding with weight loss indicated a palatal defect with a prominent tissue mass at the caudomedial margin of the defect. At necropsy, the condition was determined to be bilateral hypoplasia of the soft palate.     

Persistent dorsal displacement of the soft palate attributable to a frenulum of the epiglottis in a racing Thoroughbred

CASE DESCRIPTION: A 7-year-old sexually intact male Thoroughbred racehorse was evaluated because of exercise intolerance, respiratory tract noise, and coughing when eating. CLINICAL FINDINGS: A persistent dorsal displacement of the soft palate was identified during endoscopic examination of the upper portions of the respiratory tract. Radiography of the pharyngeal and laryngeal regions revealed a hypoplastic epiglottis that was ventral to, and not in contact with, the soft palate. The horse was anesthetized, and an oral endoscopic examination revealed a subepiglottic frenulum that had resulted in the dorsal displacement of the soft palate. TREATMENT AND OUTCOME: The frenulum was transected transendoscopically by use of a diode laser. Twenty-four hours following surgery, repeat endoscopic and radiographic examinations revealed that the epiglottis had returned to its correct anatomic position in relation to the soft palate. Four weeks after surgery, endoscopy of the upper portions of the airway revealed recurrence of the dorsal displacement of the soft palate. CLINICAL RELEVANCE: A subepiglottic frenulum should be considered as a cause of persistent dorsal displacement of the soft palate in horses. An endoscopic examination of the oropharyngeal region should be performed in horses prior to undertaking any surgical interventions to treat persistent dorsal displacement of the soft palate.     

A novel approach for palatal cyst ablation in a horse

An 18-month-old Welsh Cob was referred to a university teaching hospital for investigation of abnormal respiratory noise at rest. Video-endoscopy revealed persistent displacement of the soft palate with bilateral 2–3 cm diameter raised areas on either side of the midline. Neither trans-nasal nor oral approaches allowed sufficient visualisation of the caudal soft palate for diagnosis or treatment. Latero-lateral radiographs showed a blunted, thickened caudal edge of the soft palate consistent with a palatal cyst. Two palatal cysts were thermally ablated with a transendoscopic diode laser via temporary tracheotomy. The horse recovered uneventfully and resolution of the displacement was evident on follow-up endoscopy 11 months later. This report documents a novel surgical approach to the caudal aspect of the soft palate.