Therapeutics prior to mesenchymal stromal cell therapy improves outcome in equine orthopedic injuries.

OBJECTIVE: Mesenchymal stromal (stem) cells (MSCs) have been studied to treat many common orthopedic injuries in horses. However, there is limited information available on when and how to use this treatment effectively. The aim of this retrospective study is to report case features, treatment protocols, and clinical outcomes in horses treated with MSCs. ANIMALS: 65 horses presenting with tendinous, ligamentous, and articular injuries, and treated with MSCs prepared by a single laboratory between 2016 and 2019. Outcome information was available for 26 horses. PROCEDURES: Signalment, clinical signs, diagnostic methods, treatment protocol features (prior and concurrent therapies, cell origin, dose, application site and number), and effective outcomes were analyzed. The analysis was focused on comparing the effect of different MSC treatment protocols (eg, autologous vs allogeneic) on outcome rather than the effectiveness of MSC treatment. RESULTS: MSC treatment resulted in 59.1% (clinical lameness) to 76.9% (imaging structure) improvement in horses with diverse ages, breeds, sex, and lesions. The use of other therapeutic methods before MSC application (eg, anti-inflammatories, shockwave, laser, icing, resting, bandage and stack wrap, intra-articular injections, and/or surgical debridement) was shown to be statistically more effective compared to MSCs used as the primary therapeutic procedure (P < .05). Autologous versus allogeneic treatment outcomes were not significantly different. CLINICAL RELEVANCE: A prospective MSC treatment study with standardization and controls to evaluate the different features of MSC treatment protocols is needed. The various case presentations and treatment protocols evaluated can be used to inform practitioners who are currently using MSCs in clinical practice.

Medial malleolus fragmentation following talocalcaneal arthrodesis by a dorsomedial approach in a horse.

A 16-year-old, Quarter Horse mare was presented for a 3/5 right hind lameness associated with osteoarthritis of the talocalcaneal joint (TCLJ). Positron emission tomography (PET) and computed tomography (CT) demonstrated marked increased uptake of 18F-sodium fluoride and bone remodeling at the medial facet of the TCLJ, respectively. Under general anesthesia 2 cortical screws (4.5 and 5.5 mm) were placed in neutral fashion via an arthrotomy from dorsomedial to plantaromedial through the medial facet of the TCLJ followed by copious lavage of the tarsocrural joint. Eight weeks after surgery, observable effusion of the tarsocrural joint was present and lameness had worsened. Radiographic examination revealed a fragmented medial malleolus of the tibia, likely secondary to repetitive trauma of the screw heads during tarsal flexion. Repeated CT showed partial fusion of the TCLJ. Both screws were removed and the tarsocrural joint was thoroughly lavaged arthroscopically. At a 20-month recheck the lameness had not improved, and ultrasound examination revealed severe thickening of the TCLJ capsule. Recheck examination 48 mo after surgery showed complete fusion of the TCLJ and resolution of the lameness. Key clinical message: Diagnosis of osteoarthritis of the TCLJ is challenging. Management by arthrodesis using a dorsomedial approach can result in fragmentation of the medial malleolus, with secondary synovitis and capsulitis of the tarsocrural joint.

Fragmentation de la malléole médiale suite à une arthrodèse talo-calcanéenne par voie dorsomédiale chez un cheval. Une jument Quarter Horse âgée de 16 ans a été présentée pour une boiterie postérieure droite de 3/5 associée à une arthrose de l'articulation talo-calcanéenne (TCLJ). La tomographie par émission de positrons (TEP) et la tomodensitométrie (CT) ont démontré une augmentation marquée de l'absorption du fluorure de sodium-18F et un remodelage osseux significatif au niveau de la facette médiale du TCLJ, respectivement. Sous anesthésie générale, deux vis corticales (4,5 et 5,5 mm) ont été placées de façon neutre via une arthrotomie dorsomédiale à plantaro-médiale à travers la face médiale du TCLJ suivie d'un lavage abondant de l'articulation tarsocrurale. Huit semaines après la chirurgie, un épanchement significatif de l'articulation tarso-crurale était présent et la boiterie s'était aggravée. L'examen radiographique a révélé une malléole médiale du tibia fragmentée, probablement secondaire à un traumatisme répétitif des têtes de vis lors de la flexion du tarse. La tomodensitométrie répétée a montré une fusion partielle du TCLJ. Les deux vis ont été retirées et l'articulation tarso-crurale a été soigneusement lavée par arthroscopie. Lors d'un nouveau contrôle après 20 mois, la boiterie ne s'était pas améliorée, et l'échographie a révélé un épaississement sévère de la capsule TCLJ. Un nouvel examen 48 mois après la chirurgie a montré une fusion complète du TCLJ et une résolution de la boiterie.Message clinique clé :Le diagnostic de l'arthrose du TCLJ est difficile. La prise en charge par arthrodèse par voie dorso-médiale peut entraîner une fragmentation de la malléole médiale, avec synovite secondaire et capsulite de l'articulation tarso-crurale.(Traduit par Dr Serge Messier).

Factors affecting successful ultrasound-guided injection into the podotrochlear bursa using a palmarolateral approach.

OBJECTIVE: To evaluate potential factors affecting ultrasonographic visibility of the suprasesamoidean region and the success rate of ultrasound-guided injection (USGI) into the podotrochlear bursa (PB). STUDY DESIGN: Ex vivo, experimental study. ANIMALS: 24 cadaveric forelimbs. METHODS: The PB was distended with 0, 1, or 2 ml of iodinated contrast material and saline under radiographic guidance. Ultrasonographic visibility of the suprasesamoidean region and PB was graded before and after distention with the limb loaded in three positions (vertical, cranial and caudal). Two operators with different experience levels performed an USGI into the PB with methylene blue (12 limbs each [four limbs with each of three volumes of PB distension]) using a palmarolateral approach and caudal foot placement. Limbs were frozen and sectioned to assess accuracy. RESULTS: Ultrasonographic visibility scores of the suprasesamoidean region (p = .0081) and PB (p < .0001) were improved using a caudal foot placement. Higher visibility grades of the suprasesamoidean region were associated with higher injection success rate (p = .047). The injection success was 75% (9/12) for the experienced versus 41.6% (5/12) for the less experienced operator (p = .14). PB distension improved visibility during caudal foot placement (p = .013) but not injection success rates (p = .78). CONCLUSION: Caudal foot placement enhances visibility of the PB and suprasesamoidean region. Limbs with poor visibility of the suprasesamoidean region are more likely to undergo a failed USGI. CLINICAL SIGNIFICANCE: The lateral USGI approach should be performed only in cases of optimal visibility. The reported success rates support the technical challenge of PB injections.

Do allogeneic bone marrow derived mesenchymal stem cells diminish the inflammatory response to lipopolysaccharide infusion in horses? A pilot study.

Endotoxemia is a leading cause of morbidity and mortality in the equine industry, with colic being the most common cause of endotoxemia in horses. The objective of this study was to evaluate the safety and potential efficacy of a single dose of allogeneic equine bone marrow derived mesenchymal stem cells (BM-MSCs) in horses after the IV administration of lipopolysaccharide (LPS). Six horses were administered an IV infusion of 30 ng/kg LPS (O55:B5 Escherichia coli) in 500 ml saline over 30 min. Immediately after infusion test horses (n = 3) were administered 100 × 106 allogeneic BM-MSCs diluted in saline IV and control horses (n = 3) were administered saline. Clinicopathological data, pro-inflammatory cytokine measurements and sCD14 concentrations were compared between groups. No adverse reactions were observed in horses administered BM-MSCs intravenously. There were no significant differences between test and control horses with regard to clinicopathological values or pro-inflammatory cytokine production. At no time point did concentrations of sCD14 exceed the reference range in any horse. Results suggest that administration of a single IV dose of freshly cultured MSCs is safe and well-tolerated in horses with induced endotoxemia. Further study to evaluate their efficacy as a potential therapeutic in a larger number of horses with clinical disease is required.

Evaluation of the caudal cervical articular process joints by using a needle arthroscope in standing horses.

OBJECTIVE: To determine the safety and efficacy of caudal cervical articular process joint arthroscopy by using needle arthroscopy in standing sedated horses. STUDY DESIGN: Prospective experimental case series. ANIMALS: Six adult horses. METHODS: Three horses underwent exploration of bilateral C5-6 vertebral articular process joints and three bilateral C6-7 articular process joints by using a 1.2 × 65-mm needle arthroscope under standing sedation. The 16-gauge arthroscopic trocar and canula assembly was inserted in the desired articular process joint under ultrasonographic guidance without distention of the joint. RESULTS: All 12 articular process joints were successfully explored. Entering the joint on the first attempt was achieved in 10 of 12 joints. A craniodorsal approach for arthroscope insertion allowed evaluation of the most cranial aspect of the articular cartilage surface. Triangulation with a spinal needle was determined to be difficult and resulted in a limited space for movement. CONCLUSION: Needle arthroscopy of the caudal cervical facet joints was performed safely and efficiently in standing sedated horses. CLINICAL SIGNIFICANCE: Standing cervical articular process needle arthroscopy is a minimal morbidity technique with the potential to be an advantageous technique for the diagnosis and treatment of cervical articular process pathology.

Standing myotomy to treat fibrotic myopathy: 22 cases (2004-2016).

OBJECTIVE: To report the clinical and diagnostic findings associated with fibrotic myopathy, describe the surgical procedure for standing fibrotic myotomy and report postoperative outcomes. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Twenty-two horses. METHODS: Records were included if a clinical diagnosis of fibrotic myopathy was based on clinical and/or ultrasonographic examination and the horse was treated with myotomy of the affected muscle. Records were reviewed for signalment, preoperative findings, perioperative complications, and outcomes. Follow-up also included a phone survey of owners. RESULTS: Quarter horses represented 14 of 22 horses in this study. Ultrasonographic examination was performed in 14 of 22 horses. The semitendinosus muscle was affected in 17 of 22 horses. Other muscles affected included the semimembranosus, gracilis, and biceps femoris. Four of the 22 horses had a diagnosis of incisional postoperative complications. Eight of 12 formerly athletic horses returned to their intended athletic use. Ten of 16 owners were satisfied with the procedure. CONCLUSION: Prognosis for long-term comfort after standing fibrotic myotomy was fair with appropriate rehabilitation. Ultrasonographic examination was helpful in identifying the muscles affected and aided in surgical planning. CLINICAL IMPACT: Standing fibrotic myotomy is a technically straightforward procedure that yields a fair outcome in horses with minimal intraoperative and/or postoperative complications.

Injectable mineralized microsphere-loaded composite hydrogels for bone repair in a sheep bone defect model.

The efficacy of cell-based therapies as an alternative to autologous bone grafts requires biomaterials to localize cells at the defect and drive osteogenic differentiation. Hydrogels are ideal cell delivery vehicles that can provide instructional cues via their composition or mechanical properties but commonly lack osteoconductive components that nucleate mineral. To address this challenge, we entrapped mesenchymal stromal cells (MSCs) in a composite hydrogel based on two naturally-derived polymers (alginate and hyaluronate) containing biomineralized polymeric microspheres. Mechanical properties of the hydrogels were dependent upon composition. The presentation of the adhesive tripeptide Arginine-Glycine-Aspartic Acid (RGD) from both polymers induced greater osteogenic differentiation of ovine MSCs in vitro compared to gels formed of RGD-alginate or RGD-alginate/hyaluronate alone. We then evaluated the capacity of this construct to stimulate bone healing when transplanting autologous, culture-expanded MSCs into a surgical induced, critical-sized ovine iliac crest bone defect. At 12 weeks post-implantation, defects treated with MSCs transplanted in composite gels exhibited significant increases in blood vessel density, osteoid formation, and bone formation compared to acellular gels or untreated defects. These findings demonstrate the capacity of osteoconductive hydrogels to promote bone formation with autologous MSCs in a large animal bone defect model and provide a promising vehicle for cell-based therapies of bone healing.

Outcome following computed tomographic imaging and subsequent surgical removal of keratomas in equids: 32 cases (2005-2016).

OBJECTIVE To describe the use of CT in the diagnosis of and presurgical planning for equids with keratomas treated by hoof wall resection and determine postoperative complications and outcome. DESIGN Retrospective case series. ANIMALS 29 horses and 3 mules with keratomas treated by partial or complete hoof wall resection following CT imaging of the distal portion of the affected extremity from 2005 through 2016. PROCEDURES For each equid, physical examination and lameness evaluation were performed, followed by imaging of the distal portion of the affected extremity. A tentative diagnosis of keratoma was made given results of clinical, radiographic, and CT examination, with the definitive diagnosis made on histologic examination. After CT imaging, each equid was treated with hoof wall resection. RESULTS CT allowed accurate identification of the size and location of 35 keratomas in 33 feet. Thirty equids underwent partial and 2 underwent complete hoof wall resection for keratoma removal. Complications developed in 4 of 31 (13%) equids and included excessive granulation tissue formation at the surgical site (n = 1), excessive granulation tissue formation and worsening lameness (1), incomplete keratoma removal with suspected regrowth (1), and incomplete healing of the surgical site with distal interphalangeal joint sepsis secondary to repeated debridement of abnormal tissue (1). Twenty-eight of 31 (90%) equids returned to their previous activity level. CONCLUSIONS AND CLINICAL RELEVANCE Preoperative CT examination of the feet was useful in the diagnosis of and surgical planning for keratomas in equids. A low complication rate was achieved with the described techniques.

Use of a locking compression plate for equine proximal interphalangeal joint arthrodesis: 29 cases (2008-2014).

OBJECTIVE To describe clinical use of a locking compression plate (LCP) for proximal interphalangeal joint (PIPJ) arthrodesis in horses and compare outcomes for horses that underwent the procedure as treatment for fracture of the middle phalanx (P2) versus other causes. DESIGN Retrospective case series. ANIMALS 29 client-owned horses. PROCEDURES Medical records of 2 veterinary teaching hospitals from 2008 through 2014 were reviewed to identify horses that underwent PIPJ arthrodesis of 1 limb. Signalment, surgical, and outcome-related variables were recorded. Owners were contacted from 1 to 6 years after surgery to determine rehabilitation time, current use of the horse, and overall owner satisfaction with the procedure. Success was determined on the basis of owner satisfaction and outcome for intended use. Variables of interest were compared statistically between horses that underwent surgery for P2 fracture versus other reasons. RESULTS 14 horses underwent surgery for treatment of P2 fracture, and 15 had surgery because of osteoarthritis, subluxation, or osteochondrosis. Median convalescent time after surgery (with no riding or unrestricted exercise) was 7 months. Four horses were euthanized; of 23 known alive at follow-up, 22 were not lame, and 18 had returned to their intended use (8 and 10 at higher and lower owner-reported levels of work, respectively). Horses undergoing arthrodesis for reasons other than fracture were significantly more likely to return to their previous level of work. Twenty-two of 24 owners contacted indicated satisfaction with the procedure. CONCLUSIONS AND CLINICAL RELEVANCE Surgical arthrodesis of the PIPJ was successful in most horses of the study population. Various nuances of the system for fracture repair need to be understood prior to its use.

Safety and tracking of intrathecal allogeneic mesenchymal stem cell transplantation in healthy and diseased horses.

BACKGROUND: It is currently unknown if the intrathecal administration of a high dose of allogeneic mesenchymal stem cells (MSCs) is safe, how MSCs migrate throughout the vertebral canal after intrathecal administration, and whether MSCs are able to home to a site of injury. The aims of the study were: 1) to evaluate the safety of intrathecal injection of 100 million allogeneic adipose-derived MSCs (ASCs); 2) to assess the distribution of ASCs after atlanto-occipital (AO) and lumbosacral (LS) injection in healthy horses; and 3) to determine if ASCs homed to the site of injury in neurologically diseased horses. METHODS: Six healthy horses received 100 × 106 allogeneic ASCs via AO (n = 3) or LS injection (n = 3). For two of these horses, ASCs were radiolabeled with technetium and injected AO (n = 1) or LS (n = 1). Neurological examinations were performed daily, and blood and cerebrospinal fluid (CSF) were evaluated prior to and at 30 days after injection. Scintigraphic images were obtained immediately postinjection and at 30 mins, 1 h, 5 h, and 24 h after injection. Three horses with cervical vertebral compressive myelopathy (CVCM) received 100 × 106 allogeneic ASCs labeled with green fluorescent protein (GFP) via AO injection and were euthanized 1-2 weeks after injection for a full nervous system necropsy. CSF parameters were compared using a paired student's t test. RESULTS: There were no significant alterations in blood, CSF, or neurological examinations at any point after either AO or LS ASC injections into healthy horses. The radioactive signal could be identified all the way to the lumbar area after AO ASC injection. After LS injection, the signal extended caudally but only a minimal radioactive signal extended further cranially. GFP-labeled ASCs were not present at the site of disease at either 1 or 2 weeks following intrathecal administration. CONCLUSIONS: The intrathecal injection of allogeneic ASCs was safe and easy to perform in horses. The AO administration of ASCs resulted in better distribution within the entire subarachnoid space in healthy horses. ASCs could not be found after 7 or 15 days of injection at the site of injury in horses with CVCM.

A novel ultrasonographic assisted technique for desmotomy of the palmar/plantar annular ligament in horses.

OBJECTIVES: To describe an ultrasound assisted technique for desmotomy of the palmar/plantar annular ligament (PAL), determine its efficacy and intraoperative complications. STUDY DESIGN: Cadaveric and in vivo study. ANIMALS: Cadaveric limbs (n = 12), adult horses (n = 4), and clinical cases (n = 2). METHODS: Ultrasound assisted desmotomy of the palmar/plantar annular ligament (UAD-PAL) was performed in cadaveric limbs and in standing horses with the operated limb placed in a distal limb splint. The procedure was performed under general anesthesia and was followed by tenoscopic examination in 2 clinical cases. A hook knife was developed for the procedure. Complete transection was assessed by postmortem dissection (10 forelimbs, 10 hindlimbs) and tenoscopic examination (1 forelimb, 1 hindlimb). Thickness of PAL, surgery time, other intraoperative parameters and complications associated with the procedure were recorded. RESULTS: Complete PAL transection was accomplished in 20/22 limbs. No iatrogenic damage to adjacent intrathecal structures was identified in any case. The instrument was correctly positioned on the first attempt in 19/22 cases. The most common intraoperative complication was inadvertent subcutaneous placement of the instrument (n = 2). Significant thickening of the PAL (3 mm) was present in 1/2 limbs in which complete transection was not achieved. CONCLUSIONS: UAD-PAL with the custom-made hook knife was effective at transecting the PAL with minimal intraoperative complications. The procedure can be performed in standing sedated horses. Another method should be considered in horses with thickened PAL.

Use of a laparoscopic specimen retrieval pouch to facilitate removal of intact or fragmented cystic calculi from standing sedated horses: 8 cases (2012-2015).

OBJECTIVE To evaluate the use of a laparoscopic specimen retrieval pouch for removal of intact or fragmented cystic calculi from standing horses. DESIGN Retrospective case series. ANIMALS 8 horses (5 geldings and 3 mares) with cystic calculi. PROCEDURES Physical examination and cystoscopic, ultrasonographic, and hematologic evaluations of urinary tract function were performed for each horse. A diagnosis of cystic calculus was made on the basis of results of cystoscopy and ultrasonography. Concurrent urolithiasis or other urinary tract abnormalities identified during preoperative evaluation were recorded. Horses were sedated and placed in standing stocks, and the perineum was aseptically prepared. Direct access to the urinary bladder was gained in geldings via perineal urethrotomy or in mares by a transurethral approach. Calculi were visualized endoscopically, manipulated into the retrieval pouch, and removed intact or fragmented (for larger calculi). RESULTS For 4 geldings and 1 mare, fragmentation was necessary to facilitate calculus removal. Mean duration of surgery was 125 minutes, and trauma to the urinary bladder and urethra was limited to areas of hyperemia and submucosal petechiation. No postoperative complications were encountered for any horse. When lithotripsy was required, the retrieval pouch provided an effective means of stabilizing calculi and containing the fragments for removal. CONCLUSIONS AND CLINICAL RELEVANCE Use of the laparoscopic specimen retrieval pouch was an effective, minimally traumatic method for retrieving cystic calculi from standing horses. The pouch protected the urinary bladder and urethra from trauma during calculus removal and allowed for stabilization, containment, and fragmentation of calculi when necessary.

Scintigraphic Tracking of Allogeneic Mesenchymal Stem Cells in the Distal Limb After Intra-Arterial Injection in Standing Horses.

OBJECTIVE: To assess the feasibility of intra-arterial administration of allogeneic mesenchymal stem cells (MSC) in the median artery of standing horses and evaluate the distribution and retention of radiolabeled cells. STUDY DESIGN: In vivo experimental study. ANIMALS: Six research horses. METHODS: Technetium(99m) -HexaMethyl-Propylene-Amine Oxime-labeled MSC were injected under ultrasound guidance in the median artery of 6 front limbs of 3 horses, standing under sedation. Scintigraphic images were obtained at the time of injection, and at 1, 6, and 24 hours postinjection. Six additional limbs from 3 horses were similarly injected with unlabeled MSC. Ultrasound was performed the following day for assessment of vascular changes. RESULTS: Intra-arterial injection was performed successfully in 11 of 12 limbs. In 1 limb, partial periarterial injection compromised the success of the procedure. Homogeneous distribution of radiolabeled MSC was observed through the entire distal limb, including within the hoof. Partial venous thrombosis was found in both groups of horses, but was subjectively less severe in horses injected with unlabeled MSC. No lameness was observed. Transient swelling of the distal limb occurred in only 1 limb. CONCLUSION: Intra-arterial injection of MSC can be performed in standing horses under sedation and successfully distribute MSC to the distal limb. A risk of periarterial injection was identified but can be reduced with proper sedation, local anesthesia, and increased experience. Partial venous thrombosis was observed as a complication, but did not cause changes of clinical importance, other than rare transient swelling.

A nonterminal equine mandibular model of bone healing.

OBJECTIVES: To develop a nonterminal large animal bone defect model for assessing the efficacy of regenerative and pharmacologic treatments designed to enhance bone healing. STUDY DESIGN: In vivo experimental. SAMPLE POPULATION: Adult gelding horses (n = 6). METHODS: Under general anesthesia, using radiographic guidance, 13.5 mm diameter bilateral, full thickness mandibular defects were created in 6 horses using a custom surgical jig and coring bit. After 16 weeks, under general anesthesia, 23 mm diameter cores that encompassed the original healing defects and surrounding parent bone material were removed for evaluation. Oxytetracycline was administered 14 days before final core harvest to label bone-forming surfaces. Healing was qualitatively assessed from decalcified hematoxylin and eosin (H&E) stained and undecalcified fluorescent labeled sections. Trabecular to cortical bone fraction (Tb.V/Ct.V), bone volume fraction (BV/TV), tissue mineral density (TMD), and apparent bone mineral density (aBMD) were quantified using microcomputed tomography and compared between left and right sides using Wilcoxon signed rank test. RESULTS: BV/TV was not significantly different between left and right-sided defects. Bone deposition occurred centripetally from the border of the original defect, filling 67% ± 16% (SD) of the defect at 16 weeks. CONCLUSION: This model has potential use for comparison of regenerative and pharmacologic products aimed to augment bone healing.

Scintigraphic tracking of mesenchymal stem cells after intravenous regional limb perfusion and subcutaneous administration in the standing horse.

OBJECTIVE: To assess distribution, uptake, and persistence of radiolabeled mesenchymal stem cells (MSC) using scintigraphy after intravenous regional limb perfusion (RLP) and subcutaneous injections in standing, sedated horses. STUDY DESIGN: Experimental study. ANIMALS: Horses (n = 12). METHODS: Six horses had RLP performed through the cephalic vein on 1 limb and subcutaneous injection in the metacarpal area in the opposite limb. The other 6 horses had RLP performed through the lateral palmar digital vein and subcutaneous injection in the coronary band. A pneumatic tourniquet was used for the RLP. MSC were labeled with technetium-HMPAO. Scintigraphic images were obtained at the time of injection, 1, 6, and 24 hours later. Results of RLP were compared with results from previous studies where similar injections were performed in anesthetized horses. RESULTS: Both RLP techniques led to greater variability, lower uptake, lower persistence, and poorer distribution when compared to results previously reported for horses under general anesthesia. The subcutaneous injections in the metacarpal area and coronary band resulted in MSC loss to the general circulation but no evidence of local migration. CONCLUSION: Due to partial or complete failure of the tourniquet, RLP performed in the standing horse as described is less efficient than performed under general anesthesia. Further work is needed to optimize the use of tourniquets to perform RLP for MSC administration in standing patients. The subcutaneous injections did not result in local migration in these normal horses.

Processing of equine bone marrow using the automated MarrowXpress System: RBC depletion, volume reduction, and mononuclear cell recovery.

BACKGROUND: The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. OBJECTIVES: The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. METHODS: Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. RESULTS: For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 10(7) and after processing was significantly higher at 7.8 ± 3.3 × 10(7) (P < .0001), with a recovery of 68.5 ± 24.5% (mean ± SD). MNC count before processing was 1.1 ± 0.9 × 10(7) and after processing was significantly higher at 3.8 ± 1.9 × 10(7) (P < .0001), with a recovery 73.0 ± 31.5%. CONCLUSIONS: The MXP System can reliably reduce volume and deplete RBCs from aspirates of equine bone marrow aspirates. MNCs can be recovered in a reproducible and sterile manner. Further studies evaluating the effects of the MXP System on cell viability, identification of mesenchymal stem cells (MSCs), and the efficacy of MSC expansion are warranted.

Intradermal injections of equine allogeneic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions.

BACKGROUND AIMS. The use of allogeneic mesenchymal stem cells (MSC) to treat acute equine lesions would greatly expand equine cellular therapy options; however, the safety and antigenicity of these cells have not been well-studied. We hypothesized that equine allogeneic umbilical cord tissue (UCT)-derived MSC would not elicit acute graft rejection or a delayed-type hypersensitivity response when injected intradermally. METHODS. Six Quarterhorse yearlings received 12 intradermal injections (autologous MSC, allogeneic MSC, positive control and negative control, in triplicate) followed by the same series of 12 injections, 3-4 weeks later, at another site. Wheals were measured and palpated at 0.25, 4, 24, 48, 72 h and 7 days post-injection. Biopsies were obtained at 48 and 72 h and 7 days post-injection. Mixed leukocyte reactions were performed 1 week prior to the first injections and 3 weeks after the second injections. RESULTS. There were no adverse local or systemic responses to two intradermal injections of allogeneic MSC. MSC injection resulted in minor wheal formation, characterized by mild dermatitis, dermal edema and endothelial hyperplasia, that fully resolved by 48-72 h. No differences were noted between allogeneic and autologous MSC. The second injection of MSC did not elicit more significant physical or histomorphologic alterations compared with the first MSC injection. Neither allogeneic nor autologous UCT-derived MSC stimulated or suppressed baseline T-cell proliferation in vitro prior to or after two MSC administrations. CONCLUSIONS. Equine allogeneic UCT MSC may be safely administered intradermally on multiple occasions without eliciting a measurable cellular immune response.

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses.

BACKGROUND AIMS: The development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horses. METHODS: Sixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injection. RESULTS: MSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSC. CONCLUSIONS: The healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.

Osteogenic comparison of expanded and uncultured adipose stromal cells.

BACKGROUND AIMS: Adipose stromal cells (ASC) are a promising alternative to progenitor cells from other tissue compartments because of their multipotential and capacity to retrieve significantly more progenitor cells. Initial cell samples are heterogeneous, containing a collection of cells that may contribute to tissue repair, but the sample becomes more homogeneous with each passage. Therefore, we hypothesized that the osteogenic potential of culture-expanded ASC would differ from uncultured ASC. METHODS: Adipose tissue was collected from a yearling colt, and ASC were isolated and expanded using standard protocols or prepared by a commercial vendor using proprietary technology (proprietary stromal vascular fraction, SVFp). Cells were seeded on collagen sponges and maintained in osteogenic culture conditions for up to 21 days to assess osteogenic potential. The ability of each population to stimulate neovascularization and bone healing was determined upon implanting cell-loaded sponges into a rodent calvarial bone defect. Neovascularization was measured 3 weeks post-implantation, while bone formation was monitored over 12 weeks using in vivo microcomputed tomography (microCT). RESULTS: SVFp exhibited increased intracellular alkaline phosphatase activity compared with cultured ASC but proliferated minimally. Histologic analysis of explanted tissues demonstrated greater vascularization in defects treated with cultured ASC compared with SVFp. We detected increases in bone volume for defects treated with cultured cells while observing similar values for bone mineral density, regardless of cell type. CONCLUSIONS: These results suggest that expanded ASC are advantageous for neovascularization and bone healing in this model compared with SVFp, and provide additional evidence of the utility of ASC in bone repair.