John F. Peroni

The Regenerative Medicine Laboratory: Facilitating Stem Cell Therapy for Equine Disease

This article focuses on the emerging field of equine regenerative medicine with an emphasis on the use of mesenchymal stem cells (MSCs) for orthopedic diseases. We detail laboratory procedures and protocols for tissue handling and MSC isolation, characterization, expansion, and cryopreservation from bone marrow, fat, and placental tissues. We provide an overview of current clinical uses for equine MSCs and how MSCs function to heal tissues. Current laboratory practices in equine regenerative medicine mirror those in the human field. However, the translational use of autologous and allogeneic MSCs for patient therapy far exceeds what is currently permitted in human medicine.

Black walnut extract-induced laminitis in horses is associated with heterogeneous dysfunction of the laminar microvasculature.

REASONS FOR PERFORMING STUDY Equine laminitis purportedly involves haemodynamic dysfunction at the level of the laminar vasculature. However, to date, no studies have been performed characterising the function of laminar arteries and veins during the prodromal stages of equine laminitis. HYPOTHESIS That the prodromal stages of laminitis are associated with contractile dysfunction of the equine laminar vasculature. OBJECTIVE To assess contractile function of laminar arteries and veins to phenylephrine (PE) and 5-hydroxytryptamine (5-HT). METHODS Horses were administered black walnut heartwood extract (BWHE) or water (control horses) via nasogastric intubation. After euthanasia, laminar vessels (100-800 microm internal diameter) were isolated and mounted on small vessel myographs to assess contractile function. RESULTS Contractile responses to PE or 5-HT were identical in laminar arteries isolated from either control horses or those administered BWHE. In contrast, responses to PE or 5-HT were significantly reduced in laminar veins isolated from BWHE-administered horses when compared with laminar veins isolated from control horses. CONCLUSIONS AND POTENTIAL RELEVANCE These results are consistent with the prodromal stages of laminitis being associated with selective dysfunction of laminar veins. Further studies are required to discern the precise nature of this dysfunction and its potential relevance to the pathogenesis of acute laminitis in the horse and possible therapeutic targets for treatment.

Rapid healing of femoral defects in rats with low dose sustained BMP2 expression from PEGDA hydrogel microspheres.

Current strategies for bone regeneration after traumatic injury often fail to provide adequate healing and integration. Here, we combined the poly (ethylene glycol) diacrylate (PEGDA) hydrogel with allogeneic “carrier” cells transduced with an adenovirus expressing BMP2. The system is unique in that the biomaterial encapsulates the cells, shielding them and thus suppressing destructive inflammatory processes. Using this system, complete healing of a 5 mm‐long femur defect in a rat model occurs in under 3 weeks, through secretion of 100‐fold lower levels of protein as compared to doses of recombinant BMP2 protein used in studies which lead to healing in 2–3 months. New bone formation was evaluated radiographically, histologically, and biomechanically at 2, 3, 6, 9, and 12 weeks after surgery. Rapid bone formation bridged the defect area and reliably integrated into the adjacent skeletal bone as early as 2 weeks. At 3 weeks, biomechanical analysis showed the new bone to possess 79% of torsional strength of the intact contralateral femur. Histological evaluation showed normal bone healing, with no infiltration of inflammatory cells with the bone being stable approximately 1 year later. We propose that these osteoinductive microspheres offer a more efficacious and safer clinical option over the use of rhBMP2.

Anti-inflammatory and immunomodulatory activities of stem cells.

The recent interest in equine stem cell biology and the rapid increase in experimental data highlight the growing attention that this topic has been receiving over the past few years. Within the field of stem cell biology, the relevance of immunobiology is of particular intrigue. It appears that optimal and effective stem cell therapy for equine patients will require a thorough analysis of the immune properties of stem cells as well as their response to immune mediators. The main goal of this review is to discuss the biology of adult mesenchymal stem cells in the context of immunology.

Thromboxane and isoprostanes as inflammatory and vasoactive mediators in black walnut heartwood extract induced equine laminitis

Inflammation and vascular dysfunction occur concurrently during the prodromal stages of equine laminitis. The aim of this study was to provide insights into the role that thromboxane and isoprostanes may play in the development of black walnut heartwood extract (BWHE)-induced laminitis. Horses were divided into two groups, either control or BWHE-administered horses. Plasma concentrations of thromboxane increased transiently after administration of BWHE and coincided with the nadir in white blood cell counts, whereas plasma concentrations of iso-prostaglandin PGF(2alpha) (iso-PGF(2alpha)) did not change in either group. At 12h (for the control group) or Obel grade 1 laminitis (for the BWHE group) the horses were euthanized and laminar tissue collected. Laminar arteries and veins were used in functional studies with vasoconstrictor substances and tissue samples were used for the determination of laminar iso-PGF(2alpha) concentrations. Laminar tissue concentrations of iso-PGF(2alpha) were significantly greater in BWHE horses when compared to control horses. In parallel studies concentrations of iso-PGF(2alpha) in laminar tissue samples obtained 1.5 and 3h after administration of BWHE were indistinguishable from those for control horses at 3 or 12h after administration of an equal volume of water. Laminar vessel constrictor responses to either a thromboxane mimetic (U46619), iso-prostaglandin PGE(2) (iso-PGE(2)) or iso-PGF(2alpha) were determined using small vessel myographs. In some vessels, the effects of putative prostanoid and thromboxane receptor antagonists, SQ 29,548, SC-19220 and AH 6809, upon contractile responses were determined. In control horses, U46619, iso-PGF(2alpha) and iso-PGE(2) more potently and efficaciously constricted laminar veins when compared to laminar arteries. Responses of laminar veins from BWHE horses to iso-PGE(2) were similar to those of laminar veins from control horses, whereas iso-PGF(2alpha) elicited significantly greater responses in laminar veins from BWHE horses when compared to controls. In contrast, responses to U46619 were smaller in laminar veins isolated from BWHE horses when compared to those in laminar veins from control horses. In the presence of SQ 29,548, iso-PGF(2alpha) elicited a small dilation in laminar veins from control horses, which was not apparent in laminar veins from BWHE horses. These results are consistent with both systemic and local inflammatory events occurring during the prodromal stages of BWHE-induced laminitis. Because laminar veins are sensitive to thromboxane and isoprostanes, these substances may act as conduits between the inflammatory and vascular events occurring in laminitis and may be therapeutic targets for this crippling condition.

Equine laminitis: A journey to the dark side of venous

Equine laminitis is a crippling condition that continues to defy repeated efforts to delineate the precise mechanisms involved and develop effective therapeutic strategies for use in the clinic. In this article, the possible role of dysfunction of the laminar vasculature is discussed, with particular emphasis on the venous side of the laminar microvasculature and the possible role(s) that metabolic syndrome and thrombosis may play in the dysfunction observed in the laminar microvasculature during the development of laminitis.

Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury.

The goal of this study was to establish an SPIO-based cell-tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri-lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post-injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration.

Primary lacrimal gland adenocarcinoma of the third eyelid in a horse

A 5-year-old Draft Horse gelding presented for evaluation of a large, fleshy, ulcerated third eyelid mass OD of 3 weeks duration. Complete ophthalmic examination, ocular ultrasound and skull radiographs revealed a large soft-tissue mass involving the entire third eyelid OD and extending into the ventral right orbit to the level of the globe equator. No other abnormalities were noted on physical or ophthalmic examination. Surgical removal via exenteration was performed 3 months after initial presentation. A lacrimal adenocarcinoma of the third eyelid was diagnosed based on histopathology. Concurrent asymptomatic intra-ductal and intra-acinar Demodex caballi parasites were found in the eyelid sebaceous glands, likely as an incidental finding. No tumor recurrence or metastasis has occurred 12 months after excision. To the authors knowledge, this case is the first reported primary lacrimal adenocarcinoma in a horse. Complete surgical excision was curative.

Resection and Anastomosis of the Descending Colon in 43 Horses

Objectives: To determine (1) the short- (to hospital discharge) and long- (>6 months) term survival, (2) factors associated with short-term survival, and (3) the perioperative course for horses with resection and anastomosis of the descending colon. Study Design: Multicentered case series. Animals: Horses (n=43) that had descending colon resection and anastomosis. Methods: Medical records (January 1995–June 2009) of 7 equine referral hospitals were reviewed for horses that had descending colon resection and anastomosis and were recovered from anesthesia. Retrieved data included history, results of clinical and clinicopathologic examinations, surgical findings, postsurgical treatment and complications, and short-term survival (hospital discharge). Long-term survival was defined as survival ≥6 months after hospital discharge. Results: Of 43 horses, 36 (84%) were discharged from the hospital. Twenty-eight of 30 horses with follow-up information survived ≥6 months. No significant associations between perioperative factors and short-term survival were identified. Lesions included strangulating lipoma (n=27), postfoaling trauma (4), infarction (4), intraluminal obstruction (2), and other (6). Common postoperative complications included fever and diarrhea. During hospitalization 7 horses were euthanatized or died because of septic peritonitis (3), endotoxemia (3), and colic and ileus (1). Conclusions: Descending colon resection and anastomosis has a favorable prognosis for hospital discharge and survival ≥6 months. The most common cause of small colon incarceration was strangulating lipoma. Clinical Relevance: Complications include postoperative fever and diarrhea but the prognosis is good after small colon resection and anastomosis.OBJECTIVES To determine (1) the short- (to hospital discharge) and long- (>6 months) term survival, (2) factors associated with short-term survival, and (3) the perioperative course for horses with resection and anastomosis of the descending colon. STUDY DESIGN Multicentered case series. ANIMALS Horses (n=43) that had descending colon resection and anastomosis. METHODS Medical records (January 1995-June 2009) of 7 equine referral hospitals were reviewed for horses that had descending colon resection and anastomosis and were recovered from anesthesia. Retrieved data included history, results of clinical and clinicopathologic examinations, surgical findings, postsurgical treatment and complications, and short-term survival (hospital discharge). Long-term survival was defined as survival > or =6 months after hospital discharge. RESULTS Of 43 horses, 36 (84%) were discharged from the hospital. Twenty-eight of 30 horses with follow-up information survived > or =6 months. No significant associations between perioperative factors and short-term survival were identified. Lesions included strangulating lipoma (n=27), postfoaling trauma (4), infarction (4), intraluminal obstruction (2), and other (6). Common postoperative complications included fever and diarrhea. During hospitalization 7 horses were euthanatized or died because of septic peritonitis (3), endotoxemia (3), and colic and ileus (1). CONCLUSIONS Descending colon resection and anastomosis has a favorable prognosis for hospital discharge and survival > or =6 months. The most common cause of small colon incarceration was strangulating lipoma. CLINICAL RELEVANCE Complications include postoperative fever and diarrhea but the prognosis is good after small colon resection and anastomosis.

Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs.