Larry D. Galuppo

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.

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.

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 99mTc‐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.

Identification of variables that optimize isolation and culture of multipotent mesenchymal stem cells from equine umbilical-cord blood

OBJECTIVE-To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics. SAMPLE POPULATION-UCB samples from 79 Thoroughbred and Quarter Horse mares. PROCEDURES-UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-sample factors were analyzed to determine their influence on the success of MSC isolation and monolayer expansion. RESULTS-MSCs capable of multilineage in vitro differentiation were expanded from > 80% of UCB samples. Automated UCB processing and temperature-controlled shipping facilitated sterile and standardized RBC reduction and NC enrichment from UCB samples. The number of NCs after UCB samples were processed was the sole variable that predicted successful MSC expansion. The UCB-derived MSCs and NCs were successfully cryopreserved and thawed with no decrease in cell recovery, viability, or MSC proliferation. The use of fibronectin-coated culture plates and reduction of incubator oxygen tension from 20% to 5% improved the MSC isolation rate. Some UCB-derived MSC clones proliferated for > 20 passages before senescence. Onset of senescence was associated with specific immunocytochemical changes. CONCLUSIONS AND CLINICAL RELEVANCE-Equine UCB samples appeared to be a rich source of readily obtainable, highly proliferative MSCs that could be banked for therapeutic use.

Distribution and persistence of technetium‐99 hexamethyl propylene amine oxime‐labelled bone marrow‐derived mesenchymal stem cells in experimentally induced tendon lesions after intratendinous injection and regional perfusion of the equine distal limb

REASONS FOR PERFORMING STUDY Intralesional (i.l.) injection is currently the most commonly used technique for stem cell therapy in equine tendon injury. A comparison of different techniques of injection of mesenchymal stem cells for the treatment of tendon lesions is required. OBJECTIVES We hypothesised that vascular perfusion of the equine distal limb with mesenchymal stem cells (MSCs) would result in preferential distribution of MSCs to acute tendon injuries. STUDY DESIGN In vivo experimental study. METHODS Lesions were surgically induced in forelimb superficial digital flexor tendons of 8 horses. Three or 10 days after lesion induction, technetium-99 hexamethyl propylene amine oxime-labelled MSCs were injected via i.v. or intra-arterial (i.a.) regional limb perfusion (RLP) at the level of the distal antebrachium and compared to i.l. injection. Mesenchymal stem cell persistence and distribution within the forelimb and tendon lesions was assessed with scintigraphy for 24 h. RESULTS Lesion uptake was higher with i.l. injection than with RLP, but MSC persistence decreased similarly over time in all 3 techniques. Intra-arterial RLP resulted in a better distribution of MSCs and a higher uptake at the lesion site than i.v. RLP. Limbs perfused i.a. on Day 10 showed greater accumulation of MSCs in the lesion than limbs perfused on Day 3. Arterial thrombosis occurred in 50% of the i.v. RLP limbs and in 100% of the i.a. RLP limbs, which led to clinical complications in one horse. CONCLUSIONS AND POTENTIAL RELEVANCE Compared with i.l. injection, RLP results in lower uptake but similar persistence of MSCs at the site of tendon lesions. A time dependent accumulation of MSCs was identified with i.a. RLP. The i.a. RLP appears more advantageous than the i.v. RLP in terms of distribution and uptake. However, the described i.a. technique produced arterial thrombosis and thus cannot currently be recommended for clinical use.

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.

Collection of equine cord blood and placental tissues in 40 Thoroughbred mares

REASONS FOR PERFORMING STUDY Stem cells derived from umbilical cord tissue (UCT) and umbilical cord blood (UCB) in human subjects and horses can be obtained in a minimally invasive fashion with successful propagation of mesenchymal stem cells (MSCs). Currently there are no detailed protocols documenting a procedure to harvest UCB and UCT safely for equine stem cell propagation. HYPOTHESIS UCB and UCT could be collected without harm to mare or foal. OBJECTIVES To develop a standard and safe method for UCB and UCT collection, and prospectively to compare foal and mare health between groups of animals where tissue was and was not collected. METHODS This study was conducted at a Thoroughbred breeding facility in central California in 2008. UCB and UCT were collected from 40 mare and foal pairs. Clinical parameters including time for foal to stand and nurse, time for mare to pass the placenta, and foal haematology data at age 24 h were documented and compared to a control group, consisting of the succeeding 40 mare and foal pairs. RESULTS UCB was obtained successfully from 36 of 40 (90%) mares and UCT from 38 of 40 (95%) mares. Bacterial contamination was documented in 6 out of 36 (16.6%) UCB samples. There were no significant differences in time to stand or nurse for foals or time to pass the placenta for mares, between the experimental and control groups. There were no clinically relevant differences identified in haematological data obtained from foals with and without UCB collection. CONCLUSIONS UCB and UCT can be harvested safely without harm to mares or foals. POTENTIAL RELEVANCE UCB and UCT samples collected in an inherently contaminated environment can be successfully disinfected and transported with minimal bacterial overgrowth for use in cell culture to isolate MSCs.

Evaluation of four biodegradable, injectable bone cements in an experimental drill hole model in sheep.

Four cement applications were tested in this investigation. Two dicalcium phosphate dihydrate (DCPD-brushite) hydraulic cements, an apatite hydraulic fiber loaded cement, and a calcium sulfate cement (Plaster of Paris) were implanted in epiphyseal and metaphyseal cylindrical bone defects in sheep. The in vivo study was performed to assess the biocompatibility and bone remodeling of four cement formulations. After time periods of 2, 4, and 6 months, the cement samples were clinically and histologically evaluated. Histomorphometrically, the amount of new bone formation, fibrous tissue, and bone marrow and the area of remaining cement were measured. In all specimens, no signs of inflammation were detectable either macroscopically or microscopically. Cements differed mainly in their resorption time. Calcium sulfate was already completely resorbed at 2 months and showed a variable amount of new bone formation and/or fibrous tissue in the original drill hole over all time periods. The two DCPD cements in contrast were degraded to a large amount at 6 months, whereas the apatite was almost unchanged over all time periods.

Results of computed tomography in horses with ethmoid hematoma: 16 cases (1993–2005)

OBJECTIVE To determine whether CT provides unique information about the treatment or prognosis for horses with ethmoid hematoma (EH). DESIGN Retrospective case series. ANIMALS 16 horses with EH. PROCEDURES Horses with a diagnosis of EH that had undergone a diagnostic CT study were included. Clinical features, treatment, outcome, radiographic and CT images, and histologic specimens were reviewed. RESULTS CT provided new diagnostic information that affected treatment in 10 of 16 horses. Bilateral disease occurred in 8 of 16 horses and was undetected in 5 horses prior to CT. Paranasal sinus involvement occurred in all horses, but was incompletely defined prior to CT in 7 of 16 horses. The sphenopalatine sinus was affected in 6 of 16 horses as detected on CT; 4 of 6 of these were bilaterally affected. Medical and surgical treatments were performed. Six of 10 horses had a successful outcome, with recurrence in 4 of 10. Five of 6 patients in which treatment addressed all lesion sites identified by CT had a successful outcome. Bilateral disease did not confer a poor prognosis when all affected sites were treated. Sphenopalatine sinus involvement may have been associated with recurrence. CONCLUSIONS AND CLINICAL RELEVANCE CT provided anatomic information that may facilitate effective treatment of horses with EH, particularly in patients with bilateral disease and paranasal sinus involvement. Computed tomography is recommended for patients in which the lesion cannot be viewed endoscopically, when sinus involvement or multifocal disease are suspected, or when the lesion has been unresponsive to treatment.

Autologous point-of-care cellular therapies variably induce equine mesenchymal stem cell migration, proliferation and cytokine expression

REASONS FOR PERFORMING STUDY Autologous cellular therapy products including adipose-derived stromal vascular fraction (SVF), bone marrow mononuclear cells (BMMNs), cord blood mononuclear cells (CBMNs) and platelet rich plasma are options for treatment of acute orthopaedic lesions while mesenchymal stem cells (MSCs) are culture expanded. These products may contribute to healing by secreting matrix proteins or growth factors, but they may also act on endogenous MSCs to facilitate healing. OBJECTIVES To determine the effects of cell therapy products on MSCs function in vitro. The hypothesis was that cell therapy products promote MSCs functions including proliferation, migration and mediator release. METHODS Fat, bone marrow (BM), cord blood and platelets were obtained from 6 Quarter Horses. The BM-MSCs and their autologous cell therapy products were co-incubated in transwells. Mesenchymal stem cells proliferation, migration, gene expression and cytokine concentrations were determined. RESULTS All cell therapy products increased MSCs proliferation, but SVF induced significantly more proliferation than any other product. Also SVF elicited more MSCs chemotaxis and, along with BMMNs, significantly more MSCs chemoinvasion. Cord blood mononuclear cells stimulated MSCs to produce high concentrations of interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), and prostaglandin E(2) (PGE(2)). Stromal vascular fraction and platelet lysate did not stimulate MSCs but SVF and platelet lysate themselves contained high concentrations of PGE(2) and IL-6 (SVF) and TGF-β1 (platelet lysate). CONCLUSIONS Autologous cell products variably stimulate MSCs functions with 2 primary patterns apparent. Products either contained preformed mediators that may have intrinsic healing function, or products stimulated MSCs to secrete mediators. POTENTIAL RELEVANCE The specific clinical indications for these products may differ to include administration as a sole treatment modality prior to MSCs injection for intrinsic cell and cytokine activity (i.e. SVF) or administration concurrently with MSCs to activate MSCs for treatment of chronic lesions (i.e. CBMNs).