IntroductionAutologous mesenchymal stem cell (MSC) injection into naturally-occurring equine tendon injuries has been shown to be safe and efficacious and protocols inform translation of the technique into humans. Efficient transfer of cells from the laboratory into tissue requires well-validated transport and implantation techniques.MethodsCell viability in a range of media was determined over 72 hours and after injection through a 19G, 21G or 23G needle. Viability, proliferation and apoptosis were analysed using TrypanBlue, alamarBlue® and AnnexinV assays.ResultsCell viability was similar in all re-suspension media following 24 hour storage, however cell death was most rapid in bone marrow aspirate, platelet-rich plasma and serum after longer storage. Cryogenic media exhibited greatest viability regardless of storage time. Cell proliferation after 24 and 72 hour storage was similar for all media, except after 24 hours in serum wherein proliferation was enhanced. MSC tri-lineage differentiation and viability did not significantly change when extruded through 19G, 21G or 23G needles, but 21G and 23G needles significantly increased apoptotic cells compared to 19G and non-injected controls. All gauges induced a decrease in metabolic activity immediately post-injection but cells recovered by 2 hours.ConclusionsThese data indicate storage and injection influence viability and subsequent cell behaviour and provide recommendations for MSC therapy that implantation of cells should occur within 24 hours of recovery from culture, using larger needle bores.

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IntroductionAutologous mesenchymal stem cells (MSCs) are an attractive concept in regenerative medicine, but their mechanism of action remains poorly defined. No immune response is reported after in vivo injection of allogeneic equine MSCs or embryo-derived stem cells (ESCs) into the equine tendon, which may be due to the cells’ immune-privileged properties. This study further investigates these properties to determine their potential for clinical application in other tissues.MethodsMitomycin C-treated MSCs, ESCs, or differentiated ESCs (dESCs) were cultured with allogeneic equine peripheral blood mononuclear cells (PBMCs), and their effect on PBMC proliferation, in the presence or absence of interferon-gamma (IFN-γ) was determined. MSCs and super-antigen (sAg)-stimulated PBMCs were co-cultured directly or indirectly in transwells, and PBMC proliferation examined. Media from MSC culture were harvested and used for PBMC culture; subsequent PBMC proliferation and gene expression were evaluated and media assayed for IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 and IL-6 proteins with enzyme-linked immunosorbent assay (ELISA).ResultsCo-culture of PBMCs with ESCs or dESCs did not affect baseline proliferation, whereas co-culture with MSCs significantly suppressed baseline proliferation. Stimulation of PBMC proliferation by using super-antigens (sAgs) was also suppressed by co-culture with MSCs. Inhibition was greatest with direct contact, but significant inhibition was produced in transwell culture and by using MSC-conditioned media, suggesting that soluble factors play a role in MSC-mediated immune suppression. The MSCs constitutively secrete IL-6, even in the absence of co-culture with PBMCs. MSC-conditioned media also brought about a change in the cytokine-expression profile of sAg-stimulated PBMCs, significantly reducing PBMC expression of IL-6, IFN-γ, and TNF-α.ConclusionsEquine MSCs and ESCs possess a degree of innate immune privilege, and MSCs secrete soluble factors that suppress PBMC proliferation and alter cytokine expression. These properties may make possible the future clinical use of allogeneic stem cells to help standardize and broaden the scope of treatment of tissue injuries.

The horse is an attractive model for many human age-related degenerative diseases of the musculoskeletal system because it is a large animal species that both ages and exercises, and develops naturally occurring injuries with many similarities to the human counterpart. It therefore represents an ideal species to use as a 'proving ground' for new therapies, most notably regenerative medicine. Regenerative techniques using cell-based therapies for the treatment of equine musculoskeletal disease have been in use for over a decade. This review article provides a summary overview of the sources, current challenges and problems surrounding the use of stem cell and non-cell-based therapy in regenerative medicine in horses and is based on presentations from a recent Havemeyer symposium on equine regenerative medicine where speakers are selected from leading authorities in both equine and human regenerative medicine fields from 10 different countries.

The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1b (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or tumor necrosis factor-a (TNFa), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro-matrix metalloproteinases (MMP)-2 but not pro-MMP-9, as assessed by gelatin zymography. APC did not directly activate pro-MMP-13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10 À 5 M and 10 À10 M. High concentrations significantly increased GAG release from IL-1þAPC-treated explants. With the exception of MPA at 10 À10 M, all concentrations of corticosteroids caused significant decreases in IL-1þAPC-driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP-1, -3, and -13 mRNA. The collagenolysis associated with IL-1þAPC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively. ß