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Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model.

ABSTRACT: BACKGROUND:Mesenchymal stromal cells (MSC) have shown promising results in the treatment of tendinopathy in equine medicine, making this therapeutic approach seem favorable for translation to human medicine. Having demonstrated that MSC engraft within the tendon lesions after local injection in an equine model, we hypothesized that they would improve tendon healing superior to serum injection alone. METHODS:Quadrilateral tendon lesions were induced in six horses by mechanical tissue disruption combined with collagenase application 3 weeks before treatment. Adipose-derived MSC suspended in serum or serum alone were then injected intralesionally. Clinical examinations, ultrasound and magnetic resonance imaging were performed over 24 weeks. Tendon biopsies for histological assessment were taken from the hindlimbs 3 weeks after treatment. Horses were sacrificed after 24 weeks and forelimb tendons were subjected to macroscopic and histological examination as well as analysis of musculoskeletal marker expression. RESULTS:Tendons injected with MSC showed a transient increase in inflammation and lesion size, as indicated by clinical and imaging parameters between week 3 and 6 (p?

SUBMITTER: Ahrberg AB

PROVIDER: S-EPMC6052633 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model.

Ahrberg A B AB Horstmeier C C Berner D D Brehm W W Gittel C C Hillmann A A Josten C C Rossi G G Schubert S S Winter K K Burk J J

BMC musculoskeletal disorders 20180718 1

<h4>Background</h4>Mesenchymal stromal cells (MSC) have shown promising results in the treatment of tendinopathy in equine medicine, making this therapeutic approach seem favorable for translation to human medicine. Having demonstrated that MSC engraft within the tendon lesions after local injection in an equine model, we hypothesized that they would improve tendon healing superior to serum injection alone.<h4>Methods</h4>Quadrilateral tendon lesions were induced in six horses by mechanical tiss ...[more]

PMID: 30021608

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Project description:BackgroundAdipose tissue is a promising source of mesenchymal stromal cells (MSCs) for the treatment of tendon disease. The goal of this study was to assess the effect of a single intralesional implantation of adipose tissue-derived mesenchymal stromal cells (AT-MSCs) on artificial lesions in equine superficial digital flexor tendons (SDFTs).MethodsDuring this randomized, controlled, blinded experimental study, either autologous cultured AT-MSCs suspended in autologous inactivated serum (AT-MSC-serum) or autologous inactivated serum (serum) were injected intralesionally 2 weeks after surgical creation of centrally located SDFT lesions in both forelimbs of nine horses. Healing was assessed clinically and with ultrasound (standard B-mode and ultrasound tissue characterization) at regular intervals over 24 weeks. After euthanasia of the horses the SDFTs were examined histologically, biochemically and by means of biomechanical testing.ResultsAT-MSC implantation did not substantially influence clinical and ultrasonographic parameters. Histology, biochemical and biomechanical characteristics of the repair tissue did not differ significantly between treatment modalities after 24 weeks. Compared with macroscopically normal tendon tissue, the content of the mature collagen crosslink hydroxylysylpyridinoline did not differ after AT-MSC-serum treatment (p?=?0.074) while it was significantly lower (p?=?0.027) in lesions treated with serum alone. Stress at failure (p?=?0.048) and the modulus of elasticity (p?=?0.001) were significantly lower after AT-MSC-serum treatment than in normal tendon tissue.ConclusionsThe effect of a single intralesional injection of cultured AT-MSCs suspended in autologous inactivated serum was not superior to treatment of surgically created SDFT lesions with autologous inactivated serum alone in a surgical model of tendinopathy over an observation period of 22 weeks. AT-MSC treatment might have a positive influence on collagen crosslinking of remodelling scar tissue. Controlled long-term studies including naturally occurring tendinopathies are necessary to verify the effects of AT-MSCs on tendon disease.

Project description:BackgroundThe clinical outcomes following intrasynovial flexor tendon repair are highly variable. Excessive inflammation is a principal factor underlying the formation of adhesions at the repair surface and affecting matrix regeneration at the repair center that limit tendon excursion and impair tendon healing. A previous in-vitro study revealed that adipose-derived mesenchymal stromal cells (ASCs) modulate tendon fibroblast response to macrophage-induced inflammation. The goal of the current study was therefore to explore the effectiveness of autologous ASCs on the inflammatory stage of intrasynovial tendon healing in vivo using a clinically relevant animal model.MethodsZone II flexor tendon transections and suture repairs were performed in a canine model. Autologous ASC sheets were delivered to the surface of repaired tendons. Seven days after repair, the effects of ASCs on tendon healing, with a focus on the inflammatory response, were evaluated using gene expression assays, immunostaining, and histological assessments.ResultsASCs delivered via the cell sheet infiltrated the host tendon, including the repair surface and the space between the tendon ends, as viewed histologically by tracking GFP-expressing ASCs. Gene expression results demonstrated that ASCs promoted a regenerative/anti-inflammatory M2 macrophage phenotype and regulated tendon matrix remodeling. Specifically, there were significant increases in M2-stimulator (IL-4), marker (CD163 and MRC1), and effector (VEGF) gene expression in ASC-sheet treated tendons compared with nontreated tendons. When examining changes in extracellular matrix expression, tendon injury caused a significant increase in scar-associated COL3A1 expression and reductions in COL2A1 and ACAN expression. The ASC treatment effectively counteracted these changes, returning the expression levels of these genes closer to normal. Immunostaining further confirmed that ASC treatment increased CD163+ M2 cells in the repaired tendons and suppressed cell apoptosis at the repair site.ConclusionsThis study provides a novel approach for delivering ASCs with outcomes indicating potential for substantial modulation of the inflammatory environment and enhancement of tendon healing after flexor tendon repair.

Project description:Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte-macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.

Project description:BackgroundMesenchymal stromal cells (MSCs) are believed to be hypoimmunogeneic with potential use for allogeneic administration.MethodsBone marrow was harvested from Connemara (n = 1), Standardbred (n = 6), and Thoroughbred (n = 3) horses. MSCs were grouped by their level of expression of major histocompatibility factor II (MHC II). MSCs were then sub-grouped by those MSCs derived from universal blood donor horses. MSCs were isolated and cultured using media containing fetal bovine serum until adequate numbers were acquired. The MSCs were cultured in xenogen-free media for 48 h prior to use and during all assays. Autologous and allogeneic MSCs were then directly co-cultured with responder leukocytes from the Connemara horse in varying concentrations of MSCs to leukocytes (1:1, 1:10, and 1:100). MSCs were also cultured with complement present and heat-inactivated complement to determine whether complement alone would decrease MSC viability. MSCs underwent haplotyping of their equine leukocyte antigen (ELA) to determine whether the MHC factors were matched or mismatched between the donor MSCs and the responder leukocytes.ResultsAll allogeneic MSCs were found to be ELA mismatched with the responder leukocytes. MHC II-low and universal blood donor MSCs caused no peripheral blood mononuclear cell (PBMC) proliferation, no increase in B cells, and no activation of CD8 lymphocytes. Universal blood donor MSCs stimulated a significant increase in the number of T regulatory cells. Neutrophil interaction with MSCs showed that universal blood donor and MHC II-high allogeneic MSCs at the 6 h time point in co-culture caused greater neutrophil activation than the other co-culture groups. Complement-mediated cytotoxicity did not consistently cause MSC death in cultures with active complement as compared to those with inactivated complement. Gene expression assays revealed that the universal blood donor group and the MHC II-low MSCs were more metabolically active both in the anabolic and catabolic gene categories when cultured with allogeneic lymphocytes as compared to the other co-cultures. These upregulated genes included CD59, FGF-2, HGF, IDO, IL-10, IL-RA, IL-2, SOX2, TGF-β1, ADAMSTS-4, ADAMSTS-5, CCL2, CXCLB/IL-8, IFNγ, IL-1β, and TNFα.ConclusionsMHC II-low MSCs are the most appropriate type of allogeneic MSC to prevent activation of the innate and cell-mediated component of the adaptive immune systems and have increased gene expression as compared to other allogeneic MSCs.

Project description:BackgroundHuman autologous serum (AS) and umbilical cord serum (UCS) both contain growth and neurotrophic factors that promote corneal healing.AimOur objectives were to compare equine AS and UCS cytokine and growth factor profiles and to assess the safety and clinical feasibility of the therapeutic use of UCS eye drops in cases of spontaneous complex ulcers.Study designProspective clinical trial.MethodsVitamin A insulin growth factor, platelet-derived growth factor-BB, transforming growth factor (TGF)-β1 (enzyme-linked immunosorbent assay), interleukin (IL)-1β, IL-6, interferon-γ, and monocyte chemoattractant protein 1 concentrations were determined in 10 AS collected from different horses and 10 UCS sampled at delivery. Six client-owned horses presenting with complex non-healing corneal defects of >5 mm2 were included in a clinical trial and treated with conventional therapy and conditioned UCS drops for 8-15 days. Ulcer surface and time to complete epithelialization were recorded.ResultsMedian concentrations of vitamin A, insulin growth factor, and platelet-derived growth factor-BB were not significantly different in AS compared with UCS (respectively, 14.5 vs. 12.05 μg/ml; 107.8 vs. 107.3 pg/ml; and 369.1 vs. 924.2 pg/ml). TGF-β1 median concentration in UCS was significantly higher than in AS (3,245 vs. 2571pg/ml) (p = 0.04). IL-1β, IL-6, interferon-γ, and monocyte chemoattractant protein 1 concentrations were variable in AS and undetectable in UCS. The corneal median ulcerative area was 37.2 mm2 (6.28-57.14 mm2) and had a duration of 4-186 days (median 19 days). All lesions healed within 13-42 days (median 17 days). No adverse effects nor recurrences within 1 month were noticed.LimitationsThe sample size was small. Spontaneous corneal epithelial defects presented with variable clinical characteristics. There were no age-matched control horses to assess corneal healing time and rate.Conclusion and clinical significanceEquine UCS may be beneficial, as it contains no pro-inflammatory cytokines and a greater concentration of TGF-β1 compared with AS. Topical UCS appears safe and may potentially be used as adjunctive therapy for equine complex non-healing ulcers.

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Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model.

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Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model.

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