Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression in three-dimensional culture which can be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signaling.

We investigated how Interleukin 1 beta (IL-1ß) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-KB) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1ß for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-KB inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF-KB-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1ß increased NF-KB-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF-KB signaling. Administering direct pharmacological inhibitors of NF-KB did reduce NF-KB-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1ß. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1ß which can only be rescued by blockade of interleukin 1 receptor, but not NF-KB, signalling.

Equine Fetal, Adult, and Embryonic Stem Cell-Derived Tenocytes Are All Immune Privileged but Exhibit Different Immune Suppressive Properties In Vitro.

In horses and humans, tendon injuries are a significant problem. Not only can they occur in both athletes and nonathletes, they require lengthy periods of recuperation and undergo poor natural regeneration, which leads to high reinjury rates. Embryonic stem cells (ESCs) may provide a renewable source of allogeneic cells to use in clinical applications to aid tissue regeneration. Equine ESCs can undergo tenocyte differentiation in vivo and in vitro, but the immune properties of tenocytes isolated from either ESCs or tissues have not previously been characterized. Here, we demonstrate that equine tenocytes derived from fetal and adult tendon tissue and ESCs express robust levels of major histocompatibility complex (MHC) I but no MHC II in response to inflammatory cytokine interferon gamma (IFNγ). However, MHC expression does not affect their allorecognition by peripheral blood mononuclear cells in vitro. Adult and fetal tenocytes remain immune privileged and strongly immune suppressive in both the presence and absence of exogenously applied IFNγ. In contrast, ESC-derived tenocytes are immune privileged even in the presence of IFNγ, but they are only weakly immune suppressive in the presence but not in the absence of exogenously applied IFNγ. This is despite ESC-tenocytes expressing a number of genes involved in immune modulation at significantly higher levels than those expressed by adult and fetal tenocytes when in standard, nonstimulated monolayer culture. Together, this work suggests that, similar to other fibroblasts, tenocytes have immune modulatory properties, and that culture-expanded tenocytes derived from primary tissues or ESCs may be safe to use in clinical transplantations to injured tendons of unrelated animals.

Biocompatible Three-Dimensional Printed Thermoplastic Scaffold for Osteoblast Differentiation of Equine Induced Pluripotent Stem Cells.

Horses, like humans, can experience bone fractures and due to their large size and the need to bear weight on all limbs during the recovery period, they can be difficult to treat. Surgical techniques to improve fracture repair are improving, but to date, regenerative medicine technologies to aid fracture healing are not commonly applied in horses. We have previously demonstrated that equine induced pluripotent stem cells (iPSCs) can be differentiated into bone forming osteoblasts in 2D culture. In this study, we report on the use of a thermoplastic, 3D-printed polymer to provide a scaffold for successful, in vitro osteoblast differentiation of equine iPSCs. The scaffold provides a transparent, cost-effective solution to allow the analysis of osteoblast differentiation using live-cell imaging, immunohistochemistry, and quantitative polymerase chain reaction. This in vitro study demonstrates the future feasibility of generating 3D bone constructs through the cell seeding of scaffolds to use in regenerative medicine strategies to improve fracture repair in a relevant, large animal model.