Zonal characterization and differential trilineage potentials of equine intrasynovial deep digital flexor tendon-derived cells

Abstract

Background Intrasynovial deep digital flexor tendon (DDFT) injuries occur frequently and are often implicated in cases of navicular disease with poor outcomes and reinjuries. Cell-based approaches to tendon healing are gaining traction in veterinary medicine and ultimately may contribute to improved DDFT healing in horses. However, a better understanding of the innate cellular characteristics of equine DDFT is necessary for developing improved therapeutic strategies. Additionally, fibrocartilaginous, intrasynovial tendons like the DDFT are common sites of injury and share a poor prognosis across species, offering translational applications of this research. The objective of this study is to isolate and characterize tendon-derived cells (TDC) from intrasynovial DDFT harvested from within the equine forelimb podotrochlear bursa. TDC from the fibrocartilaginous and tendinous zones are separately isolated and assessed. Flow cytometry is performed for mesenchymal stem cell (MSC) surface markers (CD 29, CD 44, CD 90). Basal tenogenic, osteogenic and chondrogenic markers are assessed via quantitative real time-PCR, and standard trilineage differentiation is performed with third passage TDC from the fibrocartilaginous (fTDC) and tendinous (tTDC) zones of DDFT. Results Low-density plating isolated homogenous TDC populations from both zones. During monolayer passage, both TDC subpopulations exhibited clonogenicity, high in vitro proliferation rate, and fibroblast-like morphology. fTDC and tTDC were positive for MSC surface markers CD90 and CD29 and negative for CD44. There were no significant differences in basal tenogenic, osteogenic or chondrogenic marker expression between zones. While fTDC were largely restricted to chondrogenic differentiation, tTDC underwent osteogenic and chondrogenic differentiation. Both TDC subpopulations displayed weak adipogenic differentiation potentials. Conclusions TDC at the level of the podotrochlear bursa, that potentially could be targeted for enhancing DDFT injury healing in horses were identified and characterized. Pending further investigation, promoting chondrogenic properties in cells administered exogenously into the intrasynovial space may be beneficial for intrasynovial tendon regeneration.

Interleukin-1β and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro

“…DDFT and NBF cells were seeded at 5,000 cells/ cm 2 in monolayer cultures in basal expansion medium and maintained at 37 °C, 95% air/5% CO 2 in a humidified incubator. 18,21 Non-adherent cells were removed on day 2 and the resulting colonyforming units trypsinized (0.05% Trypsin-EDTA, Gibco) on days 7-9. The cells were subsequently expanded in monolayers until 80% confluence was reached and passaged twice.…”

“…DDFT and NBF cells were maintained as aggregate cultures to support their chondrogenic phenotype. 18,21…”

“…Primer specificity was confirmed by cloning and sequencing the amplicons during optimization experiments, as previously described. 18,21 PCR amplifications were catalyzed by Taq DNA polymerase (ABI QuantStudio 3™, Thermo Fisher Scientific) in the presence of SYBR green. Relative gene expression was quantified using the 2 -ΔΔCT method, 28 normalized to the expression of the reference gene, elongation factor-1α (EF1α; Appendix), and expressed as fold change from the respective DDFT or NBF cells maintained in chondrogenic medium alone (control).…”

The roles and mechanisms of the NF-κB signaling pathway in tendon disorders

“…However, tendon injuries in all species are challenging as their limited regenerative ability makes tendon repair ineffective, resulting in the formation of inferior scar tissue ( 4 , 5 ). Moreover, scar-mediated healing is particularly problematic in the flexor tendon as excessive scar tissue can form adhesion between the tendon, sheath, and surrounding tissue, further disrupting tendon function ( 6 ). On the other hand, the accumulation of microdamage during overloading after unsuccessful tendon regenerative healing is a predisposing factor for the progression of degenerative tendinopathy ( 7 ).…”