# Mechanobiological Strategies to Enhance Ovine (Ovis aries) Adipose-Derived Stem Cells Tendon Plasticity for Regenerative Medicine and Tissue Engineering Applications

## TL;DR

This study explores how stem cells from sheep fat can be guided to become tendon cells using different lab techniques, showing that a tendon-like scaffold is more effective than a growth medium.

## Contribution

The study demonstrates that ovine adipose-derived stem cells can be effectively guided toward tendon differentiation using a biomimetic scaffold.

## Key findings

- Ovine adipose-derived stem cells can be expanded in culture without losing their stem cell properties.
- Seeding cells on a tendon-like scaffold leads to more effective tendon differentiation compared to using a conditioned medium.
- The scaffold-induced differentiation involves changes in gene and protein expression, including YAP activation and SOX9 downregulation.

## Abstract

Tendon injuries are known to be difficult to heal, making effective treatments crucial. This study examines the potential of stem cells derived from sheep fat tissue for tendon repair, since little information is available on their tenogenic potential. The focus of this study was to understand how these stem cells can be induced to develop into tendon cells. Initially, the cells were grown in the lab to ensure that their essential properties were maintained. Two methods were then employed to promote the acquisition of tendon-like characteristics: exposing the cells to a tenogenic conditioned medium or seeding them on a scaffold that mimics a tendon structure. The results indicated that these stem cells could be expanded without aging or changes in their characteristics. When exposed to the tendon medium, the cells were able to start the tendon differentiation process but were not able to complete it. Conversely, when seeded on the scaffold, the cells changed their naïve genetic and protein profile and acquired that of the typical tendon cells. This suggests that these stem cells can be addressed towards tendon cells, being more effective when seeded on a tendon biomimetic scaffold. These findings highlight the potential use of these stem cell treatments for tendon injuries, particularly in veterinary medicine.

Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their phenotypic properties pre-transplantation. Moreover, their tenogenic potential was assessed using two in vitro-validated approaches: (1) teno-inductive conditioned media (CM) derived from a co-culture between ovine amniotic stem cells and fetal tendon explants, and (2) short- (48 h) and long-term (14 days) seeding on highly aligned PLGA (ha-PLGA) electrospun scaffold. Our findings indicate that oADSCs can be expanded without senescence and can maintain the expression of stemness (Sox2, Oct4, Nanog) and mesenchymal (CD29, CD166, CD44, CD90) markers while remaining negative for hematopoietic (CD31, CD45) and MHC-II antigens. Of note, oADSCs’ tendon differentiation potential greatly depended on the in vitro strategy. oADSCs exposed to CM significantly upregulated tendon-related genes (COL1, TNMD, THBS4) but failed to accumulate TNMD protein at 14 days of culture. Conversely, oADSCs seeded on ha-PLGA fleeces quickly upregulated the tendon-related genes (48 h) and in 14 days accumulated high levels of the TNMD protein into the cytoplasm of ADSCs, displaying a tenocyte-like morphology. This mechano-sensing cellular response involved a complete SOX9 downregulation accompanied by YAP activation, highlighting the efficacy of biophysical stimuli in promoting tenogenic differentiation. These findings underscore oADSCs’ long-term self-renewal and tendon differentiative potential, thus opening their use in a preclinical setting to develop innovative stem cell-based and tissue engineering protocols for tendon regeneration, applied to the veterinary field.