# Combined TGF-β3 and FGF-2 Stimulation Enhances Chondrogenic Potential of Ovine Bone Marrow-Derived MSCs

## TL;DR

Combining TGF-β3 and FGF-2 improves the ability of sheep bone marrow stem cells to form cartilage, offering a better method for tissue engineering.

## Contribution

The study demonstrates a synergistic effect of TGF-β3 and FGF-2 on enhancing chondrogenesis in ovine MSCs.

## Key findings

- Combined TGF-β3 and FGF-2 increased cell proliferation and chondrogenic gene expression.
- The cytokine combination led to higher glycosaminoglycan deposition and a regenerative secretome.
- TGF-β3 enhanced immunomodulatory and angiogenic properties, while FGF-2 balanced the secretome.

## Abstract

Mesenchymal stem cells (MSCs) represent a promising cell source for cartilage tissue engineering due to their chondrogenic potential. However, current differentiation protocols result in limited efficiency. This study assessed the combined effects of transforming growth factor-beta 3 (TGF-β3) and fibroblast growth factor-2 (FGF-2) on the morphology, proliferation, chondrogenic differentiation, chondrogenic gene expression, and cytokine profile of ovine bone marrow-derived MSCs (BM-MSCs). BM-MSCs were cultured under four conditions: control (αMEM) or αMEM supplemented with FGF-2, TGF-β3, or TGF-β3 + FGF-2. Morphological and proliferation analyses, Alcian blue staining in 2D and 3D, and real-time PCR for early (Chad, Comp, and Sox 5) and late (Agg, Col IX, Sox 9, and Fmod) chondrogenic markers were performed. Cytokine secretion profiles were analyzed using multiplex assay. TGF-β3 induced morphological changes indicative of early chondrogenesis, while FGF-2 enhanced proliferation. The combination of both cytokines led to a synergistic increase in cell proliferation, early and late chondrogenic gene expression, and glycosaminoglycans (GAG) deposition. Cytokine analysis revealed that TGF-β3 enhanced the immunomodulatory and angiogenic profile of BM-MSCs, whereas co-treatment with FGF-2 yielded a balanced and potentially regenerative secretome. Dual stimulation with TGF-β3 and FGF-2 significantly improves the chondrogenic differentiation of ovine BM-MSCs by enhancing both molecular and functional markers of cartilage formation.