(K-403) Exploring the impact of TGFβ1 on mesenchymal stem cells: implications for tendon and regulation of collagen crosslinking

Tendon is a collagen-rich tissue that attaches muscle to bone and transfers mechanical forces. Tendon injuries are common, heal poorly, and current treatments do not result in a complete return to function. Crosslinking of the collagen molecules is a major contributor to tendon strength (1), and is regulated by the enzyme, lysyl oxidase (LOX). Mesenchymal stem cells (MSCs) have been explored in regenerative therapies to improve tendon healing but have not yet resulted in a full return to mechanical function. A limited understanding of how LOX production by MSCs is regulated remains a challenge for improving tendon function following an injury. To reduce this gap in knowledge, we aim to understand how LOX production by MSCs is regulated by transforming growth factor (TGF)β1. TGFβ1 is known to increase LOX expression by fibroblasts in mechanically injured medial collateral ligament and anterior cruciate ligament (2). TGFβ1 is also released during tendon injury, resulting in tendon cell death, and may contribute to scar formation during healing (3, 4). It is this TGFβ1-enhanced environment where MSCs may be applied in a regenerative medicine approach for tendon healing, but how MSCs respond to TGFβ1 is not clear. Therefore, the objective of this study was to determine how TGFβ1 impacts MSC behavior and LOX production. Further, in a preliminary study, Akt cell signaling was explored as a potential mechanism regulating LOX production by MSCs in response to TGFβ1.

Murine MSCs (C3H10T1/2, ATCC, Manassas VA) were seeded in 12-well plates in standard culture medium (DMEM, 10% Fetal Bovine Serum, and 1% Penicillin/streptomycin) at either 5,000, 10,000, or 20,000 cells/cm². After 24 hours, the medium was changed to a low serum medium (1% FBS), and after an additional 24 hours treated with TGFβ1 (Peprotech, Cranbury, NJ) at either 1, 10, or 50 ng/mL, along with their respective vehicle controls of citric acid. Cell morphology was examined by phase contrast imaging at 1, 3, and 7 days. To evaluate how Akt activation impacts LOX production, MSCs were seeded in 12-well plates at 20,000 cells/cm² and treated with an Akt inhibitor, MK-2206 (MedChem Express, Monmouth Junction, NJ) at 500 nM, or DMSO as the vehicle control, in environments with TGFβ1 at 0.1 ng/mL. MSCs were collected for protein quantification using western blotting after 1 day of treatment.

At day 1 (Fig 1), TGFβ1 appeared to enhance MSCs proliferation in all seeding densities, except 5,000 cells/cm². However, by day 3 (Fig 2) and day 7 (Fig 3), TGFβ1 appeared to be cytotoxic for MSCs when seeded at the lower cell-seeding densities of 5,000 and 10,000 cells/cm². MSCs were more viable at a higher cell-seeding density of 20,000 cells/cm² when treated with either 1, 10, or 50 ng/mL TGFβ1 (Fig 2-3). Effects of cell-seeding density on cell viability had not previously been explored in these C3H10T1/2 MSCs, but prior studies observed that a TGFβ1 concentration as low as 4 ng/mL proved to be cytotoxic in murine tenocytes (4). However, in these prior studies, the cell seeding density was not clear. With the findings from our own cell-density experiment, MSCs appear to withstand TGFβ1 concentrations up to 50 ng/mL, but only while seeded at a higher cell seeding density of 20,000 cells/cm². At a higher cell-seeding density of 20,000 cells/cm², cell morphology is also observed to be more fibroblastic with TGFβ1 treatments. Our pilot study found that Akt phosphorylation with TGFβ1 could be inhibited with MK-2206. Further inhibition of Akt phosphorylation was not observed to impact LOX production within these MSCs (Fig 4). Future studies and exploration of alternative cell-signaling pathways are needed to further understand the role TGFβ1 has in LOX production within MSCs. These results improve our understanding of the factors that impact MSC behavior and LOX production to ultimately control collagen crosslinking and improve the mechanical function of tendon after an injury.

This project supported by National Science Foundation (NSF) Grant #2145004.

1) Marturano JE et al., (2013) Characterization of mechanical and biochemical properties of developing embryonic tendon. Proc Natl Acad Sci U S A. 110(16), 6370–6375.  https://doi.org/10.1073/pnas.1300135110

2) Xie J et al., (2013) TGF-beta1 induces the different expressions of lysyl oxidases and matrix metalloproteinases in anterior cruciate ligament and medial collateral ligament fibroblasts after mechanical injury. Journal of Biomechanics. 46(5):890-898. https://doi.org/10.1016/j.jbiomech.2012.12.019

3) Yu HB et al., (2022) TGFβ1-transfected tendon stem cells promote tendon fibrosis. Journal of Orthopaedic Surgery and Research. 17, 358. https://doi.org/10.1186/s13018-022-03241-y

4) Maeda T et al., (2011) Conversion of Mechanical Force into TGF-β-Mediated Biochemical Signals. Current Biology. 21(11):933-941. https://doi.org/10.1016/j.cub.2011.04.007