(K-401) Evaluating tissue formation in mesenchymal stem cell self-assembled neotendons

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row K - Poster # 401

Presenting Author(s)

CM

Christina Mai

Undergraduate Researcher
University of Idaho
MOSCOW, Idaho

Co-Author(s)

CM

Colin R. Marchus, n/a

Graduate Researcher
University of Idaho, Idaho, United States
Peter J. Wieber

Undergraduate Researcher
University of Idaho
Moscow, Idaho, United States

Primary Investigator(s)

NS

Nathan Schiele

Associate Professor
University of Idaho, Idaho, United States

Introduction:: Tendon connects muscle to bone and transmits mechanical force in the body. Unfortunately, tendon injuries are prevalent, and due to limited treatment options, there is a need to develop new regenerative therapies. A major challenge in developing these approaches is a limited understanding of the cellular regulators that impact tenogenic differentiation (i.e., tenogenesis) and direct tendon tissue formation. In our prior studies, we identified that treatment of mesenchymal stem cells (MSCs) with transforming growth factor (TGF)-β2 enhanced tenogenic marker production and the enzyme involved in collagen crosslinking, lysyl oxidase (LOX). At the same time, TGF-β2 treatment decreased protein levels of the cell-cell junction protein, N-cadherin1. However, these prior studies were conducted when the MSCs were grown in an artificial 2-dimensional (2D) culture; the roles that N-cadherin and LOX play in early tendon formation in a more physiologically relevant 3D tissue are unknown. Therefore, the objective of this study was to determine how TGF-β2 impacts N-cadherin and LOX levels in MSCs that are directed to form 3D neotendons using a novel culture system that enables cells to self-assemble.

Materials and Methods:: To direct MSCs to self-assemble and form 3D neotendons, a 3D printed mold to form a well (~ 8 mm X 4 mm X 4 mm) and base with two anchor points was developed and placed in the bottom of a 12-well plate. A 2% agarose solution filled the well to provide a non-adherent surface2. Murine C3H10T1/2 MSCs were plated at 3x106 cells per well. After 48 hours, MSCs were treated with 50 ng/mL TGF-β2 or the vehicle control (sterile water). Neotendons were then fixed in 10% formalin and then stained with DAPI and FITC-phalloidin to visualize cell nuclei and actin cytoskeleton, respectively. Western blotting was used to evaluate LOX and N-cadherin at 1, 3, 7 and 14 days.

Results, Conclusions, and Discussions:: MSCs self-assembled to form neotendons as seen by the cell contraction around the anchor points that appeared similar for vehicle control (VC)- and TGF-β2-treated groups (Fig 1A). However, at day 3, TGF-β2-treated neotendons showed increased actin cytoskeleton organization and alignment, compared to the VC (Fig 1B). The high cell density and fibrous actin network formation with TGF-β2 appeared to mimic what had been previously seen in embryonic chicken tendon3. Western blotting showed that N-cadherin levels were maintained between TGF-β2-treated and VC groups throughout all timepoints (p >0.05), suggesting cell-cell connections may be involved in initial tissue formation. LOX was not readily detectable by western blot until day 14. At day 14, LOX levels increased in TGF-β2 treated neotendons, compared to VC (p = 0.02) (Fig 1C), suggesting neotendons are beginning to mature and form crosslinked collagen. Future work will identify what role N-cadherin is playing in neotendon formation and explore the development of a crosslinked collagen network. TGF-β2 impacts cell morphology and LOX levels in MSCs that are directed to form 3D neotendons, but N-cadherin levels are maintained. This study provides the foundation for understanding engineering tendon formation, which could ultimately be used to advance regenerative medicine.

Acknowledgements (Optional): : This project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408 and NSF Grant #2145004.

References (Optional): : 1) Theodossiou SK, et al, , Tissue Barriers 2020;8:1695491 2) Schiele NR, et al. Tissue Eng Part A 2013;1:1223–32. 3) Schiele NR, et al. . J Orthop Res Off Publ Orthop Res Soc 2015;33:874–81.