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Tissue Engineering

Tissue Engineering - Poster Session E

Poster AA15 - Assessing the impact of dynamic tensile loading on mesenchymal stem cell self-assembled neotendons

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Tendons are musculoskeletal tissues that transfer forces between muscle and bone and are essential for movement within the body. Unfortunately, tendons are frequently injured and due to their poor healing ability, re-rupture is common. Treatment options are limited, and the standard surgical methods result in scar tissue that causes decreased mechanical function. A major hurdle in developing new regenerative therapies is a lack of understanding of the process by which tendons form. To better understand the mechanisms that regulate tendon formation, our lab has recently been developing a three-dimensional (3D), scaffold-free cell self-assembly culture method to direct mesenchymal stem cells (MSCs) to form neotendons in vitro. This scaffold-free cell self-assembly methods mimics the high cell density found in the early stages of embryonic tendon development. Our lab has also previously found that treating MSCs with transforming growth factor (TGF)β2 increases tenogenic markers in 2D, but it is unknown how TGFβ2 and tensile mechanical loading impact tissue assembly by MSCs in 3D. Thus, the objective of this study was to evaluate how TGFβ2 and cyclic tendon loading impacts the early stages of neotendon formation by MSCs.

Materials and

Methods:  Murine C3H10T1/2 MSCs are formed into neotendons using a scaffold-free, directed cell self-assembly approach. Briefly, base plates with two posts for cells to anchor around were printed in PLA using a 3D printer, sterilized, and inserted into the bottom of a 12-well plate. A custom 3D printed mold was then placed over the base plate (Figure 1A, steps 1-3) then the well was filled with 2% agarose (Figure 1A, step 4). Agarose does not contain cell attachment sites, which direct cells to form cell-to-cell adhesions. After agarose gelation, the mold was removed to develop an 8-mm long, 4-mm wide, and 4-mm deep well. 3 x 106 MSCs were seeded into each well (Figure 1A, steps 5-7), and full culture medium was added after 1 hour (Figure 1A, step 8). After 48 hours of culture, the culture medium was supplemented with 50 ng/mL TGFβ2 (Peprotech) and cultured for an additional 24 hours. Neotendons were then loaded into a custom bioreactor (Figure 1B), and cyclically loaded in tension (1% strain at 1 Hz for 4 hours), and then fixed in 10% formalin 24 hours after loading (n=3). Controls were treated identically, but not loaded (static). Neotendons were imaged using second harmonic generation (SHG), and then stained for confocal fluorescence microscopy. DAPI was used to visualized the cell nuclei and FITC-phalloidin to visualize the actin cytoskeleton.

Results, Conclusions, and Discussions: MSCs self-assembled into a cohesive tissue structure ~ 6 mm in length between the posts (Figure 1B,1), that could withstand being removed from the 12-well plate, mounted on the bioreactor, and mechanically loaded in tension (Figure 1B,4). Given that there is no exogenous extracellular matrix or scaffold and the structure is highly cellular, the ability of the neotendon to be mechanically loaded is promising for future studies to explore initial stages of tissue formation. SHG imaging was performed on neotendons to detect collagen fibril formation. While, there may be early formation of collagen fibrils in the static controls (Figure 1C, arrow), these early stage tissues likely have very limited collagen fibril formation and require additional time in culture. Interestingly, none of the loaded neotendons appeared to show any signs of collagen fibrils, which could indicate that loading may induce some collagen remodeling, but this needs to be investigated further. We also suspect that it may still be too early to consistently see collagen formation. Confocal fluorescent imaging was used to evaluate cell morphology in the neotendons. The actin cytoskeleton network appeared to have a more pronounced and aligned fiber-like structure in the loaded neotendons, compared to the static controls (Figure 1C). Cyclic loading applied to these early stage neotendons may be playing a role in directing cell organization, which could impact tissue assembly. Muscle contractions that occur throughout embryonic tendon formation in vivo are critical to normal tendon maturation, and loading may be playing a similar role in vitro. Future work will aim to evaluate the mechanically regulated mechanisms that impact actin organization and tissue formation. Our lab also previously found that treating MSCs with TGFβ2 increases the production of lysyl oxidase (LOX), an enzyme involved in collagen crosslinking (Pancheri, 2024). Future work will evaluate how mechanical stimuli impacts LOX production in these neotendons. In conclusion, we developed a method to form and mechanically stimulate cell self-assembled neotendons , and our initial findings show that actin alignment may increase with cyclic loading. We can use this method to better understand the process by which tendons form and become functional tissues.

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.