Hydrogel dimensionality directs mechanics-dependent nascent matrix deposition by tendon fibroblasts

Tendons are the tough, rope-like tissues that connect muscle to bone, allowing us to move with strength and precision. Inside these tissues, specialized cells called tendon fibroblasts act as the construction crew, constantly building and repairing the extracellular matrix. This matrix is a complex scaffold of proteins, primarily collagen, that gives the tendon its structure and ability to withstand force. For these cells to do their job correctly, they must be able to sense the physical environment around them. They feel the stiffness of the materials they are embedded in and adjust their behavior accordingly. If the environment is too soft or too hard, or if the cells are trapped in a way that prevents them from moving, the construction work can go wrong, leading to injury or poor healing. Understanding exactly how these cells interpret their physical surroundings is crucial for developing better treatments for tendon injuries and for designing artificial materials that can help repair damaged tissue.

Scientists have long known that the stiffness of a surface influences how cells behave, but the rules change depending on whether the cells are sitting on a flat surface or floating inside a three-dimensional gel. In a flat, two-dimensional world, cells generally spread out more and become more active when placed on a stiffer surface. However, when cells are trapped inside a three-dimensional gel, the rules often flip: a stiffer gel can actually squeeze the cell, making it harder for it to spread and do its work. A new study by researchers at the University of Pennsylvania and the University of Michigan set out to see how these different rules apply to tendon fibroblasts and, more specifically, how they affect the new material these cells build. The team wanted to know if the dimensionality of the environment—flat versus three-dimensional—changes the way tendon cells respond to stiffness and deposit their new matrix.

To investigate this, the researchers used a specialized type of gel made from hyaluronic acid, a natural substance found in the body, which they modified to allow them to tune its stiffness precisely. They grew tendon fibroblasts from mouse tail tendons and placed them in two different setups. In the first setup, the cells were seeded on top of flat gels with stiffness levels of 10 and 25 kilopascals. In the second setup, the cells were encapsulated inside three-dimensional gels with stiffness levels of 5, 10, and 50 kilopascals. To see exactly what new material the cells were making, the scientists used a clever labeling technique. They fed the cells a sugar molecule that the cells incorporated into their newly built proteins. Later, they added a glowing tag that attached specifically to this sugar, allowing them to see the fresh, nascent matrix as it was deposited around the cells.

In the flat, two-dimensional cultures, the results followed the expected pattern. When the cells were on the stiffer gel, they spread out more, becoming flatter and larger. This spreading was accompanied by a change in the cell's internal signaling, specifically the movement of a protein called YAP into the cell's nucleus, which acts as a switch for growth and matrix production. As a result, the cells on the stiffer 25-kilopascal gel deposited significantly more new matrix than those on the softer 10-kilopascal gel. The researchers observed that the new matrix started as small dots near the cell but eventually grew into continuous, fibrous networks, a process that happened over time regardless of the stiffness, though the total amount was higher on the stiffer surface.

The story changed completely when the cells were placed inside the three-dimensional gels. Here, the relationship between stiffness and cell behavior was reversed. The cells in the softer gels, at 5 and 10 kilopascals, were able to spread slightly more and showed higher levels of the YAP protein in their nuclei compared to the cells in the stiffest 50-kilopascal gel. The cells in the stiff 3D gel remained small and round, trapped by the tight network of the material. Consequently, the amount of new matrix they deposited was lower in the stiff gel than in the softer ones. Interestingly, the cells in the 10-kilopascal gel produced the most new matrix of all, even more than those in the 5-kilopascal gel, suggesting that there is an optimal stiffness for matrix production in three dimensions that is distinct from the trend seen in flat cultures.

The researchers also looked at the genetic instructions inside the cells to understand what they were planning to build. They found that the cells in the softer 3D gels turned on genes associated with healthy tendon formation, such as those for collagen type I and tenascin C. In contrast, the cells in the stiff 50-kilopascal gel turned down these tendon-specific genes and instead showed signs of a different, less tendon-like state. They produced less of the specific collagen that forms the pericellular environment of tendon cells and more of a different type of collagen usually found in basement membranes. This shift in genetic programming confirmed that the physical confinement of the stiff gel was fundamentally altering the cell's identity and its ability to function as a tendon builder.

The study highlights that the dimensionality of the environment is a critical factor in how tendon cells sense and respond to their surroundings. While a stiffer surface encourages matrix production in a flat culture, a stiffer three-dimensional gel can actually suppress it by physically restricting the cell and changing its genetic program. This finding suggests that simply making a material stiffer is not always the answer for encouraging tissue repair. Instead, the design of biomaterials for tendon healing must account for the three-dimensional nature of the tissue, ensuring that the material is soft enough to allow the cells to spread and activate their tendon-building genes, yet structured enough to support the formation of a robust new matrix. By mapping these complex interactions, the researchers have provided a clearer guide for creating artificial environments that can effectively support the body's natural repair mechanisms.