Functional Changes to Achilles Tendon and Enthesis in a Mouse Model of an Adolescent Masculine Gender-Affirming Hormone Treatment.

Many transgender youth seek gender affirming care, such as puberty suppression, to prolong decision-making and to align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g., exercise) yet if and how tendon adaptation is influenced by sex and gender affirming hormone therapy during growth remains unknown. The goal of this study was to understand the how pubertal suppression influences the structural and functional properties of the Achilles tendon using an established mouse model of transmasculine gender affirming hormone therapy. C57BL/6N female-born mice were assigned to experimental groups to mimic gender-affirming hormone therapy in human adolescents, and treatment was initiated prior to the onset of puberty (at postnatal day 26, P26). Experimental groups included controls and mice serially treated with gonadotropin release hormone analogue (GnRHa), delayed Testosterone (T), or GnRHa followed by T. We found that puberty suppression using GnRHa, with and without T, improved the overall tendon load capacity in female-born mice. Treatment with T resulted in an increase in the maximum load that tendon can withstand before failure. Additionally, we found that GnRHa, but not T, treatment resulted in a significant increase in cell density at the Achilles enthesis.

Engineered Microenvironmental Cues from Fiber-Reinforced Hydrogel Composites Drive Tenogenesis and Aligned Collagen Deposition.

Effective tendon regeneration following injury is contingent on appropriate differentiation of recruited cells and deposition of mature, aligned, collagenous extracellular matrix that can withstand the extreme mechanical demands placed on the tissue. As such, myriad biomaterial approaches have been explored to provide biochemical and physical cues that encourage tenogenesis and template aligned matrix deposition in lieu of dysfunctional scar tissue formation. Fiber-reinforced hydrogels present an ideal biomaterial system toward this end given their transdermal injectability, tunable stiffness over a range amenable to tenogenic differentiation of progenitors, and capacity for modular inclusion of biochemical cues. Here, tunable and modular, fiber-reinforced, synthetic hydrogels are employed to elucidate salient microenvironmental determinants of tenogenesis and aligned collagen deposition by tendon progenitor cells. Transforming growth factor ß3 drives a cell fate switch toward pro-regenerative or pro-fibrotic phenotypes, which can be biased toward the former by culture in softer microenvironments or inhibition of the RhoA/ROCK activity. Furthermore, studies demonstrate that topographical anisotropy in fiber-reinforced hydrogels critically mediates the alignment of de novo collagen fibrils, reflecting native tendon architecture. These findings inform the design of cell-free, injectable, synthetic hydrogels for tendon tissue regeneration and, likely, that of a range of load-bearing connective tissues.

Akt signaling is activated by TGFß2 and impacts tenogenic induction of mesenchymal stem cells.

BACKGROUND: Tissue engineered and regenerative approaches for treating tendon injuries are challenged by the limited information on the cellular signaling pathways driving tenogenic differentiation of stem cells. Members of the transforming growth factor (TGF) ß family, particularly TGFß2, play a role in tenogenesis, which may proceed via Smad-mediated signaling. However, recent evidence suggests some aspects of tenogenesis may be independent of Smad signaling, and other pathways potentially involved in tenogenesis are understudied. Here, we examined the role of Akt/mTORC1/P70S6K signaling in early TGFß2-induced tenogenesis of mesenchymal stem cells (MSCs) and evaluated TGFß2-induced tenogenic differentiation when Smad3 is inhibited. METHODS: Mouse MSCs were treated with TGFß2 to induce tenogenesis, and Akt or Smad3 signaling was chemically inhibited using the Akt inhibitor, MK-2206, or the Smad3 inhibitor, SIS3. Effects of TGFß2 alone and in combination with these inhibitors on the activation of Akt signaling and its downstream targets mTOR and P70S6K were quantified using western blot analysis, and cell morphology was assessed using confocal microscopy. Levels of the tendon marker protein, tenomodulin, were also assessed. RESULTS: TGFß2 alone activated Akt signaling during early tenogenic induction. Chemically inhibiting Akt prevented increases in tenomodulin and attenuated tenogenic morphology of the MSCs in response to TGFß2. Chemically inhibiting Smad3 did not prevent tenogenesis, but appeared to accelerate it. MSCs treated with both TGFß2 and SIS3 produced significantly higher levels of tenomodulin at 7 days and morphology appeared tenogenic, with localized cell alignment and elongation. Finally, inhibiting Smad3 did not appear to impact Akt signaling, suggesting that Akt may allow TGFß2-induced tenogenesis to proceed during disruption of Smad3 signaling. CONCLUSIONS: These findings show that Akt signaling plays a role in TGFß2-induced tenogenesis and that tenogenesis of MSCs can be initiated by TGFß2 during disruption of Smad3 signaling. These findings provide new insights into the signaling pathways that regulate tenogenic induction in stem cells.