(M-510) Recruitment and the role of a novel skeletal stem cell for tendon wound repair

Introduction:: Tendon injuries are the most common orthopedic disorders (up to 46% of musculoskeletal injuries)¹ and contribute to significant morbidity and healthcare costs, but the tissue tends to heal poorly^2,3. Although stem cell therapy has emerged as a promising modality for tissue repair⁴, little is known about the endogenous tendon stem cells. This paucity of information has hindered progress in therapies for tendon repair. We recently identified recruitment into injured mouse tendons of a new population of stem cells that, unlike previous studies, are not derived from tendon ensheathing tissues^5,6. Through inducible gene deletion, we find that most if not all of the recruited cells arise from Hoxa11 and Sox9 lineages. Interestingly, both markers overlap in the limb perichondrium/periosteum at postnatal stages, bringing us one step closer to identify their endogenous origin and molecular characteristics. Of note, recent studies have demonstrated the presence of Hoxa11-expressing skeletal stem cells in this region that contribute to bone growth and fracture healing^7,8. Here, we further delineate the Hoxa11-lineage origin for the recruited cells in acute tendon injury model and show that inhibition of the cell recruitment greatly impaired tendon wound healing, suggesting the importance of Hoxa11 stem cells in tendon wound repair.


Materials and Methods:: For all studies, 2-month-old adult mice underwent partial-width transection of the right Achilles tendon (without surgical repair) in accordance with IACUC guidelines. Mice underwent sham surgery were used as the uninjured control. Some mice also carry (a) Hoxa11GFP to mark periosteal skeletal stem cells⁷ and (b) Scleraxis-Cre;Rosa26-tdTomato to mark adult tendon cells⁵. Hoxa11CreERT⁸ mouse was used for tamoxifen-inducible lineage tracing and targeted gene deletion studies. Blockade of Hoxa11 stem cell recruitment was achieved by pharmacological (global) TGFβ inhibition and inducible genetic deletion of TGFβ signaling specifically in Hoxa11^lin cells (Tgfbr2^f/-;Hoxa11CreERT)⁵.


Results, Conclusions, and Discussions:: Results
Using the Hoxa11GFP reporter⁷ to mark periosteal Hoxa11 stem cells, we found a massive recruitment of Hoxa11GFP cells into the wound site following partial transection of mouse Achilles tendons (Fig.1a-c). Notably, once in the injured tendon, the recruited Hoxa11GFP cells expressed tendon markers collagen I and tenomodulin (Fig.1d,e), indicating their differentiation abilities and a possible involvement in tendon wound healing. We next determined if these cells persist in the healing tendon by lineage tracing. Briefly, Hoxa11CreERT;Rosa26-tdTomato mice were given tamoxifen i.p injection (2.5mg/mouse/injection) at days 3 and 2 prior to injury to initiate lineage labeling (Fig.2a). One month post-injury, the wound site was populated by a large number of Hoxa11-lineage (Hoxa11^lin) cells (Fig.2b), strongly suggesting the involvement of Hoxa11 stem cells in tendon healing. To determine the functional role of Hoxa11 stem cells, we next evaluated the impact of inhibition of stem cell recruitment on tendon healing (Fig.3). Excitingly, our preliminary results suggest that depletion of Hoxa11^lin cell recruitment significantly impaired Achilles tendon healing as indicated by collagen disorganization and a marked increase in small diameter immature collagen fibrils (Fig.4), suggesting the importance of Hoxa11 stem cells in tendon wound repair.

Conclusions
We identified a novel stem cell population of skeletal origin that plays an important role in extracellular matrix remodeling during tendon healing.

Discussions
Our findings highlight the exciting possibility that Hoxa11 cells may not only act as skeletal stem cells, but rather may have a broader implication in musculoskeletal (tendon) biology and healing. Intriguingly, Hoxa11 stem cells also contribute to ectopic bone formation in injured tendons⁹. Studies have shown that different populations of somatic stem cells within the musculoskeletal system may be closely related. For example, during bone regeneration, stem cells are mobilized not only from bone compartments, but also from adjacent skeletal muscle¹⁰. More recently, it was found that stem cells in periosteum and skeletal muscle share a common molecular response to bone injury¹¹. These findings, together with our identification of Hoxa11 stem cell recruitment in injured tendons, suggest their potential involvement in musculoskeletal repair and may open a new conceptual framework for the healing processes.


Acknowledgements (Optional): : This work was supported by the grants from NIH (R01AR055973) and Shriners Hospitals for Children (SHC 85410-POR-14).


References (Optional): : [1] Balvin et al. Front Vet Sci, 2015. [2] Sprague et al. Br J Sports Med, 2018. [3] Maffulli et al. Clin Sports Med, 2003. [4] Naji et al. Cell Mol Life Sci, 2019. [5] Tan et al. Stem Cell Reports, 2021. [6] Tan et al. GRC Musculoskeletal Biology and Bioengineering abstract, 2022. [7] Rux et al. Dev Cell, 2019. [8] Pineault et al. Nat Commun, 2019. [9] Pagani et al. Stem Cell Reports, 2021. [10] Julien et al. Nat Commun, 2021. [11] Julien et al. J Bone Miner Res, 2022.