Researchers have found that dormant progenitor cells hiding in skeletal muscle migrate to fracture sites and turn into bone-forming osteoblasts. The findings from a mouse study are published in Bone Research. The discovery identifies fibroadipogenic progenitors as a druggable target that could speed fracture healing or curb pathological bone growth after severe trauma.
Bone healing has long been framed as a project run largely by the skeleton itself: periosteal cells, marrow stromal cells and resident osteoblasts doing the heavy lifting while surrounding soft tissue plays a supporting role. A study led by Dr Ugur M. Ayturk, an Assistant Scientist in the Musculoskeletal Integrity Program at Hospital for Special Surgery and an Assistant Professor of Physiology and Biophysics in Orthopaedic Surgery at Weill Cornell Medicine, USA, complicates that picture. Using a new genetic tracing tool, the team shows that a population of cells resident in skeletal muscle, and largely inactive under normal conditions, is mobilised after fracture and becomes a substantial source of the osteoblasts that rebuild damaged bone.
Speaking about the findings, Dr Ayturk said the results show that “extra-skeletal cells, particularly FAPs [fibroadipogenic progenitors], recruited to help repair bone fractures and could represent a promising therapeutic target to enhance fracture healing.” Because the same cells also drive heterotopic ossification, the abnormal bone growth that can follow blast injuries, burns and major orthopaedic trauma, the work points towards a single cell population that might eventually be pharmacologically dialled up to accelerate union in slow-healing fractures, or dialled down to prevent unwanted bone forming in soft tissue.
Tracking a hidden progenitor pool
FAPs reside within skeletal muscle and had previously been characterised chiefly for their role in muscle maintenance and fatty degeneration after injury. Superficial periosteal cells occupy the connective tissue layer covering the outer bone surface.
Both populations had been suspected of contributing to skeletal repair, but tools to track them with precision were lacking, since markers such as Pdgfra and Ly6a/Sca1 are expressed broadly across mesenchymal and even some haematopoietic lineages.
To solve this specificity problem, the researchers identified Clec3b, the gene encoding the secreted protein tetranectin, as a marker uniquely restricted to these Pdgfra+ Ly6a/Sca1+ cells. They engineered a tamoxifen-inducible Clec3b.CreERT2 mouse line that permanently labels Clec3b-expressing cells with a fluorescent tag, allowing their fate to be followed over weeks and months. Under baseline conditions, Clec3b-lineage cells were found almost exclusively in muscle and the outer periosteum, and were entirely absent from bone marrow, the endosteum and growth plates. The authors write in the paper that Clec3b-expression “marks FAPs and superficial periosteal cells, which are osteogenically dormant under normal conditions but can be recruited to assist with the repair of bone fractures.”
That dormancy ended abruptly after injury. Following stabilised femoral fracture surgery, labelled cells accumulated progressively in the fibrotic callus over the first ten to fourteen days, then began expressing osteoblast markers. By 21 days, an average of 28% of osteocalcin-positive osteoblasts in the healing callus were of Clec3b lineage, a figure the authors suggest may be an underestimate given the inefficiency of tamoxifen-driven recombination. A subset of labelled cells also converted into bone marrow stromal cells, contributing to the reconstruction of the marrow cavity within the newly formed bone. Notably, cells switched off Clec3b expression as they differentiated, indicating the marker specifically tags the quiescent progenitor state rather than the mature osteoblast.
Muscle, not periosteum, supplies the cells
A central question was which tissue, muscle or periosteum, contributes more to this regenerative response. Several experiments converged on muscle as the dominant source. When the periosteum was surgically stripped before a drill-hole injury was created, Clec3b-lineage cells still migrated to the wound and differentiated into osteoblasts, osteocytes and marrow stromal cells, demonstrating that periosteal tissue was not required for their recruitment. Conversely, in vivo mechanical loading of the tibia, a stimulus known to activate periosteal osteogenesis without causing injury, failed to produce any labelled osteocytes.
The most direct test came from bone graft transplantation. Femoral grafts taken from labelled donor mice, with or without their surrounding muscle intact, were implanted into femoral defects in unlabelled littermates. Grafts that retained muscle produced significantly more labelled cells in the resulting callus than grafts without muscle, and both graft types went on to form osteoblasts and osteocytes bearing the Clec3b lineage tag. Together with single-cell RNA sequencing data confirming the emergence of osteoblast and stromal transcriptional signatures from the labelled population, the findings indicate that “Clec3b+ FAPs rather than Clec3b+ periosteal cells are mobilized to become
osteoblasts,” as the authors put it in their abstract, at least under the fracture and BMP2 (bone morphogenetic protein 2)-stimulation conditions tested.
A double-edged role in heterotopic ossification
The same muscle-resident cells that assist fracture repair also participate in heterotopic ossification, the ectopic bone formation that can complicate blast wounds, spinal cord injury and severe soft-tissue trauma. Intramuscular injection of recombinant human BMP2 produced dense ectopic bone nodules in which Clec3b-lineage cells were abundant, many having become mature osteoblasts and osteocytes. A tendon-injury model, created by severing the Achilles tendon, produced similar ectopic mineralisation around the calcaneus and ankle joint, again populated by labelled cells, some of which had passed through a cartilage intermediate.
Depletion experiments reinforced the functional importance of this population. Using a diphtheria toxin receptor system to selectively ablate Clec3b-lineage cells during fracture healing, the researchers achieved a 65% reduction in labelled cells and observed significantly lower callus bone volume and a trend towards reduced bone volume fraction, without any change in overall callus size. Genetic deletion of ß-catenin, the master transcription factor of the WNT signalling pathway known to govern osteoprogenitor activation, produced a comparable reduction in mineralisation. Dr Ayturk summarised the result: “When we blocked a key pathway required for bone formation or depleted these cells, both fracture healing and abnormal bone growth were significantly reduced. This finding highlights their important role in both processes.”
The relationship with heterotopic ossification proved more nuanced. Depleting Clec3b+ cells before BMP2 injection reduced the total volume of ectopic bone, but continuous depletion after BMP2 delivery had the opposite effect, trending towards increased bone mass. The authors interpret this as evidence that ablating the primary progenitor pool, once mineralisation is already under way, mobilises a compensatory population of unlabelled cells rather than halting ossification outright, a distinction with direct bearing on how any future intervention would need to be timed relative to injury.
Distinct behaviour and open questions
Clec3b-lineage cells also behaved differently from previously characterised skeletal stem and progenitor populations, such as those marked by Ctsk, Gli1 or Acta2/ aSMA. During fracture repair, fewer than 10% of Sox9-positive chondrocytes in the cartilaginous callus derived from the labelled lineage, indicating minimal contribution to cartilage formation in that context. Yet the same cells readily formed cartilage when stimulated by BMP2 in the heterotopic ossification model, and in ex vivo pellet culture, showing that their chondrogenic capacity is context-dependent rather than absent altogether. The authors note this discrepancy may reflect the differing molecular cues present in fracture versus BMP2-driven environments, and flag it as an area requiring further mechanistic dissection.
The authors conclude that “Clec3b+ cells (and likely their FAP subset) are important contributors to fracture healing in a WNT-dependent manner,” and that “understanding the pathways that regulate Clec3b+ cells and their fate decisions may suggest new strategies to treat patients suffering from musculoskeletal trauma.” For clinicians managing delayed union, nonunion or post-traumatic heterotopic bone, the work offers a specific cellular target, sitting in muscle rather than bone, that had not previously been isolated with this degree of precision.
Reference:
Aydin, E., Moore, J. A., Bubnovich, M., et al. (2026). Clec3b+ extraskeletal cells regulate fracture healing and heterotopic ossification. Bone Research, 14, 70. https://doi.org/10.1038/s41413-026-00532-6
