Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon

Abstract

The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomato^fl/fl ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1^fl/fl ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1^fl/fl ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.

Keywords: Adult stem cells; Bone; Fibrodysplasia ossificans progressiva; Heterotopic ossification; Osteoblast; Progenitor cells; Skeleton; Tissue specific stem cells.

Figure 1

Scleraxis-lineage cells contribute to all phases of trauma-induced heterotopic ossification (tHO). (A) Scleraxis lineage (Scx-cre/ROSA26^mTmG) defines the Achilles Tendon (eGFP+) prior to injury; (B) Scleraxis lineage (Scx-cre/ROSA26^mTmG) defines both fibroproliferative (eGFP+; white arrow) and chondroid (eGFP+; yellow arrow) tHO after injury; (C) Experimental set up of tamoxifen induction in Scx-creERT2/tdTomato^fl/fl mice with burn/tenotomy (tHO) or BMP-induced models (bHO); (D) Localization of adult pre-injury scleraxis-lineage restricted cells (tdTomato+) cells in the fibroproliferative and (tdTomato+; white arrow), chondroid (tdTomato+; yellow arrow) cells of cartilaginous tHO and in the endosteal cells (tdTomato+; green arrow) of late-ossified tHO; (E) Quantification of the adult pre-injury scleraxis-lineage restricted fraction of in the fibroproliferative, chondroid, and endosteal cells in tHO.

Figure 2

Scleraxis-lineage cells defined in the uninjured adult (tdTomato+) contribute directly to the endochondral anlagen in both trauma-induced (tHO) and BMP-scaffold driven (bHO) heterotopic ossification. (A) Representative H&E demonstrating areas of tHO in Scx-creERT2/tdTomato^fl/fl mice; (B) Expression of SOX9 (green) by tdTomato+ cells in tHO; (C) Expression of OSX (green) by tdTomato+ cells in tHO; (D) Percent of Scx-creERT2 cells (tdTomato+) expressing SOX9 or OSX in tHO (5 high power fields); (E) Representative H&E demonstrating areas of bHO in Scx-creERT2/tdTomato^fl/fl mice; (F) Expression of SOX9 (green) by tdTomato+ cells in bHO; Expression of OSX (green) by tdTomato+ cells in bHO; (G) Percent of Scx-creERT2 cells (tdTomato+) expressing SOX9 or OSX in bHO (5 high power fields).

Figure 3

Scleraxis-lineage cells defined in the uninjured adult (Scx-creERT2/tdTomato^fl/fl) display mesenchymal cell markers in both trauma-induced (tHO) and BMP-scaffold (bHO) heterotopic ossification. (A) Scx-creERT2 cells (tdTomato+) express PDGFRα in tHO; (B) Scx-creERT2 cells (tdTomato+) express S100A4 in tHO; (C) Scx-creERT2 cells (tdTomato+) express SCA1 in tHO; (D) Percent of Scx-creERT2 cells (tdTomato+) which express mesenchymal markers in tHO (5 high power fields); (E) Scx-creERT2 cells (tdTomato+) express PDGFRα in bHO; (F) Scx-creERT2 cells (tdTomato+) express S100A4 in bHO; (G) Scx-creERT2 cells (tdTomato+) express Sca1 in bHO; (H) Percent of Scx-creERT2 cells (tdTomato+) expressing mesenchymal markers in bHO (5 high power fields).

Figure 4

Scleraxis lineage-restricted caACVR1 expression causes tendon and intramuscular HO. (A) Schematic showing spontaneous HO generation by scleraxis-lineage cells expressing caACVR1 (Scx-cre/caACVR1^fl/wt) and by pre-injury scleraxis-lineage cells after cardiotoxin injury (Scx-creERT2/caACVR1^fl/wt); (B) Photograph of Scx-cre/caACVR1^fl/wt mouse showing ectopic bone formation at the hindlimb; (C) Whole body 3D MicroCT reconstruction confirming ectopic bone formation at the hindlimbs Scx-cre/caACVR1^fl/wt mouse; (D) Axial, sagittal, and serial cross sections of microCT showing ectopic bone at the distal Achilles’ tendon; (E) 3D MicroCT reconstruction showing HO in the hamstring muscle of Scx-creERT2/caACVR1^fl/wt mice induced with tamoxifen and later injected with cardiotoxin with serial cross sections; H&E, Alcian blue, and Alizarin red staining of HO after cardiotoxin injection in non-decalcified sections of intramuscular HO in Scx-creERT2/caACVR1^fl/wt mouse; (F) immunofluorescent staining for scleraxis in muscle of Scx-creERT2/caACVR1^fl/wt mice in the presence or absence of cardiotoxin.