AAV-BMP4 induces heterotopic ossification in nude mouse The gelatin sponge adsorbed with the 15 μL AAV-BMP4 virus solution was embedded in the skeletal muscle pocket of the nude mouse bilaterally. In 2 weeks, the heterotopic ossification was established locally (Figure 2A–C). The process of heterotopic ossification was observed through endochondral bone formation (Figure 2D–F).  "

BMP4 induces the disintegration of multinuclear myotubes and promotes the proliferation of mononuclear myogenic cells  After C2C12 formed myotubes on Day 9 (Figure 3A–E), BMP4 treatment led to the disintegration of multinuclear myotubes, but promoted the proliferation of mononuclear cells (Figure 3F–J, Figure 4). In addition, according to our previous studies, BMP4 also promotes the osteogenesis of myogenic cells[23]."

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Firborgenic cells displayed more prone to chondrogenic differentiation, while myognic cells showed more readily to osteogenic differentiation  As shown in Figure 5A–B, mixed cells of FDC and L6 in various ratios of cell number displayed differential chondrogenic potential under the treatment of BMP4. The more FDC fibrogenic cells in the mixed cells, the more positive alcian blue and safarin O staining it had (Figure 5D–E). Mixed cells of FDC and L6 in various ratios of cell number displayed differential osteogenic potential under the treatment of BMP4. As shown in Figure 5C–F, the more L6 myogenic cells in the mixed cells, the more positive ALP staining it had. "

Tie2+ cells are not progenitors for heterotopic ossification in our study Next, we investigated the fate of endothelial cells during heterotopic ossification. Tie2+ is predominantly expressed by vascular endothelial cells, and is widely used as a marker for endothelial cells[23]. Tie2-Cre transgenic mice have been used by several scientists for lineage tracing to understand the progression of heterotopic ossification[24]; they reported that Tie2+ cells could differentiate into chondrocytes or osteocytes during heterotopic ossification induced by BMP4. However, different conclusions were reached in our study. In our experiment, FVB/N-TgN (TIE2-LacZ) 182Sato mice was purchased from The Jackson Laboratory (Bar Harbor, ME) and used express β-galactosidase driven by the endothelial-specific TIE2 promoter. As Tie2-Cre transgenic mice were reported to have nonspecific recombination, the FVB/N-TgN (TIE2-LacZ) 182Sato mouse was used to study the role of Tie2+ cells in the heterotopic ossification process. We embedded the AAV-BMP4 sponge into the muscle of the FVB/N-TgN (TIE2-LacZ) 182Sato transgenic mice. On days 10 and 14, the ectopic bone formed in the muscle was harvested for X-gal staining (Figure 6). Without AAV-BMP4 injection, endothelial cells were distributed between the myofibers (Figure 6A). Round staining indicated that these Tie2+ cells were distributed in the blood vessel. After 10 days of induction, chondrocytes were observed in the muscles (Figure 6B). However, these chondrocytes were not stained with X-gal, indicating that Tie2+ cells were not the original cells for chondrocytes in heterotopic ossification. However, we observed X-gal-stained cells along the chondrocytes and between the myofibers. After 14 days of induction, bone trabeculae were observed (Figure 6C); however, there were no Tie2+ cells present, although stained cells were observed in the bone marrow-like region. Overall, using the transgenic mouse, we found that endothelial cells, which were Tie2 positive, were not progenitors for heterotopic ossification, but were involved in the process. "