(A) Two months after implantation, the bone mineral density (BMD) of heterotopic ossification implants was analyzed by 3DVR software. Cross-section CT images and three-dimensional reconstruction images scanned by CBCT showed that si-JNK attenuated the bone mineral density (BMD) and volume of radiographic ectopic calcification in the musculature of mice compared to the BMP9 transfection group. (B) Quantitative analysis of BMD of ossification area in implanted region from 3D images reconstructed by an i-CAT system. n = 12; ‘‘*”, p<0.01 (vs. control group); ‘‘#”, p<0.01 (vs. BMP9 group). (C) H&E staining of the sections of the implants suggested that two months after transplantation, immature woven trabecular bone containing blood vessels, bone marrow tissues, osteocytes, and osteoblasts, were formed in the BMP9 transfection group, in which both endochondral ossification and subperiosteal ossification were involved in the formation of intramuscular ectopic bone. Gene silencing of JNKs inhibited osteogenic differentiation and mineralization of hPDLSCs stimulated by BMP9 in vivo. There were few new bone-like tissues consisting of bone matrix and osteoblasts, and more chondrocytes present in the si-JNK group compared to the BMP9 group.