Dysregulation of local stem/progenitor cells as a common cellular mechanism for heterotopic ossification

Abstract

Heterotopic ossification (HO), the abnormal formation of true marrow-containing bone within extraskeletal soft tissues, is a serious bony disorder that may be either acquired or hereditary. We utilized an animal model of the genetic disorder fibrodysplasia ossificans progressiva to examine the cellular mechanisms underlying HO. We found that HO in these animals was triggered by soft tissue injuries and that the effects were mediated by macrophages. Spreading of HO beyond the initial injury site was mediated by an abnormal adaptive immune system. These observations suggest that dysregulation of local stem/progenitor cells could be a common cellular mechanism for typical HO irrespective of the signal initiating the bone formation.

Figure 1

Muscle or skin injuries facilitate HO formation. (A–D): Typical x-ray images taken 3 weeks after cardiotoxin injection. (A): WT mouse injected with PBS. (B): WT mouse injected with CTX. (C): Nse-BMP4 mouse injected with PBS. (D): Nse-BMP4 mouse injected with CTX. White arrow in (D) points to the HO. (E, F): Hematoxylin and eosin (HE) staining of HO sections from CTX-injected mice (3 weeks after injection). (E): Low-power image demonstrates HO located within muscle and surrounded by fibroproliferative lesions. (F): Higher power image shows the prominent muscle degeneration, profound fibroproliferative lesions, and HO with marrow. (G, H): Seven days after CTX injection, Nse-BMP4 and WT mice showed drastically different histological features. (G): WT mice show significant muscle regeneration. Note the newly generated muscle cells that have centrally localized nuclei. (H): In contrast, Nse-BMP4 mice have profound fibroproliferative lesions with chondrocyte formation at this time point. (I, J): Typical x-ray images of WT and Nse-BMP4 mice taken 3 weeks after skin injuries. White arrow in (J) indicates HO at the injury site. (K, L): HE staining of the injury site from an Nse-BMP4 mouse (3 weeks after injury). (K): Low-power image shows the general morphology and the features of HO generated by skin injury. Black arrow indicates the injury site. Notice the fibroproliferative lesions, the chondrocyte formation, and the nascent trabeculae-like structure at one edge. (L): High-power image of the nascent trabeculae-like structure. (M): Quantitative summary of muscle and skin injuries experiments. Bar = 500 μm in (E) and (K). Bar = 100 μm in (F)–(H) and (L). Abbreviations: BMP, bone morphogenetic proteins; CTX, cardiotoxin; HO, heterotopic ossification; Nse, neuron-specific enolase; PBS, phosphate buffered saline; WT, wild type.

Figure 2

Macrophage depletion inhibits HO formation. (A): Western blotting shows that BMP4 is expressed by thioglycollate-elicited peritoneal macrophages and that the transgene expression level is higher in inbred Nse-BMP4 mice than in Nse-BMP4/CD11b-DTR double transgenic mice. Each lane represents an individual mouse. β-Actin acts as the loading control. (B): BMP4 protein expression normalized to actin expression in WT, Nse-BMP4/CD11b-DTR, and inbred Nse-BMP4 mice. (C): Effect of macrophage depletion and transgene dosage. The percentages of mice that developed HO at different time points are depicted. Nse-BMP4 mice with clodronate (CLO, heavy green line) or without (phosphate buffered saline (PBS), heavy black line), Nse-BMP4/CD11b-DTR with DT (light blue line) or without (PBS, light black line) are presented. All other control groups, including WT mice with CLO or without (PBS), CD11b-DTR single transgenic mice with DT or without (PBS), never generate HO and are depicted. The thickness of the lines is proportional to the level of BMP4 transgene expression. Comparing the heavy green line with the heavy black line reveals the inhibitory effect of dlodronate treatment. Comparing the light blue line with the light black line reveals the inhibitory effect of DT treatment. Comparing the heavy black line with the light black line reveals the transgene dosage effect. The horizontal line at the bottom indicates the period of the injection following the injuries. Abbreviations: *, differs from WT by ANOVA at p < .05; **, differs from WT by ANOVA at p < .01; BMP, bone morphogenetic proteins; CLO, clodronate; DT, diphtheria toxin; DTR, DT receptor; HO, heterotopic ossification; Nse, neuron-specific enolase; WT, wild type.

Figure 3

The adaptive immune system plays a key role in the spread of heterotopic ossification (HO). X-ray images (3 months after skin injury of right hind limb) demonstrate that the size of HO in Nse-BMP4/RAG(±) mice (A, B) is larger and spreads to a far remote location (left side) compared to that of Nse-BMP4;RAG(−/−) mice (C, D). White arrows indicate the HO. Abbreviations: BMP, bone morphogenetic proteins; Nse, neuron-specific enolase; RAG, recombination activating gene 1.

Figure 4

Five tested cre lines worked properly, but none of these cre-labeled cell populations made a significant contribution to heterotopic ossification. (A): Schematic mating and tracing strategy. (B, C): Typical image of X-gal staining of section from injured triple transgenic mice carrying CD19-cre with fast nuclear red counterstaining (A). Note that none of the cells in fibroproliferative lesions are LacZ positive, although in the same animal the CD19-cre mediated recombination worked properly in the spleen, which contains many LacZ positive cells in the germinal center (C). (D): Typical image of X-gal staining of section from injured triple transgenic mice carry Myf5-cre. Notice that none of the cells in fibroproliferative lesions was LacZ positive. Although the Myf5-cre mediated recombination worked efficiently in neighboring muscle cells. (E, F): Typical image of X-gal staining of section from injured triple transgenic mice carrying Lyz-cre (E). Note that no cells in fibroproliferative lesions are LacZ positive, although in the same animal the Lyz-cre mediated recombination worked in the spleen in which many cells in red pulp are LacZ positive. (F). (G): Typical image of X-gal staining of section from injured triple transgenic mice carrying Nestin-cre. Many subcutaneous muscle cells are LacZ positive (black), whereas there are no cells in the HO that are LacZ positive. Green or yellow florescence is autoflorescence, which is present in ossificated tissues and the surface of the skin. (H, I): Typical image of X-gal staining of section from injured triple transgenic mice carrying LCK-cre. (H): Note that no cells in fibroproliferative lesions are LacZ positive, although in the same animal the LCK-cre mediated recombination worked properly in the thymus in which many medullary cells are LacZ positive (I). Abbreviations: BMP, bone morphogenetic proteins; Nse, neuron-specific enolase.

Figure 5

Current working model. See Discussion section for the details.