Gdf5 progenitors give rise to fibrocartilage cells that mineralize via hedgehog signaling to form the zonal enthesis

Abstract

The sequence of events that leads to the formation of a functionally graded enthesis is not clearly defined. The current study demonstrates that clonal expansion of Gdf5 progenitors contributes to linear growth of the enthesis. Prior to mineralization, Col1+ cells in the enthesis appose Col2+ cells of the underlying primary cartilage. At the onset of enthesis mineralization, cells at the base of the enthesis express alkaline phosphatase, Indian hedgehog, and ColX as they mineralize. The mineralization front then extends towards the tendon midsubstance as cells above the front become encapsulated in mineralized fibrocartilage over time. The hedgehog (Hh) pathway regulates this process, as Hh-responsive Gli1+ cells within the developing enthesis mature from unmineralized to mineralized fibrochondrocytes in response to activated signaling. Hh signaling is required for mineralization, as tissue-specific deletion of its obligate transducer Smoothened in the developing tendon and enthesis cells leads to significant reductions in the apposition of mineralized fibrocartilage. Together, these findings provide a spatiotemporal map of events – from expansion of the embryonic progenitor pool to synthesis of the collagen template and finally mineralization of this template – that leads to the formation of the mature zonal enthesis. These results can inform future tendon-to-bone repair strategies to create a mechanically functional enthesis in which tendon collagen fibers are anchored to bone through mineralized fibrocartilage.

Coordinated development of the limb musculoskeletal system: Tendon and muscle patterning and integration with the skeleton

“…Recent studies showed that enthesis fibrocartilage cells are specified during embryonic stages, beginning at E14.5–15.5. The fibrocartilage transitional zone of the enthesis emerges from a specialized subpopulation of cells (likely derived from the Scx/Sox9 pool) that can be detected based on their hedgehog signaling activity (Gli1+) (Dyment et al, 2015; Schwartz et al, 2015). Gli1+ cells specified during embryonic stages ultimately give rise to the entire mature enthesis.…”

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

“…This step is critical to fix the soluble cytoplasmic GFP of transgenic animals within the limits of the cell. We have utilized the cryotape for a wide range of tissue types [5][6][7][8][9][10][11][12][13] . Laboratory personnel with limited histological experience can produce high-quality sections with this method.…”

“…Recently, we have published a new high-throughput cryohistological method for assessing several response measures within a given section from mineralized tissue [5][6][7][8][9][10][11][12][13][14] . The process involves stabilizing the cryosection with frozen cryotape, adhering the taped section rigidly to a microscope slide, and conducting several rounds of staining and imaging on each section.…”

The role of bone sialoprotein in the tendon–bone insertion

“…The distribution of mineral and orientation of the collagen fibers in the enthesis has been shown to influence its mechanical properties [19,24,25,26,27]. Recent studies have indicated that the Indian Hedgehog signaling pathway is involved in enthesis development and mineralization of the CFC, in part because its ablation results in decreased mineralization and less collagen imbedded in the mineralized tissues [28,29,30,31]. Similarly, alkaline phosphatase, whose expression is correlated to mineral formation, is expressed by the differentiated fibrocartilaginous cells [29].…”