Kuntz, A. F. | Search bioRxiv

bioRxiv2022

Targeting the Hedgehog Signaling Pathway to Improve Tendon-to-Bone Integration

ObjectiveWhile the role of Hedgehog (Hh) signaling in promoting zonal fibrocartilage production during development is well-established, whether this pathway can be leveraged to improve tendon-to-bone repair in adults is unknown. Our objective was to genetically and pharmacologically stimulate the Hh pathway in cells that give rise to zonal fibrocartilaginous attachments to promote tendon-to-bone integration. DesignHh signaling was stimulated genetically, via constitutive Smo (SmoM2 construct) activation of activated bone marrow stromal cells, or pharmacologically, via systemic agonist delivery, to mice following anterior cruciate ligament reconstruction (ACLR). To assess tunnel integration, we measured mineralized fibrocartilage (MFC) formation in these mice 28 days post-surgery and performed tunnel pullout testing. ResultsHh pathway-related genes increased in cells forming the zonal attachments in WT mice. Both genetic and pharmacologic stimulation of the Hh pathway increased MFC formation and integration strength 28 days post-surgery. We next conducted studies to define the role of Hh in specific stages of the tunnel integration process. We found Hh agonist treatment increased proliferation of the progenitor pool in the first week post-surgery. Additionally, genetic stimulation led to continued MFC production in later stages of the integration process. These results indicate that Hh signaling plays an important biphasic role in cell proliferation and differentiation towards fibrochondrocytes following ACLR. ConclusionThis study reveals a biphasic role for Hh signaling during the tendon-to-bone integration process after ACLR. In addition, the Hh pathway is a promising therapeutic target to improve tendon-to-bone repair outcomes.

bioengineering↗

bioRxiv2019

Bone marrow stromal cells from a GDF5 origin produce zonal tendon-to-bone attachments following anterior cruciate ligament reconstruction

Following anterior cruciate ligament (ACL) injury, the ligament is often reconstructed with a tendon graft passed through bone marrow tunnels. This procedure results in a staged repair response where cell death occurs in the tendon graft, the graft is repopulated by host cells outside the graft, and tendon-to-bone attachments form at the graft/bone interface. While this healing process is well appreciated, the biological mechanisms that regulate it including the cellular origin of the repair response is poorly understood. Embryonic progenitor cells expressing growth and differentiation factor 5 (GDF5) give rise to several mesenchymal tissues in the joint and epiphyses. Therefore, we hypothesized that cells from a GDF5 origin, even in the adult tissue, would give rise to cells that contribute to the stages of repair following ACL reconstruction. ACLs were reconstructed in Gdf5Cre;R26R-tdTomato lineage tracing mice to monitor the contribution of Gdf5Cre;tdTom+ cells to graft revitalization and examine the extent to which these cells are capable of creating mineralized attachments within the bone tunnels. Anterior-posterior drawer tests were used to establish the stability of the knee following the procedure and demonstrated 58% restoration in anterior-posterior stability. Following reconstruction, Gdf5Cre;tdTom+ cells within the bone marrow expanded by 135-fold compared to intact controls in response to the injury. These cells migrated to the tendon graft interface, repopulated regions of the graft, and initiated tendon-to-bone attachments. These cells continued to organize and mature the attachments yielding a zonal insertion site at 4 weeks with collagen fibers spanning across unmineralized and mineralized fibrocartilage and anchored to adjacent bone. The zonal attachment possessed organized tidemarks with concentrated alkaline phosphatase activity similar to normal tendon or ligament entheses. This study established that mesenchymal cells from a GDF5 origin contribute to the creation of zonal tendon-to-bone attachments within bone tunnels following ACL reconstruction. Future studies will target this cell population to modulate the repair response in order to better understand key biological mechanisms that regulate tendon-to-bone repair.

bioengineering↗