Notch-Wnt signal crosstalk regulates proliferation and differentiation of osteoprogenitor cells during intramembranous bone healing.

Adult bone regeneration is orchestrated by the precise actions of osteoprogenitor cells (OPCs). However, the mechanisms by which OPC proliferation and differentiation are linked and thereby regulated are yet to be defined. Here, we present evidence that during intramembranous bone formation OPC proliferation is controlled by Notch signaling, while differentiation is initiated by activation of canonical Wnt signaling. The temporospatial separation of Notch and Wnt signal activation during the early stages of bone regeneration suggests crosstalk between the two pathways. In vitro and in vivo manipulation of the two essential pathways demonstrate that Wnt activation leads to initiation of osteogenic differentiation and at the same time inhibits Notch signaling, which results in termination of the proliferative phase. Here, we establish canonical Wnt signaling as a key regulator that facilitates the crosstalk between OPC proliferation and differentiation during intramembranous, primary bone healing.

Temporary inhibition of the plasminogen activator inhibits periosteal chondrogenesis and promotes periosteal osteogenesis during appendicular bone fracture healing.

INTRODUCTION: Aminocaproic acid is approved as an anti-fibrinolytic for use in joint replacement and spinal fusion surgeries to limit perioperative blood loss. Previous animal studies have demonstrated a pro-osteogenic effect of aminocaproic acid in spine fusion models. Here, we tested if aminocaproic acid enhances appendicular bone healing and we sought to uncover the effect of aminocaproic acid on osteoprogenitor cells (OPCs) during bone regeneration. METHODS: We employed a well-established murine femur fracture model in adult C57BL/6J mice after receiving two peri-operative injections of aminocaproic acid. Routine histological assays, biomechanical testing and micro-CT analyses were utilized to assess callus volume, and strength, progenitor cell proliferation, differentiation, and remodeling in vivo. Two disparate ectopic transplantation models were used to study the effect of the growth factor milieu within the early fracture hematoma on osteoprogenitor cell fate decisions. RESULTS: Aminocaproic acid treated femur fractures healed with a significantly smaller cartilaginous callus, and this effect was also observed in the ectopic transplantation assays. We hypothesized that aminocaproic acid treatment resulted in a stabilization of the early fracture hematoma, leading to a change in the growth factor milieu created by the early hematoma. Gene and protein expression analysis confirmed that aminocaproic acid treatment resulted in an increase in Wnt and BMP signaling and a decrease in TGF-ß-signaling, resulting in a shift from chondrogenic to osteogenic differentiation in this model of endochondral bone formation. CONCLUSION: These experiments demonstrate for the first time that inhibition of the plasminogen activator during fracture healing using aminocaproic acid leads to a change in cell fate decision of periosteal osteoprogenitor cells, with a predominance of osteogenic differentiation, resulting in a larger and stronger bony callus. These findings may offer a promising new use of aminocaproic acid, which is already FDA-approved and offers a very safe risk profile.