Hypoxia and Reactive Oxygen Species Homeostasis in Mesenchymal Progenitor Cells Define a Molecular Mechanism for Fracture Nonunion

Abstract

Fracture nonunion is a major complication of bone fracture regeneration and repair. The molecular mechanisms that result in fracture nonunion appearance are not fully determined. We hypothesized that fracture nonunion results from the failure of hypoxia and hematoma, the primary signals in response to bone injury, to trigger Bmp2 expression by mesenchymal progenitor cells (MSCs). Using a model of nonstabilized fracture healing in transgenic 5 0 Bmp2BAC mice we determined that Bmp2 expression appears in close association with hypoxic tissue and hematoma during the early phases of fracture healing. In addition, BMP2 expression is induced when human periosteum explants are exposed to hypoxia ex vivo. Transient interference of hypoxia signaling in vivo with PX-12, a thioredoxin inhibitor, results in reduced Bmp2 expression, impaired fracture callus formation and atrophic-like nonunion by a HIF-1a independent mechanism. In isolated human periosteum-derived MSCs, BMP2 expression could be induced with the addition of platelets concentrate lysate but not with hypoxia treatment, confirming HIF-1a-independent BMP2 expression. Interestingly, in isolated human periosteum-derived mesenchymal progenitor cells, inhibition of BMP2 expression by PX-12 is accomplished only under hypoxic conditions seemingly through dis-regulation of reactive oxygen species (ROS) levels. In conclusion, we provide evidence of a molecular mechanism of hypoxia-dependent BMP2 expression in MSCs where interference with ROS homeostasis specifies fracture nonunion-like appearance in vivo through inhibition of Bmp2 expression. STEM CELLS 2016;34:2342-2353 SIGNIFICANCE STATEMENTFracture nonunion is a major cause of chronic pain and disability. However, the molecular mechanisms that result in impaired fracture healing are not yet determined. The regenerative capability of bone tissue resides in the mesenchymal progenitor cells (MSCs) that can differentiate into chondrocytes and osteoblasts in response to several stimuli derived of bone injury. Here we demonstrate that fracture-healing initiation is highly dependent on the management by MSCs of hypoxiaderived intracellular oxidative stress. This study identifies a mechanism for fracture nonunion appearance that may allow the prevention and non-surgical treatment of fracture nonunions.

Modulation of Synovial Fluid‐Derived Mesenchymal Stem Cells by Intra‐Articular and Intraosseous Platelet Rich Plasma Administration

“…Mesenchymal lineage differentiation assays were carried out as described in Muinos-López et al 2016 [33]. Briefly, SF-derived cells were assessed between passages 2 and 5 to confirm their osteogenic, adipogenic, and chondrogenic capacity.…”

Redox regulation in regenerative medicine and tissue engineering: The paradox of oxygen

“…In the heart, Nrf2 prevents pathological hypertension‐induced cardiac remodelling and concomitant heart failure (Zhou, Sun, Zhang, & Zheng, ) and is essential for cardiac repair after injury (Tao et al, ). Finally, Nrf2 upregulation is detected during bone fracture repair in rats (Komatsu & Hadjiargyrou, ), and inhibition of thioredoxin, one of the essential endogenous antioxidant systems mediated by Nrf2, reduced fracture healing (Muinos‐Lopez et al, ). All in all, targeting Nrf2 is a promising strategy to enhance the regenerative capacity of numerous tissues.…”

Stimulating Fracture Healing in Ischemic Environments: Does Oxygen Direct Stem Cell Fate during Fracture Healing?

“…From these experiments, we concluded that oxygen levels were important for fracture healing generally. Indeed, others have shown that transient hypoxia may actually stimulate fracture repair (Muinos-Lopez et al, 2016). …”