Immunolocalization of BMPs, BMP antagonists, receptors, and effectors during fracture repair

Abstract

Bone morphogenetic proteins (BMPs) are potent bone inducers used clinically to enhance fracture repair. BMPs have been shown to be produced in the fracture callus; however, the comparative expression of BMPs and BMP signaling components has only been partially examined at the cellular level. The aim of the present study was to establish a detailed spatiotemporal localization of BMPs and BMP signaling components in mouse models of stabilized and nonstabilized fractures. During healing of nonstabilized fractures, which occurs via endochondral ossification, BMP2, 3, 4, 5, and 8, noggin, BMPRIA, BMPRII, and pSmad 1/5/8 were immunolocalized in the activated periosteum as early as 3 days after fracture. BMP2, 4, 5, 6, 7, and 8 and noggin were also found in isolated inflammatory cells within granulation tissue during the early stages of repair, but not BMP receptors and effectors. During the soft callus phase of repair, all BMPs and BMP signaling components were detected in chondrocytes with various intensities of staining depending on the stage of chondrocyte differentiation and their location in the callus. The strongest staining was observed in hypertrophic chondrocytes with decreased intensity during the hard callus phase of repair. All BMPs and components of the BMP pathway were detected in osteoblasts and osteocytes within new bone, with strongest intensity of immunoreaction reported during the early soft callus phase followed by decreasing intensity during the hard callus phase of repair. Most components of the BMP pathway were also detected in endothelial cells associated with new bone. In stabilized fractures that heal strictly via intramembranous ossification, BMPs and BMP antagonists were detected in isolated inflammatory cells and BMP signaling components were not detectable in osteoblasts or osteocytes within new bone. In conclusion, the BMP signaling pathway is primarily activated during fracture healing via endochondral ossification, suggesting that this pathway may influence the mode of healing during the recruitment of skeletal progenitors.

Introduction

Fracture healing involves the synchronized effects of many local and systemic regulatory factors including growth and differentiation factors, and extracellular matrix components [1], [2], [3]. The interaction of these factors causes undifferentiated mesenchymal cells to migrate, proliferate, and differentiate at the fracture site. The recruitment of stem cells in response to these growth factors is a key step to assure the success of bone regeneration. In a significant amount of cases, however, impaired healing requires surgical intervention combined with the implantation of osteoconductive materials and osteoinductive factors [4], [5]. Bone morphogenetic proteins (BMPs), which belong to the TGF-β superfamily, play important roles in skeletogenesis and are now used as therapeutic agents for treating skeletal diseases and injuries [6], [7], [8]. Since their clinical efficacy is sometimes limited, we would benefit from a better understanding of BMP functions during fracture healing. Functional and expression analyses of BMPs and their inhibitors have been reported [9], [10], [11], [12], but we still lack a detailed localization of BMP signaling components during fracture repair.

BMP signaling is mediated through serine–threonine transmembrane receptors (BMPR) type I and type II, which subsequently phosphorylate the receptor-regulated Smads (R-Smads) 1, 5, and 8 [13], [14], [15]. Once activated, the R-Smads undergo heterodimerization with Smad 4 (co-Smad) in the cytoplasm and the complex translocates into the nucleus, where it regulates gene transcription. BMP signal transduction is regulated by the inhibitory Smads, which are potent intracellular antagonists that inhibit BMP signaling at different levels in the cell [13], [14], [16]. BMP signaling is also influenced by extracellular antagonists such as noggin, which bind to BMPs and prevent interaction with receptors [17], [18]. Alternatively, BMP3 is an antagonist, which inhibits BMP2 activity by activating TGF-β/activin pathway [19], [20]. To better relate the in vivo functions of BMPs during fracture repair with the cellular localization of BMPs and BMP signaling components during the course of healing, we performed a detailed spatiotemporal localization of BMPs (2, 4, 5, 6, 7, 8), BMP extracellular antagonists (noggin, BMP3), BMP type I (BMPRIA, BMPRIB), and BMP type II (BMPRII) receptors, as well as the activated form of BMP receptor-regulated Smads (pSmad 1-5-8) during fracture healing in mice. Since the balance between chondrogenesis and osteogenesis in the fracture callus depends on the mechanical environment, we also examined if activation of the BMP pathway differs in mechanically stable and unstable fractures.