Abstract
Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.
Highlights
bone morphogenetic protein (BMP) have been found to regulate processes as disparate as embryonic dorso-ventral patterning, neuronal differentiation, cardiomyogenesis, thymocyte differentiation, and cranial suture fusion (6 –10)
Evaluation of BMP Signaling—The effects of noggin suppression on endogenously produced BMP activity were evaluated by quantitative real-time RT-PCR (QRT-PCR) analysis of the signaling intermediates Smad1 and 5
Transcript levels in cells expressing noggin-directed small interfering RNA (siRNA) constructs, a control siRNA, or cells undergoing vehicle only sham infection were compared with baseline Smad expression in unperturbed MC3T3-E1 preosteoblasts
Summary
Validation of Noggin siRNA Constructs—Early passage MC3T3-E1 preosteoblasts and first passage primary calvarial osteoblasts were expanded on 12-well tissue culture plates and grown to subconfluence prior to retroviral infection. Noggin-directed siRNA constructs demonstrating significant transcript suppression by QRT-PCR analysis were evaluated for protein suppression in MC3T3-E1 preosteoblasts and primary calvarial osteoblasts. Transcript levels in cells expressing noggin-directed siRNA constructs, a control siRNA, or cells undergoing vehicle only sham infection were compared with baseline Smad expression in unperturbed MC3T3-E1 preosteoblasts. Smad transcript levels were determined in a separate group of cells cultured in standard growth media supplemented with 1 ng/ml rhBMP-4 (R & D Systems) for 24 h. MC3T3-E1 preosteoblasts infected with a control GFP-targeted siRNA construct demonstrated no significant change in noggin level when compared with controls. Only siRNA constructs 1 and 2 were selected for further analyses
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