Impaired Intramembranous Bone Formation during Bone Repair in the Absence of Tumor Necrosis Factor-Alpha Signaling
Authors: Gerstenfeld L.C. , Cho T.-J. , Kon T. , Aizawa T. , Cruceta J. , Graves B.D. , Einhorn T.A. 
Published: April 2003

Tumor necrosis factor-alpha (TNF-α) is known to mediate bone resorption; however, its role in osteogenesis has not been fully elucidated. In order to investigate the direct role of TNF-α signaling in the recruitment and differentiation of osteoblasts, two separate models of bone repair were used, marrow ablation and simple transverse fractures. These models were carried out in the tibiae of both wild-type and knock-out mice in which both TNF-α receptors (p55<sup>–/–</sup>/p75<sup>–/–</sup>) had been ablated. Marrow ablation is a unique model in which robust intramembranous bone formation is induced without an endochondral component, followed by remodeling and restoration of the original trabecular architecture of the bone marrow. In contrast, fracture repair proceeds concurrently through both endochondral and intramembranous processes of new bone tissue formation. In both models of bone repair, healing was delayed in the TNF-α receptor (p55<sup>–/–</sup>/p75<sup>–/–</sup>) deficient mice. In the marrow ablation model, young osteoblasts were recruited into the marrow space by day three in the wild-type mice, while the TNF-α (p55<sup>–/–</sup>/p75<sup>–/–</sup>) mice had only granulation tissue in the marrow cavity. Type I collagen and osteocalcin mRNA expressions were reduced ∼30 and ∼50%, respectively, of the control values in the TNF-α receptor ablated mice. In the fracture repair model there was almost a complete absence of the initial intramembranous bone formation on the periosteal surface in the TNF-α (p55<sup>–/–</sup>/p75<sup>–/–</sup>) mice. As healing progressed however, the callus tissues were greatly enlarged, and there was a delay in hypertrophy of the chondrocytes and the resorption of cartilage tissue. While during the initial period of fracture repair there was a marked reduction in the expression of both type I collagen and osteocalcin mRNAs in the TNF-α (p55<sup>–/–</sup>/p75<sup>–/–</sup>) mice, levels of these mRNAs were elevated by ∼10–20% over the wild type at the later time points in the absence of endochondral resorption of the callus. The lack of inhibition of osteogenesis during endochondral resorption suggests that a different set of signals are involved in the recruitment of osteogenic cells during endochondral repair then during intramembranous bone formation. Co-culture of chondrocytes with a mesenchymal stem cell line was carried out to examine if chondrocytes themselves produced paracrine factors that promote osteogenic differentiation. These experiments demonstrated that chondrocytes do indeed produce factors that promoted osteogenic differentiation. In summary, the results presented here suggest that TNF-α plays a crucial role in promoting postnatal bone repair through the induction of osteoprogenitor cell recruitment or osteogenic cell activation in the context of intramembranous bone formation. These results further suggest that the signals that promote osteogenesis during endochondral bone formation are different from those involved in intramembranous bone formation.