Tibial pseudarthrosis causes substantial morbidity in patients with neurofibromatosis type 1 (NF1). We studied tibial pseudarthrosis tissue from patients with NF1 and found elevated levels of b-catenin compared to unaffected bone. To elucidate the role of b-catenin in fracture healing, we used a surgically induced tibial fracture model in conditional knockout (KO) Nfl (Nf1 flox/flox ) mice. When treated with a Cre-expressing adenovirus (Ad-Cre), there was a localized knockdown of Nf1 in the healing fracture and a subsequent development of a fibrous pseudarthrosis. Consistent with human data, elevated b-catenin levels were found in the murine fracture sites. The increased fibrous tissue at the fracture site was rescued by local treatment with a Wingless-type MMTV integration site (Wnt) antagonist, Dickkopf-1 (Dkk1). The murine pseudarthrosis phenotype was also rescued by conditional b-catenin gene inactivation. The number of colony-forming unit osteoblasts (CFU-Os), a surrogate marker of undifferentiated mesenchymal cells able to differentiate to osteoblasts, correlated with the capacity to form bone at the fracture site. Our findings indicate that the protein level of b-catenin must be precisely regulated for normal osteoblast differentiation. An up-regulation of b-catenin in NF1 causes a shift away from osteoblastic differentiation resulting in a pseudarthrosis in vivo. These results support the notion that pharmacological modulation of b-catenin can be used to treat pseudarthrosis in patients with NF1.-Ghadakzadeh, S., Kannu, P., Whetstone, H., Howard A., Alman, B. A. b-catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications. FASEB J. 30, 3227-3237 (2016). www.fasebj.orgNeurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by activated RAS signaling and mutations that dysregulate the NF1 protein (neurofibromin). NF1-related skeletal abnormalities are an important cause of morbidity with osteoporosis, scoliosis, and tibial dysplasia affecting more than half of affected individuals. Tibial dysplasia typically starts with anterolateral tibial bowing, characteristically progressing to a fracture that will not heal, termed a pseudarthrosis. Normal fracture healing is characterized by the deposition of new bone from osteoblasts, followed by a period of remodeling in which osteoclasts are active. At the tibial pseudarthrosis fracture site, however, is fibrous hamartoma tissue contain cells that do not undergo osteoblastic differentiation form. There is also an increase in osteoclastogenic activity at the fracture site, when compared to the healing in a normal fractured tibia (1-4). Because tibial pseudarthrosis is refractive to medical treatment, the only management options are surgical. For those continuing to experience chronic pain and impaired mobility, an amputation may be required if other treatments are not effective.Germline heterozygous mutations of NF1 cause NF1. In some but not all NF1-related tibial pseudarthrosis, a second hit to the NF1 gen...
Silencing gene expression through a sequence-specific manner can be achieved by small interfering RNAs (siRNAs). The discovery of this process has opened the doors to the development of siRNA therapeutics. Although several preclinical and clinical studies have shown great promise in the treatment of neurological disorders, cancers, dominant disorders, and viral infections with siRNA, siRNA therapy is still gaining ground in musculoskeletal tissue repair and bone regeneration. Here we present a comprehensive review of the literature to summarize different siRNA delivery strategies utilized to enhance bone regeneration. With advancement in understanding the targetable biological pathways involved in bone regeneration and also the rapid progress in siRNA technologies, application of siRNA for bone regeneration has great therapeutic potential. High rates of musculoskeletal injuries and diseases, and their inevitable consequences, impose a huge financial burden on individuals and healthcare systems worldwide.
Several types of serious bone defects would not heal without invasive clinical intervention. One approach to such defects is to enhance the capacity of bone-formation cells. Exogenous bone morphogenetic proteins (BMP) have been utilized to positively regulate matrix mineralization and osteoblastogenesis, however, numerous adverse effects are associated with this approach. Noggin, a potent antagonist of BMPs, is an ideal candidate to target and decrease the need for supraphysiological doses of BMPs. In the current research we report a novel siRNA-mediated gene knock-down strategy to down-regulate Noggin. We utilized a lipid nanoparticle (LNP) delivery strategy in pre-osteoblastic rat cells. In vitro LNP-siRNA treatment caused inconsequential cell toxicity and transfection was achieved in over 85% of cells. Noggin siRNA treatment successfully down-regulated cellular Noggin protein levels and enhanced BMP signal activity which in turn resulted in significantly increased osteoblast differentiation and extracellular matrix mineralization evidenced by histological assessments. Gene expression analysis showed that targeting Noggin specifically in bone cells would not lead to a compensatory effect from other BMP negative regulators such as Gremlin and Chordin. The results from this study support the notion that novel therapeutics targeting Noggin have the clinically relevant potential to enhance bone formation without the need for toxic doses of exogenous BMPs. Such treatments will undeniably provide safe and economical treatments for individuals whose poor bone repair results in permanent morbidity and disability.
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