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Combined MEK inhibition and BMP2 treatment promotes osteoblast differentiation and bone healing in Nf1Osx -/- mice.

ABSTRACT: Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase-activating protein neurofibromin. Non-bone union after fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hematopoietic cells to TGF?. However, clinically relevant pharmacological approaches to augment bone union in these patients remain limited. In this study, we report the generation of a novel conditional mutant mouse line used to model NF1 pseudoarthrosis, in which Nf1 can be ablated in an inducible fashion in osteoprogenitors of postnatal mice, thus circumventing the dwarfism associated with previous mouse models where Nf1 is ablated in embryonic mesenchymal cell lineages. An ex vivo-based cell culture approach based on the use of Nf1(flox/flox) bone marrow stromal cells showed that loss of Nf1 impairs osteoprogenitor cell differentiation in a cell-autonomous manner, independent of developmental growth plate-derived or paracrine/hormonal influences. In addition, in vitro gene expression and differentiation assays indicated that chronic ERK activation in Nf1-deficient osteoprogenitors blunts the pro-osteogenic property of BMP2, based on the observation that only combination treatment with BMP2 and MEK inhibition promoted the differentiation of Nf1-deficient osteoprogenitors. The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1osx (-/-) mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol-based delivery method. Collectively, these results provide novel evidence for a cell-autonomous role of neurofibromin in osteoprogenitor cells and insights about a novel targeted approach for the treatment of NF1 pseudoarthrosis.

SUBMITTER: de la Croix Ndong J

PROVIDER: S-EPMC4280331 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Combined MEK inhibition and BMP2 treatment promotes osteoblast differentiation and bone healing in Nf1Osx -/- mice.

de la Croix Ndong Jean J Stevens David M DM Vignaux Guillaume G Uppuganti Sasidhar S Perrien Daniel S DS Yang Xiangli X Nyman Jeffry S JS Harth Eva E Elefteriou Florent F Elefteriou Florent F

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20150101 1

Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase-activating protein neurofibromin. Non-bone union after fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hemat ...[more]

PMID: 25043591

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Project description:Critical-sized cranial bone defect remains a great clinical challenge. With advantages in regenerative medicine, injectable hydrogels incorporated with bioactive molecules show great potential in promoting cranial bone repair. Recently, we developed a dual delivery system by sequential release of bone morphogenetic protein 2 (BMP2) followed by insulin-like growth factor 1 (IGF1) in microparticles (MPs), and an injectable alginate/collagen (alg/col)-based hydrogel. In this study, we aim to evaluate the effect of dual delivery of BMP2 and IGF1 in MPs through the injectable hydrogel in critical-sized cranial bone defect healing. The gelatin MPs loaded with BMP2 and poly(lactic-co-glycolic acid)-poly(ethylene glycol)-carboxyl (PLGA-PEG-COOH) MPs loaded with IGF1 were prepared, respectively. The encapsulation efficiency and release profile of growth factors in MPs were measured. A cranial defect model was applied to evaluate the efficacy of the dual delivery system in bone regeneration. Adult Sprague Dawley rats were subjected to osteotomy to make an ⌀8-mm cranial defect. The injectable hydrogel containing MPs loaded with BMP2 (2 μg), IGF1 (2 μg), or a combination of BMP2 (1 μg) and IGF1 (1 μg) were injected to the defect site. New bone formation was evaluated by microcomputed tomography, histological analysis, and immunohistochemistry after 4 or 8 weeks. Data showed that dual delivery of the low-dose BMP2 and IGF1 in MPs through alg/col-based hydrogel successfully restored cranial bone as early as 4 weeks after implantation, whose effect was comparable to the single delivery of high-dose BMP2 in MPs. In conclusion, this study suggests that dual delivery of BMP2 and IGF1 in MPs in alg/col-based hydrogel achieves early bone regeneration in critical-sized bone defect, with advantage in reducing the dose of BMP2. Impact Statement Sequential release of bone morphogenetic protein 2 (BMP2) followed by insulin-like growth factor 1 (IGF1) in two different microparticles promotes critical-sized bone defect healing. This dual delivery system reduces the dose of BMP2 by supplementing IGF1, which may diminish the potential side effects of BMP2.

Project description:Background: Bone grafts are in high demand due to the increase in the cases of bone defects mainly caused by trauma, old age, and disease-related bone damages. Tissue-engineered calcium phosphate (CaP) biomaterials match the major inorganic contents of bone, thereby could be the potential bone graft substitute. However, CaP-bone grafts lack the osteoinductivity that is vital for effective bone regeneration. In this study, we aimed to test the bone defect healing potential of biomimetically fabricated low dose BMP2-doped CaP (BMP2.BioCaP) grafts in a large animal model. Methods: Low dose BMP2 was doped internally (BMP2-int.BioCaP) or on the surface of CaP (BMP2-sur.BioCaP) grafts during the fabrication process. Our previous study showed the robust bone regenerative potential of BMP2-int.BioCaP and BMP2-sur.BioCaP grafts in the rat ectopic model. In this study, we investigated the bone defect healing potential of BMP2.BioCaP grafts in sheep humerus/femoral defects, as well as compared with that of autologous bone graft and clinically used deproteinized bovine bone (DBB) xenograft. Results: Different ways of BMP2 doping did not affect the surface morphology and degradation properties of the graft materials. Micro-CT and histology results showed robustly higher bone defect-healing potential of the BMP2.BioCaP grafts compared to clinically used DBB grafts. The bone defect healing potential of BMP2.BioCaP grafts was as effective as that of the autologous bone graft. Although, BMP2-int.BioCaP doped half the amount of BMP2 compared to BMP2-sur.BioCaP, its' bone defect healing potential was even robust. The BMP2.BioCaP grafts showed less immunogenicity compared to BioCaP or DBB grafts. The volume density of blood vessel-like and bone marrow-like structures in both BMP2.BioCaP graft groups were in a similar extent to the autologous group. Meticulous observation of higher magnification histological images showed active bone regeneration and remodeling during bone defect healing in BMP2.BioCaP graft groups. Conclusion: The robust bone regenerative potential of BMP2.BioCaP grafts in the ectopic model and in-situ bone defects in small and large animals warrant the pre-clinical studies on large animal critical-sized segmental bone defects.

Dataset Information

Combined MEK inhibition and BMP2 treatment promotes osteoblast differentiation and bone healing in Nf1Osx -/- mice.

Publications

Combined MEK inhibition and BMP2 treatment promotes osteoblast differentiation and bone healing in Nf1Osx -/- mice.

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