Joshua L. Crapps

Introduction. Delayed union and nonunion bone fractures fail to fuse by 6 or 9 months post-injury, respectively, with no progression for an additional 3 months in those classified as nonunion.¹ Of the 7.9 million annual fractures in the United States, 600,000 exhibit delayed union and 100,000 progress to nonunion.^1-3 The current standard of care, autologous bone grafting, is associated with pain, hematoma, donor site fracture, and infection.^3,4 Studies have shown that bone morphogenetic protein 2 (BMP-2) stimulates osteoblast differentiation and bone repair through various signaling pathways, including those involving intracellular Smad proteins 1/5/8.^3,5-7 Research has also shown Nel-Like Molecule 1 (NELL-1) enhances BMP-2 stimulated bone repair and represses associated side-effects^8,9. Methods. Mouse ST2 stromal cells and murine bone marrow stem cells (BMSCs) were cultured in either control medium (Cm-Control) or adipocyte conditioned medium (CM-Adipo).⁵  Human adipose stem cells (HASCs) were cultured in basic medium (BM) or osteogenic medium (OM) with or without BMP-2.⁷.Human BMSCs, M2-10B4 cells, and MC3T3-E1 pre-osteoblasts were cultured in media containing BMP-2 and/or NELL-1.^8,9. Quantitative real-time polymerase chain reaction (qRT-PCR), Alkaline phosphatase (ALP) assays, Alizarin red staining, Malachite green staining, and Oil Red O staining determined osteogenic and adipogenic gene markers and cell differentiation. Western blot assays were performed using antibodies specific to Smad 1/5/8, NF-κB p65, Ppar-γ, C/Ebpa, β-cantenin, Pit-1, or Pit-2, according to specific cell lines and cultures.^5,7,8 Mid-diaphyseal femur defects were created in male Lewis rats, implanted with BMP-2 and/or NELL-1, and analyzed with three-dimensional microcomputer tomography (microCT) and X-ray imaging.⁸ Results. Cells in CM-Adipo showed reduced ALP activity, impaired osteogenic gene transcription and Smad 1/5/8 phosphorylation, and activation of the NF-κB signaling pathway. Addition of BAY11-7082, an NF- κB inhibitor, restored osteogenesis⁵.  HASCs cultured in OM with BMP-2 showed the greatest ALP activity and osteogenesis while those in BM tended toward adipogenesis.⁷ Treatment with NELL-1 and BMP-2 together exhibited the greatest strength of new bone, osteogenesis, adipogenic gene downregulation, osteogenic gene transcription, and increased nuclear β-cantenin.⁸ NELL-1 did not increase gene transcription for sodium dependent phosphate transporters Pit-1 and Pit-2, however its activity significantly decreased with the addition of transporter antibodies.⁹ Conclusions. Studies show that the osteoblast’s environment effects differentiation and the BMP-2 pathway. Adipose tissue can inhibit osteogenesis through the activation of the NF-κB pathway. The addition of NELL-1 to BMP-2 treatment stimulates osteogenesis and inhibits adipogenesis for improved bone repair by increasing nuclear β-cantenin and activating Pit-1 and Pit-2 phosphate transporters.

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