Abstract
Craniofacial bone defects are observed in a variety of clinical situations, and their reconstructions require coordinated coupling between angiogenesis and osteogenesis. In this study, we explored the effects of cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1), a synthetic and soluble variant of angiopoietin 1, on bone morphogenetic protein 2 (BMP2)-induced cranial bone regeneration, and recruitment and osteogenic differentiation of perivascular pericytes. A critical-size calvarial defect was created in the C57BL/6 mouse and COMP-Ang1 and/or BMP2 proteins were delivered into the defects with absorbable collagen sponges. After 3 weeks, bone regeneration was evaluated using micro-computed tomography and histologic examination. Pericyte recruitment into the defects was examined using immunofluorescence staining with anti-NG2 and anti-CD31 antibodies. In vitro recruitment and osteoblastic differentiation of pericyte cells were assessed with Boyden chamber assay, staining of calcified nodules, RT-PCR and Western blot analyses. Combined administration of COMP-Ang1 and BMP2 synergistically enhanced bone repair along with the increased population of CD31 (an endothelial cell marker) and NG2 (a specific marker of pericyte) positive cells. In vitro cultures of pericytes consistently showed that pericyte infiltration into the membrane pore of Boyden chamber was more enhanced by the combination treatment. In addition, the combination further increased the osteoblast-specific gene expression, including bone sialoprotein (BSP), osteocalcin (OCN) and osterix (OSX), phosphorylation of Smad/1/5/8, and mineralized nodule formation. COMP-Ang1 can enhance BMP2-induced cranial bone regeneration with increased pericyte recruitment. Combined delivery of the proteins might be a therapeutic strategy to repair cranial bone damage.
Highlights
Repair of bone defects requires a coordinated coupling between osteogenesis and angiogenesis for regeneration [1]
We focused on the combined effect of recombinant COMP-Ang1 and bone morphogenetic protein 2 (BMP2) proteins on orthotopic bone regeneration in critical-sized cranial defects
Fold changes of fluorescence intensity were presented in Fig 3B; for CD31, 1.18 ± 0.62, 2.99 ± 0.54, and 4.74 ± 0.86 in COMP-Ang1, BMP2, and COMP-Ang1/ BMP2, respectively (p < 0.01); for NG2, 1.35 ± 0.84, 4.31 ± 1.33, and 7.56 ± 1.18 (p < 0.01); for merging, 2.84 ± 1.10, 6.97 ± 0.79, 10.95 ± 0.99, respectively (p < 0.01).These results suggested that the enhanced effects of COMP-Ang1 and BMP2 on bone repair might be related to increased angiogenesis and pericyte recruitment
Summary
Repair of bone defects requires a coordinated coupling between osteogenesis and angiogenesis for regeneration [1]. It involves a multistep process that includes migration, proliferation, and differentiation of several types of cells such as endothelial cells, fibroblasts, osteoblasts, osteoclasts and pericytes within the bone microenvironment [2, 3]. The formation of vascular wall contributes to migration of osteoblast progenitor cells such as pericytes into target site [4]. Mesenchymal stem cells (MSCs) and pericyte progenitors are both perivascular cells with similar multipotent properties regardless of tissue of origin; they are shown to be capable of differentiating into osteoblasts, chondrocytes, adipocytes and fibroblasts under special stimulations [5, 6]. A growing interest exists in the recruitment, proliferation, and osteoblastic differentiation of pericytes for therapeutic bone regeneration. VEGF-A and angiopoietin-1 (Ang-1) act on pericytes in an autocrine and paracrine manner to stimulate their proliferation and migration with activation of their receptors [9, 10]
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