Bone morphogenetic protein-9 effectively induces osteogenic differentiation of reversibly immortalized calvarial mesenchymal progenitor cells

Abstract

Critical-sized craniofacial defect repair represents a significant challenge to reconstructive surgeons. Many strategies have been employed in an effort to achieve both a functionally and cosmetically acceptable outcome. Bone morphogenetic proteins (BMPs) provide a robust osteoinductive cue to stimulate bony growth and remodeling. Previous studies have suggested that the BMP-9 isoform is particularly effective in promoting osteogenic differentiation of mesenchymal progenitor cells. The aim of this study is to characterize the osteogenic capacity of BMP-9 on calvarial mesenchymal progenitor cell differentiation. Reversibly immortalized murine calvarial progenitor cells (iCALs) were infected with adenoviral vectors encoding BMP-9 or GFP and assessed for early and late stages of osteogenic differentiation in vitro and for osteogenic differentiation via in vivo stem cell implantation studies. Significant elevations in alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA transcription, osteopontin (OPN) protein expression, and matrix mineralization were detected in BMP-treated cells compared to control. Specifically, ALP activity was elevated on days 3, 7, 9, 11, and 13 post-infection and OCN mRNA expression was elevated on days 8, 10, and 14 in treated cells. Additionally, treatment groups demonstrated increased OPN protein expression on day 10 and matrix mineralization on day 14 post-infection relative to control groups. BMP-9 also facilitated the formation of new bone in vivo as detailed by gross, microcomputed tomography, and histological analyses. Therefore, we concluded that BMP-9 significantly stimulates osteogenic differentiation in iCALs, and should be considered an effective agent for calvarial tissue regeneration.

Keywords: Bone morphogenetic protein (BMP); Bone morphogenetic protein-9 (BMP-9); Immortalized calvarial cell (iCAL); Mesenchymal progenitor cell; Osteogenic capacity; Osteogenic differentiation.

Fig. 1

Detection of early markers of osteogenic differentiation. (A) Fluorescent microscopy imaging of green fluorescent protein (GFP) expression at 24 h post-adenoviral infection of calvarial cells in GFP (control) and BMP-9 groups. (B) Alkaline phosphatase activity is a well-established marker of early osteogenic differentiation. Average relative alkaline phosphatase activity in calvarial cells on days 3, 7, 9, 11, and 13 post-adenoviral infection with BMP-9 (blue bars) or GFP (red bars) is shown. Experiments were performed in triplicate. Error bars represent standard error.*P < 0.05, **P < 0.01.

Fig. 2

Detection of late markers of osteogenic differentiation. (A) mRNA expression levels of osteocalcin were measured in calvarial cells on days 8, 10, and 14 post-adenoviral infection with BMP-9 or GFP. Internal control with GAPDH is also shown. (B) Immunohistochemical staining for osteopontin expression in AdBMP-9 and negative control (NC) groups at 10 days post infection. (C) Matrix mineralization (Alizarin red S staining) on post-infection day 14 in cells treated with AdGFP and AdBMP-9.

Fig. 3

Stem cell implantation assay to assess in vivo effects of AdBMP-9 infection. (A) Schematic diagram of experimentation. (B) Digital photography of representative mice after sacrifice. (C) Representative gross specimens after retrieval. (D) Using microCT imaging analysis, ectopic osseous nodules (denoted by arrow) derived from BMP-9-treated cells that were injected into mice are similar in character and appearance to native bone harvested after sacrifice of animals.

Fig. 4

iCALs treated with BMP-9 form more mature ectopic bone than iCALs treated with GFP. (A) Representative hematoxylin and eosin staining of 0.1 mm cross sections of harvested specimens reveal dense trabecular bone in experimental groups versus fibrous cell reaction present in the control group (GFP). Top panel, lower power; bottom panel, higher power. (B) Representative trichrome staining of 0.1 mm cross sections of harvested specimens reveal matrix mineralization in the osseous nodules produced from iCALs treated with BMP-9. From top to bottom, low to high power.