Matthews B G, Roguljic H, Franceschetti T, Roeder E, Matic I, Vidovic I, Joshi P, Kum K-Y, Kalajzic I
Department of Reconstructive Sciences, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT, USA.
Division of Pediatric Dentistry, Department of Craniofacial Sciences, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT, USA.
J Periodontal Res. 2016 Jun;51(3):304-12. doi: 10.1111/jre.12309. Epub 2015 Jul 27.
Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly than bone, does not have vasculature or innervation and does not undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-green fluorescent protein (OC-GFP) transgenic mice.
OC-GFP reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not of periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP(+) cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real-time PCR and immunostaining or in situ hybridization.
Microarray analysis identified 95 genes that were expressed at least two-fold higher in cementoblasts than in osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real-time PCR confirmed that expression of the Wnt inhibitors Wnt inhibitory factor 1 (Wif1) and secreted frizzled-related protein 1 (Sfrp1) was elevated in cementoblasts compared with osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor BARX homeobox 1 (Barx1) was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 was expressed in apical cementoblasts and cementocytes, but not in osteoblasts or odontoblasts.
The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts.
牙骨质和骨是相似的矿化组织，但牙骨质的积累比骨慢得多，没有血管或神经支配，也不发生重塑。尽管存在这些差异，但尚无成熟的标志物来区分成牙骨质细胞与其他成熟的矿化细胞，如成骨细胞和成牙本质细胞。本研究的目的是通过对直接从骨钙素绿色荧光蛋白（OC-GFP）转基因小鼠分离的细胞群体进行基因谱分析，评估成牙骨质细胞与成骨细胞之间基因表达的差异。
使用OC-GFP报告基因小鼠，因为它们在牙周组织中显示成牙骨质细胞、成骨细胞和成牙本质细胞有标记，但牙周膜成纤维细胞没有标记。我们对从磨牙根表面消化的细胞进行分选，以分离OC-GFP(+)成牙骨质细胞。从颅骨消化物中分离成骨细胞。进行微阵列分析，并通过实时PCR、免疫染色或原位杂交对选定结果进行确认。
微阵列分析确定了95个基因，这些基因在成牙骨质细胞中的表达比在成骨细胞中至少高两倍。我们的分析表明，Wnt信号通路受到不同调节，与骨骼发育相关的基因也是如此。实时PCR证实，与成骨细胞相比，成牙骨质细胞中Wnt抑制剂Wnt抑制因子1（Wif1）和分泌型卷曲相关蛋白1（Sfrp1）的表达升高，且Wif1表达定位于根尖区域。此外，转录因子BARX同源盒1（Barx1）在成牙骨质细胞中的表达水平更高，免疫组织化学表明BARX1在根尖成牙骨质细胞和牙骨质细胞中表达，但在成骨细胞或成牙本质细胞中不表达。
OC-GFP小鼠为选择性分离成牙骨质细胞提供了一个良好的模型，并有助于鉴定成牙骨质细胞与成骨细胞之间差异表达的基因。
