Delivery of Apical Mesenchymal Stem Cells into Root Canals of Mature Teeth

Abstract

Regenerative endodontic procedures are stem cell-based treatments for immature teeth with pulp necrosis. The translation of regenerative endodontic procedures into treating mature teeth depends, among other factors, on the availability and delivery of mesenchymal stem cells (MSCs) into the root canal system. The aim of this clinical study was to evaluate whether evoked bleeding from the periapical tissues elicits the influx of MSCs into the root canal system in mature teeth with apical lesions. Participants included in this study (N = 20) were referred for endodontic treatment of mature teeth with apical lesions. Following chemomechanical debridement, intracanal bleeding from the periapical tissues was achieved, and intracanal blood samples were collected. A positive blood aspirate was also collected in the cartridges during local anesthesia. Total RNA was isolated and used as a template in quantitative reverse transcription polymerase chain reactions using MSC-specific arrays. Data were analyzed with the Wilcoxon signed-rank test, and correlation between gene expression and sex or age was tested with Spearman's rank correlation coefficient test. In addition, MSCs were isolated from an intracanal bleeding sample and subjected to flow cytometry and quantitative osteogenesis assay. Last, the presence and distribution of MSCs within periradicular lesions were evaluated with immunohistochemistry (n = 4). The MSC markers CD73, CD90, CD105, and CD146 were significantly upregulated, with median fold change values of 2.9, 31.7, 4.6, and 6.8, respectively. Conversely, the negative marker for MSCs, CD45, was significantly downregulated (median, -2.7). There was no correlation with age, sex, tooth type, or treatment for any of the evaluated genes. Isolated intracanal cells coexpressed MSC markers and demonstrated robust mineralizing differentiation potential. Finally, immunohistochemical analysis revealed that MSCs were found compartmentalized mainly within vasculature structures located in periapical lesions. Collectively, findings indicate that the evoked-bleeding technique delivers MSCs into the root canal system in mature teeth with apical lesions.

Keywords: adult; bleeding; cell therapy; endodontics; periapical periodontitis; tissue engineering.

Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

© International & American Associations for Dental Research 2015.

Figures

Figure 1.

Real-time quantitative reverse transcription polymerase chain reaction analysis; fold change in gene expression for the mesenchymal stem cell (MSC) markers CD73, CD90, CD105, CD146, and CD45. Data are presented as median fold change in gene expression in the intracanal blood samples normalized to the systemic blood levels for each marker. The positive MSC markers CD73, CD90, CD105, and CD146 were significantly upregulated. The negative MSC marker CD45 was significantly downregulated. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 2.

Scatter plots showing the expression of mesenchymal stem cell markers by multicolor flow cytometry analysis. The majority of cells isolated from intracanal blood following overinstrumentation of the periapical tissues coexpress mesenchymal stem cell markers. After doublet discrimination, CD45-negative cells were analyzed for CD73, CD90, CD105, and CD146 using gates based on the unstained control. CD73 (A) and CD90 (B) were found to be expressed in >95% of cells, whereas CD105 (C) and CD146 (D) were expressed in 87% and 77% of cells, respectively. (E) Of the CD73 and CD90 coexpressing cells, 9% also expressed CD146 but not CD105 (Q1); 67% expressed both CD105 and CD146 (Q2); 17% expressed only CD105 but not CD146 (Q3); and 7% did not express either CD105 or CD146. SSC, side scatter.

Figure 3.

Mesenchymal stem cells isolated from an intracanal blood following overinstrumentation of the periapical tissues demonstrated robust mineralizing differentiation potential. Cells were cultured for 2 wk in basal or osteogenic media. Alizarin red staining was performed (A) and quantified (B). Cells cultured in osteogenic media (osteogenesis group) expressed significantly higher mineralization activity as compared with cells cultured in standard culture media (control group). The mineralization activity was measured by absorbance at 405 nm. Data are presented as mean absorbance (n = 6). ***P < 0.001, Student’st test.

Figure 4.

Immunohistochemistry and confocal microscopy. Cells expressing mesenchymal stem cell markers CD90, CD105, and CD146 (green) are mostly colocalized with von Willebrand factor–identified (red) endothelial cells (arrows) in periapical lesions (A, C, E) and normal periapex (B, D, F), while CD146 is also expressed in vascular-associated smooth muscle (E, F; asterisks). In contrast, CD45 (green) is expressed in immune cells (arrowheads) in periapical lesion (G) and normal periapex (H). Nuclei are identified by TO-PRO-3 staining (blue), and scale bar in panel H applies to all images.