Tracking of Stem Cells from Human Exfoliated Deciduous Teeth Labeled with Molday ION Rhodamine-B during Periodontal Bone Regeneration in Rats

Abstract

Background and objectives: Chronic periodontitis can lead to alveolar bone resorption and eventually tooth loss. Stem cells from exfoliated deciduous teeth (SHED) are appropriate bone regeneration seed cells. To track the survival, migration, and differentiation of the transplanted SHED, we used super paramagnetic iron oxide particles (SPIO) Molday ION Rhodamine-B (MIRB) to label and monitor the transplanted cells while repairing periodontal bone defects.

Methods and results: We determined an appropriate dose of MIRB for labeling SHED by examining the growth and osteogenic differentiation of labeled SHED. Finally, SHED was labeled with 25 μg Fe/ml MIRB before being transplanted into rats. Magnetic resonance imaging was used to track SHED survival and migration in vivo due to a low-intensity signal artifact caused by MIRB. HE and immunohistochemical analyses revealed that both MIRB-labeled and unlabeled SHED could promote periodontal bone regeneration. The colocalization of hNUC and MIRB demonstrated that SHED transplanted into rats could survive in vivo. Furthermore, some MIRB-positive cells expressed the osteoblast and osteocyte markers OCN and DMP1, respectively. Enzyme-linked immunosorbent assay revealed that SHED could secrete protein factors, such as IGF-1, OCN, ALP, IL-4, VEGF, and bFGF, which promote bone regeneration. Immunofluorescence staining revealed that the transplanted SHED was surrounded by a large number of host-derived Runx2- and Col II-positive cells that played important roles in the bone healing process.

Conclusions: SHED could promote periodontal bone regeneration in rats, and the survival of SHED could be tracked in vivo by labeling them with MIRB. SHED are likely to promote bone healing through both direct differentiation and paracrine mechanisms.

Keywords: Magnetic resonance imaging; Periodontal bone defect; Stem cells from human exfoliated deciduous teeth (SHED); Transplanted cells tracking.

Fig. 1

SHED culture and osteoinductive treatment (A). The morphology of SHED isolated from the dental pulp tissue of children. (B) Alizarin Red S staining of SHED after osteoinductive treatment. (C, D) Changes in the expression of Runx2 and Alp before and after osteoinductive treatment. The scale bar indicates 100 μm.

Fig. 2

Detection of morphology, iron content, and live cell ratio of SHED treated with various concentrations of MIRB (A). Morphology of SHED labeled with various concentrations of MIRB. SHED nuclei are stained with DAPI (blue); the cytoskeleton is stained with FITC-phalloidin (green); and MIRB particles are shown with Rhodamine B (red). (B) Prussian blue staining was used to detect Fe^3＋. (C) Iron content analysis of SHED. (D) Percentage of live cells was analyzed with Trypan blue staining. *p<0.05. The scale bar indicates 100 μm.

Fig. 3

Determination of the optimal MIRB concentration for use (A). Growth curve of SHED labeled with various concentrations of MIRB. (B, C) Alizarin Red S staining and quantitative analysis of SHED labeled with various concentrations of MIRB after osteoinductive treatment. The scale bar indicates 100 μm. (D) Sig-nal intensity images of 1×10⁶ SHED labeled with various concentrations of MIRB in vitro by MRI.

Fig. 4

Construction of the periodontal bone defect in rats (A). The location and size of the defect in the rat mandible. (B) The created periodontal fenestration defect. (C) Illustration of fibrin gel containing MIRB-labeled SHED for transplantation.

Fig. 5

MRI analysis of the healing ratio of periodontal bone defects and tracking the survival of transplanted cells (A). MRI images of rats at 0, 3, 6, and 9 weeks after surgery. Red arrows indicate the low-intensity artifact signal caused by MIRB. (B) The average healing percent of bone tissue at each time point was calculated according to the results of the MRI. Healing percent=(total defect area − present defect area)/total defect area. ^%,&,*: SHED (MIRB) group is compared with SHED, Fibrin, and PBS groups, respectively. ^#,$: SHED group is compared with Fibrin and PBS groups, respectively. ^%,&,*,#,$p<0.05. ^{%%,&&,**,##,$$}p<0.01.

Fig. 6

The effects of SHED (MIRB-labeled and unlabeled) on bone regeneration (A). HE staining of the defected area 4 weeks after surgery. (A’) The higher magnified images in the boxed regions of (A). (B) Quantitative analysis of newly formed bone areas in four groups by HE staining. (C) Immunohistochemical staining of OCN 4 weeks after surgery. (C’) The higher magnified images in the boxed regions of (C). (D) Quantitative analysis of OCN expression in four groups. (E) Immunohistochemical staining of Runx2 2 weeks after surgery. (E’) The higher magnified images in the boxed regions of (E). (F) Comparison of the number of Runx2^＋ cells in four groups. The black scale bars represent 100 μm, and the white scale bars represent 50 μm. NS: not significant. *p<0.05, **p<0.01.

Fig. 7

Colocalization of MIRB and hNUC, OCN, and DMP1 in vivo (A). Immunohistochemical analysis of hNUC 4 weeks after transplantation. (B, C) Immunofluorescence colocalization of MIRB with OCN and DMP1, respectively, 4 weeks after the surgery. The position indicated by the arrow is the hNUC and MIRB double-positive cells. The bars represent 100 μm.

Fig. 8

Detection of protein factors secreted by SHED and the impact of transplanted SHED on their surrounding host cells (A). ELISA test of proteins factors secreted by SHED cultured in serum-free medium. **p<0.01, ***p<0.0001. (B, C) Immunofluorescence colocalization of MIRB with Runx2 and Col II, respectively, 2 weeks after the surgery. (B’, C’) The higher magnified images in the boxed regions of (B, C), respectively. The bars represent 100 μm.