Evaluation of bone regeneration potential of dental follicle stem cells for treatment of craniofacial defects

Abstract

Background aims: Stem cell-based tissue regeneration offers potential for treatment of craniofacial bone defects. The dental follicle, a loose connective tissue surrounding the unerupted tooth, has been shown to contain progenitor/stem cells. Dental follicle stem cells (DFSCs) have strong osteogenesis capability, which makes them suitable for repairing skeletal defects. The objective of this study was to evaluate bone regeneration capability of DFSCs loaded into polycaprolactone (PCL) scaffold for treatment of craniofacial defects.

Methods: DFSCs were isolated from the first mandibular molars of postnatal Sprague-Dawley rats and seeded into the PCL scaffold. Cell attachment and cell viability on the scaffold were examined with the use of scanning electron microscopy and alamar blue reduction assay. For in vivo transplantation, critical-size defects were created on the skulls of 5-month-old immunocompetent rats, and the cell-scaffold constructs were transplanted into the defects.

Results: Skulls were collected at 4 and 8 weeks after transplantation, and bone regeneration in the defects was evaluated with the use of micro-computed tomography and histological analysis. Scanning electron microscopy and Alamar blue assay demonstrated attachment and proliferation of DFSCs in the PCL scaffold. Bone regeneration was observed in the defects treated with DFSC transplantation but not in the controls without DFSC transplant. Transplanting DFSC-PCL with or without osteogenic induction before transplantation achieved approximately 50% bone regeneration at 8 weeks. Formation of woven bone was observed in the DFSC-PCL treatment group. Similar results were seen when osteogenic-induced DFSC-PCL was transplanted to the critical-size defects.

Conclusions: This study demonstrated that transplantation of DFSCs seeded into PCL scaffolds can be used to repair craniofacial defects.

Keywords: bone regeneration; craniofacial defects; dental follicle stem cells; stem cell transplantation.

Figure 1

(A) In vitro assessment of osteogenic differentiation potential of the four DFSC cultures established from 4 different litters of rat pups at passage 3. Cells were induced for osteogenic differentiation for 2 weeks, and stained with 1% Alizarin Red solution showing positive staining covering the entire wells, indicating strong osteogenic capability of the cultures that were later used for experiments in this study. (B) Alkaline phosphatase (ALP) assay was performed, and a significant increase of ALP activity was seen in the DFSCs after 2 weeks of osteogenic induction as compared to the control without osteogenic induction.

Figure 2

Surgical procedures for transplantation of DFSCs to treat critical-size defects on rat calvarial bone. (A) Experimental rat was anaesthetized using isoflurane inhalation. (B) Skin in the incision area was shaved and disinfected with Povidone-iodine solution. (C) A midline incision was created from the nasofrontal area to the anterior area of the occipital protuberance. (D) Two 5 mm diameter defects were created using trephine bur. (E) Scaffold implants were placed into the defects. A photograph of the scaffold was shown on the lower right corner. (F) The scalp was closed using Michael clips.

Figure 3

Evaluation of DFSC attachment on PCL scaffold by SEM. Scaffold structure at various magnifications without loading DFSCs (A-C), 500 × (A), 1000× (B) and 3000× (C). Various magnifications of PCL scaffold microphotographs showing attachment of DFSCs (arrows) on the scaffold (D-F), 500 × (D), 1000× (E) and 3000× (F). Note three-dimensional porous structure for attachment of DFSCs.

Figure 4

Assessment of viability and proliferation of DFSCs on PCL using Alamar blue assay. Note that a significant increase of Alamar blue reduction (Mean ± SE) was observed at day 3 compared to day 1. Single (*) asterisk indicates significant difference at P≤0.05 (N=4).

Figure 5

Micro-CT scanning to evaluate bone regeneration after 4 and 8 weeks of DFSC transplantation (N=4). Note the lack of bone regeneration in the defects of all treatment groups at 4 weeks (upper panel). In contrast, bone regeneration appeared at 8 weeks (Lower panel). No bone formation was seen in the controls without DFSCs, whereas in the defects treated with PCL plus DFSCs or PCL plus iDFSCs, new bone formation filled almost half of the defects.

Figure 6

Histological evaluation of bone regeneration after 4 weeks post-transplantation of DFSCs with H & E staining (N=4). Low magnification micrographs showing the entire cross section of the defects (left panel), and higher magnification micrographs showing the middle of the defects (right panel). Note that no new bone formation was seen in the defects in all treatments. Fibrous tissues were seen within the defects.

Figure 7

Histological evaluation of bone regeneration after 8 weeks post-transplantation of DFSCs (N=4). Low magnification H & E stained micrographs showing the entire defects (A-D). Note that no bone regeneration was seen in the empty control (A) and in the PCL scaffold without DFSCs (B). Substantial bone formation was seen in the defects treated with either PCL plus DFSCs (C) or PLC plus iDFSCs (D). Formation of woven bone can be seen in these treatments at higher magnifications (E, F, G, H, I and J). Sections were also stained with Masson Trichrome to confirm new bone formation (K and L). Bone histomorphometric analysis of histological sections showed no significant difference in new bone formation between DFSC and iDFSC treatments at 8 week after transplantation (M).