Stem cell-based tissue regeneration offers potential for treatment of craniofacial bone defects. The dental follicle (DF), a loose connective tissue surrounding unerupted tooth, has been shown to contain progenitor/stem cells. Dental follicle stem cells (DFSCs) possess 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. DFSCs were isolated from the first mandibular molars of postnatal Sprague Dawley rats and seeded into PCL scaffold. Cell attachment and cell viability on the scaffold were examined using scanning electron microscopy (SEM) and Alamar blue reduction assay. For in vivo transplantation, critical-size defects (CSD) were created on the skulls of 5 month-old immunocompetent rats, and the cell–scaffold constructs were transplanted into the defects. Skulls were collected at 4 and 8 weeks post-transplantation, and bone regeneration in the defects was evaluated with micro-CT and histological analysis. SEM 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 prior to 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 CSD. This study demonstrated that transplantation of DFSCs seeded into PCL scaffolds can be used to repair craniofacial defects.
In this study, we developed an injectable in situ forming hydrogel/microparticle system consisting of two drugs, melatonin and methylprednisolone, to investigate the capability of the system for chondrogenesis in vitro and in vivo. The chemical, mechanical, and rheological properties of the hydrogel/microparticle were investigated. For in vitro evaluation, the adipose-derived stem cells might be mixed with hydrogel/microparticles, then cellular viability was analyzed by acridine orange/propidium iodide and 4′,6-diamidino-2-phenylindole staining and also dimethylmethylene blue assay were conducted to find the amount of proteoglycan. The real-time polymerase chain reaction for aggrecan, sex-determining region Y-Box 9, collagen I (COL1), and COL2 gene expression was performed after 14 and 21 days. For evaluation of cartilage regeneration, the samples were implanted in rabbit knees with cartilaginous experimental defects. Defects were created in both knees of three groups of rabbits. Group 1 was the control with no injection, and Groups 2 and 3 were loaded with hydrogel/cell and hydrogel/microparticle/cell; respectively. Then, after 3 and 6 months, histological evaluations of the defected sites were carried out. The amount of glycosaminoglycans after 14 and 21 days increased significantly in hydrogels/microparticles loaded with cells. The expression of marker genes was also significant in hydrogels/microparticles loaded with cells. According to histology analysis, the hydrogels/microparticles loaded with cells showed the best cartilage regeneration. Overall, our study revealed that the developed injectable hydrogel/microparticle can be used for cartilage regeneration.
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