Yinzhong Duan

Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/ periodontal ligament-like tissues

BACKGROUND AND OBJECTIVE Limitations of current periodontal regeneration modalities in both predictability and extent of healing response, especially on new cementum and attachment formation, underscore the importance of restoring or providing a microenvironment that is capable of promoting the differentiation of periodontal ligament stem cells (PDLSCs) towards cementoblast-like cells and the formation of cementum/periodontal ligament-like tissues. The aim of this study was to investigate the biological effect of conditioned medium from developing apical tooth germ cells (APTG-CM) on the differentiation and cementogenesis of PDLSCs both in vitro and in vivo. MATERIAL AND METHODS Using the limiting dilution technique, single-colony-derived human PDLSCs were isolated and expanded to obtain homogeneous populations of PDLSCs. Morphological appearance, cell cycle analysis, bromodeoxyuridine incorporation, alkaline phosphatase (ALP) activity, mineralization behavior, gene expression of cementoblast phenotype and in vivo differentiation capacities of PDLSCs co-cultured with APTG-CM were evaluated. RESULTS The induced PDLSCs exhibited several characteristics of cementoblast lineages, as indicated by the morphological changes, increased proliferation, high ALP activity, and the expression of cementum-related genes and calcified nodule formation in vitro. When transplanted into immunocompromised mice, the induced PDLSCs showed tissue-regenerative capacity to produce cementum/periodontal ligament-like structures, characterized by a layer of cementum-like mineralized tissues and associated periodontal ligament-like collagen fibers connecting with the newly formed cementum-like deposits, whereas control, untreated PDLSCs transplants mainly formed connective tissues. CONCLUSION Our findings suggest that APTG-CM is able to provide a cementogenic microenvironment and induce differentiation of PDLSCs along the cementoblastic lineage. This has important implications for periodontal engineering.

Differentiation of dermal multipotent cells into odontogenic lineage induced by embryonic and neonatal tooth germ cell-conditioned medium.

Stem cell-based therapy represents a novel and more advantageous modality of treatment for tooth defect or loss. However, this strategy is challenged in the circumstances where tooth-derived stem cells are not readily accessible. In present study we sought to explore the possibility of utilizing dermal multipotent cells (DMCs) easily available from skin tissue for odontogenic induction. Using the limiting dilution technique, colony-forming cell population was isolated and characterized by proliferative activity and multilineage differentiation potential. By exposure to conditioned medium of embryonic and neonatal tooth germ cells in culture, the proliferation and mineralization activity of DMCs was elevated, while the embryonic tooth germ cell-conditioned medium (ETGC-CM) produced more significant effects. Meanwhile, ETGC-CM-treated DMCs phenocopied the odontoblasts in vitro as indicated by specific lineage markers. Following in vivo transplantation as cell pellet, ETGC-CM-treated DMCs were capable of producing blocks of mineralized tissues, which resembled those of dental pulp stem cell (DPSC) explants in the same subcutaneous pockets environment. These observations suggest that although more sufficient and continuous inductive microenvironment may be needed for undifferentiated DMCs to perform as odontoblasts, ETGC-CM-treated DMCs indeed acquire properties as those of DPSCs. Our work highlights the potential utility of DMCs as an alternative candidate cell source in hopes of developing more practical strategy of tooth regeneration research and offering promising opportunities for therapeutic approach.

The apical region of developing tooth root constitutes a complex and maintains the ability to generate root and periodontium-like tissues

BACKGROUND AND OBJECTIVE Mammalian tooth root development is a long-term process during which root elongates along the apical direction and is accompanied with the formation of periodontium. Considering the heterogeneous apical region of developing root as a functional entity, we observed and characterized the developing apical complex of rat molar root. The aim of the present study was to investigate the characteristics and developmental capability of developing apical complex in situ and in vitro. MATERIAL AND METHODS Histological analysis of rat developing apical complex was performed using hematoxylin and eosin staining and immunohistochemical staining. Cell counting, bromodeoxyuridine incorporation, flow cytometry assays and western blot analyses were performed to assess the proliferation potential of developing apical complex cells in vitro, and its mineralization potential was investigated by alkaline phosphatase activity, alizarin red staining and reverse transcription-polymerase chain reaction analysis. In vivo transplantation of both developing apical complex tissues and cells were used to characterize the differentiation capacity of developing apical complex cells. Dental pulp cells were used as a control in this study. RESULTS Isolated developing apical complex maintained the developmental capability to form tooth root/periodontal complex ectopically. Developing apical complex cells exhibited relatively higher proliferation and mineralization potential compared with dental pulp cells in culture. When cultured developing apical complex cells were putatively depleted of Hertwigs epithelial root sheath cells, only osteodentin-like tissues and fibrous connective tissues formed in cell-scaffold explants. CONCLUSION The sustainable development ability of developing apical complex qualifies it as the growth center of tooth root and as a promising candidate source of cells for tooth root and periodontal regeneration.

Periapical Follicle Stem Cell: A Promising Candidate for Cementum/Periodontal Ligament Regeneration and Bio-Root Engineering

Mesenchymal stem cell (MSC)-mediated tissue regeneration offers opportunities to regenerate a bio-root and its associated periodontal tissues to restore tooth loss. Previously, we proved that the apical end of developing root was acting as a promising candidate cell source for root/periodontal tissue (R/PT) regeneration. In the present study, we investigated the properties of periapical follicle stem cells (PAFSCs) isolated from the apical end of developing root of human third molars at the root-developing stage and evaluated the potential application of these cells for cementum/periodontal ligament (PDL) regeneration and bio-root engineering. Putative PAFSCs were isolated and subcultured until 20th passage. Cell characteristics of PAFSCs at early or late passage were evaluated and compared with periodontal ligament stem cells (PDLSCs) via a series of histological, cellular, and molecular analyses. PAFSCs at early passage presented crucial stem cell properties and showed a higher proliferation rate than PDLSCs in vitro. Meanwhile, PAFSCs also showed the tissue-regenerative capacity to produce a typical cementum/PDL-like complex in vivo. During long-term passage, both cell populations changed in morphology and gradually lost their stem cell properties. The alkaline phosphatase (ALP) activity and expression of mineralization-related genes markedly declined as more passages were carried out, which might lead to the loss of tissue-regenerative capacity of these 2 groups of cells in vivo. Our findings suggest that developing tissue-derived PAFSCs are a distinctive cell population from PDLSCs and might be a promising candidate for bio-root engineering.

Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages.

Background information. Although the mechanism of cementogenesis is an area full of debate, the DFCs (dental follicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs come into contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentin or dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentin non‐collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages.

Activation of cannabinoid receptor CB2 regulates osteogenic and osteoclastogenic gene expression in human periodontal ligament cells

BACKGROUND AND OBJECTIVE Cannabinoid receptor CB2, expressed in osteoblasts and osteoclasts, plays a crucial role in the regulation of bone metabolism. Since periodontal ligament (PDL) cells can differentiate into osteoblasts, this study was undertaken to investigate CB2 expression and the effect of CB2 activation on osteogenic differentiation of PDL cells. MATERIAL AND METHODS Human PDL (hPDL) cells were obtained from extracted teeth of periodontally healthy subjects. Expression of CB2 was observed in hPDL cells by RT-PCR, Western blotting and immunofluorescence assay. Then hPDL cells were treated with a CB2-specific agonist, HU-308 (10(-7) m), for 12, 24, 48 or 72 h. The mRNA expressions of osteogenic genes, such as runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OC) and collagen type I (COL I), and osteoclastogenic genes, including osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL), were examined using quantitative real-time PCR analysis. A mineralization assay was performed in hPDL cells in mineralization conditions with or without HU-308. RESULTS Expression of CB2 mRNA and protein was detected in hPDL cells. HU-308 enhanced the mRNA levels of the above osteogenic genes. Expression of the OPG gene was up-regulated, whereas RANKL gene expression was down-regulated, contributing to the elevated OPG/RANKL ratio. Accelerated mineralization was observed in hPDL cells in mineralization conditions with HU-308. CONCLUSION Our findings demonstrate that activation of CB2 is able to enhance osteogenic differentiation of hPDL cells and potentially create a favorable osteogenic microenvironment. This implies that CB2 might play an important role in alveolar bone metabolism.

Porcine tooth germ cell conditioned medium can induce odontogenic differentiation of human dental pulp stem cells

It is suggested that the differentiation of tooth‐derived stem cells is modulated by the local microenvironment in which they reside. Previous studies have indicated that tooth germ cell‐conditioned medium (TGC‐CM) holds the potential to induce dental pulp stem cells (DPSCs) to differentiate into the odontogenic lineage. Nevertheless, human TGC‐CM (hTGC‐CM) is not feasible in practical application, so we conjectured that xenogenic TGC‐CM might exert a similar influence on human dental stem cells. In this study, we chose swine as the xenogenic origin and compared the effect of porcine tooth germ cell‐conditioned medium (pTGC‐CM) with its human counterpart on human DPSCs. Morphological appearance, colony‐forming assay, in vitro multipotential ability, protein and gene expression of the odontogenic phenotype and the in vivo differentiation capacity of DPSCs were evaluated. The results showed that pTGC‐CM exerted a similar effect to hTGC‐CM in inducing human DPSCs to present odontogenic changes, which were indicated by remarkable morphological changes, higher multipotential capability and the expression of some odontogenic markers in gene and protein levels. Besides, the in vivo results showed that pTGC‐CM‐treated DPSCs, similar to hTGC‐CM‐treated DPSCs, could form a more regular dentine–pulp complex. Our data provided the first evidence that pTGC‐CM is able to exert almost the same effect on DPSCs with hTGC‐CM. The observations suggest that the application of xenogenic TGC‐CM may facilitate generating bioengineered teeth from tooth‐derived stem cells in future. Copyright

Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells possess the ability of self‐renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum‐free conditioned medium (ASF–CM), we successfully generated ameloblast‐like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF–CM supplemented with BMP4 (ASF–BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast‐specific as well as odontoblast‐specific genes. On the other hand, culturing mouse iPS cells in ASF–CM supplemented with noggin (ASF–noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF–BMP4 to differentiate into ameloblast‐like and odontoblast‐like cells. The results of our study raise the possibility of using patient‐specific iPS cells for tooth regeneration in the future. Copyright

The Application of Bolton’s Ratios in Orthodontic Treatment Planning for Chinese Patients

Aim: To investigate the application of Bolton’s ratios in the proper diagnosis and treatment planning for Chinese orthodontic patients with congenitally missing mandibular incisor. Materials and Methods: Twenty-seven Chinese cases (males 10, females 17) with congenitally missing mandibular incisor and class I molar relationship were recruited in this study. Guided by the anterior Bolton’s ratios, three therapeutic strategies (each for 9 patients) were carried out according to different indications, which included enamel stripping, prosthetic restoration and extraction therapy. Results: After treatment, all the patients achieved good occlusion with normal Bolton’s ratios and the clinical results were satisfied. Conclusion: It suggested that the application of Bolton’s ratios in orthodontic treatment planning for Chinese patients with mandibular congenital missing incisor might be clinically beneficial for optimum treatment outcomes.