Clinical Potential and Current Progress of Dental Pulp Stem Cells for Various Systemic Diseases in Regenerative Medicine: A Concise Review.

Dental pulp stem cells (DPSCs) are mesenchymal stem cells (MSCs) that have multipotent differentiation and a self-renewal ability. They have been useful not only for dental diseases, but also for systemic diseases. Extensive studies have suggested that DPSCs are effective for various diseases, such as spinal cord injuries, Parkinson's disease, Alzheimer's disease, cerebral ischemia, myocardial infarction, muscular dystrophy, diabetes, liver diseases, eye diseases, immune diseases, and oral diseases. DPSCs have the potential for use in a cell-therapeutic paradigm shift to treat these diseases. It has also been reported that DPSCs have higher regenerative potential than the bone marrow-derived mesenchymal stem cells known as representative MSCs. Therefore, DPSCs have recently gathered much attention. In this review, the therapeutic potential of DPSCs, the latest progress in the pre-clinical study for treatment of these various systemic diseases, and the clinical applications of DPSCs in regenerative medicine, are all summarized. Although challenges, including mechanisms of the effects and establishment of cell processing and transplantation methods for clinical use, still remain, DPSCs could be promising stem cells sources for various clinical applications, because of their easy isolation by a noninvasive procedure without ethical concerns.

Evaluation of perforated demineralized dentin scaffold on bone regeneration in critical-size sheep iliac defects.

OBJECTIVES: Regenerating critical-size bone injury is a major problem that continues to inspire the design of new graft materials. Therefore, tissue engineering has become a novel approach for targeting bone regeneration applications. Human teeth are a rich source of stem cells, matrix, trace metal ions, and growth factors. A vital tooth-derived demineralized dentin matrix is acid-insoluble and composed of cross-linked collagen with growth factors. In this study, we recycled human non-functional tooth into a unique geometric dentin scaffold, entitled perforated root-demineralized dentin matrix (PR-DDM). The aim of this study was to evaluate the feasibility of PR-DDM as the scaffold for regenerating bone in critical-size iliac defects. MATERIAL AND METHODS: Artificial macro-pores (1 mm in diameter) were added to human vital wisdom tooth after removing the enamel and pulp portions. The modified tooth was demineralized in 0.34 N HNO3 for 30 min and is referred to as PR-DDM scaffold. Critical-size defect (10 mm × 15 mm × 9 mm Ø) was created in the iliac crest of six adult sheep. The in vivo bone regeneration by the scaffold was evaluated by micro-CT, 3D micro-CT, and histological examination at 2 and 4 months post-implantation. RESULTS: PR-DDM exhibited better bone ingrowth, especially in the artificial macro-pores. The results of micro-CT and 3D micro-CT revealed good union between scaffold and native bone. New bone formation was observed in almost all portions of PR-DDM. Higher bone volume inside the scaffold was detected at 4 months compared with 2 months. New bone ingrowth was ankylosed with PR-DDM, and both osteoinduction and osteoconduction capability of PR-DDM were confirmed histologically. The ratio of new bone formation was higher at 4 months compared with 2 months by histomorphometric analysis. CONCLUSIONS: Altogether, these results demonstrated that the human tooth-derived graft material with a unique geometric structure, PR-DDM, contributed to active bone ingrowth in critical-size bone defects. This novel scaffold may have great utility in the near-future clinical application.

Immediate Tooth Autotransplantation with Root Canal Filling and Partially Demineralized Dentin/Cementum Matrix into Congenital Missing Tooth Region.

This clinical report describes immediate tooth auto-transplantation with an autograft of partially demineralized dentin/cementum matrix (pDDM), based on an orthodontic treatment plan for a 16-year-old male patient with a congenital missing tooth (#45). First, vital teeth (#14, #24) were extracted, and root canal filling (#14) was immediately performed with the support of a fixation device. Simultaneously, the tooth (#24) was crushed in an electric mill for 1 min, and the crushed granules were partially demineralized in 2% HNO3 solution for 20 min as the graft material. Next, the donor tooth was transplanted into the created socket (#45), and stabilized using an enamel bonding agent. The wet pDDM was loaded into the location of the congenital missing tooth, and the flap was repositioned. The bonding agent for stabilization was removed at 28 days, and also small contact points between the transplanted tooth and the upper premolar (#14) were added using photopolymerizable composite resin. X-ray photos were taken sequentially, and there were no postoperative complications. The radiographic images showed that the periodontal ligament space and alveolar ridge line could be observed at 18 months. The pDDM was harmonized with the mandible, and the remodeled bone-like shadow was observed in the graft region. We concluded that immediate tooth transplantation with root canal fillings and autogenous pDDM may be a valuable alternative to dental implanting or bridge formation for patients with a congenital missing tooth, followed by orthodontic treatment.

Contribution to Bone Formation of the Schneiderian Membrane after Sinus Augmentation: A Histological Study in Rabbits.

AIM: to evaluate bone formation in close contact with the sinus mucosa after different periods from sinus augmentation and the influence on healing of the presence of an inward dis-placed bone window. MATERIAL AND METHODS: Eighteen rabbits were included in the experiment. A trap-door technique was applied at the test sites, and the bony window was elevated inward (inward window; IW) together with the sinus mucosa. At the control sites, the bony window was removed before the elevation of the sinus mucosa. The elevated space was filled with deproteinized bovine bone mineral (DBBM) and both access windows were covered with a collagen membrane. Histometric measurements were performed subjacent the sinus mucosa after 2, 4, and 8 weeks of healing. RESULTS: Very few sinuses presented small percentages of new bone in close contact with the sinus mucosa in the various period examined. The presence of bone in the neighbor areas might have influenced bone formation close to the sinus mucosa. The inward displaced bone window supported bone formation close to the sinus mucosa only in the earliest period of healing, while the bone walls increased their influence over time. The lack of increased new bone percentage over time in the most central regions of the elevated sinus mucosa do not support the hypothesis that the sinus mucosa may express its potential in bone formation. It can be speculated that the new bone found in the intermediate and middle regions of the control sites in the earliest period of healing might be due to residual of bone from the osteotomy. CONCLUSIONS: Very small amounts of new bone were found subjacent to the sinus mucosa, mostly formed from the bone walls, the inward displaced bone window or from possible bone residues from the osteotomy procedures. The lack of increased new bone percentage over time in the most central regions of the elevated sinus mucosa indicates that the contribution to bone formation provided by the sinus mucosa is limited.

Accuracy and dimensional reproducibility by model scanning, intraoral scanning, and CBCT imaging for digital implant dentistry.

BACKGROUND: Recently, it has become possible to analyze implant placement position using the digital matching data of optical impression data of the oral cavity or plaster models with cone beam computed tomography (CBCT) data, and create a highly accurate surgical guide. It has been reported that CBCT measurements were smaller than the actual values, termed shrinkage. Matching of digital data is reliable when the plaster model or intraoral impression values show shrinkage at the same rate as the CBCT data. However, if the shrinkage rate is significantly different, the obtained digital data become unreliable. To clarify digital matching reliability, we examined dimensional reproducibility and shrinkage in measurements obtained with a model scanner, intra-oral scanner (iOS), and CBCT. MATERIALS AND METHODS: Three implants that were arranged in a triangle were fixed in an acrylic plate. The distance between each implants were measured using model scanner, iOS, and CBCT. The actual size measured by electronic caliper was regarded as control. RESULTS: All values measured with CBCT were significantly smaller than that of model scanner, iOS, and control (p<0.001). The model scanner shrinkage was 0.37-0.39%, iOS shrinkage was 0.9-1.4%, and CBCT shrinkage was 1.8-6.9%. There were statistically significant differences among the shrinkage with iOS, CBCT, and model scanner (p<0.001). CONCLUSION: Our findings showed that all measurements obtained with those modalities showed shrinkage as compared to the actual values. In addition, CBCT shrinkage was largest among three different measuring methods. They indicated that data matching between CBCT and scanner measurements requires attention in regard to the reliability of values obtained with those devices.

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

BACKGROUND: Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis. METHODS: All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies. RESULTS: A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not. CONCLUSIONS: The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

Upregulated expression of human beta defensin-1 and -3 mRNA during differentiation of keratinocyte immortalized cell lines, HaCaT and PHK16-0b.

BACKGROUND: Human beta-defensins (hBDs) belong to a group of antimicrobial peptide that are expressed in the epithelial cells. OBJECTIVE: The present study investigated mRNA expression levels of the beta-defensins, hBD-1, -2 and -3, in human keratinocytes during differentiation in vitro. METHODS: Immortalized keratinocyte cell lines, HaCaT and PHK16-0b, were used in this study; in order to stimulate differentiation, the Ca(2+) concentration in the growth media was increased from 0.3 to 1.8 mM. RESULTS: Four days after the increase, the expression levels of hBD-1 and -3 were increased in both cell lines, followed by an increase in the mRNA levels of the differentiation markers, involucrin and keratin 10. No increased expression of hBD-2 was observed. CONCLUSION: The results indicate that keratinocyte differentiation may stimulate hBD-1 and -3 expression in stratified squamous epithelia.

Effect of defensin peptides on eukaryotic cells: primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines.

BACKGROUND: Although the usefulness of the antimicrobial peptides known as defensins has been suggested against oral and skin infections, possible adverse effects of the defensins on the host should be understood before clinical applications can be contemplated. OBJECTIVE: In the present study, we investigated how alpha-defensin (HNP-1) and beta-defensins (hBD-1, -2, -3) affect cells including primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines, SCC-9 and KB. METHOD: Cell proliferation was assessed by the direct cell counting and XTT assay. RESULTS: We found that alpha-defensin promotes proliferation of the epithelial cells at low concentration but has a cytotoxic effect at high concentration. In contrast, beta-defensins have little effect on these cells at any concentration, suggesting that beta-defensins may have no adverse effects on the host. CONCLUSION: Therefore, in terms of host response beta-defensins may be more suitable antimicrobial agents for clinical applications than alpha-defensins.

Atypical carcinoid (neuroendocrine carcinoma) of the gingiva: counterpart of a laryngeal tumor.

Intraoral localization of neuroendocrine carcinoma, usually called Merkel cell carcinoma, is extremely rare. A case of neuroendocrine carcinoma that was a counterpart of laryngeal neuroendocrine carcinoma but was not a Merkel cell carcinoma, occurring at the mandibular gingiva in a 69-year-old Japanese man, is described. The tumor formed a cauliflower-like mass, measuring 20 x 20 mm, with a small area of necrosis. A computed tomography image showed metastasis in the right submandibular lymph node. Histopathologically, the tumor was composed of immature, small round cells that formed anastomosing trabecular nests. Few mitotic and no necrotic features were observed in the nests. Immunohistochemical studies showed positive staining for chromogranin, synaptophysin and neuron-specific enolase in the tumor nests. We diagnosed it as an atypical carcinoid (neuroendocrine carcinoma), a counterpart to the same type of tumor occurring in the larynx. The present case is an extremely rare case of neuroendocrine carcinoma without the feature of Merkel cell carcinoma arising from the gingiva.

Upregulation of human beta-defensin 2 peptide expression in oral lichen planus, leukoplakia and candidiasis. an immunohistochemical study.

Human beta defensin 2 (hBD-2) is a major antimicrobial peptide that is produced by many types of epithelial cells, and is transcriptionally inducible by various proinflammatory agents, such as cytokines and bacteria. Although in vitro studies of the hBDs in oral epithelial cells have been well documented, only little is known about the in vivo pathological state of oral epithelium. We investigated the localization of hBD-2 peptide in tissue sections of oral lichen planus, leukoplakia, candidal leukoplakia and radicular cysts using immunohistochemistry. HBD-2 was stained in both the hyperkeratinized and the granular layers in cases of lichen planus with hyperkeratosis and leukoplakia. Expression in spinous and suprabasal layers was often strong in lichen planus. There were no significant differences in the number of S-100 positive dendritic cells between the widely stained areas and those with limited staining areas in lichen planus. In cases of candidal leukoplakia, the hyphae of candida were mainly detected on the surface of keratinization, which showed only negative or faint staining for hBD-2. These results suggest that hBD-2 is vigorously induced by lichen planus-related inflammation and that it plays an important role in protection from Candida albicans infection; however, it is not a strong chemotactic attractant for Langerhans cells in pathological conditions of oral epithelium.

Expression of MIP-3alpha/CCL20, a macrophage inflammatory protein in oral squamous cell carcinoma.

We have examined the expression of MIP-3alpha/CCL20 in oral squamous cell carcinoma (SCC) in vivo and in vitro. In addition, we have investigated whether the expression of MIP-3alpha/CCL20 is regulated by bacterial infection and inflammatory cytokines. In order to determine the mRNA level of MIP-3alpha, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with LightCycler using the double-stranded DNA dye, SYBR Green I. Oral epithelial cells and six SCC cell lines (SCC-9, SAS, BSC-OF, HSC-4, HSC, Ca9-22) were found to express MIP-3alpha mRNA. The expression of MIP-3alpha was upregulated by infection with Actinobacillus actinomycetemcomitans and by stimulation with lipopolysaccharide and TNF-alpha. By in situ hybridization, the detectable MIP-3alpha expression in SCC was localized primarily at the epithelial pearls corresponding to the spinous layer. These results suggest that MIP-3alpha contributes to the oral immunoresponse to bacterial infection, and may be involved in the growth of SCC.

Correlated expression of human beta defensin-1, -2 and -3 mRNAs in gingival tissues of young children.

OBJECTIVE: Human beta-defensins (hBDs) are a group of antimicrobial peptides, expressed by the epithelial cells of many organs including gingival epithelium. The present study examined correlation between the gene expressions of hBD-1, -2, -3 mRNAs and the inflammatory cytokines in human gingival tissues. STUDY DESIGN: The gingival tissues were obtained from surgical discards from 20 different patients (age range, 5-13 years). The expression levels of mRNAs were evaluated by quantitative RT-PCR with LightCycler. The mRNA expression levels were normalized with those of keratin 10 mRNA. The data were statistically analysed using Person's correlation coefficient. RESULTS: The expression levels of hBD-1,-2 and -3 were significantly correlated with each other and also correlated with that of TNF-alpha. CONCLUSIONS: The results indicate that the expression levels of hBDs vary from one individual to another.

Ameloblastic carcinoma ex ameloblastoma: report of a case-possible involvement of CpG island hypermethylation of the p16 gene in malignant transformation.

Ameloblastic carcinoma is described in the updated World Health Organization (WHO) classification as a rare malignant lesion. Ameloblastic carcinoma meeting the WHO criteria may arise either as a result of malignant change in a pre-existing benign ameloblastoma (carcinoma ex ameloblastoma) or as a primary malignant ameloblastoma not preceded by an ordinary ameloblastoma (de novo carcinoma). We report a case of ameloblastic carcinoma ex ameloblastoma and examine how this case underwent malignant transformation. The DNA was extracted separately from benign and malignant areas in paraffin sections of the tumor. Direct sequencing showed no genetic mutation of exons 5-8 of the p53 gene. Hypermethylation of CpG islands of the p16 gene was detected in the malignant parts of the tumor. The results indicate that hypermethylation of p16 may have been involved in the malignant transformation of the ameloblastoma in the present case.

Localization of human beta-defensin 3 mRNA in normal oral epithelium, leukoplakia, and lichen planus: an in situ hybridization study.

Human beta-defensin 3 (hBD-3), an antimicrobial peptide, is produced by various epithelial and some nonepithelial tissues. hBD-3 mRNA is widely expressed in oral tissues, including oral epithelium and the salivary glands. Although the localization of hBD-1 and hBD-2 has been well demonstrated in tissue sections, the localization pattern of hBD-3 has not yet been shown. In the present study, we investigated the expression pattern of hBD-3 mRNA by in situ hybridization using specific RNA probes; the signal for hBD-3 was detected in upper spinous and granular layers in normal oral epithelium. In cases of leukoplakia, a strong signal of hBD-3 mRNA was observed in the granular layer. In lichen planus, the signal was strongly detected in the spinous and suprabasal layers. The signals were stronger than those of either normal oral epithelium or leukoplakia. The results indicate that the localization pattern of hBD-3 is very similar to that of hBD-2. hBD-2 and hBD-3 may function together or compensate each other for expressional loss.