Comparative gene expression analysis of stemness between periodontal ligament and umbilical cord tissues in humans.

Background/purpose: Due to their regenerative potential, periodontal ligament (PDL) and umbilical cord (UBC) tissues are an attractive potential mesenchymal stem cells (MSCs) source. This study compared the expression patterns of genes related to stemness between fresh PDL and UBC tissues. Materials and methods: PDL tissues were collected from 38 permanent premolars extracted for orthodontic purposes, and UBC tissues were obtained from three newborns. Each sample was immediately frozen to prevent RNA degradation. cDNA microarray analysis, quantitative real-time polymerase chain reaction (PCR), and immunohistochemical staining were performed. Gene expression patterns associated with dental stemness (DS) and induced pluripotent stemness (iPS) were compared between PDL and UBC tissues. Results: In the cDNA microarray analyses, the expressions of most iPS genes were greater in the PDL than in the UBC. Meanwhile, the expressions of most DS genes were greater in the UBC than in the PDL. Quantitative real-time PCR analyses showed that the expression levels of matrix metallopeptidase 13 (MMP13), ADAM metallopeptidase domain 22 (ADAM22), vascular cell adhesion protein 1 (VCAM1), and kruppel-like factor 4 (KLF4) genes were greater in the PDL than in the UBC, while the expressions of melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) were greater in the UBC than in the PDL. Conclusion: These results suggest that UBC and PDL tissues showed slightly different expression patterns of genes related to stemness, which warrants further investigation to use these tissues for future regeneration and implantation therapies.

Assessment of dental caries lesion activity status using quantitative parameters obtained from the quantitative light-induced fluorescence method and difference of microbial distribution in primary molars.

BACKGROUND: This study aimed to assess differences in quantitative measures obtained from the quantitative light-induced fluorescence method and microbial composition of carious dentin and saliva according to the activity status of caries lesions in primary molars. METHODS: A total of 34 teeth from 34 children were evaluated in this study. The activity status of carious lesions was classified using the International Caries Classification and Management System criteria (active or inactive). Images of the carious lesions were captured using a quantitative light-induced fluorescence device for quantitative analyses. Carious dentin and saliva were collected to detect and quantify selected bacterial species (S. mutans, S. sobrinus, Lactobacillus species, F. nucleatum, P. nigrescence, P. intermedia) and C. albicans by quantitative polymerase chain reaction. Mann-Whitney U tests were performed to evaluate differences in quantitative measures from quantitative light-induced fluorescence, the microbial composition of carious dentin, and saliva according to the activity status of carious lesions. RESULTS: Red fluorescence values (∆R, ∆Rmax) from the quantitative light-induced fluorescence method were significantly higher in active lesions (∆R, p = 0.009; ∆Rmax, p = 0.014). The quantitative mean levels of Lactobacillus species (p = 0.010) in carious dentin and S. sobrinus (p = 0.017) in saliva were significantly higher in the active-lesion group. CONCLUSIONS: Quantitative measures related to red fluorescence from the quantitative light-induced fluorescence method, levels of Lactobacillus species from carious dentin, and levels of S. sobrinus from saliva were associated with caries lesion activity.

Selective Recognition of Phosphatidylinositol Phosphate Receptors by C-Terminal Tail of Mitotic Kinesin-like Protein 2 (MKlp2).

The mitotic kinesin-like protein 2 (MKlp2) plays a key role in the proper completion of cytokinetic abscission. Specifically, the C-terminal tail of MKlp2 (CTM peptides) offers a stable tethering on the plasma membrane and microtubule cytoskeleton in the midbody during abscission. However, little is known about the underlying mechanism of how the CTM peptides bind to the plasma membrane of the intercellular bridge. Herein, we identify the specific molecular interaction between the CTM peptides and phosphatidylinositol phosphate (PIP) receptors using quartz crystal microbalance-dissipation and atomic force microscopy force spectroscopic measurements. To systematically examine the effects of amino acids, we designed a series of synthetic 33-mer peptides derived from the wild-type (CTM1). First, we evaluated the peptide binding amount caused by electrostatic interactions based on 100% zwitterionic and 30% negatively charged model membranes, whereby the nonspecific attractions were nearly proportional to the net charge of peptides. Upon incubating with PIP-containing model membranes, the wild-type CTM1 and its truncated mutation showed significant PI(3)P-specific binding, which was evidenced by a 15-fold higher binding mass and 6-fold stronger adhesion force compared to other negatively charged membranes. The extent of the specific binding was predominantly dependent on the existence of S21, whereby substitution or deletion of S21 significantly hindered the binding affinity. Taken together, our findings based on a correlative measurement platform enabled the quantification of the nonelectrostatic, selective binding interactions of the C-terminal of MKlp2 to certain PIP receptors and contributed to understanding the molecular mechanisms on complete cytokinetic abscission in cells.

General gene expression patterns and stemness of the gingiva and dental pulp.

BACKGROUND/PURPOSE: Due to the unique properties of healing processes and cellular differentiation, the gingiva and dental pulp have attracted attention as a potential source of mesenchymal stem cells (MSCs). The purpose of this study was to obtain molecular-level information on these tissues in terms of their function and differentiation processes and investigate stemness. MATERIALS AND METHODS: Healthy gingival tissues were collected from patients (n = 9; aged 7-12 years) who underwent simple surgical procedures, and normal dental pulp tissues were obtained from patients (n = 25; aged 11-25 years) undergoing tooth extraction for orthodontic reasons. Complementary DNA microarray, qRT-qPCR, and immunohistochemical staining were performed to assess general and MSC gene expression patterns. RESULTS: In the gingival tissue, genes related to keratinization, the formation of epithelial cells and ectoderm, and immune and/or inflammatory responses were highly expressed. Meanwhile, in the dental pulp tissue, genes related to ion transport, neuronal development and axon guidance, bone and enamel mineralization, extracellular matrix organization, and angiogenesis were highly expressed. When focusing on the expression of MSC genes, induced pluripotent stem (iPS) cell genes, such as Sox2, c-Myc, and KLF4, were expressed at higher levels in the gingival tissue, whereas dental stem cell genes, such as NT5E and VCAM1, were expressed in dental pulp tissue. CONCLUSION: We found different general and MSC gene expression patterns between the gingival and dental pulp tissue. These results have implications for future regenerative medicine, considering the application of gingival tissue as a potential source of iPS cells.

Modifications of Rect-Spring to Enhance the Engagement of Ectopically Entrapped Molars with 2 Case Reports.

The Rect-spring appliance, used for the management of ectopically erupting molars, shows weak retention on mesially tilted molars. We present three modifications of the appliance for better engagement and their advantages. We describe cases of two 7-year-old patients with ectopically erupting maxillary first molars with a 2.2 mm and 2.5 mm depth of entrapment, respectively. The modified Rect-spring (mRS) was inserted between the ectopically erupting first molar and adjacent primary second molar, and exerted a distalization force with an interproximal wedging effect at the same time. After 3 months, the ectopically erupting first molars were successfully brought into proper occlusion. No discomfort was reported. The mRS is suitable for various locking cases except for severely tilted molars without requiring any laboratory procedures. We suggest it as the first choice for unlocking the first molars.

The Piston Elastic: A Novel Device for Treating Entrapped Ectopic Permanent Molars.

Ectopic eruption of the permanent molar may absorb the distal root of the primary second molar and may result in a decreased arch length or delayed eruption of the permanent tooth, requiring timely treatment. Therefore, we devised an effective and convenient method to unlock the entrapped tooth using a novel device called a "piston-elastic". This case report aims to explain the design and clinical application of this piston-elastic and to describe successful cases. Three patients (aged 6, 13, and 16 years) with ectopically erupted maxillary and mandibular molars, respectively, were treated with a piston-elastic. It was bound to the locked molar to improve the eruption path. After a certain time period, the repulsive force pushed the surface of the adjacent tooth, improving the eruption path of the entrapped tooth. The piston-elastic is a novel device that simply and effectively changes the direction of eruption of ectopically entrapped molars. As it can be manufactured and attached to the chair side, impression acquisition on a model cast and laboratory procedures are unnecessary. Compared to existing methods, the piston-elastic can be easily produced and delivered, causes little irritation, and is inexpensive.

An Analysis of the Temporomandibular Joint Range of Motion and Related Factors in Children and Adolescents.

This study was designed to establish safe guidelines for pediatric dental practice regarding temporomandibular joint (TMJ) range of motion (ROM) and mouth area (MA). A total of 438 children aged 3-15 years old of homogenous ethnicity participated in the study; the distribution of participants was approximately equal (sex; n = 15; age, n = 30). Maximum mouth opening (MMO), body height, weight, and age of each participant were recorded, and the TMJ ROM including anterior and lateral movements, MA, and mouth width were documented. Males showed higher mouth width, MMO, and MA values than females. MMO and MA increased with age, height, and weight in a statistically significant manner. MMO of 40 mm is reached by the age of 5.2 years, at a height of 105.9 cm and a weight of 18.6 kg. MMO showed a moderate correlation with age, height, weight, and mouth width, and MA moderately correlated with mouth width. Anterior and lateral movements did not show any close relation to these aforementioned factors. The findings of this study suggest that forcible mouth opening over 40 mm should be more cautiously considered, especially in children shorter than 105 cm, lighter than 18 kg and in children under 5 years old.

In Vivo Evaluation of Decellularized Human Tooth Scaffold for Dental Tissue Regeneration.

Conventional root canal treatment may result in loss of tooth vitality, which can lead to unfavorable treatment outcomes. Notably, a ceased tooth development of immature permanent teeth with open apices, regeneration of periodontal ligaments (PDL), and pulp is highly expected healing process. For regeneration, the scaffold is one of the critical components that carry biological benefits. Therefore, this study evaluated a decellularized human tooth as a scaffold for the PDL and pulp tissue regeneration. A tooth scaffold was fabricated using an effective decellularization method as reported in previous studies. PDL stem cells (PDLSCs) and dental pulp stem cells (DPSCs) obtained from human permanent teeth were inoculated onto decellularized scaffolds, then cultured to transplant into immunosuppressed mouse. After 9 weeks, PDLSCs and DPSCs that were inoculated onto decellularized tooth scaffolds and cultured in an in vivo demonstrated successful differentiation. In PDLSCs, a regeneration of the cementum/PDL complex could be expected. In DPSCs, the expression of genes related to revascularization and the hard tissue regeneration showed the possibility of pulp regeneration. This study suggested that the potential possible application of decellularized human tooth could be a scaffold in regeneration PDL and pulp tissue along with PDLSCs and DPSCs, respectively, as a novel treatment method.

Production of protein hydrolysate from Protaetia brevitarsis seulensis (Kolbe) larvae by enzyme treatment under high pressure.

To improve the industrial use of health-functional materials based on edible insects, the objective of this study was to establish optimal conditions for improving the quality of Protaetia brevitarsis seulensis larval (PBSL) hydrolysates. PBSL was extracted using four methodologies: atmospheric pressure 50 °C-water extraction, atmospheric pressure 95 °C-water extraction, atmospheric pressure 50 °C-water enzymatic hydrolysis, and enzyme treatment under high pressure (HPE). The quality characteristics of soluble solid content, extraction yield, total protein content, protein yield, protein content with low molecular weight (LMW) (< 1kD), and the amino acid composition of hydrolysates were compared based on the different methods. All of the quality characteristics were found to be higher for HPE extracts than for the other extracts. Under optimized HPE conditions, extraction yield, protein yield, protein content with LMW, amino acid content and the content of essential amino acids increased by 3.4, 4.4 1.4 1.5, and 1.3 times respectively, compared to the other methods.

Anti-Photoaging Effect of Plant Extract Fermented with Lactobacillus buchneri on CCD-986sk Fibroblasts and HaCaT Keratinocytes.

Ultraviolet (UV) exposure triggers the abnormal production of reactive oxygen (ROS) species and the expression of matrix metalloproteinases (MMPs) that are responsible for photoaging. Probiotics are widely used in healthcare and for immune enhancement. One probiotic, Lactobacillus buchneri is found in Kimchi. This study was aimed at assessing the anti-photoaging effect of plant extracts fermented with L. buchneri (PELB) to develop functional cosmetics. We investigated the anti-photoaging effect of PELB in a UVB-induced photoaging in vitro model and selected effective extracts using the elastase inhibition assay, ELISA for Type I procollagen and collagenase-1, and quantitative real time PCR. Normal human dermal fibroblasts and epidermal keratinocytes were pre-treated with PELB and exposed to UVB. We found that PELB decreased elastase activity and increased type I collagen expression in a UVB-induced photoaging in vitro model. In addition, PELB greatly reduced collagenase activity and MMP mRNA levels in a UVB-induced photoaging in vitro model. Furthermore, PELB promoted the expression of moisture factor and anti-oxidant enzymes in a UVB-induced photoaging in vitro model. These results indicated that the PELB could be potential candidates for the protective effects against UVB-induced photoaging. Overall, these results suggest that PELB might be useful natural components of cosmetic products.

Changing the Angulation of the Tooth Germ in the Bony Crypt: A Case Report.

An ankylosed primary molar may cause rotation or ectopic impaction of succedaneous premolar. When conventional treatment modalities such as observation, surgical exposure with or without orthodontic traction, and autotransplantation are not possible, the simple surgical relocation method could be an alternative treatment option for a lingually rotated premolar during the tooth germ stage before opting to extraction. In the case reported herein, the lingually rotated permanent mandibular second premolar tooth germ was surgically relocated within its bony crypt. Continued root development and spontaneous eruption were observed without complications during the 3.5-year follow-up period.

The Effect of MMP-13, MMP-12, and AMBN on Gingival Enlargement and Root Deformation In a New Type of Gingival Fibromatosis.

This case compared gene-expression between a new type of idiopathic gingival fibromatosis (IGF) and normal gingiva, to clarify the nature of the gingival overgrowth and dental anomaly. A 6-year-old girl with generalized gingival overgrowth and root deformations was diagnosed with IGF. Gene expression profiles were compared between normal gingiva (N=9) and one IGF gingiva using cDNA microarray. Genes related to regulation of cell proliferation and proteolytic degradation were expressed strongly in IGF. MMP-13 and MMP-12 expression were 120 times and 96 times lower in IGF, respectively, whereas AMBN expression was 79 times higher. RT-PCR and immunohistochemical staining supported the microarray results. Reduced proteolytic activity due to low MMP-13 and MMP-12 expression appears to be a potential mechanism for gingival overgrowth. Genetic investigations, such as expression levels of MMP-13, MMP-12, and AMBN, may enable classification of a new syndrome characterized by gingival enlargement with abnormal root development.

Cytokine Expression of Stem Cells Originating from the Apical Complex and Coronal Pulp of Immature Teeth.

INTRODUCTION: The aim of this study was to measure and compare the expression levels of cytokines from developing apical complex cells (DACCs) and dental pulp stem cells (DPSCs) of the immature tooth. METHODS: DPSC-conditioned medium (CM) and DACCs-CM were obtained from human young teeth, and 174 cytokines secreted from each CM were identified and compared. A cytokine membrane array and enzyme-linked immunosorbent assay were used to measure and compare the expression levels of the cytokines. Immunocytochemistry targeting insulin-like growth factor-1 and neurotrophin-3 was additionally performed. RESULTS: There were statistically significant differences in the expression levels of 25 cytokines: 22 and 3 were expressed more strongly in DPSCs-CM and DACCs-CM, respectively. Odontoblast differentiation-related cytokines were more strongly expressed in DPSCs-CM, while cell-proliferation-related cytokines were more strongly expressed in DACCs-CM. Proinflammatory and anti-inflammatory cytokines were predominantly expressed in DPSCs-CM and DACCs-CM, respectively. CONCLUSIONS: DPSCs may exert a stronger paracrine effect than DACCs on regeneration of the dentin-pulp complex, in terms of odontoblast differentiation.

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition.

There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein-phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH-PI(4,5)P2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH-PI(4,5)P2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein-phosphoinositide interactions.

Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy.

BACKGROUND: Cell fixation is an essential step to preserve cell samples for a wide range of biological assays involving histochemical and cytochemical analysis. Paraformaldehyde (PFA) has been widely used as a cross-linking fixation agent. It has been empirically recognized in a gold standard protocol that the PFA concentration for cell fixation, CPFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of CPFA is optimized. METHODS: Here, we investigated the mechanical properties of cell fixation as a function of CPFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of CPFA (0-10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells. RESULTS: We found that both Young's modulus, E, and the fluctuation amplitude of apical cell membrane, am, were almost constant in a lower CPFA (<10-4%). Interestingly, in an intermediate CPFA between 10-1 and 4%, E dramatically increased whereas am abruptly decreased, indicating that entire cells begin to fix at CPFA = ca. 10-1%. Moreover, these quantities were unchanged in a higher CPFA (>4%), indicating that the cell fixation is stabilized at CPFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols. CONCLUSIONS: Taken together, these findings offer a deeper understanding of how varying PFA concentrations influence the mechanical properties of cells and suggest new avenues for establishing refined cell fixation protocols.

Biological efficacy of two mineral trioxide aggregate (MTA)-based materials in a canine model of pulpotomy.

The aim of this study was to compare the biocompatibility of Endocem Zr® and ProRoot MTA® by histopathologic analysis in a canine model of pulpotomy. This study utilized 39 teeth of two beagle dogs. The exposed pulp tissues were treated by pulpotomy using ProRoot MTA (n=19) or Endocem Zr (n=20). After 8 weeks, the teeth were extracted and processed with hematoxylin-eosin staining for histologic evaluation. Most of the specimens in both groups developed a calcific barrier at the pulp amputation site and formed an odontoblast layer. However, some of the Endocem Zr specimens showed less calcific barrier formation with a greater inflammatory response and less odontoblast layer formation when compared with the ProRoot MTA specimens. ProRoot MTA and Endocem Zr specimens developed a calcific barrier; however, ProRoot MTA was more biocompatible than Endocem Zr.

Prevalence of delayed tooth development and its relation to tooth agenesis in Korean children.

OBJECTIVE: The aim of this study was to investigate the epidemiology of delayed tooth development (DTD) and the link between DTD and tooth agenesis (TA). DESIGN: The dental maturity of all of the developing permanent teeth of 4611 children (2417 males and 2194 females) was evaluated from panoramic radiographs. The prevalence of DTD and TA was analyzed, and gender difference for DTS and TA was investigated. The correlation of DTD and TA was investigated in intra-fields and inter-fields. RESULTS: The total prevalence of DTD among the 4611 children was 3.40%. The maxillary second premolar was the most frequently delayed tooth (1.02%), followed by the maxillary second molar (0.88%) and the mandibular second premolar (0.74%). DTD significantly correlated with TA in both intra-fields and inter-fields (p<0.05). CONCLUSIONS: The field of delayed development exhibited a significant correlation with that of TA.

Comparison of Stemness and Gene Expression between Gingiva and Dental Follicles in Children.

The aim of this study was to compare the differential gene expression and stemness in the human gingiva and dental follicles (DFs) according to their biological characteristics. Gingiva (n = 9) and DFs (n = 9) were collected from 18 children. Comparative gene expression profiles were collected using cDNA microarray. The expression of development, chemotaxis, mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSs) related genes was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Histological analysis was performed using hematoxylin-eosin and immunohistochemical staining. Gingiva had greater expression of genes related to keratinization, ectodermal development, and chemotaxis whereas DFs exhibited higher expression levels of genes related to tooth and embryo development. qRT-PCR analysis showed that the expression levels of iPSc factors including SOX2, KLF4, and C-MYC were 58.5 ± 26.3, 12.4 ± 3.5, and 12.2 ± 1.9 times higher in gingiva and VCAM1 (CD146) and ALCAM (CD166) were 33.5 ± 6.9 and 4.3 ± 0.8 times higher in DFs. Genes related to MSCs markers including CD13, CD34, CD73, CD90, and CD105 were expressed at higher levels in DFs. The results of qRT-PCR and IHC staining supported the microarray analysis results. Interestingly, this study demonstrated transcription factors of iPS cells were expressed at higher levels in the gingiva. Given the minimal surgical discomfort and simple accessibility, gingiva is a good candidate stem cell source in regenerative dentistry.

Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.

Comparative analysis of secretory factors from permanent- and deciduous-teeth periodontal ligament cells.

OBJECTIVE: Studies of regenerative therapies have focused on the paracrine effects of mesenchymal stem cells, but little has been revealed about the humoral factors of periodontal ligament (PDL) stem cells. The aim of this study was to identify and compare the secretory factors of human permanent- and deciduous-teeth PDL cells (P-PDL and D-PDL cells, respectively) in order to understand the characteristics of these cells and their potential applications in regenerative therapies. DESIGN: Conditioned media were collected from P-PDL and D-PDL cells (P-PDL-CM and D-PDL-CM, respectively). These media were analyzed with high-performance liquid-chromatography-coupled electrospray ionization tandem mass spectrometry and a cytokine membrane assay. In addition, Western blot analysis was performed to verify the differences between the two media. RESULTS: Cytokines related to neurogenesis (NT-3 and NT-4) and angiogenesis-related cytokines (EGF and IGF-1) were identified in P-PDL-CM. The expression levels of immune-response-related cytokines (interleukins I, II, and IV) and secreted proteins related to tissue degradation and catalytic activities (matrix metallopeptidase 1 (MMP1), Proteasome subunit, alpha type, 1 (PSMA1), and cullin 7 (CUL7)) were higher in D-PDL-CM. Vasorin (VASN) was expressed more strongly in P-PDL-CM, but tudor domain containing 7 (TDRD7) was expressed more strongly in D-PDL-CM in Western blot analysis. CONCLUSION: The cytokine expressions of the two cell types showed different patterns, especially in neurogenesis and immune responses. P-PDL cells are more suitable candidates for applications in regenerative therapies.