Morphological analysis of anterior permanent dentition in a Chinese population using cone-beam computed tomography.

PURPOSE: Morphological analysis of permanent anterior dentition is essential for achieving an ideal treatment outcome and avoiding unnecessary failure. This study aimed to analyze the morphologies of anterior teeth in the Chinese population in depth. METHODS: In this retrospective study, 4309 anterior teeth from 401 Chinese patients were investigated using cone-beam computed tomography (CBCT) from 2019-2021. We summarized the morphological characteristics of the anterior teeth in terms of the root length, cementoenamel junction curvature (CEJ-C), root furcation and canal variations. RESULTS: We found that the root lengths of the maxillary anterior incisors were similar (13.3 mm), while the root lengths of the mandibular central (12.2 mm) and lateral incisors (13.4 mm) varied significantly (p < .0001). Both the maxillary (16.6 mm) and mandibular canines (15.5 mm) were found to have greater root lengths than the corresponding incisors (p < .0001). The CEJ-C was significantly greater around incisors (2.5 mm) than around the canines (2.0 mm) in the maxilla (p < .0001), while the curvature remained similar in mandibular anterior teeth (1.8 mm). Root furcation was observed in mandibular canines and lateral incisors. Moreover, all types of Vertucci's classification in anterior dentitions were observed, while two other new types were found. Among them, the maxilla was only observed to exhibit types I, II, III, and ST II, while the mandible was found to exhibit almost all types. However, Type I still accounts for the majority of dentitions. CONCLUSIONS: Morphological analysis of permanent anterior dentition revealed diversity in the tooth length, CEJ-C, furcation proportion, and canal variations. In general, mandibular anterior teeth showed a more complex structure than maxillary teeth.

CGFe and TGF-ß1 enhance viability and osteogenic differentiation of human dental pulp stem cells through the MAPK pathway.

The present study aimed to evaluate the effects of concentrated growth factor exudate (CGFe) and TGF-ß1 on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs). CGFe was prepared from the peripheral blood of healthy donors (obtained with informed consent). STRO-1+ hDPSCs were isolated from dental pulp tissues and treated in four groups: i) Control; ii) TGF-ß1 (1 ng/ml); iii) 100% CGFe; and iv) TGF-ß1 (1 ng/ml) + 100% CGFe group. hDPSC viability was measured via MTT assay. The osteogenic differentiation of hDPSCs was quantified via alkaline phosphatase (ALP) activity, western blotting and reverse transcription-quantitative PCR assays. CGFe and TGF-ß1 enhanced hDPSC viability, upregulated ALP activity, upregulated the expression of phosphorylated (p)-ERK1/2, p-JNK and p-p38 in hDPSCs, and promoted transcription and protein expression of osteogenic-related genes (bone sialoprotein, Runt-related transcription factor 2 and osteocalcin) in hDPSCs. The present study demonstrated that CGFe and TGF-ß1 facilitated the viability and osteogenic differentiation of hDPSCs potentially through activation of the MAPK signaling pathway.

Double-edged effects of interferons on the regulation of cancer-immunity cycle.

Interferons (IFNs) are a large family of pleiotropic cytokines that regulate both innate and adaptive immunity and show anti-cancer effects in various cancer types. Moreover, it was revealed that IFN signaling plays critical roles in the success of cancer therapy strategies, thereby enhancing their therapeutic effects. However, IFNs have minimal or even adverse effects on cancer eradication, and mediate cancer immune escape in some instances. Thus, IFNs have a double-edged effect on the cancer immune response. Recent studies suggest that IFNs regulate each step of the cancer immunity-cycle, consisting of cancer antigen release, presentation of antigens and activation of T cells, trafficking and infiltration of effector T cells into the tumor microenvironment, and recognition and killing of cancer cells, which contributes to our understanding of the mechanisms of IFNs in regulating cancer immunity. In this review, we focus on IFNs and cancer immunity and elaborate on the roles of IFNs in regulating the cancer-immunity cycle.

Proliferation and pluripotency potential of ectomesenchymal cells derived from first branchial arch.

Cranial neural crest-derived ectomesenchymal cells are multipotential progenitors that contribute to various tissue types during embryogenesis. Their potential to be expanded in culture as a monolayer and to be induced into different cell lineages in vitro has not been previously reported in detail. In this study, the ectomesenchymal cells in the first branchial arch were enzymatically isolated from the mandibular processes of BALB/c mice and were maintained in an intact state in a medium containing leukaemia inhibitory factor. Here, we first evaluated the proliferative activity of the cells after the third passage, using bromodeoxyuridine labelling and in situ hybridization of telomerase mRNA. Positive staining for expression of HNK-1, S-100 and vimentin confirmed that the population of stem cells originated from the ectomesenchyme, which did not express cytokeratin. Then we investigated the molecular and cellular characteristics of the ectomesenchymal cells during their differentiation towards neurogenic, endothelial, myogenic and odontogenic lineages. Expression of multiple lineage-specific genes and proteins was detected by utilizing a range of molecular and biochemical approaches when the cells were transferred to inductive medium. Histological and immunohistochemical analysis of the induced cells at various intervals indicated obvious phenotypic alteration and presence of specific proteins for the differentiated lineages, for example nestin, factor VIII, alpha-SMA and dentin sialophosphoprotein (DSPP), respectively. Correlatively, results of reverse transcription-PCR corroborated at mRNA level the expression of the characteristic molecules during differentiation. Therefore, it is suggested that the ectomesenchymal cells derived from the first branchial arch may represent a novel source of multipotential stem cells capable of undergoing expansion and variant differentiation in vitro.

[A study on transfection of green fluorescence protein gene into human adipose stromal cells in vitro].

OBJECTIVE: To make a comparison on the efficiency of two methods for transfecting Green Fluorescence Protein gene into human adipose tissue-derived stromal cells, and to study the biological properties and multipotential differentiation of gene-transfected cells. METHODS: The human subcutaneous adipose tissue was obtained, digested with one volume of collagenase type I, and then cultured with BGJb medium. After subculture and expansion, the human adipose tissue-derived stromal cells infected with Ad-GFP or liposome were observed and analyzed with fluorescence microscopy and flow cytometry to assess transfection efficiency. The growth curve of transfected adipose tissue-derived stromal cells was protracted. The adipose tissue-derived storomal cells were induced to differentiate into osteoblasts, and non-transfected cells were set as control. RESULTS: 42.5% +/- 1.5 of the human adipose tissue-derived stomal cells infected with Ad-GFP were found to express GFP at a level higher than that of the control of liposome (11.40%). Infected adipose tissue-derived stromal cells were noted to form mineralized nodes by the use of Alizarin Red stain. CONCLUSION: The human adipose tissue-derived stromal cells infected with Ad-GFP can express higher level of GFP, and can maintain the ability of proliferation and differentiation as the non-infected human adipose tissue-derived stromal cells do. The infected adipose tissue-derived stromal cells with Ad-GFP can track the change of adipose tissue-derived stromal cells in the study of multipotential differentiation and can serve as cellular vehicles for systemic gene delivery.

[Cultivation and morphological characteristics of rat adipose tissue-derived vascular endothelial cells in vitro].

The subcutaneous adipose tissue from the inguen of four Sprague-Dawley rats was obtained, then digested with one volume of collagenase type I and cultured with BGJb medium. The obtained adipose stromal cells were induced in human endothelial-SFM for 7 d. The cells were observed under inverted microscope every day and identified by transmission electron microscope and immunocytochemical staining with factor VIII antigen. The results showed the induced cells uniformly had characteristic cobblestone morphology of endothelial cells. Factor VIII antigen staining was positive in cytoplasm. Under transmission electron microscope, the cells displayed many finger like microvilli and numerous lysosomes, mitochondria, a few coarse endoplasmic reticulum and Weibel-Palade bodies. The characteristics of the rat adipose tissue-derived endothelial cells were consistent with those of vascular endothelial cells derived from other tissues. It seems that subcutaneous adipose tissue may represent a new alternative source of endogenous vascular endothelial cells.

[Comparison of periodontal indices and Porphyromonas gingivalis between conventional and self-ligating brackets].

OBJECTIVE: To compare the periodontal indices and Porphyromonas gingivalis (P. gingivalis) between the use of self-ligating brackets and conventional brackets. METHODS: Thirty patients were divided into 2 groups(n=15). Self-ligating brackets were used in the experimental group. Conventional brackets were used in the control group. Clinical periodontal indices, including plaque index (PLI), gingival index (GI) and probing depth (PD) of observed teeth were examined at three different time points: Before orthodontic treatment, the first month after treatment and the third month after treatment. Subgingival plaques were collected simultaneously at each time point. The number of total bacteria and P. gingivalis in each sample were detectd and quantitated by real-time quantitative polymerase chain reaction, the percentage of P. gingivalis in total bacteria was obtained. RESULTS: Before treatment, the periodontal indices and the percentage of P. gingivalis in total bacteria had no difference between the two groups (P>0.05). After 1 and 3 months respectively, the periodontal indices and the percentage of P. gingivalis in total bacteria increased with time (P<0.05) and were obviously lower than those of the control group (P<0.05). CONCLUSION: Compared with conventional brackets, the self-ligating brackets are better for periodontal health. But it is adverse effect on oral health.

[Psychological impact on the adolescent patients at the beginning of the fixed orthodontic treatment].

OBJECTIVE: To assess whether there is a short negative psychological influence on adolescent patients at the beginning of the fixed orthodontic treatment. METHODS: 150 patients (average 14.8 years old) were selected. All the patients accepted the fixed appliance treatment. They completed a questionnaire regarding anxiety and depression at the first day when they came to the hospital (T1) and 7 days after fixed appliance insertion (T2). 129 effective questionnaires were received. The scales of anxiety and depression of subjects were assessed according to the questionnaires. RESULTS: Comparing the scales of questionnaires before treatment (T1) and 7 days after placement of fixed appliance (T2), there was a significant increase in anxiety and depression scales in female patients, extraction cases and patients who were unwilling to see an orthodontist. CONCLUSION: There is a certain extent of negative psychological influence on adolescent patients during fixed orthodontic treatment. At the first week after the placement of fixed appliance, three kinds of subjects, female patients, extraction cases and patients who were unwilling to see an orthodontist would suffer from anxiety and depression in emotional reflection.

Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein.

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-gamma2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.

Multilineage differentiation of adipose-derived stromal cells from GFP transgenic mice.

Functional engineering of musculoskeletal tissues generally involves rapid expansion of progenitor cells in vitro while retaining their potential for further differentiation and then induction in specific culture conditions. The autologous adipose-derived stromal cells (ASCs) are considered to contain pluripotent mesenchymal stem cells. Imaging with expression of green fluorescent protein (GFP) facilitates the detailed research on ASCs physiological behavior during differentiation into a variety of cell lineages both in vitro and in vivo. In this study, we aimed to confirm the trans-germ plasticity of homogeneously marked ASCs from GFP transgenic mice. Simultaneously, the term and intensity of GFP expression in ASCs were also focused on during variant inductions, when cells were incubated with multiple growth factors and adjuvant. ASCs were harvested from inguinal fat pads of transgenic nude mice, passaged 3 times in monolayer cultures, and then transferred to osteogenic, adipogenic, neurogenic, and myogenic medium. The morphological characterization of inductive cells was observed using phase-contrast microscopy and histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. The expression of marker genes or proteins was measured using RT-PCR and immunocytochemical analysis. Collagen type I, osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, peroxisome proliferator-activated receptor(PPAR)-gamma2 and lipoprotein lipase (LPL) were positive in adipogenic ones, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) were positive in neurogenic ones, and alpha-smooth muscle actin (alpha-SMA) was positive in myogenic ones. Moreover, the results of fluorescence microscopic imaging suggested that there was no significant decline of GFP expression during ASCs differentiation and the level of GFP maintained stable till differentiated ASCs showed apoptotic phenotype. So the endogenous GFP and multilineage potential of transgenic ASCs had no influences on each other. Since the population of GFP ASCs can be easily identified, it is proposed that they may be promising candidate seed cells for further studies on ASCs tissue engineering, especially the study on engineered tissues formed in vivo.

Characterization of ectomesenchymal cells isolated from the first branchial arch during multilineage differentiation.

Ectomesenchymal cells isolated from the first branchial arch have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. This study was aimed to confirm the plasticity of multilineage differentiation with molecular and cellular characterization. Monolayer cultures of ectomesenchymal cells harvested from the first branchial arch primordia in embryonic day 9.5 BALB/c mice were passaged 3 times before analysis. Staining with antibodies against S-100, p75 and vimentin suggested that the population of stem cells originated from ectomesenchyme, with few contaminating cells stained for cytokeratin. Then, cells were transferred to adipogenic, osteogenic, chondrogenic and odontogenic media. The initiation of controlled differentiation was determined with histological assays, and the expression of tissue-specific genes was detected using immunocytochemical staining and reverse transcription polymerase chain reaction. The adipogenic ectomesenchymal cells showed accumulation of lipid vacuoles and expression of lipoprotein lipase and peroxisome proliferator-activated receptor gamma(2). Following osteoinduction, the fibroblast-like cells became cuboidal and formed mineralized nodules. In addition, the expression of mRNA encoding osteocalcin and osteopontin proved osteogenesis at the molecular level. Chondrogenic lineage expressed collagen type II, aggrecan and Sox9 with a low level of collagen type I in monolayer culture. Odontogenesis was determined by dentin sialophosphoprotein, collagen type I and dentin matrix protein 1 expression. Therefore, we have demonstrated that ectomesenchymal cells from the first branchial arch are capable of extensive multilineage differentiation in vitro, controllable by the culture environment. This makes them a relevant and valuable source of stem cells for research of craniofacial development and tissue engineering of restoration.

[Clinical study on reconstruction of hemifacial atrophy with serratus anterior free-muscle flap].

OBJECTIVE: To study the method of treating hemifacial atrophy with free serratus muscle flap. METHODS: Three patients diagnosed as having serious hemifacial atrophy was treated with free serratus muscle flap. The root of the flap was thoracodorsal artery and thoracodorsal vein, which was anastomosed with superficial temporal artery and vein, facial artery and vein, lingual artery and vein, and so on. During the operation, long thoracic nerve was dissected and anastomosed with facial nerve. The sizes of the flaps were 12 cm x 8 cm - 16 cm x 12 cm. RESULTS: All free-muscle flaps healed well after the transplant. The face and buccal area looked chubby and rounded. There were no obvious protuberance and discontentment on the buccal area. The shoulders of all patients moved well. The facial contour of the patients recovered well during the follow-up period (1-3 years). CONCLUSION: The method has a good result. The long-term effect needs further study.

[A study on the chondrogenesis of the compound of alginate gelatin and bone marrow stromal cells in vivo].

OBJECTIVE: To investigate the chondrogenisis by alginate gelatin and rats' bone marrow stromal cells (BMSCs) chondrogenicly induced in vitro. METHODS: Thirty-two male adult SD rats were assigned randomly to experimental and control groups. In experimental group, bone marrow was obtained from the right tibias of all the rats. After expanding and culturing 3 passages, induced BMSCs by chondrogenic culture medium for 10 days. Suspended induced cells in alginate gelatin, and injected the complex into the hypodermic tissue of the backs of rats autogenously. In control group only alginate gelatins were injected. The grafts were taken out for examinations 4 and 8 weeks after the operations. RESULTS: Considerable cartilage appeared in experimental group 8 weeks after operations. Regular HE staining and alcian blue staining showed a great deal of cartilage holding chondrocyte masses surrounded by abundant matrix. Alginate gelatin decompounded obviously, and the rest distributed among newly formed cartilage. No cartilage appeared in control group all through. CONCLUSION: BMSCs and alginate gelatin have a beautiful future in cartilage tissue engineering.

Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation.

Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs' physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, and Western Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes.

[A study on transfecting green fluorescent protein gene to rat bone marrow mesenchymal stem cells].

OBJECTIVE: To study an efficient method to transfect green fluorescent protein gene (GFP) to rat bone marrow mesenchymal stem cells (MSCs) and to determine the biological properties and differentiation potency of transfected MSCs. METHODS: SD rats' bone marrow MSCs were separated and purified in vitro. After subculture and expansion, MSCs infected with Adenoviral vector (Ad-GFP) or transfected with liposome were observed, and their transfection efficiency was assessed with flow cytometry. The MSCs expressing GFP gene were induced to differentiate to osteoblast, and non-transfected MSCs were set as control. RESULTS: Ad-GFP delivered GFP gene with high efficiency to rat MSCs. (41.3 +/- 1.4)% of MSCs infected with Ad-GFP expressed GFP gene, which was much higher than the control (12.5%). Expression of GFP gene of infected MSCs maintained stable from 1 to 6 weeks after infection. Infected MSCs possessed the same alkaline phosphatase activation as non-infected MSCs, and formed mineralized mouldes. CONCLUSIONS: The infected MSCs with Ad-GFP expressed GFP with much higher efficiency than liposome transfection, and maintained the same ability of proliferation and differentiation as non-infected MSCs. Transfection with Ad-GFP is a highly effective method for labeling MSCs.

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo.

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo. When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo, the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo. These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo, however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.

[Study on multi-lineage potential of bone marrow mesenchymal stem cells derived from green fluorescent protein transgenic mice].

OBJECTIVE: To study the multi-lineage potential of bone marrow mesenchymal stem cells (MSCs) derived from transgenic mice with green fluorescent protein (GFP) gene in vitro. METHODS: A 6-week-old GFP transgenic mouse was executed by dislocation of cervical vertebra, and the marrow in tibia and thighbone was washed out with asepsis. The limited cell strains of MSCs derived from GFP transgenic mice (GFP-MSCs) were obtained with density gradient centrifugation. The passage 3 GFP-MSCs were induced to differentiate into osteoblast, adippcyte, neuron with solution of calcium induction medium, adipogenic medium and neural induction medium respectively. After being calcium-induced, the activity of alkaline phosphatase on GFP-MSCs was determined by micro-plate reader, and alizarin red staining was performed to test the formation of calcium concentration. The adipo-induced MSCs were detected with oil red O staining. Immunocytochemical staining was performed to detect the expression of NSE on neuron-induced MSCs. RESULTS: The ALP activity of GFP-MSCs heightened gradually along with being calcium-induced, and alizarin red staining showed positive. Oil red O staining of adipo-induced cells and NSE immunocytochemical staining of neuron-induced cells demonstrated positive. CONCLUSION: The limited cell strain of GFP-MSCs possesses multi-lineage potential, which can be used as an efficient tracking facility for studying the mechanism of multi-lineage potential on the MSCs.

[Establishment of a culture system of chick embryo for mouse tooth germ development].

OBJECTIVE: To establish a new culture system for mouse tooth germs in chick embryo. METHODS: The mandibular first molar germ fragments of 15 embryonic days' Kunming mouse embryo were implanted into the lateral mesenchyme of 4-5 days' chick embryo wing buds in ove. Eggs were reincubated and implanted tissues were examined by histochemistry. RESULTS: The cultured tooth germ development continued from cap stage to latest bell stage. The ameloblast and the odontoblast all differentiated maturely and secreted matrix. CONCLUSION: 4-5 days' wing buds chick embryo could serve as developing the mouse tooth germs and demonstrate well physiological process of differentiation and morphogenesis.