Human dental pulp stem cells expressing transforming growth factor ß3 transgene for cartilage-like tissue engineering.

BACKGROUND AIMS: The aim of this study was to engineer sizable three-dimensional cartilage-like constructs using stem cells isolated from human dental pulp stem cells (DPSCs). METHODS: Human DPSCs were isolated from teeth extracted for orthodontic treatment and enriched further using immuno-magnetic bead selection for stem cell marker CD146. Chondrogenic lineage differentiation of DPSCs induced using recombinant transforming growth factor ß3 (TGFß3) was verified by pellet culture. Because the use of recombinant proteins is associated with rapid degradation and difficult in vivo administration, we constructed the recombinant adeno-associated viral vector encoding human TGFß3 and determined the best multiplicity of infection for DPSCs. Transduced DPSCs were seeded on poly-l-lactic acid/polyethylene glycol (PLLA/PEG) electrospun fiber scaffolds demonstrating proper attachment, proliferation and viability as shown by scanning electron microscopy micrographs and CCK-8 cell counting kit. Scaffolds seeded with DPSCs were implanted in the back of nude mice. RESULTS: Transduced DPSCs highly expressed human TGFß3 for up to 48 days and expressed chondrogenic markers collagen IIa1, Sox9 and aggrecan, as verified by immunohistochemistry and messenger RNA (mRNA). Immunohistochemistry for TGFß3/DPSC constructs (n = 5/group) showed cartilage-like matrix formation with glycosaminoglycans. In vivo constructs with TGFß3/DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSC constructs (n = 5/group). Western blot analysis confirmed this expression pattern on the protein level (n = 3/group). CONCLUSIONS: Immuno-selected DPSCs can be successfully differentiated toward chondrogenic lineage, while expressing the chondrogenic inducing factor. Seeded on PLLA/PEG electrospun scaffold, human DPSCs formed three-dimensional cartilage constructs that could prove useful in future treatment of cartilage defects.

Evaluation of the long-term storage stability of saliva as a source of human DNA.

OBJECTIVES: The objectives of this paper are to determine the storage stability of saliva at 37 °C over an 18-month period, and its influence on the DNA yield, purity, PCR protocols and genotyping efficacy. MATERIALS AND METHODS: Of the 60 participants, blood samples were obtained from 10 and saliva from 50. Samples were subjected to different storage conditions: DNA extracted immediately; DNA extracted following storage at 37 °C for 1, 6, 12 and 18 months. Subsequently, DNA yield, OD(260/280) and OD(260/230) ratios were measured. The isolated DNA was used to amplify exons 0-7 of the RUNX2 gene and subsequently sequenced. Furthermore, 25 SNPs were genotyped. RESULTS: The mean DNA yield, OD(260/280) and OD(260/230) ratios obtained from blood were 67.4 ng/µl, 1.8 ± 0.05 and 1.8 ± 0.4 respectively. DNA yield obtained from saliva was significantly higher than blood (p < 0.0001), ranging from 97.4 to 125.8 ng/µl while the OD(260/280) ratio ranged from 1.8 ± 0.13 to 1.9 ± 0.1. The success rates for the 25 SNPs ranged from 98 to 100 % for blood and 96-99 % for saliva samples with the genotype frequencies in Hardy-Weinberg equilibrium (>0.01). CONCLUSIONS: Saliva can be stored at 37 °C for 18 months without compromising its quality and ability to endure genetic analyses. CLINICAL RELEVANCE: Saliva is a viable source of human DNA to facilitate the feasibility of large-scale genetic studies.

The effect of naringin on early growth and development of the spheno-occipital synchondrosis as measured by the expression of PTHrP and Sox9--an in vitro model.

The aim of this study was to assess the effect of the flavonoid naringin on the growth of the spheno-occipital synchondrosis by quantifying the levels of expression of Sox9 and PTHrP in an in vitro mouse model. Fifty 1-day-old BALB/c mice were randomly assigned to experimental or control groups, and each group equally divided into five time frames (6, 24, 48, 72 and 168 hours). The mice were sacrificed with phenobarbitone sodium, and the spheno-occipital synchondroses dissected and cultured in control or experimental medium, with the experimental medium supplemented with 0.1 µm naringin. Sections of the specimens underwent immunohistochemical staining for Sox9 and PTHrP, and the amount of expression was quantified using true-colour RGB (red-green-blue) computer-assisted image-analysing system with digital imaging. Data analysis showed there was a significant increase of expression of Sox9 at 6 and 24 hours (P < 0.001) between experimental and control groups, however, there was no significant difference between the levels of expression of PTHrP between experimental and control groups at any of the time frames. There was a very weak correlation found in this study between the expression of PTHrP and Sox9. In conclusion, naringin enhances the growth of the spheno-occipital synchondrosis through over expression of Sox9. This is a successful in vitro model to study factors regulating the growth of the spheno-occipital synchondrosis.

Reliability of panoramic radiographs for identifying supernumerary teeth in children.

OBJECTIVES. To evaluate the reliability of panoramic radiographs (PRs) for identifying supernumerary teeth (ST) and to determine whether the level of dental training of the observer influenced the identification of ST. METHODS. Seventy-five PRs were randomly selected from the patient records and 18 examiners independently rated 25 radiographs each, for specific risk factors as well as for a measure of adequacy. Subsequently, the results were paired with those of the other examiners who assessed the same set of PRs. Descriptive statistics were computed using Fisher's exact test, and kappa statistics were used to assess the inter- and intra-observer reliability. RESULTS. Four hundred and fifty PRs were available for analysis. The overall sensitivity and specificity figures were 50% and 98.3%, whereas the positive and negative predictive values were 90.6% and 83.6%, respectively. The sensitivity figures for Junior House Dental Officers and Postgraduate Paediatric Dental Trainees were 39.2% and 60.8%, whereas the specificity figures were 99.4% and 95% with slight inter-examiner and moderate intra-examiner reliability. CONCLUSIONS. Panoramic radiographs are unreliable for identifying ST, and higher level of dental training is essential for identifying ST.

Biobased poly(propylene sebacate) as shape memory polymer with tunable switching temperature for potential biomedical applications.

From the point of better biocompatibility and sustainability, biobased shape memory polymers (SMPs) are highly desired. We used 1,3-propanediol, sebacic acid, and itaconic acid, which have been industrially produced via fermentation or extraction with large quantities as the main raw materials for the synthesis of biobased poly(propylene sebacate). Diethylene glycol was used to tailor the flexibility of the polyester. The resulted polyesters were found to be promising SMPs with excellent shape recovery and fixity (near 100% and independent of thermomechanical cycles). The switching temperature and recovery speed of the SMPs are tunable by controlling the composition of the polyesters and their curing extent. The continuously changed switching temperature ranging from 12 to 54 °C was realized. Such temperature range is typical for biomedical applications in the human body. The molecular and crystalline structures were explored to correlate to the shape memory behavior. The combination of potential biocompatibility and biodegradability of the biobased SMPs makes them suitable for fabricating biomedical devices.

Analysis of genetic polymorphisms in skeletal Class I crowding.

INTRODUCTION: Dental crowding is a problem for both adolescents and adults in modern society. The purpose of this research was to identify single nucleotide polymorphisms (SNPs) responsible for crowding in subjects with skeletal Class I relationships. METHODS: The case subjects consisted of healthy Chinese people living in Hong Kong with skeletal Class I relationships and at least 5 mm of crowding in either arch. The control subjects met the same requirements but lacked crowding or spacing. SNP genotyping was performed on the MassARRAY platform. The chi-square test was used to compare genotype and allele type distributions between the case and the control groups. Logistic regression was used to calculate odds ratios with 95% confidence intervals, and the effects of age and sex for each SNP. Analyses of linkage disequilibrium and haplotype associations between SNPs were performed with software. RESULTS: Five SNPs were found to be significantly different in genotype or allele type distributions. SNP rs372024 was significantly associated with crowding (P = 0.004). Two SNPs, rs3764746 and rs3795170, on the EDA gene were found to be associated marginally. SNPs rs1005464 and rs15705 also exhibited marginal association with crowding. The effects of associated SNPs remained significant after adjustments for age and sex factors. CONCLUSIONS: This study suggests an association for the genes EDA and XEDAR in dental crowding in the Hong Kong Chinese population.

Comparison of oral impacts experienced by patients treated with labial or customized lingual fixed orthodontic appliances.

INTRODUCTION: Our objective was to compare the oral impacts experienced by patients treated with labial or customized lingual fixed orthodontic appliances. METHODS: This was an age- and sex-matched prospective longitudinal study of 60 adult patients treated with either labial or customized lingual fixed orthodontic appliances over a 3-month period. Ratings of oral impacts experienced and satisfaction were made on visual analog scales at 3 time points after appliance fixation. Variations in oral impacts and satisfaction over the trajectory of treatment were assessed. Area-under-the-curve analyses were conducted to assess variations in oral impacts and satisfaction between the groups. RESULTS: All patients experienced oral impact disturbances, although these disturbances decreased over time (P < 0.001). Patients treated with customized lingual appliances reported more oral discomfort (P < 0.001), dietary changes (P < 0.001), swallowing difficulty (P < 0.001), speech disturbances (P < 0.001), and social problems (P < 0.001) than did those in the other group. There was no significant difference between the groups regarding ratings of oral self-care, mastication, and satisfaction level of treatment (P > 0.05). CONCLUSIONS: The findings indicate that oral impacts are commonly experienced during both labial and customized lingual fixed orthodontic therapies. However, the oral impacts decreased over the observational period. Patients treated with customized lingual appliances experienced more oral impacts. Both groups had similar levels of treatment satisfaction.

Novel method of cell-free in vitro synthesis of the human fibroblast growth factor 1 gene.

Recombinant DNA projects generally involve cell-based gene cloning. However, because template DNA is not always readily available, in vitro chemical synthesis of complete genes from DNA oligonucleotides is becoming the preferred method for cloning. This article describes a new, rapid procedure based on Taq polymerase for the precise assembly of DNA oligonucleotides to yield the complete human fibroblast growth factor 1 (FGF1) gene, which is 468 bp long and has a G+C content of 51.5%. The new method involved two steps: (1) the design of the DNA oligonucleotides to be assembled and (2) the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene. The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure. This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.

Evaluation of bone thickness around the mental foramen for potential fixation of a bone-borne functional appliance: a computer tomography scan study.

AIM: A mandible bone-borne Herbst appliance (MBBHA) would avoid the proclination of the lower incisors that occurs with any teeth-borne functional appliance. But mapping of the bone characteristics at potential fixation areas around the mental foramen has not been carried out so far. The aim of this computer tomographic (CT) study was to evaluate bone thickness at specific positions around the mental foramen. MATERIAL AND METHODS: CT scans of 60 randomly chosen adult Hong Kong Chinese subjects (mean age 28 ± 6.3 years) were used to measure the bi-cortical bone thickness in the mandible in the mental foramen area. The thickness of buccal and lingual cortical and cancellous bone was assessed at the following locations: 10 mm (A10 mm) and 5 mm (A5 mm) anterior, 10 mm (P10 mm) and 5 mm (P5 mm) posterior, and 5 mm (Inf5 mm) below the mental foramen. RESULTS: The amount of buccal cortical bone thickness ranged between 1.89 mm, 10 mm anterior of the mental foramen, and 2.16 mm, 10 mm posterior to its location. At the A10 mm level, cortical thickness showed a marginal statistically significant difference between A5 and A10 mm. The total amount of bone thickness ranged from 10.19 to 12.06 mm. CONCLUSION: At the locations studied around the mental foramen, a mean bicortical bone thickness of 10-12 mm was measured. No large variation in the thickness was found between bicortical bone thicknesses in the measured locations around the mental foramen. Thorough evaluation on a case-by-case basis is advisable.

Systemic effect of Fructus Psoraleae extract on bone in mice.

Fructus Psoraleae extract is used in China for the treatment of bone diseases. The objective of the study was to investigate the systemic effect of Fructus Psoraleae extract consumption on bone histomorphology. Sixteen 8-week-old male BALB/c mice were divided into control and experimental groups. In the control group, eight mice were fed daily with distilled water. In the experimental group, eight mice were fed daily with distilled water mixed with Fructus Psoraleae extract. The mice were kept for 5 weeks and then killed. Using micro-computed tomography, 20 micro-tomographic slices with a separation of 0.25 mm were acquired to cover the proximal end of the left tibia of each mouse. Quantitative morphometry of the bone structure was performed. The results showed that consumption of Fructus Psoraleae extract significantly increased the bone volume/tissue volume ratio by 11.8%. The bone trabeculae increased by 7.1% in thickness so that the bone density was increased. To conclude, Fructus Psoraleae extract taken orally increases bone density and alters bone histomorphology.

Management of Severe Class II Malocclusion With Sequential Fixed Functional and Orthodontic Appliances: A Case for MOrthRCSEd Examination.

Fixed functional appliance is an effective way of treating skeletal Class II malocclusion in children and adolescents. The following case report documented a 13-year-old boy with 15 mm overjet treated by a phase I 12-month growth modification therapy using Herbst appliance with Hyrax palatal expander and high pull headgear in a stepwise mandibular advancement protocol followed by a phase II pre-adjusted edgewise appliance therapy. It is one of the cases submitted for the Membership of Orthodontics Examination of the Royal College of Surgeons of Edinburgh.

A comparison of pain experienced by patients treated with labial and lingual orthodontic appliances.

SUMMARY: The aim of this prospective longitudinal study was to compare pain experiences among Chinese adult patients treated with labial and lingual orthodontic appliances. Sixty patients, 30 with labial appliances (18 females and 12 males, mean age 20.33 years, SD +/- 4.205) and 30 with lingual appliances (22 females and 8 males, mean age 21.63 years, SD +/- 2.236), rated their overall pain experience on a 100 mm visual analogue scale (VAS) at three time points: 1 week (T(1)), 1 month (T(2)), and 3 months (T(3)) after bracket placement. In addition, on a separate 100 mm VAS, they rated their pain experience at the locations of the tongue, lips, cheeks, gums, face, and jaw at T(1), T(2), and T(3). Changes in pain VAS were conducted using Friedman analysis of variance, area under the curve (AUC) analysis and the data were compared using a t-test. There was no significant difference in global ratings of pain among those treated with labial or lingual appliances (P > 0.05). Among both groups, global ratings of pain decreased over the study period (P < 0.001). Patients treated with lingual appliances reported higher ratings of tongue pain (P < 0.001), while those treated with labial appliances reported higher ratings of lip (P < 0.001) and cheek (P < 0.001) pain. The findings indicate that patients treated with labial and lingual appliances rate similarly the level of overall pain they experience during treatment. Ratings of overall pain experienced decreased for both treatment groups with time. However, ratings of pain differed at various sites with respect to the type of orthodontic appliance. These findings have implications in informing patients' treatment decision-making processes regarding labial and lingual appliances and in the management of discomfort associated with different treatment modalities.

Use of cervical vertebral maturation to determine skeletal age.

INTRODUCTION: The purpose of this study was to evaluate the validity of the cervical vertebral maturation (CVM) method as an indicator of skeletal age in the circumpubertal period by correlating it to the hand-wrist method (HWM). METHODS: Hand-wrist and lateral cephalometric radiographs of 400 Chinese subjects were randomly selected. Their ages were 10 to 15 years for girls and 12 to 17 years for boys, so they were within the circumpubertal period. Skeletal ages were assessed according to the CVM method and the HWM. RESULTS: The CVM was significantly correlated with HWM skeletal age (Spearman r = 0.9521 [boys] and 0.9408 [girls]). All patients in cervical vertebral stage 3 of the CVM corresponded to stages MP3-FG or MP3-G (around the peak of the growth spurt) in the HWM. CONCLUSIONS: The CVM is a valid indicator of skeletal growth during the circumpubertal period, providing information for timing of growth modification.

The mechanism of action of Lipiburn on fat metabolism.

Paeoniflorin (PF) is the active ingredient in Lipiburn, a fat loss supplement. PF is a component in Paeonia Lactiflora with multiple medicinal uses. Here, we studied the effect of Lipiburn on fat metabolism in differentiated 3T3-L1 adipocytes. Adipocytes exposed to Lipiburn exhibited significant increase in expression of beta-adrenergic receptors, HSL levels, and cAMP and exhibited increase in glycerol release. The data show that Lipiburn affects fat metabolism through the cAMP route by activating HSL which in turn breaks down triglycerides into glycerol and fatty acids.

Use of miniscrews and miniplates in orthodontics.

Paradigms have started to shift in the orthodontic world since the introduction of mini-implants in the anchorage armamentarium. For example, miniscrews have allowed the management of wider discrepancies than those treatable by conventional biomechanics because force can be applied directly from the bone-borne anchor unit. Therefore, miniscrews not only free orthodontists from anchorage-demanding cases, but they also enable clinicians to have good control over tooth movement in 3 dimensions. This review will illustrate the versatility of skeletal anchorage provided by mini-implants in the correction of malocclusion, focusing on orthodontic or orthopedic movements in the 3 spatial planes. Adjunctive orthodontic treatments in adult periodontal conditions and treatment for impacted teeth with the aid of miniscrews will also be discussed.

The inhibitory effects of naringin on the growth of periodontal pathogens in vitro.

Naringin is a flavonoid that is commonly found in grapefruits. The objective of this study was to evaluate the effects of naringin on the growth of periodontal pathogens such as A. actinomycetemcomitans and P. gingivalis in vitro. For comparison, the effects of naringin on several oral microbes were also studied. Different concentrations of naringin solution were added to calibrated suspensions of A. actinomycetemcomitans and P. gingivalis. All the suspensions were incubated for 3, 6 and 24 h in an anaerobic chamber at 37 degrees C. At each time point, selected dilutions from each culture broth were plated on blood agar plates. Colonies recovered on blood agar were visually counted on days 3 and 5, respectively. A. actinomycetemcomitans showed a significant decrease (p < 0.05) in viable counts after 3 h when naringin was added at baseline. P. gingivalis also showed a marked growth reduction in the presence of naringin, and no colony forming units could be observed after 24 h. Naringin also had an inhibitory effect against all bacteria and yeasts tested. The results suggest that naringin possesses significant antimicrobial properties on periodontal pathogens in vitro. It also has an inhibitory effect on some common oral microorganisms in low concentrations.

Condylar growth and mandibular positioning with stepwise vs maximum advancement.

INTRODUCTION: The aim of this study was to compare the effects of several Class II malocclusion treatments on condylar growth and positioning of the mandible. METHODS: The material comprised series of lateral cephalograms obtained at the start, after about 6 months, and after about 12 months of treatment from 3 groups of consecutively treated patients who used a headgear-activator with stepwise mandibular advancement (HGA-S), a headgear-activator with maximum jumping of the mandible (HGA-M), and a headgear-Herbst appliance with stepwise advancement (HGH-S), respectively. Six-month growth data from matched controls were used to calculate the net treatment effects. RESULTS: Mandibular prognathism was enhanced after stepwise advancement but not after maximum jumping, and only during the initial phase of therapy; the effect was significantly greater for the fixed functional appliance than for the removable functional appliance. Lower-facial height was increased by the HGA-S, unchanged by the HGA-M, and restrained by the HGH-S. The low construction bite of the HGH-S meant that the extent of bone apposition on the posterior and superior parts of the condyle was similar, whereas the high construction bite of the HGA-S and the HGA-M meant that the effect on the superior part was greater, but only significantly so after stepwise advancement. CONCLUSIONS: The mode of jumping, the vertical opening, and whether the functional appliance is fixed or removable affect the amount and pattern of condylar growth, and the position of the mandible.

Gene therapy to enhance condylar growth using rAAV-VEGF.

OBJECTIVE: To test the hypothesis that the introduction of specific vascular growth inducting genes would favorably affect mandibular condylar growth in Sprague-Dawley (SD) rats over a limited experimental period. Therefore, the aim of this study is to examine the effect of gene therapy on condylar growth by means of a morphological assessment. MATERIALS AND METHODS: Ninety 35-day-old female SD rats were randomly divided into three groups, which received any of the injections of recombinant adeno-associated virus mediated vascular endothelial growth factor (rAAV-VEGF), rAAV mediated enhanced green fluorescence protein (rAAV-eGFP), or phosphate-buffered saline (PBS) into both mandibular condyles. Each group of rats was sacrificed on the following experimental days: 7, 14, 21, 30, and 60. Left halves of the mandibles were isolated and digital pictures were obtained in a standardized manner. RESULTS: The length of condylar process (B-F) as well as mandibular length (A-F) significantly increased on day 30 and continued to increase until the end of the experiment. Moreover, the width of condyle (Q-R) had increased significantly from day 30 and lasted to day 60. Condylar length (C-D) was found to be significantly longer on day 60. CONCLUSIONS: Gene therapy with VEGF stimulates condylar growth at will. The rAAV-VEGF is an excellent candidate for future gene therapy to induce mandibular growth.

Temporal expression of SOX9 and type II collagen in spheno-occipital synchondrosis of mice after mechanical tension stimuli.

OBJECTIVE: To associate the expressions of SOX9 and type II collagen during growth in the synchondrosis with and without tensile stress in order to understand the role of these factors in the growth of cartilage in spheno-occipital synchondrosis. MATERIALS AND METHODS: Sixty 1-day-old male BALB/c mice were randomly divided into experimental and control groups. Each group was subdivided again into five different time points which were 6, 24, 48, 72, and 168 hours. Each subgroup consisted of five mice. Each mouse was sacrificed using an overdose of pentobarbitone sodium. The synchondroses were aseptically removed and incubated in a 24-well plate with or without tensile stress in tissue culture. Tissue sections were stained immunohistochemically to quantitatively analyze the expression of SOX9 and type II collagen. RESULTS: There was a statistically significant increase of 57% (P < .001) in the expression of SOX9 between the experimental and control groups at 24 hours, followed by a significant increase of 44.4% (P < .001) in the expression of type II collagen at 72 hours. CONCLUSIONS: SOX9 may play an important role for early differentiation of chondrocytes and increase the expression of type II collagen, a major component of the extracellular matrix, during the growth of cartilage in the spheno-occipital synchondrosis.