Peijun Lyu

Photobiomodulation of human adipose-derived stem cells using 810nm and 980nm lasers operates via different mechanisms of action.

Photobiomodulation (PBM) using red or near-infrared (NIR) light has been used to stimulate the proliferation and differentiation of adipose-derived stem cells. The use of NIR wavelengths such as 810nm is reasonably well accepted to stimulate mitochondrial activity and ATP production via absorption of photons by cytochrome c oxidase. However, the mechanism of action of 980nm is less well understood. Here we study the effects of both wavelengths (810nm and 980nm) on adipose-derived stem cells in vitro. Both wavelengths showed a biphasic dose response, but 810nm had a peak dose response at 3J/cm2 for stimulation of proliferation at 24h, while the peak dose for 980nm was 10-100 times lower at 0.03 or 0.3J/cm2. Moreover, 980nm (but not 810nm) increased cytosolic calcium while decreasing mitochondrial calcium. The effects of 980nm could be blocked by calcium channel blockers (capsazepine for TRPV1 and SKF96365 for TRPC channels), which had no effect on 810nm. To test the hypothesis that the chromophore for 980nm was intracellular water, which could possibly form a microscopic temperature gradient upon laser irradiation, we added cold medium (4°C) during the light exposure, or pre-incubated the cells at 42°C, both of which abrogated the effect of 980nm but not 810nm. We conclude that 980nm affects temperature-gated calcium ion channels, while 810nm largely affects mitochondrial cytochrome c oxidase.

Photobiomodulation (blue and green light) encourages osteoblastic-differentiation of human adipose-derived stem cells: role of intracellular calcium and light-gated ion channels

Human adipose-derived stem cells (hASCs) have the potential to differentiate into several different cell types including osteoblasts. Photobiomodulation (PBM) or low level laser therapy (LLLT) using red or near-infrared wavelengths has been reported to have effects on both proliferation and osteogenic differentiation of stem cells. We examined the effects of delivering four different wavelengths (420 nm, 540 nm, 660 nm, 810 nm) at the same dose (3 J/cm2) five times (every two days) on hASCs cultured in osteogenic medium over three weeks. We measured expression of the following transcription factors by RT-PCR: RUNX2, osterix, and the osteoblast protein, osteocalcin. The 420 nm and 540 nm wavelengths were more effective in stimulating osteoblast differentiation compared to 660 nm and 810 nm. Intracellular calcium was higher after 420 nm and 540 nm, and could be inhibited by capsazepine and SKF96365, which also inhibited osteogenic differentiation. We hypothesize that activation of light-gated calcium ion channels by blue and green light could explain our results.

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

Red (660 nm) or near-infrared (810 nm) photobiomodulation stimulates, while blue (415 nm), green (540 nm) light inhibits proliferation in human adipose-derived stem cells

We previously showed that blue (415 nm) and green (540 nm) wavelengths were more effective in stimulating osteoblast differentiation of human adipose-derived stem cells (hASC), compared to red (660 nm) and near-infrared (NIR, 810 nm). Intracellular calcium was higher after blue/green, and could be inhibited by the ion channel blocker, capsazepine. In the present study we asked what was the effect of these four wavelengths on proliferation of the hASC? When cultured in proliferation medium there was a clear difference between blue/green which inhibited proliferation and red/NIR which stimulated proliferation, all at 3 J/cm2. Blue/green reduced cellular ATP, while red/NIR increased ATP in a biphasic manner. Blue/green produced a bigger increase in intracellular calcium and reactive oxygen species (ROS). Blue/green reduced mitochondrial membrane potential (MMP) and lowered intracellular pH, while red/NIR had the opposite effect. Transient receptor potential vanilloid 1 (TRPV1) ion channel was expressed in hADSC, and the TRPV1 ligand capsaicin (5uM) stimulated proliferation, which could be abrogated by capsazepine. The inhibition of proliferation caused by blue/green could also be abrogated by capsazepine, and by the antioxidant, N-acetylcysteine. The data suggest that blue/green light inhibits proliferation by activating TRPV1, and increasing calcium and ROS.

Influence of object translucency on the scanning accuracy of a powder-free intraoral scanner: A laboratory study

Statement of problem. Limited information is available regarding the influence of object translucency on the scanning accuracy of a powder‐free intraoral scanner. Purpose. The purpose of this in vitro study was to evaluate the scanning accuracy of a confocal microscopy principle powder‐free intraoral scanner on ceramic copings and to analyze the relationship between scanning accuracy and object translucency. Methods. Six slice specimens (12×10 mm) and 6 offset copings (1.00‐mm thickness) were made from different translucent homogeneous ceramic blocks (CEREC Blocs, S0‐M to S5‐M, highest to lowest translucency). The primary sintered zirconia offset coping was produced in the same way as the control. Optical parameters related to the translucency of each slice were measured with a spectrophotometer. Three‐dimensional (3D) datasets of the surface morphology of offset copings were obtained by using the intraoral scanner. The same white wax resin bases were used for registration. Quantitative parameters of scanning trueness and precision were measured. One‐way ANOVA was used to analyze the values of each parameter among the 6 ceramic blocks. Bivariate correlation was used to analyze the relationships between each parameter of scanning accuracy and translucency (&agr;=.05). Results. Translucent copings showed a positive 3D bias (S0‐M to S5‐M: 0.149 ±0.038 mm to 0.068 ±0.020 mm), a narrower collar diameter (Dd=−0.067 mm), larger convergence angle (&Dgr;&PHgr;=2.79 degrees), and larger curvature radius of the internal gingivoaxial corner (&Dgr;&rgr;=0.236 mm). The smaller the percentage sum of scattering and absorption, the greater was the occurrence of scanning bias (r=−0.918) and curvature (r=−0.935) decrease. Conclusions. Use of the tested powder‐free intraoral scanner, higher translucency objects (greater translucency than S1‐M/A1C) resulted in lower scanning accuracy and morphological changes. Therefore, more suitable methods of measurement are still required.

An automatic tooth preparation technique: A preliminary study

The aim of this study is to validate the feasibility and accuracy of a new automatic tooth preparation technique in dental healthcare. An automatic tooth preparation robotic device with three-dimensional motion planning software was developed, which controlled an ultra-short pulse laser (USPL) beam (wavelength 1,064 nm, pulse width 15 ps, output power 30 W, and repeat frequency rate 100 kHz) to complete the tooth preparation process. A total of 15 freshly extracted human intact first molars were collected and fixed into a phantom head, and the target preparation shapes of these molars were designed using customised computer-aided design (CAD) software. The accuracy of tooth preparation was evaluated using the Geomagic Studio and Imageware software, and the preparing time of each tooth was recorded. Compared with the target preparation shape, the average shape error of the 15 prepared molars was 0.05–0.17 mm, the preparation depth error of the occlusal surface was approximately 0.097 mm, and the error of the convergence angle was approximately 1.0°. The average preparation time was 17 minutes. These results validated the accuracy and feasibility of the automatic tooth preparation technique.

Comparative Evaluation of the Artefacts Index of Dental Materials on Two-Dimensional Cone-beam Computed Tomography.

The aim of the study was to propose the artefact index on cone-beam computed tomography (CBCT) images of clinical prosthodontics materials, and to compare the effect of the artefacts on CBCT image clarity of normal oral tissues. Seven spheres of different materials were secured on the centre of a resin baseboard, respectively, and four human molars in vitro were placed at 10 mm front, back, left and right of the sphere. The board was scanned using CBCT with the same setting. 10 tomographic images from each of the seven data sets with clear artefacts was selected. The grayscale measuring tool of Photoshop software was used to measure the grayscale (G0) within the boundary of tomographic image and the grayscales of the streaky artefacts that were 1 mm and 20 mm outside the circular boundary (G1 and G2). The arc length, L1, of the circular boundary with artefacts was measured; the circumference, L2, was calculated. The artefact index, A, was determined as (G1/G0) × 0.5 + (G2/G1) × 0.4 + (L2/L1) × 0.1. The artefact index A can comprehensively represent the effect of artefacts on CBCT image clarity for oral tissue.

A quantitative study of 3D-scanning frequency and Δd of tracking points on the tooth surface

Micro-movement of human jaws in the resting state might influence the accuracy of direct three-dimensional (3D) measurement. Providing a reference for sampling frequency settings of intraoral scanning systems to overcome this influence is important. In this study, we measured micro-movement, or change in distance (∆d), as the change in position of a single tracking point from one sampling time point to another in five human subjects. ∆d of tracking points on incisors at 7 sampling frequencies was judged against the clinical accuracy requirement to select proper sampling frequency settings. The curve equation was then fit quantitatively between ∆d median and the sampling frequency to predict the trend of ∆d with increasing f. The difference of ∆d among the subjects and the difference between upper and lower incisor feature points of the same subject were analyzed by a non-parametric test (α = 0.05). Significant differences of incisor feature points were noted among different subjects and between upper and lower jaws of the same subject (P < 0.01). Overall, ∆d decreased with increasing frequency. When the frequency was 60 Hz, ∆d nearly reached the clinical accuracy requirement. Frequencies higher than 60 Hz did not significantly decrease Δd further.

Quantitative evaluation of three-dimensional facial scanners measurement accuracy for facial deformity

Objective: To evaluate the measurement accuracy of three-dimensional (3D) facial scanners for facial deformity patients from oral clinic. Methods: 10 patients in different types of facial deformity from oral clinical were included. Three 3D digital face models for each patient were obtained by three facial scanners separately (line laser scanner from Faro for reference, stereophotography scanner from 3dMD and structured light scanner from FaceScan for test). For each patient, registration based on Iterative Closest Point (ICP) algorithm was executed to align two test models (3dMD data & Facescan data) to the reference models (Faro data in high accuracy) respectively. The same boundaries on each pair models (one test and one reference models) were obtained by projection function in Geomagic Stuido 2012 software for trimming overlapping region, then 3D average measurement errors (3D errors) were calculated for each pair models also by the software. Paired t-test analysis was adopted to compare the 3D errors of two test facial scanners (10 data for each group). 3D profile measurement accuracy (3D accuracy) that is integrated embodied by average value and standard deviation of 10 patients’ 3D errors were obtained by surveying analysis for each test scanner finally. Results: 3D accuracies of 2 test facial scanners in this study for facial deformity were 0.44±0.08 mm and 0.43±0.05 mm. The result of structured light scanner was slightly better than stereophotography scanner. No statistical difference between them. Conclusions: Both test facial scanners could meet the accuracy requirement (0.5mm) of 3D facial data acquisition for oral clinic facial deformity patients in this study. Their practical measurement accuracies were all slightly lower than their nominal accuracies.

Detection of cervical lymph node metastasis from oral cavity cancer using a non-radiating, noninvasive digital infrared thermal imaging system

This study aimed to evaluate the diagnostic performance of a non-radiating, noninvasive infrared (IR) thermal imaging system in the detection of cervical lymph node metastasis from oral cavity cancer. In this prospective clinical trial, a total of 90 oral cavity cancer patients suspected of having cervical lymph node metastasis underwent IR imaging of the neck prior to neck dissection. Analysis of the IR images was performed by two methods: manual qualitative analysis and automatic analysis by an entropy-gradient support vector machine (EGSVM). The efficacies of the EGSVM-based infrared thermal imaging system and contrast-enhanced computed tomography (CT) were compared by using the Noninferiority Testing. Compared with manual qualitative analysis, the EGSVM-based automatic analysis had a higher sensitivity (84.8% vs. 71.7%), specificity (77.3% vs. 72.7%), accuracy (81.1% vs. 72.2%), positive predictive value (79.6% vs. 73.3%) and negative predictive value (82.9% vs. 71.1%). The EGSVM-based infrared thermal imaging system was noninferior to contrast-enhanced CT (P < 0.05). The EGSVM-based infrared thermal imaging system showed a trend of higher sensitivity, whereas contrast-enhanced CT showed a trend of higher specificity. The EGSVM-based infrared thermal imaging system is a promising non-radiating, noninvasive tool for the detection of lymph node metastasis from oral cavity cancer.