Chung-Chen Yao

Landmark identification errors on cone-beam computed tomography-derived cephalograms and conventional digital cephalograms.

INTRODUCTION In this study, we investigated the landmark identification errors on cone-beam computed tomography (CBCT)-derived cephalograms and conventional digital cephalograms. METHODS Twenty patients who had both a CBCT-derived cephalogram and a conventional digital cephalogram were recruited. Twenty commonly used lateral cephalometric landmarks and 2 fiducial points were identified on each cephalogram by 11 observers at 2 time points. The mean positions of the landmarks identified by all observers were used as the best estimate to calculate the landmark identification errors. In addition to univariate analysis, regression analysis of landmark identification errors was conducted for identifying the predicting variables of the observed landmark identification errors. To properly handle the multilayer correlations among the clustered observations, a marginal multiple linear regression model was fitted to our correlated data by using the well-known generalized estimating equations method. In addition to image modality, many variables potentially affecting landmark identification errors were considered, including location and characteristics of the landmark, seniority of the observer, and patient information (sex, age, metallic dental restorations, and facial asymmetry). RESULTS Image modality was not the significant variable in the final generalized estimating equations model. The regression coefficient estimates of the significant landmarks for the overall identification error ranged from -0.99 (Or) to 1.42 mm (Ba). The difficulty of identifying landmarks on structural images with multiple overlapping--eg, Or, U1R, L1R, Po, Ba, UMo, and LMo--increased the identification error by 1.17 mm. In the CBCT modality, the identification errors significantly decreased at Ba (-0.76 mm). CONCLUSIONS The overall landmark identification errors on CBCT-derived cephalograms were comparable to those on conventional digital cephalograms, and Ba was more reliable on CBCT-derived cephalograms.

Glucosamine promotes osteogenic differentiation of dental pulp stem cells through modulating the level of the transforming growth factor-β type I receptor†

Dental pulp stem cells (DPSCs) are clonogenic, self‐renewing, and multi‐potential DPSCs capable of differentiating into osteoblasts. In this study, primary cell cultures were obtained from human dental pulp tissue of developing third molars, and flow cytometry was used to sort the subpopulation of DPSCs with STRO‐1 and CD146 double‐positive expression (denoted “DPSCs”). It was noted that DPSCs exhibited superior clonogenic potential and osteogenic differentiation capability than the dental pulp cell subpopulation with STRO‐1 and CD146 double‐negative expression (denoted DPCs). Furthermore, a low concentration (0.005 mg/ml) of exogenous glucosamine (GlcN) was effective in promoting the early osteogenic differentiation of DPSCs through the transforming growth factor‐β receptor (TGF‐βr) type I and Smads signal pathways, which upregulated the Runt‐related transcription factor 2/core‐binding factor alpha1 (Runx2/Cbfa1) and alkaline phosphatase at both the mRNA and protein levels. In the presence of osteogenic supplements, GlcN‐treated DPSCs produced more mineralized‐matrix deposition than did the untreated groups. Taken together, this study demonstrates the capacity of GlcN to promote the osteogenic differentiation of human DPSCs, and the underlying mechanism involves a TGF‐βr‐dependent Smad signal pathway. J. Cell. Physiol. 225: 140–151, 2010.

Relationship of Chondrocyte Apoptosis to Matrix Degradation and Swelling Potential of Osteoarthritic Cartilage

BACKGROUND AND PURPOSE Softening of cartilage is the initial degenerative step of osteoarthritic cartilage by matrix degradation and corruption of interconnection of the collagen fibrillar network. The purpose of this study was to investigate the correlation of chondrocyte apoptosis, matrix degradation, and the corruption of collagen architecture in the development of severe swelling of osteoarthritic cartilage. METHODS Twenty osteoarthritic and 7 normal femoral neck fractured cartilage samples were obtained from patients with knee osteoarthritis and normal patients with femoral neck fracture at the time of total hip joint replacement surgery. Apoptosis was verified by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end-labeling) staining and structural changes were observed under phase-contrast microscopy. Matrix degradation was evaluated by histochemical analysis of proteoglycans. Swelling tests were performed by immersing the cartilage slices in hypotonic solution. The results of ultrastructural study of collagen architecture of osteoarthritic cartilage performed by scanning electron microscopy before and after swelling were compared. RESULTS Matrix degradation was most prominent in the middle zone of osteoarthritic cartilage. The percentage of chondrocytes in osteoarthritic cartilage showing apoptosis ranged from 15 to 20% (average, 18%; standard deviation (SD) = 3.2%) and was correlated with the extent of structural changes and matrix degradation. The swelling strain of the osteoarthritic cartilage varied from 120 to 200% (average, 160%; SD = 40%) depending on the degree of matrix degradation and structural changes. The loss of interconnectivity of collagen fibrillar architecture was correlated with the increased swelling potential of osteoarthritic cartilage. CONCLUSION This study demonstrated that chondrocyte apoptosis was correlated with matrix degradation and the corruption of fibrillar architecture and that the extent of these manifestations correlated with the swelling potential of osteoarthritic cartilage. These findings also emphasize the importance of the fibrillar architecture in maintaining the mechanical properties of cartilage.

Differential regulation of collagen, lysyl oxidase and MMP-2 in human periodontal ligament cells by low- and high-level mechanical stretching

BACKGROUND AND OBJECTIVE Mechanical stretching modulates extracellular matrix (ECM) protein synthesis by periodontal ligament (PDL) cells. However, the mechanoregulation of lysyl oxidase (LOX), a key enzyme for collagen cross-linking, is not fully understood. In the present study, we hypothesized that low-level and high-level mechanical stretching differentially regulates collagen deposition and the expression of LOX and the enzymes responsible for ECM degradation, such as MMP-2 in PDL cells. MATERIAL AND METHODS Human PDL cells were cultured on flexible-bottom culture plates and subjected to cyclic mechanical stretching (3% and 10% elongation at 0.1 Hz) for 24 and 48 h in a Flexercell FX-4000 strain unit. The levels of expression of type I collagen alpha 1 (COL1A1), type III collagen alpha 1 (COL3A1), lysyl oxidase (LOX), MMP2 and TIMP2 mRNAs were analyzed using an RT-PCR technique. The cell layer and the culture medium were separately collected and processed for detection of the following ECM-related molecules: (i) total collagen content using a Sircol dye-binding method; (ii) LOX protein expression by western blotting; (iii) LOX activity using a fluorometric assay; and (iv) MMP-2 enzyme activity by gelatin zymography. RESULTS Low-level (3%) mechanical stretching of PDL cells upregulated the expression of COL1A1, COL3A1 and LOX mRNAs, enhanced the production of collagen and increased the LOX activity but did not change the level of expression of MMP2 or TIMP2 mRNA. The collagen content and LOX activity showed obvious elevation in the medium, but not in the cell layer. High-level (10%) mechanical stretching downregulated COL1A1 mRNA but upregulated COL3A1 mRNA; however, the effect on COL3A1 was smaller, and occurred earlier, compared with the effect on the COL1A1 gene. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs but did not change collagen production or LOX activity. Moreover, high-level mechanical stretching increased the level of pro-MMP-2, especially in the cell layer. CONCLUSIONS This study substantiates the mechanoregulation of the expression of ECM-related molecules in PDL cells. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs, but did not affect collagen production or LOX activity. In addition to increasing the transcription of COL1A1, COL3A1 and LOX genes, low-level mechanical stretching enhanced total collagen production and LOX activity, which should favor ECM stabilization. As an effective regulator of ECM remodeling, mechanical stretching can be exploited in periodontal regeneration and ligament tissue engineering via application of appropriate mechanical stimulation.

A new classification of mandibular asymmetry and evaluation of surgical-orthodontic treatment outcomes in Class III malocclusion

INTRODUCTION Facial asymmetry is a common manifestation in patients with Class III malocclusion. The aims of this study were to classify mandibular asymmetry in Class III patients and to evaluate treatment outcomes according to different characteristics of asymmetry. MATERIALS AND METHODS Three dimensional cone-beam CT images of 38 patients were analyzed for menton deviation and discrepancies between bilateral structures of mandibular ramus and body. The patients were classified into 3 groups. Groups 1 and 2 exhibited a larger distance of ramus to midsagittal plane on menton-deviated side. In group 1, menton deviation was greater than ramus asymmetry and the condition was reversed for group 2. Group 3 had menton deviation contralateral to the side with larger transverse ramus distance. The features of asymmetry were delineated and the outcomes after surgical-orthodontic treatment were analyzed. RESULTS Group 1 exhibited a roll rotation of mandibular structures. Mandibular deviation of group 2 patients was more of a horizontal shift nature rather than rotation. Group 3 patients displayed a yaw rotation of mandible to the side with lesser growth in body and ramus. After treatment, menton deviation and body asymmetry were significantly improved in all 3 groups, but the effect of therapy on ramus asymmetry was less predictable, especially for group 3. CONCLUSIONS The classification system is simple and clinically useful and could form a base for future studies on facial asymmetry.

Cephalometric Analysis Using Digital Radiography Acquired by a Storage Phosphor Imaging System-A Comparison of Reading soft- and hard-copies

Traditional cephalometric analysis is done on a transparent blue film which is called a hard-copy. The aim of this study was to explore differences in cephalometric measurements between monitor displays (i.e., soft-copies) and hard-copies of digital cephalometric images acquired by a storage phosphor (SP) imaging system. Ten digital cephalograms and their conventional counterparts (a hard-copy on a transparent blue film) were obtained using an SP imaging system and a dye-sublimation printer. Twelve orthodontic residents identified 19 cephalometric landmarks on monitor-displayed soft-copies of digital cephalograms with computer-aided methods, as well as on their counterpart hard-copies using conventional methods. To assess differences between measurements obtained from soft- and hard- copies, 27 cephalometric items generated by a computer-aided digital cephalometric analysis system were analyzed to determine the interobserver errors and absolute differences in the measured values. The influence of the level of cephalometric experience of the observer on measurement differences between soft-and hard-copies was also assessed. Overall interobserver errors of 27 commonly used cephalometric measurements were smaller for the soft-copy compared to the hard-copy of SP digital cephalometric images (0.20~1.78 units for soft-copies, and 0.34~2.72 units for hard-copies). Differences in measurements between the soft-and hard-copies were more significant for linear than angular measurements, particularly items referring to the Frankfort horizontal plane. The overall differences were clinically acceptable and not substantially influenced by the cephalometric experience of the observers.

Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding: HSIEH et al.

Multicellular three‐dimensional (3D) spheroids allow intimate cell–cell communication and cell–extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two‐dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia‐inducible factor 1‐α (HIF‐1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF‐1α and VEGF. The treatment with YC‐1 (inhibitor to HIF‐1α) blocked the upregulation of VEGF, indicating a downstream event to HIF‐1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y‐27632 (inhibitor to Rho‐associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho‐associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF‐1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC‐based research and application.

Chondrocyte Apoptosis and Structural Changes of Cartilage under Biomechanical Compression-A Preliminary Study

The purpose of this study was to investigate degeneration-related ultrastructural changes of cartilages with molecular and biomechanical factors involved in matrix degradation under load. Immature pig front knee joints were used for experiments. Explant culture of the cartilage specimen were under compressive force with 30 MPa with 20% strain for 24 hours by FlexorCell. Control groups were prepared without loading. Structural study with Nomarski light microscopy and ultrastructural observation with scanning electron microscopy were performed. The analysis of chondrocyte apoptosis by TUNELs method combined with corresponding view of phase contrast were performed as well. The immunochemical study of proteoglycan was investigated to show the matrix degradation. Our study demonstrated that the percentage of chondrocyte apoptosis was higher in the loaded cartilage than in the control group. A higher level of proteolglycan degradation was detected in the loaded cartilage compared to the control. The collagen fibrillar architecture of the loaded cartilage was shown to exhibit less interconnectivity under SEM investigation. This study has provided a new understanding of the way in which the chondrocytes and proteoglycans involved in the chondrocyte-matrix interaction, and how this relates to changes in fibrillar architecture under load.