Yong-Jin Chen

The combined use of cell sheet fragments of periodontal ligament stem cells and platelet-rich fibrin granules for avulsed tooth reimplantation.

The aim of this study was to construct a cell transplant method consisting of cell sheet fragments of periodontal ligament stem cells (PDLSCs) and platelet-rich fibrin (PRF) granules to enhance periodontal healing in avulsed tooth reimplantation. To test this concept in vitro, human PDLSCs were isolated and characterized by colony forming unit assay, cell surface marker characterizations, and their osteogenic/adipogenic differentiation potential. The biological effects of autologous PRF as a growth factor-enriched endogenous scaffold on human PDLSCs were then investigated and quantified for statistical analyses, including cell viability and proliferation, alkaline phosphatase (ALP) activity, and the gene expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col-I), and cementum protein 23 (CP23). It was found that the PRF induced a significant and continuous stimulation of proliferation in human PDLSCs throughout the 7-day incubation period. Furthermore, the PRF suppressed the osteoblastic differentiation of PDLSCs by decreasing ALP activity and the gene expression of BSP and OCN while up-regulating the mRNA expression levels of Col-I and CP23 during the testing period. To assess the potential application of the PDLSCs/PRF construct in tooth reimplantation, 36 incisors were extracted from 6 dogs. The incisors then underwent 2 h of dry storage and were randomly divided into four groups receiving different strategies of reimplantation, where the avulsed teeth were reimplanted with the use of the autologous PDLSCs/PRF construct (cell sheet fragments in combination with PRF granules), with the use of autologous PDLSCs or PRF alone, or without adjuvant use of PRF or PDLSCs. Eight weeks post-reimplantation, the PDLSCs/PRF group achieved a more effective periodontal healing, characterized by the regeneration of PDL-like tissues and a reduction of ankylosis and inflammation, compared with the other testing groups. These overall results suggest that the PDLSCs/PRF construct may be a useful tool for alveolar surgery that has the potential to improve the clinical outcomes in future avulsed tooth reimplantations.

Oxidative damage and HSP70 expression in masseter muscle induced by psychological stress in rats

Psychological stressors are generally associated with masseter muscle dysfunction and disorders in emotional response. In addition, oxidative states and HSP70 expression, which are involved in the physical and pathological changes of the masseter muscle, could be altered in the stressed tissues and organs. However, the link between psychological stress and the redox homeostasis or the expression of HSP70 in masseter muscles in rats has not been examined. Therefore, we used a communication box paradigm to induce psychological stress in rats. The successful establishment of the animal model was evidenced by an increase in plasma corticosterone and adrenocorticotropic hormone (ACTH). Meanwhile, the stressed rats showed a decrease in the number of entries on open arms, percentage of time spent in open arms, and distance moved in the elevated plus-maze test. The stressed rats also displayed a decrease in the time spent in the center zone, active velocity, and the distance moved in the open-field test. These results demonstrate affective-like behavioral changes in the stressed rats. Moreover, compared with the control rats, a decrease in SOD, GSH-Px and catalase activities and an increase in MDA content were observed in the masseter muscles in stressed rats after 3 weeks and 5 weeks, and the HSP70 expression was elevated in muscles in the rats exposed to stress for 5 weeks. These results indicate that psychological stress induces oxidative damage and up-regulates the expression of HSP70 in masseter muscles in rats, which are associated with behavior resembling anxiety.

Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1.

Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs). The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR) experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs.

Psychological Stress Alters Ultrastructure and Energy Metabolism of Masticatory Muscle in Rats

To investigate the effects of psychological stress on the masticatory muscles of rats, a communication box was applied to induce the psychological stress (PS) in rats. The successful establishment of psychological stimulation was confirmed by elevated serum levels of adrenocorticotropic hormone (ACTH) and changed behaviors in the elevated plusmaze apparatus. The energy metabolism of the bilateral masseter muscles was tested via chemocolorimetric analysis, whereas muscle ultrastructure was assessed by electron microscopy. In comparison to the control group, the PS group showed evidence of swollen mitochondria with cristae loss and reduced matrix density in the masticatory muscles after three weeks of stimulation; after five weeks of stimulation, severe vacuolar changes to the mitochondria were observed. Increased vascular permeability of the masticatory muscle capillaries was found in the five-week PS rats. In addition, there was decreased activity of Na+-K+ATPase and Ca2+-ATPase and a simultaneous increase in the activity of lactate dehydrogenase and lactic acid in the masticatory muscles of PS rats. Together, these results indicate that psychological stress induces alterations in the ultrastructure and energy metabolism of masticatory muscles in rats.

Estrogen and Its Receptor Enhance Mechanobiological Effects in Compressed Bone Mesenchymal Stem Cells

Mechanical stimulation and estrogen have been proven to be two important factors in promoting mesenchymal stem cell activity, which is closely associated with bone formation, mass maintenance and remodeling. However, the superposition effects of mechanical stimulation and estrogen on stem cells remain unknown. It is also unclear if the estrogen receptor (ER) plays only a key role in estrogen signaling or if it is also involved in the mechanotransduction of stem cells. To investigate the role of estrogen and its receptors in the mechanobiological effects in bone mesenchymal stem cells (BMSCs), isolated mesenchymal stem cells from bone marrow were exposed to mechanical pressure under additional estrogen treatment or ER blockade. Cell proliferation was examined using an MTT assay and alkaline phosphatase (ALP) activity was determined by a modified enzyme kinetic method. Alignment of the cytoskeleton was observed by Coomassie brilliant blue staining and F-actin fluorescent staining. Cellular ultrastructure was observed under transmission electron microscope. Expression of ERα was investigated using Western blot analysis. Results indicated that mechanical pressure promoted cell proliferation, ALP activity, ERα expression and F-actin stress fiber formation. Overall, this effect was enhanced by the addition of estrogen and inhibited by ER blockade. We concluded that pressure stimulated proliferation and differentiation capability via F-actin transduction in BMSCs. The effects were enhanced by the addition of estrogen, and the ER plays an important role in regulating mechanobiological effects and the mechanotransduction processes of BMSCs.

Psychological stress induces temporary masticatory muscle mechanical sensitivity in rats.

To explore the relationship between psychological stress and masticatory muscle pain, we created a communication stress animal model to determine whether psychological stress could induce increased mechanical sensitivity in masticatory muscles and to study the changes of mechanical nociceptive thresholds after stress removal. Forty-eight male Sprague-Dawley rats were divided into a control group (CON), a foot-shocked group (FS, including 3 subgroups recorded as FS-1, FS-2, and FS-3), a psychological stress group (PS), and a drug treatment group (DT). PS and DT rats were confined in a communication box for one hour a day to observe the psychological responses of neighboring FS rats.Measurements of the mechanical nociceptive thresholds of the bilateral temporal and masseter muscles showed a stimulus-response relationship between psychological stress and muscle mechanical sensitivity. The DT rats, who received a diazepam injection, showed almost the same mechanical sensitivity of the masticatory muscles to that of the control in response to psychological stress. Fourteen days after the psychological stressor was removed, the mechanical nociceptive thresholds returned to normal. These findings suggest that psychological stress is directly related to masticatory muscle pain. Removal of the stressor could be a useful method for relieving mechanical sensitivity increase induced by psychological stress.

Development of a System to Monitor Laryngeal Movement during Swallowing Using a Bend Sensor

Background Swallowing dysfunction (also known as dysphagia), which results in a deterioration of nutritional intake, slows rehabilitation and causes aspiration pneumonia, is very common following neurological impairments. Although videofluorographic (VF) examination is widely used for detecting aspiration, an objective and non-invasive method for assessing swallowing function has yet to be established because of a lack of adequate devices and protocols. In this paper, a bend sensor whose resistance is altered by bending was introduced to monitor swallowing-related laryngeal movement. Methods Six healthy male volunteers were recruited in the present study. Specific time points on the signal waveform produced by the bend sensor were defined to describe laryngeal movement by differential analysis. Additionally, the physiological significance of the obtained waveform was confirmed by analyzing the sequential correlations between the signal waveform from the bend sensor and hyoid bone kinetics simultaneously recorded by VF. Results Seven time points were successfully defined on the signal waveform to reference laryngeal movement. Each time point was well correlated with certain VF events, with evidence of no significant time lags, and there were positive correlations between waveform time points and matched VF events. Furthermore, obvious similarities were noticed between the duration of each phase on the signal waveform and the duration of the matched hyoid bone activity. Conclusions The present monitoring system using a bend sensor might be useful for observing the temporal aspects of laryngeal movement during swallowing, and it was well coordinated with hyoid bone movement.

Psychological stress alters the ultrastructure and increases IL-1β and TNF-α in mandibular condylar cartilage

Psychological factors can be correlated with temporomandibular disorders (TMDs), but the mechanisms are unknown. In the present study, we examined the microstructural changes and expression of proinflammatory cytokines in mandibular condylar cartilage of the temporomandibular joint (TMJ) in a psychological stress animal model. Male Sprague-Dawley rats (8 weeks old, 210 ± 10 g) were randomly divided into 3 groups: psychological stress (PS, N = 48), foot shock (FS, N = 24), and control (N = 48). After inducing psychological stress using a communication box with the FS rats for 1, 3, or 5 weeks, PS rats were sacrificed and compared to their matched control littermates, which received no stress and were killed at the same times as the PS rats. Body and adrenal gland weight were measured and corticosterone and adrenocorticotropic hormone levels were determined by radioimmunoassay. After hematoxylin-eosin staining for histological observation, the ultrastructure of the TMJ was examined by scanning electron microscopy. Transcription and protein levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were evaluated by ELISA and semi-quantitative RT-PCR. The PS group showed a significantly higher adrenal gland weight after 3 weeks of stress and higher hormone levels at weeks 1, 3, and 5. Histopathological changes and thinning cartilage were apparent at weeks 3 and 5. In the PS group, TNF-α increased at 1, 3, and 5 weeks and IL-1β increased significantly after 1 and 3 weeks of stress, and then decreased to normal levels by 5 weeks. Psychological stress increased plasma hormone levels and RT-PCR indicated increased IL-1β and TNF-α expression in the TMJ in a time-dependent manner. These results suggest that cytokine up-regulation was accompanied by stress-induced cartilage degeneration in the mandibular condyle. The proinflammatory cytokines play a potential role in initiating the cartilage destruction that eventually leads to the TMDs.

Coordination in oro-pharyngeal biomechanics during human swallowing.

In swallowing, the tongue contacts against the hard palate to generate pressure for propelling a bolus from the oral cavity into the pharynx. Meanwhile, the hyoid and larynx move upward and forward to facilitate the bolus from the pharynx into the esophagus. It has been well known that sequential coordination between those actions is critical for safety accomplishment of swallowing. However, the absence of noninvasive assessment for it limits the detection to the physiological symptom of dysphagia. We applied a sensor sheet on the hard palate to measure tongue contact pressure and a bend sensor on the frontal neck to monitor the laryngeal movement, which was synchronized with hyoid motion for assessing the coordination between both actions in 14 healthy male subjects when swallowing 5ml of water. The sequential order of tongue pressure and hyoid movement was successfully displayed. Tongue pressure was produced after slight movement of the hyoid and closely to the hyoid elevation, then reached a maximum when the hyoid stabilized in the most anterior-superior position, and ceased concurrently with the onset of hyoid descent. Additionally, the synchronized data from both sensors showed positive correlations between identified time points on the laryngeal signal waveform and onset, peak and offset of tongue pressure. Our sensing system successfully showed the coordination between tongue pressure production and hyoid motion, and could be a simple and noninvasive method for clinicians to evaluate the oral and pharyngeal stages of swallowing.

Crosstalk between integrin and G protein pathways involved in mechanotransduction in mandibular condylar chondrocytes under pressure

To investigate the role of integrin and G protein pathways in the mechanotransduction process within MCCs and explore the possible crosstalk between the two traditional signal pathways, in vitro-cultured rabbit MCCs were treated with pressure. The mRNA level of alpha5beta1 integrin was determined by in situ hybridization and the distributions of vinculin, Galphaq/11 protein, F-actin and intracellular calcium were studied with a laser scanning confocal microscope. Increased integrin alpha5beta1 expression, enhanced stress fiber assembly, elevated G protein and vinculin level and up-regulated IP(3) channel sensitivity were found in the mechanotransduction process of MCCs under pressure. Furthermore, the vinculin and the Galphaq/11 were observed co-localized with each other, and the F-actin reassembly and stress fibers formation could be inhibited by intracellular calcium channel blocking, which gave direct evidence that the traditional integrin-mediated or G protein-mediated signaling pathways coordinately regulate the function of MCCs under mechanical stimulation.