Mechanotransductive Mechanisms of Biomimetic Hydrogel Cues Modulating Meckel's Cartilage Degeneration.

Meckel's cartilage, a cartilage rod present in the mandible during developmental stages, shows a unique developmental fate: while the anterior and posterior portions undergo ossification, the middle part degenerates. Previously, it was shown that a stiff environment promoted cartilage degeneration in the middle region, while a soft environment enhanced the mineralization in the anterior region of Meckel's cartilage. This study aims to elucidate the spatio-temporal changes in the mechanosensing properties of Meckel's cartilage during its early developmental stages and clarify the mechanotransduction-related mechanisms involved in its degeneration. The results show that the expression of Hippo pathway effector yes-associated protein (YAP) is only detectable in the Meckel's cartilage onward embryonic day (E)14.5, indicating that mechanosensing is dependent on the tissue developmental stage. Consistently, microenvironmental stiffness-induced cartilage degeneration can only be induced in cartilages onward E14.5, but not in those at earlier developmental stages. Expressions of integrin-ß1 and cartilage matrix-degrading enzymes, matrix metalloproteinase 1 (MMP-1) and MMP-13, are significantly enhanced in the degeneration area. Moreover, verteporfin (YAP inhibitor) and integrin-ß1 antibody block the substrate stiffness-induced degeneration by suppressing the expressions of MMP-1 and MMP-13. These data provide new insights into the interplay between biochemical and mechanical cues determining the fate of Meckel's cartilage.

Preclinical bioequivalence study of E.coli-derived rhBMP-2/ß-TCP and autogenous bone in a canine guided-bone regeneration model.

PURPOSE: Bone morphogenetic protein (BMP)-2 is a potent growth factor that is widely used in the orthopedic and dental fields for bone regeneration. However, recombinant human BMP-2 (rhBMP-2) products have not been legally approved in Japan. Recently, our research group succeeded in producing GMP-grade rhBMP-2 using the E. coli system (E-rhBMP-2) at the industrial level and developed E-rhBMP-2 adsorbed onto ß-TCP (E-rhBMP-2/ß-TCP) as an alternative material to autogenous bone grafts. Previous studies on the toxicity, pharmacokinetics, and optimal doses of E-rhBMP-2 have confirmed its safety and efficiency. However, comparative studies with standard treatment therapies are still necessary before clinical application in humans. Therefore, in this preclinical study, we compared the bone regeneration ability of E-rhBMP-2/ß-TCP and autogenous bone grafts in a canine guided-bone regeneration model. METHODS: Following extraction of the maxillary third premolar, box-type bone defects (10 mmL × 4 mmW × 9 mmH) were created in the extraction socket area and transplanted with E-rhBMP-2/ß-TCP or autogenous bone graft in a canine. After 8 weeks, micro-CT and histological analyses were performed. RESULTS: Transplantation of both E-rhBMP-2/ß-TCP and autogenous bone graft significantly promoted bone formation compared to the non-transplantation control group. The bone formation ability of E-rhBMP-2/ß-TCP was equal to that of the autogenous bone graft. Histological analysis showed that excessive infiltration of inflammatory cells and residual ß-TCP particles mostly were not observed in the E-rhBMP-2/ß-TCP transplantation group. CONCLUSION: This preclinical study demonstrated that E-rhBMP-2/ß-TCP and autogenous bone have equal potential to promote bone regeneration.

Fibronectin-induced ductal formation in salivary gland self-organization model.

BACKGROUND: Recent advances in tissue regeneration approaches including 3D organoids, were based on various 3D organogenesis models. However, 3D models are generally technique-sensitive and time-consuming. Thus, we utilized an existing model of submandibular salivary gland (SMG) to modify a simple and highly reproducible in vitro 3D culture model of primary SMG cells self-organization into a well-developed cell spheroid inside Matrigel substrate. We used this model to observe the collective multicellular behavior during spheroid formation. Further, we applied various quantitative approaches including real-time live imaging and immune histochemical image analysis to dissect the cellular dynamics during tissue patterning. RESULTS: On a time-scale of hours, we observed marked size and shape transformations in the developed 3D spheroid which resulted in a spatially-controlled growth differential from the canter to the periphery of the formed aggregates. Moreover, we investigated the effect of fibronectin (FN) on SMG cells self-organization using our simplified culture model. Interestingly, we discovered a novel role of FN in inducing duct-like elongation during initial stages of SMG bud formation. CONCLUSION: This in vitro model provides an excellent tool for analyzing the intercellular dynamics during early SMG tissue development as well as revealing a novel role of FN in SMG ductal expansion.

CCN4/WISP1 controls cutaneous wound healing by modulating proliferation, migration and ECM expression in dermal fibroblasts via α5ß1 and TNFα.

Understanding the mechanisms that control cutaneous wound healing is crucial to successfully manage repair of damaged skin. The goal of the current study was to uncover novel extracellular matrix (ECM) components that control the wound healing process. Full thickness skin defects were created in mice and used to show CCN4 up-regulation during wound-healing as early as 1 day after surgery, suggesting a role in inflammation and subsequent dermal migration and proliferation. To determine how CCN4 could regulate wound healing we used Ccn4-KO mice and showed they had delayed wound closure accompanied by reduced expression of Col1a1 and Fn mRNA. Boyden chamber assays using Ccn4-deficient dermal fibroblasts showed they have reduced migration and proliferation compared to WT counterparts. To confirm CCN4 has a role in proliferation and migration of dermal cells, siRNA knockdown and transduction of CCN4 adenoviral transduction were used and resulted in reduced or enhanced migration of human adult dermal fibroblast (hADF) cells respectively. The induced migration of the dermal fibroblasts by CCN4 appears to work via α5ß1 integrin receptors that further stimulates down-stream ERK/JNK signaling. The regulation of CCN4 by TNF-α prompted us look further at their potential relationship. Treatment of hADFs with CCN4 and TNF-α alone or together showed CCN4 counteracted the inhibition of TNF-α on COL1A1 and FN mRNA expression and the stimulation of TNF-α on MMP-1 and MMP3 mRNA expression. CCN4 appeared to counterbalance the effects of TNF-α by inhibiting downstream NF-κB/p-65 signaling. Taken together we show CCN4 stimulates dermal fibroblast cell migration, proliferation and inhibits TNF-α stimulation, all of which could regulate wound healing.

Sleep bruxism frequency and platelet serotonin transporter activities in young adult subjects.

PURPOSE: To evaluate correlations between serotonin transporter (SERT) uptake ability in human peripheral platelets and sleep bruxism (SB) frequency. METHODS: Subjects were consecutively recruited from sixth-year students at Okayama University Dental School. Subjects were excluded if they (1) were receiving orthodontic treatment, (2) had a dermatological disease, (3) had taken an antidepressant within 6 months, or (4) had used an oral appliance within 6 months. SB frequency was determined as the summary score of three consecutive night assessments using a self-contained electromyography detector/analyzer in their home. Fasting peripheral venous blood samples were collected in the morning following the final SB assessment. SERT amount and platelet number were quantified via an ELISA assay and flow cytometry, respectively. Functional SERT characterization, 5-hydroxytryptamine (5-HT) uptake, maximum velocity (V max), and an affinity constant (K m ) were assessed with a [(3)H] 5-HT uptake assay. The correlations between these variables and SB level were evaluated. RESULTS: Among 50 eligible subjects (26 males, mean age 25.4 ± 2.41 years), 7 were excluded because of venipuncture failure, smoking, and alcohol intake during the experimental period. A small but significant negative correlation between SB level and [(3)H] 5-HT uptake was observed (Spearman's correlation R (2) = 0.063, p = 0.04). However, there were no significant correlations between SB level and total platelet amount, SERT, V max, and K m values (p = 0.08, 0.12, 0.71, and 0.68, respectively). CONCLUSIONS: Platelet serotonin uptake is significantly associated with SB frequency, yet only explains a small amount of SB variability.

Efficient bone formation in a swine socket lift model using Escherichia coli-derived recombinant human bone morphogenetic protein-2 adsorbed in ß-tricalcium phosphate.

Several preclinical studies have shown that Escherichia coli-derived bone morphogenetic protein-2 (E-BMP-2) is as effective as mammalian cell-derived bone morphogenetic protein-2 (C-BMP-2) in the treatment of bone defects. However, further investigation of the effectiveness and determination of the optimal dosage of E-BMP-2 in large animals are still necessary before its full application in humans. This study investigated the efficiency of different concentrations of E-BMP-2 adsorbed in ß-TCP for bone augmentation and osseointegration of immediate dental implants in a swine socket lift model. Following exposure of the maxillary sinus lateral wall, a 3.4-mm (diameter) cavity was drilled and filled with 0.1 g of ß-TCP containing different doses of E-BMP-2 (0, 10, 30, or 100 µg/site) to lift the Schneiderian membrane. A dental implant was then immediately inserted. Bone-to-implant contact (BIC) and bone density (BD) examined via histological analysis were used as parameters to assess E-BMP-2 efficiency in bone formation. The implant stability quotient (ISQ) was measured using Osstell to determine the effect of E-BMP-2/ß-TCP on implant stability. After 8 weeks, the groups that received 30 and 100 µg of E-BMP-2 showed substantial new bone formation in the elevated space, while no bone formation was observed with ß-TCP alone. Accordingly, BIC and BD presented a dose-dependent response to increasing doses of E-BMP-2. However, there was no increase in implant stability with E-BMP-2 treatment. In conclusion, the E-BMP-2/ß-TCP combination was efficient in bone formation and osseointegration of dental implants in a socket lift model in mini-pigs.

The regenerative effects of CCN2 independent modules on chondrocytes in vitro and osteoarthritis models in vivo.

The role of CCN family proteins has been proven to be of extreme importance in the process of cartilage development and endochondral ossification. The second member, CCN2, consists of 4 conserved modules that interact with a number of cofactors to display multiple functions. Although the potentially therapeutic effect of intact CCN2 on cartilage regeneration has been indicated by a number of studies, the regenerative effect of independent modules comprising CCN2 has never been evaluated before. This study aims to discover a more robust and effective CCN2 derivative to induce regeneration through assessing the effect of CCN2 independent modules on regeneration in vitro and in vivo, in comparison to the full length CCN2. In vitro evaluation using human chondrocytic cells showed a remarkable enhancing effect of several single modules on the gene expression of cartilaginous extracellular matrix components; whereas combinations of 2 or 3 modules rather diminished such effects. Interestingly, combination of all 4 modules redeemed the effect of intact CCN2 in vitro. Suspecting the re-assembly of the 4 modules, interaction among the modules was examined by surface plasmon resonance analysis. However, the results did not support the possible formation of a tetramodular complex. Next, the thrombospondin 1 type 1 repeat module (TSP1), which was found most promising in the experiments in vitro, and the combination of 4 modules were forwarded further to in vivo confirmation using 2 rat osteoarthritis (OA) models. As a result, TSP1 displayed more prominent regenerative effects than intact CCN2 on damaged cartilage. Unexpectedly, the combination of 4 modules showed limited effects in vivo. These results indicate the utility of TSP1 in the regenerative therapeutics of OA. Possible molecular mechanism that enables conditional reconstruction of CCN2 by 4 modules is discussed as well.

Multiple sleep bruxism data collected using a self-contained EMG detector/analyzer system in asymptomatic healthy subjects.

PURPOSE: Small, self-contained electromyographic (EMG) detector/analyzer (D/A) devices have become available for the detection of jaw muscle activity events above threshold. These devices claim to be less intrusive to the subjects sleep so it is less prone to induce disturbed sleep. The objective of this study was to evaluate for night-to-night variability and examine for a systematic alteration on the first night in EMG levels. METHODS: Ten asymptomatic healthy volunteers (mean age, 26.8 ± 3.78) were recorded for six sequential nights in their home environment using EMG D/A system. The device yields a nightly EMG level above threshold score on a 0-4 level. Because the data are categorical and nonparametric, the data of the ten subjects across six nights were submitted to a Friedman repeated measures ANOVA. The significant level was set as alpha equal to 0.05. RESULTS: The median and mode values of the subjects were tabulated and analyzed and we did not find a significant difference in EMG D/A level across the six nights (p = 0.287, Kendall's coefficient of concordance = 0.124, Friedman two-way repeated measures ANOVA). The data did show clear and substantial night-to-night variability. CONCLUSION: Substantial night-to-night variability in masseter EMG activity levels was clearly observed in our subjects. There was no evidence of a suppressed or elevated first-night effect-like variability on masseter muscle EMG level seen in these subjects using a small portable self-contained EMG detector/analyzer. These data suggest that recordings should be at least 5-6-nights duration to establish a reasonable measure of an individual's average nightly masseter EMG level.