Simultaneous inhibition of transforming growth factor-β (TGF-β) type I receptors Acvr1b and Tgfbr1 signalling has been associated with excessive skeletal muscle hypertrophy in vivo. However, it remains unclear whether the increased muscle mass in vivo is a direct result of inhibition of intracellular TGF-β signalling or whether this is an indirect effect of an altered extracellular anabolic environment. Here, we tested whether individual or simultaneous knockdown of TGF-β type I receptors in C2C12 myotubes was sufficient to induce muscle hypertrophy. The expression levels of TGF-β type I receptors Acvr1b and Tgfbr1 in myotubes were knocked down individually or in combination in the absence or presence of TGF-β1 and myostatin. Knocking down either Acvr1b or Tgfbr1 did not significantly change cell phenotype. Unexpectedly, simultaneous knockdown of both receptors reduced C2C12 myotube diameter, mRNA expression levels of Hgf, Ccn2 and Mymx with or without TGF-β1 and myostatin administration. In spite of decreased phosphorylation of Smad2/3, phosphorylation of P70S6K was reduced. In addition, the gene expression level of β1-syntrophin (Sntb1), which encodes a protein associated with the dystrophin-glycoprotein complex, was increased. Parallel experiments where Sntb1 gene expression was reduced showed an increase in myotube diameter and fusion of C2C12 myoblasts. Together, these results indicate that the knockdown of both TGF-β type I receptors reduced myotube diameter. This atrophic effect was attributed to reduced protein synthesis signalling and an increased expression of β1-syntrophin. These results have implications for our fundamental understanding of how TGF-β signalling regulates skeletal muscle size.

In skeletal muscle, the Hippo effector Yap promotes satellite cell, myoblast, and rhabdomyoblast proliferation but prevents myogenic differentiation into multinucleated muscle fibres. We previously noted that Yap drives expression of the first enzyme of the serine biosynthesis pathway, phosphoglycerate dehydrogenase ( Phgdh ). Here, we examined the regulation and function of Phgdh in satellite cells and myoblasts and found that Phgdh protein increased during satellite cell activation. Analysis of published data reveal that Phgdh mRNA in mouse tibialis anterior muscle was highly expressed at day 3 of regeneration after cardiotoxin injection, when markers of proliferation are also robustly expressed and in the first week of synergist-ablated muscle. Finally, siRNA-mediated knockdown of PHGDH significantly reduced myoblast numbers and the proliferation rate. Collectively, our data suggest that Phgdh is a proliferation-enhancing metabolic enzyme that is induced when quiescent satellite cells become activated.

Transforming growth factor-β (TGF-β) signaling is associated with progressive skeletal muscle wasting and fibrosis, while double knockout of TGF-β type I receptors Acvr1b and Tgfbr1 results in hypertrophy. Gaining insights in how myofibre-specific knockout of these receptors affects muscle transcriptome, strength and mitochondrial activity could aid in the development of therapeutic interventions to improve muscle function. Here, we show that 3 months of myofibre-specific knockout of both receptors (dKO) in mice induced a 1.6-fold increase in gastrocnemius medialis mass and a 1.3-fold increase in maximal force. Soleus muscle mass and maximal force both increased 1.2-fold in dKO mice. Muscle hypertrophy in dKO mice was accompanied by a proportional increase in succinate dehydrogenase enzyme activity. Single receptor knockout caused minor phenotypical alterations. Transcriptome analyses revealed that gastrocnemius medialis had 1811 and soleus had 295 differentially expressed genes, mainly related to muscle contraction, hypertrophy, filament organization and oxidative metabolism. Hgf and Sln genes were strongly upregulated in both muscles of dKO mice, while Sntb1 was downregulated. This in combination of transcriptional changes are associated with muscle hypertrophy and increased mitochondrial biosynthesis. Our study highlights that myofibre-specific interference with both TGF-β type I receptors concurrently stimulates myofibre hypertrophy and mitochondrial activity.

AbstractBackgroundSystemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement.MethodsDifferentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100–200 ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3‐induced IL‐1β production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function.ResultsNLRP3 gene expression and protein concentrations increased in a time‐dependent and dose‐dependent manner. Intracellular IL‐1β concentration significantly increased (P < 0.0001), but significantly less with MCC950 (P = 0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24 h (P = 0.03), and myotubes remained smaller up to 72 h (P = 0.0009). Exposure of myotubes to IL‐1β caused similar alterations in cell morphology, and MCC950 mitigated these LPS‐induced differences in cell diameter. NLRP3 appeared to co‐localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P = 0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation.ConclusionsLPS‐induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association.

In order to verify the hypothesis that fibrin glue (FG) is able to seal the area of bone grafting and facilitate bone regeneration. Twenty-one Sprague–Dawley rats with critical-sized calvarial bone defects were randomly assigned to three groups: (A) co-administrated deproteinized bovine bone (DBB) and autologous bone grafts with FG [fibrin ( +)], (B) co-administrated DBB and autologous bone grafts without FG [fibrin ( −)], and (C) no graft as control. Four weeks and 8 weeks later, micro-CT analysis and histomorphometric analysis were carried out to evaluate following parameters: bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp), percentage of new bone area (Pe.NB), average thickness of bone defect (Th.BD), average thickness of basal bone (Th.BB), and percentage of new bone in center of the skull defect (Pe.NBc). BV/TV, Tb.Th, and Tb.N in fibrin ( −) group were significantly higher than that of fibrin ( +) group (p = 0.008, 0.000, 0.007, respectively) and control group (p = 0.004, 0.001, and 0.007, respectively) at 8 weeks. Pe.NB in fibrin ( −) group (33.67 ± 11.72%) was significantly higher than that of fibrin ( +) group (12.33 ± 3.21%) (p = 0.038) and control group (9.66 ± 8.50%) (p = 0.045) at 8 weeks. Pe.NBc in fibrin ( −) group (12.05 ± 3.91%) was significantly higher than that of fibrin ( +) group (4.79 ± 1.21%) (p = 0.005) and control group (0.00 ± 0.00%) (p = 0.000) at 4 weeks. Administration of both DBB and autograft stimulates calvarial bone defect regeneration, while combination of FG does not additionally accelerate new bone formation. The use of fibrin to cement traditional bone graft materials in oral clinical practice requires caution.

In skeletal muscle, transforming growth factor-β (TGF-β) family growth factors, TGF-β1 and myostatin, are involved in atrophy and muscle wasting disorders. Simultaneous interference with their signalling pathways may improve muscle function; however, little is known about their individual and combined receptor signalling. Here, we show that inhibition of TGF-β signalling by simultaneous muscle-specific knockout of TGF-β type I receptors Tgfbr1 and Acvr1b in mice, induces substantial hypertrophy, while such effect does not occur by single receptor knockout. Hypertrophy is induced by increased phosphorylation of Akt and p70S6K and reduced E3 ligases expression, while myonuclear number remains unaltered. Combined knockout of both TGF-β type I receptors increases the number of satellite cells, macrophages and improves regeneration post cardiotoxin-induced injury by stimulating myogenic differentiation. Extra cellular matrix gene expression is exclusively elevated in muscle with combined receptor knockout. Tgfbr1 and Acvr1b are synergistically involved in regulation of myofibre size, regeneration, and collagen deposition.

Skeletal muscle fibrosis and regeneration are modulated by transforming growth factor β (TGF-β) superfamily. Amongst them, TGF-β1 is a highly potent pro-fibrotic factor, while TGF-β3 has been implicated to reduce scar formation and collagen production in skin and vocal mucosa. However, little is known about the individual and combined short- and long-term effects of TGF-β1 and TGF-β3 on collagen expression in myoblasts and myotubes. Here we show that in C2C12 myoblasts TGF-β1 and/or TGF-β3 increased mRNA expression of Ctgf and Fgf-2 persistently after 3 h and of Col1A1 after 24 h, while TGF-β1+TGF-β3 mitigated these effects after 48 h incubation. Gene expression of Tgf-β1 was enhanced by TGF-β1 and/or TGF-β3 after 24 h and 48 h. However, Tgfbr1 mRNA expression was reduced at 48 h. After 48 h incubation with TGF-β1 and/or TGF-β3, Col3A1 and Col4A1 mRNA expression levels were decreased. Myoblasts produced collagen after three days incubation with TGF-β1 and/or TGF-β3 in a dose independent manner. Collagen deposition was doubled when myoblasts differentiated into myotubes and TGF-β1 and/or TGF-β3 did not stimulate collagen production any further. TGF-β type I receptor (TGFBR1) inhibitor, LY364947, suppressed TGF-βs-induced collagen production. Collagen I expression was higher in myotubes than in myoblasts. TGF-β1 and/or TGF-β3 inhibited myotube differentiation which was antagonized by LY364947. These results indicate that both C2C12 myoblasts and myotubes produce collagen. Whereas TGF-β1 and TGF-β3 individually and simultaneously stimulate collagen production in C2C12 differentiating myoblasts, in myotubes these effects are less prominent. In muscle cells, TGF-β3 is ineffective to antagonize TGF-β1-induced collagen production.

This case represents aesthetic restoration by full-contour monolithic zirconia crowns without porcelain veneer via a completely digital workflow. Virtual prostheses were designed following intraoral scanning and digital smile design (DSD) process. Polymethyl methacrylate (PMMA)-based provisional restorations were used and adjusted as guide for aesthetic and occlusal evaluation. Definite self-glazed zirconia crowns were delivered by superimposing the abutment teeth and modified temporary crowns in situ. Such novel zirconia crowns were formed by net-shape manufacturing process following biogeneric copying of the provisional crowns. The restorations represented functionally tailored hierarchical structures exhibiting superior smoothness on the surface without conventional manual work of grinding, polishing and veneering. The application of full-contour monolithic zirconia restorations in the anterior region via a completely digital workflow indicates a reliable treatment option.

Large‐volume bone defects can result from congenital malformation, trauma, infection, inflammation and cancer. At present, it remains challenging to treat these bone defects with clinically available interventions. Allografts, xenografts and most synthetic materials have no intrinsic osteoinductivity, and so an alternative approach is to functionalize the biomaterial with osteoinductive agents, such as bone morphogenetic protein 2 (BMP2). Because it has been previously demonstrated that human salivary histatin‐1 (Hst1) promotes endothelial cell adhesion, migration and angiogenesis, we examine here whether Hst1 can promote BMP2‐induced bone regeneration. Rats were given subcutaneous implants of absorbable collagen sponge membranes seeded with 0, 50, 200 or 500 μg Hst1 per sample and 0 or 2 μg BMP2 per sample. At 18 days postsurgery, rats were sacrificed, and implanted regional tissue was removed for micro computed tomography (microCT) analyses of new bone (bone volume, trabecular number and trabecular separation). Four samples per group were decalcified and subjected to immunohistochemical staining to analyze osteogenic and angiogenic markers. We observed that Hst1 increased BMP2‐induced new bone formation in a dose‐dependent manner. Co‐administration of 500 μg Hst1 and BMP2 resulted in the highest observed bone volume and trabecular number, the lowest trabecular separation and the highest expression of osteogenic markers and angiogenic markers. Our results suggest that coadministration of Hst1 may enhance BMP2‐induced osteogenesis and angiogenesis, and thus may have potential for development into a treatment for large‐volume bone defects.

This clinical case report describes the fabrication of monolithic self-glazed zirconia prostheses via a digital workflow, for full-arch rehabilitation in a patient with a severely worn dentition. Prostheses composed of self-glazed zirconia are less prone to chipping, and have a lower risk of progressive wear of the opposing natural teeth. The use of a digital workflow not only ensures a higher degree of precision in the fabrication of self-glazed zirconia prostheses, but it also greatly improves patient comfort, and reduces the clinical chairside time required for adjustments.

Cell‐based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large‐volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co‐administration of all‐trans retinoic acid (ATRA) and human salivary peptide histatin‐1 (Hst1) on the spreading and osteogenic activities of pre‐osteoblasts on bio‐inert glass surfaces. Pre‐osteoblasts (MC3T3‐E1 cell line) were seeded onto bio‐inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point‐counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, β, and γ, such as ER‐50891, LE‐135, and MM‐11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short‐term (2 h) co‐administration of 10 μm ATRA and Hst1 to pre‐osteoblasts resulted in significantly higher spreading of pre‐osteoblasts compared to ATRA or Hst1 alone. ER‐50891 and LE‐135 both nullified these effects of ATRA. Co‐administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co‐administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre‐osteoblasts on bio‐inert glass surfaces in vitro.

The amount of enamel wear on the antagonist occlusal surfaces caused by self-glazed zirconia crowns was compared with that caused by contralateral natural teeth. Thirteen self-glazed zirconia crowns were placed in situ. The impressions of self-glazed zirconia crowns, their antagonists and the control teeth were taken and scanned at baseline and 6-month follow-up. The patients were divided into two groups, the self-glazed crowns in one group were subjected to a large amount of grinding with well polishing (LaP group, n=7), while the other group required a little amount of grinding with well polishing (LiP group, n=6). Statistics were analysed by two-sided paired Student's t-tests to a significance level of p<.05. The results revealed that the maximum and mean enamel wear significantly different between the antagonists of self-glazed crowns and the control teeth (p<.05). Increased amount of enamel wear was found in LaP group (p<.05). The self-glazed zirconia crowns caused more enamel wear of antagonists than natural teeth after 6 months. Occlusal adjustment and polishing were considered as possible confounders which affected wear behaviour.

Objective:To evaluate the feasibility of the application of newly developed self-glazed zirconia crowns without veneering porcelain in the esthetic zone by following a completely digital workflow.Methods:To compare the esthetic outcomes of two patients who had been treated for esthetic restorations in the anterior maxilla.For one case,teeth were prepared with minimally invasive approach allowing for the preservation of dental pulp vitality.Monolithic self-glazed zirconia crowns with a thickness of only 0.4 mm in the labial side was prepared.For another case,teeth were prepared based on conventional preparing procedure.Two kinds of zirconia crowns were then prepared,namely monolithic self-glazed zirconia crowns without veneering porcelain and conventional laminate-structured zirconia crowns with veneering porcelain,respectively.Results:The monolithic self-glazed zirconia crowns with a thickness of only 0.6 mm in the labial side was prepared,as well as the self-glazed zirconia crowns were applying glaze on the labial surface if necessary,that can also achieve a more natural,authentic and biomimetic appearance.Conclusion:The self-glazed zirconia crowns prepared by following a completely digital workflow can obtain superior esthetic results while allowing the minimal invasive preparation and enabling the treatment of the complicated cases with high aesthetic demands.

Muscle wasting diseases are characterized by the loss of muscle mass, function and regenerative capacity, which negatively affects the quality of life. Transforming growth factor-beta (TGF-β) superfamily members, such as TGF-β, myostatin and activin A, play an important role in reducing muscle mass (atrophy), force and regenerative capacity. Targeting TGF-β signalling is a potential approach to treat muscle wasting. In vivo studies of this thesis show that simultaneous knockout of TGF-β type I receptors Acvr1b and Tgfbr1 in a skeletal muscle-specific manner induced muscle hypertrophy, improved muscle regeneration upon acute injury and increased muscle contractile force. Knockout of both receptors in mice induced more differentially expressed genes in fast-type muscle than slow-type muscle, which were related to muscle growth, contraction, cytoskeleton and metabolism. However, myofibre size increment was not proportional to the increase in contractile force in gastrocnemius. Strikingly, the increase in myofibre size was accompanied by an increase in oxidative metabolism in muscles lacking both type I receptors. In vitro studies show that myotubes produced more collagen I protein than myoblasts. Both TGF-β1 and 3 stimulated collagen production in muscle cells. Noteworthy, siRNA-mediated knockdown of both type I receptors in myotubes reduced their diameter and protein synthesis process, which was associated with the increased expression level of Sntb1 which encodes a unit of dystrophin-glycoprotein complex. Taken together, we identified the regulatory role of TGF-β signalling with respect to muscle adaptation which contributes to the development of treatments for muscle wasting.