Study protocol for periodontal tissue regeneration with a mixture of autologous adipose-derived stem cells and platelet rich plasma: A multicenter, randomized, open-label clinical trial.

Introduction: Adipose-derived stem cells (ASCs) secrete various growth factors to promote wound healing and to regenerate various tissues, such as bone, cartilage, and fat tissue. Subcutaneous adipose tissue is a considerable cell source in clinical practice and can be collected relatively easily and safely under local anesthesia. Moreover, platelet-rich plasma (PRP), a plasma component containing many platelets purified by centrifuging the collected blood, also promotes wound healing. PRP can be easily gelled and is therefore attracting attention as a scaffolding material for transplanted cells. The usefulness of a mixture of ASCs and PRP for periodontal tissue regeneration has been in vitro demonstrated in our previous study. The aim of this study is to present the protocol of translation of tissue regeneration with ASCs and PRP into practical use, evaluating its efficacy. Methods: This study is a multicenter, randomized, open-label comparative clinical trial. Fifteen patients will be randomly assigned to the treatment with mixture of ASCs and PRP or enamel matrix derivate administration into periodontal tissue defects. Increase in height of new alveolar bone in the transplanted area will be evaluated. The evaluation will be performed using dental radiographs after 36 weeks of transplantation. Occurrence of adverse events will be evaluated as secondary outcome. Results: This clinical study was initiated after meeting the regulations to be complied with, including ethical review and regulatory notifications. Conclusions: If effective, this cell therapy using autologous mesenchymal stem cells can represent a useful medical technology for regeneration of periodontal defects.

Grafting of autologous concentrated bone marrow processed using a point-of-care device for patients with osteonecrosis of the femoral head: A phase 1 feasibility and safety study.

Introduction: Along with the accumulating reports of autologous concentrated bone marrow (CBM) grafting for osteonecrosis of the femoral head (ONFH), the related medical device, a "point-of-care device" has also been recently developed. However, no study has confirmed the feasibility, safety, and efficiency of CBM grafting using a specific point-of-care device. Materials and methods: We designed this phase I, prospective clinical study to evaluate the feasibility and safety of autologous CBM grafting processed using a point-of-care device, the BioCUE system, in patients with ONFH. The primary outcomes were the safety and adverse event (AE), the secondary outcomes included pain score; hip function score; ONFH stage using X-ray; and the volume of the osteonecrotic area on 3T MRI. Besides, safety quality tests on the final product of concentrated bone marrow were performed. Results: Two patients (a 34-year-old man and a 33-year-old woman; three hips) with systemic lupus erythematosus were included. The incidence of AEs was 100% such as pain or transient fever after the operation, but all AEs were nonserious. No peri-operative complications were observed. Pain and hip function score remained unchanged from the preoperative to the postoperative observational periods. Safety quality test demonstrated were all negative or under the threshold. Conclusion: The feasibility and safety of grafting of concentrated autologous CBM in patients with ONFH using a point-of-care device were confirmed. A further clinical study aiming for the authorization of this procedure should be conducted in the future.

T1R3 homomeric sweet taste receptor negatively regulates insulin-induced glucose transport through Gαs-mediated microtubules disassembly in 3T3-L1 adipocytes.

T1R3 is a class C G protein-coupled receptor family member that forms heterodimeric umami and sweet taste receptors with T1R1 and T1R2, respectively, in the taste cells of taste buds. T1R3 is expressed in 3T3-L1 cells in homomeric form and negatively regulates adipogenesis in a Gαs-dependent but cAMP-independent manner. Although T1R3 expression is markedly upregulated during adipogenesis, its physiological role in mature adipocytes remains obscure. Here, we show that stimulation of T1R3 with sucralose or saccharin induces microtubule disassembly in differentiated 3T3-L1 adipocytes. The effect was reproduced by treatment with cholera toxin or isoproterenol but not with forskolin. Treatment with sucralose or saccharin for 3 h inhibited insulin-stimulated glucose uptake by 32% and 45% in differentiated adipocytes, respectively, similar to the inhibitory effect of nocodazole (by 33%). Isoproterenol treatment inhibited insulin-stimulated glucose transport by 26%, whereas sucralose did not affect the intrinsic activity of the glucose transporter, indicating that it inhibited insulin-induced GLUT4 translocation to the plasma membrane. Immunostaining analysis showed that insulin-stimulated GLUT4 accumulation on the plasma membrane was abrogated in sucralose-treated cells, in association with depolymerization of microtubules. Sucralose-mediated inhibition of GLUT4 translocation was reversed by the overexpression of dominant-negative Gαs (Gαs-G226A) or knockdown of Gαs. Additionally, membrane fractionation analysis showed that sucralose treatment reduced GLUT4 levels in the plasma membrane fraction from insulin-stimulated adipocytes. We have identified a novel non-gustatory role for homomeric T1R3 in adipocytes, and activation of the T1R3 receptor negatively regulates insulin action of glucose transport via Gαs-dependent microtubule disassembly.

Correction: T1R3 homomeric sweet taste receptor regulates adipogenesis through Gαs-mediated microtubules disassembly and Rho activation in 3T3-L1 cells.

[This corrects the article DOI: 10.1371/journal.pone.0176841.].

T1R3 homomeric sweet taste receptor regulates adipogenesis through Gαs-mediated microtubules disassembly and Rho activation in 3T3-L1 cells.

We previously reported that 3T3-L1 cells express a functional sweet taste receptor possibly as a T1R3 homomer that is coupled to Gs and negatively regulates adipogenesis by a Gαs-mediated but cAMP-independent mechanism. Here, we show that stimulation of this receptor with sucralose or saccharin induced disassembly of the microtubules in 3T3-L1 preadipocytes, which was attenuated by overexpression of the dominant-negative mutant of Gαs (Gαs-G226A). In contrast, overexpression of the constitutively active mutant of Gαs (Gαs-Q227L) as well as treatment with cholera toxin or isoproterenol but not with forskolin caused disassembly of the microtubules. Sweetener-induced microtubule disassembly was accompanied by activation of RhoA and Rho-associated kinase (ROCK). This was attenuated with by knockdown of GEF-H1, a microtubule-localized guanine nucleotide exchange factor for Rho GTPase. Furthermore, overexpression of the dominant-negative mutant of RhoA (RhoA-T19N) blocked sweetener-induced dephosphorylation of Akt and repression of PPARγ and C/EBPα in the early phase of adipogenic differentiation. These results suggest that the T1R3 homomeric sweet taste receptor negatively regulates adipogenesis through Gαs-mediated microtubule disassembly and consequent activation of the Rho/ROCK pathway.

A novel regulatory function of sweet taste-sensing receptor in adipogenic differentiation of 3T3-L1 cells.

BACKGROUND: Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. METHODOLOGY/PRINCIPAL FINDINGS: In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects. CONCLUSIONS: 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism.