Bio-Pulsed Stimulation Effectively Improves the Production of Avian Mesenchymal Stem Cell-Derived Extracellular Vesicles That Enhance the Bioactivity of Skin Fibroblasts and Hair Follicle Cells.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (exosomes) possess regeneration, cell proliferation, wound healing, and anti-senescence capabilities. The functions of exosomes can be modified by preconditioning MSCs through treatment with bio-pulsed reagents (Polygonum multiflorum Thunb extract). However, the beneficial effects of bio-pulsed small extracellular vesicles (sEVs) on the skin or hair remain unknown. This study investigated the in vitro mechanistic basis through which bio-pulsed sEVs enhance the bioactivity of the skin fibroblasts and hair follicle cells. Avian-derived MSCs (AMSCs) were isolated, characterized, and bio-pulsed to produce AMSC-sEVs, which were isolated, lyophilized, characterized, and analyzed. The effects of bio-pulsed AMSC-sEVs on cell proliferation, wound healing, and gene expression associated with skin and hair bioactivity were examined using human skin fibroblasts (HSFs) and follicle dermal papilla cells (HFDPCs). Bio-pulsed treatment significantly enhanced sEVs production by possibly upregulating RAB27A expression in AMSCs. Bio-pulsed AMSC-sEVs contained more exosomal proteins and RNAs than the control. Bio-pulsed AMSC-sEVs significantly augmented cell proliferation, wound healing, and gene expression in HSFs and HFDPCs. The present study investigated the role of bio-pulsed AMSC-sEVs in the bioactivity of the skin fibroblasts and hair follicle cells as mediators to offer potential health benefits for skin and hair.

Antioxidants protect against gingival overgrowth induced by cyclosporine A.

OBJECTIVE: We investigated the importance of reactive oxygen species (ROS) on developing gingival overgrowth (GO) and then introduced the antioxidant strategy to prevent, or even reduce GO. BACKGROUND: Gingival overgrowth is a common side effect of the patients receiving cyclosporine A (CsA), an immune suppressant. Although it has been broadly investigated, the exact pathogenesis of the induced GO is still uncertain. METHODS: We cultured human primary gingival fibroblasts and used animal model of GO to investigate the ameliorative effects of antioxidants on CsA-induced GO. To examine the CsA-induced oxidative stress, associated genes and protein expression, and the overgrown gingiva of rats by using immunocytochemistry, confocal laser scanning microscopy, real-time PCR, ELISA, gelatin zymography, gingival morphological, and immunohistochemical analysis. RESULTS: We found for the first time that ROS was responsible for the CsA-induced oxidative stress and TGF-ß1 expression in human primary gingival fibroblasts, as well as the GO of rats. The antioxidants (oxidative scavenger of vitamin E and an antioxidative enzyme inducer of hemin) ameliorated CsA-induced pathological and morphological alterations of GO without affected the CsA-suppressed il-2 expression in rats. CsA-induced oxidative stress, HO-1, TGF-ß1, and type II EMT were also rescued by antioxidants treatment. CONCLUSIONS: We concluded that CsA repetitively stimulating the production of ROS is the cause of CsA-GO which is ameliorated by treating antioxidants, including vitamin E and sulforaphane. Furthermore, the immunosuppressive effect of CsA is not interfered by antioxidant treatments in rats. This finding may thus help the clinician devise better prevention strategies in patients susceptible to GO.

Combined Treatment of Heteronemin and Tetrac Induces Antiproliferation in Oral Cancer Cells.

BACKGROUND: Heteronemin, a marine sesterterpenoid-type natural product, possesses an antiproliferative effect in cancer cells. In addition, heteronemin has been shown to inhibit p53 expression. Our laboratory has demonstrated that the thyroid hormone deaminated analogue, tetrac, activates p53 and induces antiproliferation in colorectal cancer. However, such drug mechanisms are still to be studied in oral cancer cells. METHODS: We investigated the antiproliferative effects by Cell Counting Kit-8 and flow cytometry. The signal transduction pathway was measured by Western blotting analyses. Quantitative PCR was used to evaluate gene expression regulated by heteronemin, 3,3',5,5'-tetraiodothyroacetic acid (tetrac), or their combined treatment in oral cancer cells. RESULTS: Heteronemin inhibited not only expression of proliferative genes and Homo Sapiens Thrombospondin 1 (THBS-1) but also cell proliferation in both OEC-M1 and SCC-25 cells. Remarkably, heteronemin increased TGF-ß1 expression in SCC-25 cells. Tetrac suppressed expression of THBS-1 but not p53 expression in both cancer cell lines. Furthermore, the synergistic effect of tetrac and heteronemin inhibited ERK1/2 activation and heteronemin also blocked STAT3 signaling. Combined treatment increased p53 protein and p53 activation accumulation although heteronemin inhibited p53 expression in both cancer cell lines. The combined treatment induced antiproliferation synergistically more than a single agent. CONCLUSIONS: Both heteronemin and tetrac inhibited ERK1/2 activation and increased p53 phosphorylation. They also inhibited THBS-1 expression. Moreover, tetrac suppressed TGF-ß expression combined with heteronemin to further enhance antiproliferation and anti-metastasis in oral cancer cells.

Leptin OB3 peptide suppresses leptin-induced signaling and progression in ovarian cancer cells.

BACKGROUND: Obesity and its comorbidities constitute a serious health burden worldwide. Leptin plays an important role in diet control; however, it has a stimulatory potential on cancer cell proliferation. The OB3 peptide, a synthetic peptide, was shown to be more active than leptin in regulating metabolism but with no mitogenic effects in cancer cells. METHODS: In this study, we investigated the proliferative effects, gene expressions and signaling pathways modulated by leptin and OB3 in human ovarian cancer cells. In addition, an animal study was performed. RESULTS: Leptin, but not OB3, induced the proliferation of ovarian cancer cells. Interestingly, OB3 blocked the leptin-induced proliferative effect when it was co-applied with leptin. Both leptin and OB3 activated the phosphatidylinositol-3-kinase (PI3K) signal transduction pathway. In addition, leptin stimulated the phosphorylation of signal transducer and activator of transcription-3 (STAT3) Tyr-705 as well as estrogen receptor (ER)α, and the expression of ERα-responsive genes. Interestingly, all leptin-induced signal activation and gene expressions were blocked by the co-incubation with OB3 and the inhibition of extracellular signal-regulated kinase (ERK)1/2. Coincidently, leptin, but not OB3, increased circulating levels of follicle-stimulating hormone (FSH) which is known to play important roles in the initiation and proliferation of ovarian cancer cells. CONCLUSIONS: In summary, our findings suggest that the OB3 peptide may prevent leptin-induced ovarian cancer initiation and progression by disrupting leptin-induced proliferative signals via STAT3 phosphorylation and ERα activation. Therefore, the OB3 peptide is a potential anticancer agent that might be employed to prevent leptin-induced cancers in obese people.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside Isolated from Polygoni Multiflori Ameliorates the Development of Periodontitis.

Periodontitis, a chronic infection by periodontopathic bacteria, induces uncontrolled inflammation, which leads to periodontal tissue destruction. 2,3,5,4'-Tetrahydroxystilbene-2-O-beta-glucoside (THSG), a polyphenol extracted from Polygoni Multiflori, reportedly has anti-inflammatory properties. In this study, we investigated the mechanisms of THSG on the Porphyromonas gingivalis-induced inflammatory responses in human gingival fibroblasts and animal modeling of ligature-induced periodontitis. Human gingival fibroblast cells were treated with lipopolysaccharide (LPS) extracted from P. gingivalis in the presence of resveratrol or THSG to analyze the expression of TNF-α, IL-1ß, and IL-6 genes. Increased AMP-activated protein kinase (AMPK) activation and SirT1 expression were induced by THSG. Treatment of THSG decreased the expression of LPS-induced inflammatory cytokines, enhanced AMPK activation, and increased the expression of SirT1. In addition, it suppressed the activation of NF-κB when cells were stimulated with P. gingivalis LPS. The anti-inflammatory effect of THSG and P. Multiflori crude extracts was reproduced in ligature-induced periodontitis animal modeling. In conclusion, THSG inhibited the inflammatory responses of P. gingivalis-stimulated human gingival fibroblasts and ameliorated ligature-induced periodontitis in animal model.

Role of Shh and TGF in cyclosporine-enhanced expression of collagen and α-SMA by gingival fibroblast.

OBJECTIVE: Cyclosporine-A (CsA)-induced gingival overgrowth may arise from an alteration in stoma matrix homeostasis. Sonic hedgehog (Shh) plays a key role during embryogenic development and fibrotic progression, and may be involved in CsA-altered gingival matrix homeostasis. METHODS: Using the reverse transcription-polymerase chain reaction and Western blot analysis, we investigated the mRNA and protein expressions of Shh, type 1 collagen (COL1), alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-ß) in human gingival fibroblasts after CsA treatments. The effect of Shh on CsA-induced alterations was further evaluated by the extra-supplement or inhibition of Shh or TGF-ß. RESULTS: Cyclosporine-A enhanced COL1, α-SMA, Shh and TGF-ß expressions in human gingival fibroblasts. The exogenous Shh/TGF-ß augmented the expression of COL1 and α-SMA, and the Shh/TGF-ß inhibition suppressed the CsA-enhanced COL1 and α-SMA expressions. Moreover, Shh mRNA and protein expressions increased if extra-supplementing the exogenous TGF-ß, whereas the CsA-upregulated Shh was mitigated by the TGF-ß pathway inhibitor. However, neither exogenous Shh nor the Shh pathway inhibitor alters TGF-ß expression or CsA-up-regulated TGF-ß expression. CONCLUSIONS: Shh, regulated by TGF-ß, mediates CsA-altered gingival matrix homeostasis.

Can chlorhexidine mouthwash twice daily ameliorate cyclosporine-induced gingival overgrowth?

BACKGROUND/PURPOSE: Gingival overgrowth can be induced in patients treated with cyclosporine-A (CsA), an immunosuppressant often used following organ transplantation. A pre-existing rat model designed to mimic CsA-induced gingival overgrowth in humans was used to test the effectiveness of frequent application of a chlorhexidine antiplaque solution in reducing the overgrowth. METHODS: Four groups of rats were fed CsA. One group received chlorhexidine mouthwash twice a day, the second group received chlorhexidine mouthwash once a day, the third group received chlorhexidine mouthwash every other day, and the fourth group did not receive chlorhexidine mouthwash all. A fifth negative control group received only mineral oil. Overgrowth was determined by measuring the changes in the gingival probing depth and the keratinized gingival width on molars. A gingival histological examination was performed. RESULTS: Rats treated with mouthwash twice daily exhibited decreased probing depths and gingival widths without statistical significance. Histological examination revealed that CsA treatment caused gingival enlargement, whereas chlorhexidine treatment twice a day diminished the enlargement. CONCLUSION: These findings suggest that chlorhexidine mouthwash used twice daily may reduce the severity of CsA-induced gingival overgrowth. Further research is warranted to determine the optimal dose and treatment regimen.

Recent advances of exosomes in soft tissue injuries in sports medicine: A critical review on biological and biomaterial applications.

Sports medicine is generally associated with soft tissue injuries including muscle injuries, meniscus and ligament injuries, tendon ruptures, tendinopathy, rotator cuff tears, and tendon-bone healing during injuries. Tendon and ligament injuries are the most common sport injuries accounting for 30-40% of all injuries. Therapies for tendon injuries can be divided into surgical and non-surgical methods. Surgical methods mainly depend on the operative procedures, the surgeons and postoperative interventions. In non-surgical methods, cell therapy with stem cells and cell-free therapy with secretome of stem cell origin are current directions. Exosomes are the main paracrine factors of mesenchymal stem cells (MSCs) containing biological components such as proteins, nucleic acids and lipids. Compared with MSCs, MSC-exosomes (MSC-exos) possess the capacity to escape phagocytosis and achieve long-term circulation. In addition, the functions of exosomes from various cell sources in soft tissue injuries in sports medicine have been gradually revealed in recent years. Along with the biological and biomaterial advances in exosomes, exosomes can be designed as drug carriers with biomaterials and exosome research is providing promising contributions in cell biology. Exosomes with biomaterial have the potential of becoming one of the novel therapeutic modalities in regenerative researches. This review summarizes the derives of exosomes in soft tissue regeneration and focuses on the biological and biomaterial mechanism and advances in exosomal therapy in soft tissue injuries.

2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside protects against neuronal cell death and traumatic brain injury-induced pathophysiology.

Traumatic brain injury (TBI) is a global health issue that affects at least 10 million people per year. Neuronal cell death and brain injury after TBI, including apoptosis, inflammation, and excitotoxicity, have led to detrimental effects in TBI. 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG), a water-soluble compound extracted from the Chinese herb Polygonum multiflorum, has been shown to exert various biological functions. However, the effects of THSG on TBI is still poorly understood. THSG reduced L-glutamate-induced DNA fragmentation and protected glial and neuronal cell death after L-glutamate stimulation. Our results also showed that TBI caused significant behavioral deficits in the performance of beam walking, mNSS, and Morris water maze tasks in a mouse model. Importantly, daily administration of THSG (60 mg/kg/day) after TBI for 21 days attenuated the injury severity score, promoted motor coordination, and improved cognitive performance post-TBI. Moreover, administration of THSG also dramatically decreased the brain lesion volume. THSG reduced TBI-induced neuronal apoptosis in the brain cortex 24 h after TBI. Furthermore, THSG increased the number of immature neurons in the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. Our results demonstrate that THSG exerts neuroprotective effects on glutamate-induced excitotoxicity and glial and neuronal cell death. The present study also demonstrated that THSG effectively protects against TBI-associated motor and cognitive impairment, at least in part, by inhibiting TBI-induced apoptosis and promoting neurogenesis.

Comparison of 2,3,5,4'-tetrahydroxystilbene-2-O-b-D-glucoside-induced proliferation and differentiation of dental pulp stem cells in 2D and 3D culture systems-gene analysis.

BACKGROUND/PURPOSE: Culture environments play a critical role in stem cell expansion. This study aimed to evaluate the effects of 2,3,5,4'-tetrahydroxystilbene-2-O-b-D-glucoside (THSG) on the proliferation and differentiation of human dental pulp stem cells (DPSCs) in 2-dimensional (2D) and 3-dimensional (3D) culture systems. MATERIALS AND METHODS: Human DPSCs were seeded in T25 flasks for 2D cultivation. For the 3D culture system, DPSCs were mixed with microcarriers and cultured in spinner flasks. Cells in both culture systems were treated with THSG, and cell proliferation was determined using a cell counter and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. In THSG-treated DPSCs, the genes associated with proliferation, adipogenesis, neurogenesis, osteogenesis, pluripotency, oncogenesis, and apoptosis were analyzed using real-time polymerase chain reactions. RESULTS: The spinner flask time-dependently improved cell numbers, cell viability, and expansion rates in THSG-treated DPSCs. In both the T25 and spinner flasks, the messenger RNA (mRNA) levels of proliferation, osteogenesis, and pluripotent-related genes had a significant maximum expression with 10 µM THSG treatment. However, 0.1 µM of THSG may be the most suitable condition for triggering neurogenesis and adipogenesis gene expression when DPSCs were cultured in spinner flasks. Furthermore, the number of oncogenes and apoptotic genes decreased considerably in the presence of THSG in both the T25 and spinner flasks. CONCLUSION: The spinner flask bioreactor combined with THSG may upregulate proliferation and lineage-specific differentiation in DPSCs. Thus, the combination can be used to mass-produce and cultivate human DPSCs for regenerative dentistry.

Cyanidin-3-O-glucoside downregulates ligation-activated endoplasmic reticulum stress and alleviates induced periodontal destruction in rats.

OBJECTIVE: Cyanidin-3-O-glucoside (C3G), a dietary anthocyanin, possesses various biological properties, including alleviating endoplasmic reticulum (ER) stress. This study examined the effect of C3G on periodontitis via ER stress in rats. DESIGN: Periodontitis was induced by placing silk sutures around maxillary second molars. C3G (0, 3, or 9 mg/kg) was fed on the day before ligation (10 rats/group). Further, 10 non-ligation control rats received deionized water. On day 8, gingivae were obtained to determine CCAAT/enhancer-binding protein homologous protein (CHOP), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), and nuclear factor-κB (NF-κB) by immunoblotting. Periodontal destruction was evaluated using micro-computed tomography (µCT) and histology. RESULTS: Gingival expression of CHOP, p-JNK/JNK, and NF-κB significantly increased in ligation rats (0 mg/kg C3G) than that in controls. However, protein expression in ligation groups presented a negative association with C3G concentration. By µCT, the distance of cemento-enamel junction to bone significantly increased in ligation groups; however, distances showed a negative association with C3G concentration. In the region of interest, bone volume and trabecular thickness and number significantly decreased in ligation groups but they were positively associated with C3G concentration. In terms of trabecular separation, opposite results were found. Histologically, infiltrated connective tissue (ICT) and periodontal destructions increased in ligation groups; however, they were negatively associated with C3G concentration. Moreover, ICT area is positively correlated with µCT- and histologically measured destructions and protein expression of CHOP, p-JNK/JNK, or NF-κB. CONCLUSION: C3G promotes favorable modulation of ER stress and alleviates destruction of periodontitis, which may imply a new strategy.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-Glucoside improves female ovarian aging.

Reduced fertility associated with normal aging may reflect the over-maturity of oocytes. It is increasingly important to reduce aging-induced infertility since recent trends show people marrying at later ages. 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside (THSG), a polyphenol extracted from Polygonum multiflorum, has been reported to have anti-inflammatory and anti-aging properties. To evaluate whether THSG can reduce aging-related ovarian damage in a female mouse model of aging, THSG was administered by gavage at a dose of 10 mg/kg twice weekly, starting at 4 weeks of age in a group of young mice. In addition, the effect of THSG in a group of aged mice was also studied in mice starting at 24 weeks of age. The number of oocytes in the THSG-fed group was higher than in the untreated control group. Although the percentage of secondary polar bodies (PB2) decreased during aging in the THSG-fed group, it decreased much more slowly than in the age-matched control group. THSG administration increased the quality of ovaries in young mice becoming aged. Western blotting analyses also indicated that CYP19, PR-B, and ER-ß expressions were significantly increased in 36-week-old mice. THSG also increased oocyte numbers in aged mice compared to mice without THSG fed. Studies of qPCR and immunohistochemistry (IHC) analyses of ovaries in the aged mice groups were conducted. THSG increased gene expression of anti-Müllerian hormone (AMH), a biomarker of oocyte number, and protein accumulation in 40-week-old mice. THSG increased the expression of pgc1α and atp6, mitochondrial biogenesis-related genes, and their protein expression. THSG also attenuated the fading rate of CYP11a and CYP19 associated with sex hormone synthesis. And THSG maintains a high level of ER-ß expression, thereby enhancing the sensitivity of estrogen. Our findings indicated that THSG increased or extended gene expression involved in ovarian maintenance and rejuvenation in young and aged mice. On the other hand, THSG treatments significantly maintained oocyte quantity and quality in both groups of young and aged mice compared to each age-matched control group. In conclusion, THSG can delay aging-related menopause, and the antioxidant properties of THSG may make it suitable for preventing aging-induced infertility.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside promotes the effects of dental pulp stem cells on rebuilding periodontal tissues in experimental periodontal defects.

BACKGROUND: This study aimed to investigate the regenerative effects of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside (THSG)-treated human dental pulp stem cells (DPSC) on the healing of experimental periodontal defects in rats. METHODS: The maxillary first molars of 30 male Sprague-Dawley rats were extracted, and after healing, bilateral periodontal defects were surgically created mesially in second molars. The defects were treated with Matrigel (as control), DPSC, or DPSC + THSG. After 2 weeks, the healed defects were evaluated using microcomputed tomography and through histological and immunohistochemical analyses. RESULTS: In the microcomputed tomography analysis, more new bone formation in the DPSC and DPSC + THSG groups was observed compared with the control group. The periodontal bone supporting ratio in site with DPSC + THSG was significantly higher than that in DPSC. Histologically, an enhanced new bone formation and more significant periodontal attachment were observed in the DPSC + THSG group. The expression levels of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), and osteopontin (OPN) in the DPSC + THSG group were significantly greater than those in other groups. CONCLUSIONS: THSG-revolutionized DPSCs significantly shortened the regenerative period of periodontal defects by enhancing the cell recruitment and possibly the angiogenesis in rat models, which illustrate the critical implications for a clinical application and provide a novel tactic for periodontitis treatment.

2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside-stimulated dental pulp stem cells-derived conditioned medium enhances cell activity and anti-inflammation.

BACKGROUND/PURPOSE: Dental pulp stem cells (DPSCs) contribute to the regeneration of various tissues and have superior proliferation, immune privilege, and anti-inflammation properties to other mesenchymal stem cells. 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) not only enhances the aforementioned properties of DPSCs but also promotes self-renewal and reprogramming-like ability. However, whether THSG enhances the aforementioned properties and abilities through direct or indirect interaction mechanisms remains unclear. To address this knowledge gap, we examined the effects of THSG-stimulated DPSC-derived conditioned medium (THSG-CM) on the activity and anti-inflammation properties of cells. MATERIALS AND METHODS: DPSCs were treated with various concentrations of THSG to produce THSG-CM, which was then collected, analyzed, and lyophilized. A cytokine profiling antibody assay was used to compare protein components between THSG-treated and nontreated CM. Human skin fibroblasts (HSFs) and human gingival fibroblasts (HGFs) were used to investigate the effect of THSG-CM on cell proliferation, anti-inflammation, and wound healing abilities; for this investigation, MTS assay, quantitative real-time PCR analysis, and 2-well silicone inserts wound model were conducted. RESULTS: We observed that THSG enhanced the secretion of growth- and immune-associated proteins in THSG-CM and increased the proliferation of HSFs and HGFs. Furthermore, THSG-CM significantly attenuated lipopolysaccharide-stimulated mRNA levels of cytokines in both cells and improved wound healing abilities. CONCLUSION: We conclude that THSG-CM had more beneficial effects on cell activity and anti-inflammation in the HSFs and HGFs than DPSC-derived CM. DPSC-derived CM can be developed into a cell-free regenerative strategy in the future, and its therapeutic efficacy may be improved by THSG-CM.

2,3,5,4'-tetrahydroxystilbene-2-O-b-D-glucoside triggers the pluripotent-like possibility of dental pulp stem cells by activating the JAK2/STAT3 axis: Preliminary observations.

Abstract. BACKGROUND/PURPOSE: Although 2,3,5,4'-Tetrahydroxystilbene-2-O-beta-glucoside (THSG) reportedly has anti-inflammatory properties, its role in inducing the dedifferentiation of human dental pulp stem cells (DPSC) into pluripotent-like stem cells remains to be determined. The purpose of this study is to evaluate the effects of THSG on the pluripotent-like possibility and mechanism of DPSC. MATERIALS AND METHODS: DPSCs were treated with THSG, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTS) assay. Real-time polymerase chain reaction was used to analyze the mRNA expression levels of pluripotency-associated genes and oncogenes and to detect telomerase activity in the cells. Embryoid body formation assay was conducted, and pluripotency-related proteins were identified using Western blotting. Data were analyzed using one-way analysis of variance. RESULTS: Cell viability, telomerase activity, and embryoid body formation were enhanced in THSG-treated DPSCs. The mRNA expression levels of pluripotent-like genes (including Nanog homeobox [NANOG], SRY-box 2 [SOX2], and POU class 5 homeobox 1 [POU5F1/OCT4]) significantly increased after THSG treatment. The expression levels of pluripotency-related genes (Janus kinase-signal transducer 2 [JAK2] and signal transducer and activator of transcription 3 [STAT3]) increased, whereas those of oncogenes (Ras, SRC, HER2, and C-sis) decreased. Furthermore, the expression levels of the phosphorylated JAK2 and STAT3 proteins significantly increased after THSG treatment. CONCLUSION: THSG treatment may enhance the pluripotent-like possibility of DPSC through the JAK2/STAT3 axis. Hence, it may be used as an alternative cell-based therapeutic strategy in regenerative dentistry.

CD147 self-regulates matrix metalloproteinase-2 release in gingival fibroblasts after coculturing with U937 monocytic cells.

BACKGROUND: Cluster of differentiation 147 (CD147) is a multifunctional glycoprotein that functions as an inducer of matrix metalloproteinase (MMP) expression in fibroblasts. Synergistically enhanced MMP-2 expression was recently observed in the coculture of human gingival fibroblasts (HGFs) and U937 human monocytic cells; however, the responsible mechanisms have not yet been fully established. The aim of this study was to evaluate the release of soluble CD147 in HGFs after coculturing with U937 cells and its functional effect on the enhancement of MMP-2 expression in HGFs. METHODS: Enzyme-linked immunosorbent assay was used to determine the amount of CD147 protein in media, whereas real-time polymerase chain reaction was performed to evaluate the mRNA levels of CD147 and MMP-2 in HGFs and U937 cells. The enzyme activities of MMP-2 released from cells were examined by zymography. Transwell coculturing and conditioned media treatments were selected to rule out the effect of direct contact of HGFs and U937 cells. RESULTS: The protein and mRNA expression of CD147 in HGFs were enhanced after transwell coculturing with U937 cells and exposure to U937-conditioned medium. MMP-2 enzyme activities in HGFs were also significantly increased by the coculturing methods. Administration of exogenous CD147 enhanced MMP-2 expression in HGFs, whereas treatment with cyclosporine-A, which inhibited CD147 expression, reduced U937-enhanced MMP-2 expression in HGFs. CONCLUSIONS: CD147 can interact with fibroblasts to stimulate the expression of MMPs associated with periodontal extracellular matrix degradation. This study has demonstrated that CD147 released from fibroblasts might play a role in monocyte-enhanced MMP-2 expression in HGFs.

NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells.

Nano-diamino-tetrac (NDAT), a tetraiodothyroxine deaminated nano-particulated analog, has shown to inhibit expression of pro-inflammatory genes. NDAT inhibits expression of programmed death-ligand 1 (PD-L1). On the other hand, in addition to inhibiting inflammatory effect, the stilbene, resveratrol induces expression of cyclooxygenase-2 (COX-2) and its accumulation. Sequentially, inducible COX-2 complexes with p53 and induces p53-dependent anti-proliferation. In current study, we investigated mechanisms involved in combined treatment of NDAT and resveratrol on anti-proliferation in human oral cancer cells. Both resveratrol and NDAT inhibited expression of pro-inflammatory IL-1ß and TNF-α. They also inhibited expression of CCND1 and PD-L1. Both resveratrol and NDAT induced BAD expression but only resveratrol induced COX-2 expression in both OEC-M1 and SCC-25 cells. Combined treatment attenuated gene expression significantly compared with resveratrol treatment in both cancer cell lines. Resveratrol reduced nuclear PD-L1 accumulation which was enhanced by a STAT3 inhibitor, S31-201 or NDAT suggesting that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. In the presence of T4, NDAT further enhanced resveratrol-induced anti-proliferation in both cancer cell lines. These findings provide a novel understanding of the inhibition of NDAT in thyroxine-induced pro-inflammatory effect on resveratrol-induced anticancer properties.

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer.

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

Dental Pulp Stem Cell Transplantation with 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside Accelerates Alveolar Bone Regeneration in Rats.

INTRODUCTION: Although the therapeutic potential of human dental pulp stem cells (hDPSCs) has been studied for bone regeneration, the therapeutic efficiency needs further consideration and examinations for clinical applications. Thus, the aims of this study were to evaluate the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) on the osteogenic differentiation of hDPSCs and to examine the therapeutic efficiency of the THSG-enhanced osseous potential of hDPSCs in alveolar bony defects of rats. METHODS: Expressions of osteogenic messenger RNAs (including ALP, RUNX2, BGLAP, and AMBN) were examined by quantitative real-time polymerase chain reaction. Alizarin red S staining was conducted to analyze THSG-induced mineralization of hDPSCs. To investigate the regenerative effects of THSG-treated hDPSCs on dental alveolar bone, bony defects were created in male Sprague-Dawley rats. Defects were treated with Matrigel (Corning Inc, Corning, NY), hDPSCs, or hDPSCs + THSG. After 2 weeks, defect healing was evaluated by micro-computed tomographic and histologic analyses. RESULTS: In the cell model, THSG induced osteogenesis-associated genes (ALP, RUNX2, and BGLAP) and an enamel-related gene (AMBN), resulting in mineralization as detected by alizarin red S staining after 2 weeks of treatment. In the animal model, THSG increased all parameters of bone formation (the relative bone volume, trabecular thickness, trabecular number, and trabecular separation) in alveolar bony defects of rats. THSG not only improved the quality of newly formed bone but also the quantity of new bone. CONCLUSIONS: These results showed important findings in revealing the THSG-enhanced osteogenic differentiation of hDPSCs and THSG-facilitated bone regeneration, which may provide an alternative option for cell-based regenerative therapy.

Accuracy of cone beam computed tomography in measuring thicknesses of hard-tissue-mimicking material adjacent to different implant thread surfaces.

BACKGROUND/PURPOSE: To evaluate the measurement accuracy of hard-tissue thicknesses adjacent to dental implants with different thread designs on images obtained from cone beam computed tomography (CBCT) using an in vitro model. MATERIALS AND METHODS: On 4 × 13-mm implant, the neck of the implant was designed with micro-threads, and the apical part was covered by macro-threads; these implants were placed in a vinyl polysiloxane block that mimicked hard-tissue. Models were prepared with various thicknesses of 2.0, 1.0, 0.5 and 0.3 mm adjacent to the dental implant. Each model was scanned using CBCT, and the thickness of the cortical bone from the outer surface of the micro-threads and macro-threads were recorded. Ground sections were prepared, and the thickness was measured with electronic calipers as the gold standard (GS) measurement. RESULTS: CBCT measurements of the micro-thread surface were consistently underestimated compared to the GS measurement when the thickness of the hard-tissue-mimicking material was ≤1.0 mm. In comparison, CBCT measurements of the macro-thread surface closely approximated the standard measurement, except when the thickness of the hard-tissue-mimicking material was 0.3 mm. The mean percentage errors from the standard measurement for the 2.0-, 1.0-, 0.5-, and 0.3-mm thickness groups were 4.8%, 16.4%, 37.8%, and 92.6%, respectively, for the micro-thread group, and were 0.6%, 2.9%, 9.5%, and 40.8%, respectively, for the macro-thread group. CONCLUSION: Within the limitations of this study, we conclude that CBCT may not produce sufficient resolution for thin sections of hard tissue-mimicking materials adjacent to micro-thread surfaces.