Amounts Of TGF-β Research Articles

Impact of using XP-endo finisher and nanobubble water during EDTA dentin conditioning on TGF-β1 release in regenerative endodontic procedures.

Transforming Growth Factor-Beta 1 (TGF-β1) plays a crucial role in the success of Regenerative Endodontic Procedures (REPs) as they directly impact the proliferation and differentiation of stem cells. TGF-β1 is released by conditioning of the dentin matrix using 17% EDTA. EDTA was found to have deleterious effects on dentin especially in immature teeth with fragile dentin walls. Decreasing the irrigation time was reported to decrease these effects. Accordingly, enhancement and activation of the EDTA solution to maintain its efficiency in TGF-β1 release from dentin and thus compensating the reduction in irrigation time was employed. EDTA solution was enhanced by adding Nanobubble (NB) water which contains oxygen filled cavities less than 200nm in diameter. Additionally, EDTA was activated with XP-endo Finisher rotary file. The aim of this study was to assess the impact of NB enhancement and/or XP-endo Finisher activation of the EDTA solution on the TGF-β1 release from dentin. Fifty standardized root segments with open apex were allocated to two main groups according to whether EDTA was enhanced with NB water or not, and within each group whether XP-endo Finisher activation was used or not in addition to a Negative Control group. The concentration of the released TGF-β1 in the root canal was measured using enzyme-linked immunosorbent assay (ELISA). The statistical analysis was done using the Shapiro- Wilk, Kolmogorov Smirnov, ANOVA and Post-hoc Tukey tests. All groups released a considerable amount of TGF-β1 with the highest values in the EDTA/NB/XP group, followed by EDTA/NB, EDTA/DW/XP, EDTA/DW and Negative Control groups respectively. The results of this study suggest that NBs can promote the success of REPs since it revealed a significant increase in the TGF-β1 release following its use in the enhancement of the EDTA solution. A comparable effect was obtained by XP-endo finisher activation of the EDTA solution. The combined use of NBs and XP-endo Finisher can be a promising addition in REPs. Accordingly, Enhancement and activation of the EDTA solution may compensate decreasing the EDTA irrigation time attempted to avoid the deleterious effect of EDTA on dentin.

The Effect of Elasticity Improvement from Acetyl Hexapeptide-8 Ampoule on UVB-Induced Mouse Skin Damage

Acetyl hexapeptide-8 (AH-8) is a well-known component of anti-aging products. AH-8 appears in a variety of cosmetic products, its skin penetration is sparsely studied and controversially discussed. This study investigated ampoule containing AH-8 as an active ingredient. The elasticity improving effect of 3% AH-8 ampoule was tested <i>in vivo</i>. To test the stability of the formulated ampoules, we performed the centrifugation, cycling and measured viscosity, and pH changes. 6 weeks-old female C3H mice were purchased from Orient Bio, Korea Inc. (Seongnam, Korea). After one week of acclimatization, the mice were randomly assigned to experimental groups (n=4 for each group); Control, UVB irradiation, Vehicle ampoule, and 3% AH-8 ampoule group. Irradiated with UVB to mouse skin, then 3% AH-8 ampoule were applied into irradiated sites for 6 weeks. Changes in the amount of TGF-β1 and collagen were measured using western blot. As a result, it was confirmed that the 3% AH-8 ampoule increased the expression of TGF-β1 and collagen. In summary, we showed that AH-8 ampoule had extremely high capability of improving elasticity when compared to vehicle ampoule containing ginkgo leaf and green tea extracts. This study revealed the efficacy of AH-8 for increasing skin elasticity and anti-aging in terms of a cosmetic active ingredient.

TGF-β in patients with hematologic malignancies

TGF-β expression is often increased in diseases such as malignancy, inflammation and fibrotic pathology. TGF-β ligand is involved in many processes of lymphoid cell development. TGF-β exerts an inhibitory stimulus on the differentiation and proliferation of stem cell progenitors. The amount of TGF-β on stem cell progenitors can determine various biological effects. For instance, if high concentrations of TGF-β have an inhibitory effect on stem cell progenitors, at low doses it can become stimulatory, especially in that group of stem cell progenitors that have a high probability of developing into myeloid cells.The aim of the study was to determine the level of TGF-β in the peripheral blood of patients with hematologic malignancies and to see its correlation with blood biomarkers of systemic inflammation.

Impaired immunomodulatory effects of seminal plasma may play a role in unexplained recurrent pregnancy loss: Results of an in vitro study

BackgroundSeminal plasma contains signaling molecules capable of modulating the maternal immune environment to support implantation and pregnancy. Prior studies indicated that seminal plasma induces changes in gene transcription of maternal immune cells. Reduced immune suppressive capacity may lead to pregnancy loss. The aim of this study was to investigate the immunomodulating effects of seminal plasma on T cells and monocytes in the context of recurrent pregnancy loss (RPL). MethodsFemale T cells and monocytes were incubated with seminal plasma of 20 males in unexplained RPL couples (RPL males) and of 11 males whose partners had ongoing pregnancies (control males). The effect of seminal plasma on messenger RNA (mRNA) expression of immune cells was measured. Levels of mRNA expression were related to key signaling molecules present in the seminal plasma. Agglomerative hierarchical cluster analysis was performed on seminal plasma expression profiles and on mRNA expression profiles. ResultsExpression of CD25 and anti-inflammatory IL-10 by female T cells was significantly lower after stimulation with seminal plasma of RPL males compared to control males. Female monocytes treated with seminal plasma of RPL males showed an immune activation signature of relatively elevated HLA-DR expression. Expression of these T cell and monocyte components was particularly correlated with the amounts of TGF-β and VEGF in the seminal plasma. ConclusionOur findings indicate that seminal plasma has immunomodulating properties on female immune cells compatible with the induction of a more regulatory phenotype, which may be impaired in cases of unexplained RPL.

Isolation of Endogenous TGF-β1 from Root Canals for Pulp Tissue Engineering: A Translational Study.

Simple SummaryTissue engineering of the dental pulp has been a goal of dental research for years. In this translational study, a chairside protocol is designed using endogenous dentin matrix proteins as signaling molecules for pulp regeneration. These bioactive molecules can be isolated from root canals by ultrasonic-activated irrigation, further processed chairside, and mixed with a hydrogel. The scaffold material is to be injected into the root canal and effect cell homing, i.e., allowing stem cells from the periapical space to migrate into the root canal. The aim of this innovative approach is the formation of an innervated and vascularized connective tissue that resembles the pulp in form and function.Cell homing for dental pulp tissue engineering has been advocated as a feasible approach to regenerate dental pulp in a clinical setting. In order to develop a translational protocol for clinical application, we wanted to determine the effects of disinfectants on the availability of growth factors from the root canal, the amount that can be obtained in this context, and whether they can be processed for use in tissue engineering procedures. The extraction of growth factors should also be confirmed in a clinical setting. Root canals were prepared in 36 extracted mature teeth, and the amount of TGF-β1 in solution was quantified after different irrigation protocols (sodium hypochlorite, chlorhexidine) and after intracanal medication (calcium hydroxide). Centrifugal filters with a cut-off of 10,000 Da and 3000 Da were used for efficient concentration, and volumes and amounts of retained TGF-β1 were measured at different time points. During conventional endodontic treatment, ethylenediaminotetraacetic acid (EDTA) solution was collected after ultrasonic activation from the root canals of mature teeth of 38 patients, and growth factor content was quantified via enzyme-linked immunosorbent assay (ELISA). Irrigation with sodium hypochlorite reduced TGF-β1 release into EDTA. This effect was partially reversed by canal enlargement after the use of sodium hypochlorite and by subsequent use of calcium hydroxide. A few minutes of centrifugation with a cut-off of 10,000 Da reduced the initial volume of the irrigant by 90% and led to a continuous increase in concentration to the same extent. Furthermore, TGF-β1 was obtained from root canals of mature teeth during endodontic treatment in quantities that have been shown to elicit desirable cellular responses in a subsequent clinical application. A mixture with a suitable scaffold material and injection into the root canal has the potential to promote dental pulp regeneration.

Anti-edema effect of Aloe vera leaf extract following traumatic brain injury: Role of pro-inflammatory cytokines.

Objective:Based on anti-inflammatory effects of Aloe vera, the effect of aqueous extract of this plant on brain edema and changes in some pro-inflammatory cytokines was investigated after traumatic brain injury (TBI).Materials and Methods:In this study, adult male Wistar rats were divided into 5 groups: Sham, TBI, vehicle (Veh), and low dose (LA) and high dose (HA) Aloe vera. The vehicle and aqueous extract of Aloe vera were injected intraperitoneally 30 min after induction of diffuse TBI by Marmarou’s method. Brain edema (brain water content), and transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6) and IL-1β levels in serum and brain were measured 24 hr after TBI induction.Results:Increased brain edema by TBI was reduced by both LA and HA (p<0.01 and p<0.05, respectively). IL-6 increased in the brain of TBI group compared to sham, and which was inhibited by both Aloe vera doses compared to Veh (p<0.001). The differences in the IL-6 serum levels among Veh, LA and HA groups were not significant. Increases in serum and brain IL-1β levels were reduced only in the HA group (p<0.001). Although only in the brain, TNF-α level increased after trauma, but both LA and HA inhibited it in a dose-dependent manner (p<0.01 and p<0.05, respectively) . The amount of TGF-β in the brain was reduced by both doses of the extract (p<0.001). Conclusion:These results indicated that Aloe vera has a neuroprotective effect induced by reducing brain edema. The probable mechanism particularly for HA is decreasing levels of pro-inflammatory cytokines such as TGF-β, TNF-α, IL-6 and IL-1β.

Ustekinumab therapy changes the transcriptional activity pattern of TGF-β1-3 genes.

IntroductionOne of the examples of genes whose expression can be altered by the action of ustekinumab is TGF-β. It is a pleiotropic cytokine whose activity affects psoriatic changes and the state of homeostasis of the whole organism.AimTo evaluate the effect of ustekinumab on the transcriptional activity of TGF-β family genes in patients with psoriatic arthritis and to check whether the results obtained can be helpful in monitoring the progress of treatment.Material and methodsFrom total PBMCs obtained from peripheral blood of 14 patients with psoriatic arthritis, total RNA was isolated. The expression level of the TGF-β1, TGF-β2 and TGF-β3 genes was determined by RT-qPCR in real time.ResultsIn all the analysed samples, the presence of mRNA of three TGF-β isoforms was quantitated in each week of therapy. TGF-β3 and the smallest TGF-β2 showed the highest expression. Statistically significant correlations were observed in the amount of TGF-β1 and TGF-β3/µg mRNA RNA, TGF-β2 and TGF-β2/µg RNA and TGF-β3 and TGF-β3/µg RNA.ConclusionsUstekinumab influences the transcriptional activity of TGF-β genes, and the changes caused have a bearing on the patient’s health.

Cross-Inhibition of Norrin and TGF-β Signaling Modulates Development of Retinal and Choroidal Vasculature.

Norrin is essential for the formation of the retinal vasculature during development and promotes its repair after damage via activation of Wnt/β-catenin signaling. Since retinal TGF-β signaling has essentially opposite effects on the retinal vasculature we investigated if and how Norrin inhibits TGF-β signaling, and vice versa. Eyes from transgenic mice with an overexpression of Norrin (βB1-Norrin) and/or active TGF-β (βB1-TGF-β1) in the lens were generated and analyzed by light microscopy, immunohistochemistry, and TUNEL. Further on, protein as well as mRNA levels were investigated by Western blot analyses and real-time RT-PCR, respectively. In βB1-TGF-β1 mice, the lack of retinal vascular development and choriocapillaris maintenance was rescued when transgenic Norrin was additionally overexpressed in the eye. In addition, retinal Wnt/β-catenin signaling and the levels of SMAD7, an inhibitor of the canonical TGF-β pathway, were substantially suppressed in retinae of βB1-TGF-β1 mice. In contrast, Norrin normalized Wnt/β-catenin signaling and SMAD7 levels in double transgenic mice. Moreover, in retinae of βB1-TGF-β1 mice, the amounts of phosphorylated SMAD3, a downstream mediator of TGF-β signaling, were increased compared to those of βB1-Norrin/βB1-TGF-β1 mice. In vitro, Norrin substantially reduced the TGF-β-mediated induction of target genes, an effect that was blocked by Dickkopf-1, a specific inhibitor of Wnt/β-catenin signaling. High amounts of TGF-β in the eye cause a substantial reduction in the activity of Wnt/β-catenin signaling. This effect is inhibited in the presence of high amounts of Norrin, which further induce the expression of SMAD7 to inhibit TGF-β signaling.

Comparision of the Effects of Leishmania Soluble Antigen (LSA) and Lipopolysaccharide (LPS) on C57BL/6 Mice Macrophage Function

Background Macrophages activation is the important anti-leishmania immune response. Different signals could affect macrophages development and functional activation. In the present study, we compared the effect of Leishmania Soluble Antigen (LSA) and Lipopolysaccharide (LPS) on peritoneal macrophage responses. Appropriate activation of macrophages depends on the signals they receive from pathogens and their different functional differentiation is crucial for anti-leishmania effects of macrophages. Materials and Methods In order to assay C57BL/6 mice macrophage function after LPS or LSA treatment, we measured phagocytic activity, cytokine pattern, and Nitric Oxide (NO) production by macrophages. Results Phase contrast microscopy showed that LPS-treated macrophages became more granular and spindle-shaped and similar to untreated macrophages, LSA-treated cells displayed round and spindle-shaped morphology. In addition, Nitric oxide assay and cytokine analysis showed that IL-6, IL-10, and TNF-α production was significantly reduced in LSA-treated macrophages in comparison with LPS-stimulated cells. It was also found that LSA-treated macrophages represented an anti-inflammatory phenotype compared with LPS-treated macrophages. Conclusion This anti-inflammatory phenotype was related to increase in IL-10/TNF- α production of LSA-treated macrophages and there was no difference in the amount of TGF-β between LSA- and LPS-treated groups.

Long-term treatment with anti-VEGF does not induce cell aging in primary retinal pigment epithelium

Anti-Vascular Endothelial Growth Factor (VEGF) therapy is given repeatedly for an extended period of time to patients when treated for age-related macular degeneration. While short-term effects of anti-VEGF agents on retinal pigment epithelial (RPE) cells have been investigated, the effects of long-term and repeated treatment on these cells are scarce. In this study, we have investigated the effects of anti-VEGF treatment after long-term, repeated treatment on cell aging and morphology. The experiments were conducted in primary porcine RPE cells passage one and two. Cells were treated with 125 μg/ml bevacizumab, ranibizumab, aflibercept or rituximab once a week for 1 day, 4 days, 7 days, 4 weeks and 12 weeks. Cell survival was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) and trypan blue exclusion assay. Activity of β-galactosidase was assessed in a commercially available assay. Influence of these compounds was investigated on the expression of cathepsin D and amyloid β, and the expression and phosphorylation of mechanistic target of rapamycin (mTOR), all proteins involved in senescence and aging, in Western blot. The secretion of Pigment Epithelium Derived Factor (PEDF) and Transforming Growth Factor (TGF)-β was investigated in Enzyme-linked Immunosorbent Assay (ELISA). The cellular morphology was investigated with electron microscopy, investigating the number and area of mitochondria and autophagosomes. Statistical analysis was conducted using a mixed linear model. Weekly treatment up to 12 weeks displayed no toxic effects on RPE cells in any of the substances tested. Ranibizumab showed a significant increase in β-galactosidase signal on day 4 (p < 0.05) and 7 (p < 0.05) after treatment. In long-term, however, ranibizumab displayed no significant difference to untreated cells. Bevacizumab displayed a significant reduction of the β-galactosidase signal after 12 weeks (p < 0.05). Aflibercept significantly decreased β-galactosidase after 1 day (p < 0.01) and 12 weeks (p < 0.05). Rituximab and bevacizumab also decreased β-galactosidase signal after 12 weeks (p < 0.05). The expression of mTOR, phospho-mTOR, amyloid β and cathepsin D was not significantly altered by any of the compounds tested. RPE cells secreted considerate amounts of TGF-β. Bevacizumab treated cells showed significantly lower TGF-β secretion than ranibizumab and rituximab (p < 0.05). In contrast, only small amounts of PEDF were secreted which were not altered by any substance tested. Ultrastructural analysis showed no alterations in mitochondria after long-term treatment with either substance. Autophagosomes were not reduced by long-term anti-VEGF treatment compared to control. However, the area of autophagosomes in bevacizumab and aflibercept treated cells was significantly less compared to both ranibizumab and rituximab treated cells (all p < 0.05). Taken together, weekly treatment with VEGF-antagonists up to 3 months does not induce premature aging in primary RPE cells in any tested compound. A significant difference can be found between bevacizumab and aflibercept on the one hand, and ranibizumab (and rituximab) on the other hand, with more autophagosomal area in ranibizumab and (rituximab). Taken together, our data provide indications for long-term safety of anti-VEGF compounds. Further research is warranted.

Effects of Two Calcium Silicate Cements on Transforming Growth Factor-β1 Secretion from Human Dental Pulp Stem Cells.

The aims of this in vitro study were to evaluate the effects of two calcium silicate based cements, Calcum-enriched Mixture (CEM) and Biodentine on proliferation of human dental pulp stem cells (hDPSCs) and the effects of proposed cements on the secretion of Transforming Growth Factor β1 (TGF-β1). The cell cultures of human Dental Pulp Stem Cells (hDPSCs) at passage 3-5 were treated with various dilutions (1/1, 1/2, 1/4, 1/8, 1/16, and 1/32) of CEM and Biodentine extracts to assess the cell proliferation using 3-(4, 5-dimethylthiazol-2-Y1)-2, 5-diphenyltetrazolium brovide (MTT) assay after 48 and 72 h. The amount of TGF-β 1 secretion were estimated after 72 h using an enzyme-linked immunosorbent assay. Data were analyzed using the one-way analysis of variance (ANOVA) followed by the Dunnett's test at the level of significance set at 0.05. CEM showed the highest rates of cell proliferation compared to Biodentine after 72 h (P<0.05). A greater amount of TGF-β1 was secreted by hDPSCs treated with Biodentine compared to CEM (P<0.05). These differences were statistically significant (P<0.05). In this in vitro study hDPSCs showed more proliferation capacity with CEM rather than Biodentine and TGF-β1 secretion rate in Biodentine was higher.

Role of TGF-β in Self-Peptide Regulation of Autoimmunity

Transforming growth factor (TGF)-β has been implicated in regulation of the immune system, including autoimmunity. We have found that TGF-β is readily produced by T cells following immunization with self-peptide epitopes that downregulate autoimmune responses in type 1 diabetes (T1D) prone nonobese diabetic (NOD) mice. These include multiple peptide epitopes derived from the islet β-cell antigens GAD65 (GAD65 p202-221, GAD65 p217-236), GAD67 (GAD67 p210-229, GAD67 p225-244), IGRP (IGRP p123-145, IGRP p195-214) and insulin B-chain (Ins. B:9-23) that protected NOD mice from T1D. Immunization of NOD mice with the self-MHC class II I-Ag7 β-chain-derived peptide, I-Aβg7 p54-76 also induced large amounts of TGF-β and also protected these mice from diabetes development. These results indicate that peptides derived from disease related self-antigens and MHC class II molecules primarily induce TGF-β producing regulatory Th3 and Tr1-like cells. TGF-β produced by these cells could enhance the differentiation of induced regulatory iTreg and iTreg17 cells to prevent induction and progression of autoimmune diseases. We therefore suggest that peripheral immune tolerance could be induced and maintained by immunization with self-peptides that induce TGF-β producing T cells.

Wound healing in Mac‐1 deficient mice

Mac-1 (CD11b/CD18) is a macrophage receptor that plays several critical roles in macrophage recruitment and activation. Because macrophages are essential for proper wound healing, the impact of Mac-1 deficiency on wound healing is of significant interest. Prior studies have shown that Mac-1-/- mice exhibit deficits in healing, including delayed wound closure in scalp and ear wounds. This study examined whether Mac-1 deficiency influences wound healing in small excisional and incisional skin wounds. Three millimeter diameter full thickness excisional wounds and incisional wounds were prepared on the dorsal skin of Mac-1 deficient (Mac-1-/- ) and wild type (WT) mice, and wound healing outcomes were examined. Mac-1 deficient mice exhibited a normal rate of wound closure, generally normal levels of total collagen, and nearly normal synthesis and distribution of collagens I and III. In incisional wounds, wound breaking strength was similar for Mac-1-/- and WT mice. Wounds of Mac-1 deficient mice displayed normal total macrophage content, although macrophage phenotype markers were skewed as compared to WT. Interestingly, amounts of TGF-β1 and its downstream signaling molecules, SMAD2 and SMAD3, were significantly decreased in the wounds of Mac-1 deficient mice compared to WT. The results suggest that Mac-1 deficiency has little impact on the healing of small excisional and incisional wounds. Moreover, the findings demonstrate that the effect of single genetic deficiencies on wound healing may markedly differ among wound models. These conclusions have implications for the interpretation of the many prior studies that utilize a single model system to examine wound healing outcomes in genetically deficient mice.

Impaired T cell function in malignant pleural effusion is caused by TGF-β derived predominantly from macrophages.

Malignant pleural effusion (MPE) is an indication of advanced cancer. Immune dysfunction often occurs in MPE. We aimed to identify the reason for impaired T cell activity in MPE from lung cancer patients and to provide clues toward potential immune therapies for MPE. The surface inhibitory molecules and cytotoxic activity of T cells in MPE and peripheral blood (PB) were analyzed using flow cytometry. Levels of inflammatory cytokines in MPE and PB were tested using ELISA. TGF-β expression in tumor-associated macrophages (TAMs) was also analyzed. The effect of TAMs on T cells was verified in vitro. Lastly, changes in T cells were evaluated following treatment with anti-TGF-β antibody. We found that expression levels of Tim-3, PD-1 and CTLA-4 in T cells from MPE were upregulated compared with those from PB, but levels of IFN-γ and Granzyme B were downregulated (p < 0.05). The amount of TGF-β was significantly higher in MPE than in PB (p < 0.05). TGF-β was mainly produced by TAMs in MPE. When T cells were co-cultured with TAMs, expression levels of Tim-3, PD-1 and CTLA-4 were significantly higher than controls, whereas levels of IFN-γ and Granzyme B were significantly decreased, in a dose-dependent manner (p < 0.05). In vitro treatment with anti-TGF-β antibody restored the impaired T cell cytotoxic activity in MPE. Our results indicate that macrophage-derived TGF-β plays an important role in impaired T cell cytotoxicity. It will therefore be valuable to develop therapeutic strategies against TGF-β pathway for MPE therapy of lung cancer.

The role of transforming growth factor beta 1 in communicating and non-communicating hydrocele

Repair of inguinal hernia and hydrocele are one of the most common operations performed by surgeons. However, the exact biological mechanism responsible for the closure of processus vaginalis (PV) is not completely understood. Transforming growth factor beta 1 (TGF-β1) is a potent fibrogenic agent and probably stimulate fibrosis and disappearing of PV. From September 2012 to December 2014, all boys from 1 to 5 years who were referred for surgery of hydrocele were divided into two groups of communicating (HC) or non-communicating hydrocele (HNC). During surgery, the fluid in the sac was aspirated and sent for biochemical evaluation including calcium, phosphorus, total protein, and TGF-β1. Finally, a biopsy of the sac was sent to the pathology. The results obtained were considered statistically significant (P<0.05). The patients were categorized into two groups of non-communicating hydrocele, including 43 patients and communicating, including 33. The patients studied were aged 1-5years (mean 33.6months). Biochemical tests on hydrocele fluid showed no significant difference in the levels of calcium, phosphorus, total protein, and bilirubin between two groups. However, mean TGF-β1 in NHC was found to be 53.45-114.28pg/ml in HC group. A statistically significant difference (P=0.04) was obtained. Furthermore, the study showed higher amounts of muscles in NHC (P<0.001). The amount of TGF-β1 was higher in HC fluid than in non-communicating. To investigate the role of cytokine in the closure of PV, further studies will be required.

Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering

Mesenchymal cells condensation is crucial in chondrogenic development. However current tissue-engineered scaffolds for chondrogenesis pay little attention to this phenomenon. In this study, we fabricate poly(l-lactide-co-glycolide) (PLGA)/poly(l-lactide) (PLLA) microfiber scaffolds and coat them with human fibroblast-derived matrix (hFDM) that is a decellularized extracellular matrix (ECM) obtained from in vitro cultured human lung fibroblasts (WI-38). Those scaffolds were then conjugated with heparin via EDC chemistry and subsequently immobilized with transforming growth factor (TGF)-β1. The amount of TGF-β1 was quantitatively measured and the release profile showed a continuous release of TGF-β1 for 4 weeks. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were seeded in four different scaffolds; control, fibronectin (FN)-coated, hFDM-coated, hFDM/TGF-β1 and subjected to chondrogenic differentiation in vitro for up to 28 days. Both hFDM and hFDM/TGF-β1 groups exhibited significantly more synthesis of glycosaminoglycan (GAG) and much better upregulation of chondrogenic markers expression. Interestingly, MSCs condensation that led to cell aggregates was clearly observed with time in the two hFDM-coated groups and the quantitative difference was obvious compared to the control and FN group. A mechanistic study in gene and protein level indicated that focal adhesion kinase (FAK) was involved at the early stage of cell adhesion and cell–cell contact-related markers, N-cadherin and neural cell adhesion molecule (NCAM), were highly up-regulated at later time point. In addition histological analysis proved that hFDM/TGF-β1 group was the most effective in forming neocartilage tissue in a rabbit articular cartilage defect model. Taken together, this study demonstrates not only the positive effect of hFDM on chondrogenesis of MSCs and cartilage repair but also provides an important insight toward the significance of in vitro mesenchymal condensation on chondrogenic development of MSCs.

Increasing proliferation of murine adipose tissue-derived mesenchymal stem cells by TNF-α plus IFN-γ

The objective of the current study was to assess the effects of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) along with their simultaneous application on proliferation and pluripotency genes of murine adipose tissue-derived mesenchymal stem cells (AT-MSCs). The proliferation, doubling time (DT), colony-forming unit–fibroblast (CFU-F), pluripotency genes expression, and proliferation-related immunomodulatory markers of MSCs were analyzed upon activation with TNF-α (10 ng/ml), IFN-γ (10 ng/ml) and both TNF-α and IFN-γ (5 ng/ml + 5 ng/ml). Pluripotency genes including Oct-4, Sox-2, and Nanog as well as proliferation-associated immunomodulatory cytokines such as insulin-like growth factor 1 (IGF-1) and transforming growth factor-β (TGF-β) expression were evaluated using real-time PCR. Surface expression of Qa2 (HLA-G) was analyzed by flow cytometry. Pretreatment of MSCs with TNF-α plus IFN-γ led to significantly increased proliferation, DT and CFU-F as well as expression of pluripotency genes in AT-MSCs (p < 0.01). MSCs expressed more IGF-1, TGF-β, and Qa2 upon activation with TNF-α plus IFN-γ and IFN-γ. MSCs expressed significantly decreased amounts of TGF-β and Qa2 in presence of TNF-α. TNF-α combined with IFN-γ may be improved the proliferation of AT-MSCs. Conversely, expanded MSCs pointed out low levels of the immunomodulatory marker, s especially Qa2 in the presence of TNF-α. In conclusion, we showed that TNF-α together with IFN-γ increased the proliferation of MSCs and slightly enhanced the expression of pluripotency genes.

Absence of TGF-β Receptor Activation by Highly Purified hCG Preparations.

Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFβR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies.

Periodontal tissue regeneration with PRP incorporated gelatin hydrogel sponges

Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5 × PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3 × PRP. In the transplantation, the size of the bone regenerated in the defect with 3 × PRP incorporated gelatin sponge was larger than that of the other group.

The preventive effect of topical zafirlukast instillation for peri-implant capsule formation in rabbits.

BackgroundCapsular contracture is the most troublesome complication in breast implant surgery. Although capsule formation can be seen as a normal reaction to a foreign body, it can induce pain, hardness, deformity, and other pathologic problems. Surgical intervention is required in severe cases, but even surgery cannot guarantee a successful outcome without recurrence. This experimental study confirms that single topical administration of leukotriene antagonist zafirlukast (Accolate, Astrazeneca) reduces peri-implant capsule formation and prevents capsular contracture.MethodsTwelve smooth-surfaced cohesive gel implants were implanted in New Zealand White rabbits. These miniature implants were designed to be identical to currently used products for breast augmentation. The rabbits were divided into 2 groups. In the experimental group (n=6), the implant and normal saline with zafirlukast were inserted in the submuscular pocket. In the control group (n=6), the implant and normal saline alone were used. Two months later, the implants with peri-implant capsule were excised. We evaluated capsule thickness and collagen pattern and performed immunohistochemical staining of myofibroblasts, transforming growth factor (TGF)-β1, 2.ResultsThe thickness of the capsules in the experimental group was reduced in both dorsal and ventral directions. The collagen pattern showed parallel alignment with low density, and the number of myofibroblasts as well as the amounts of TGF-β1 and TGF-β2 were reduced in the experimental group.ConclusionsWe suggest that single topical administration of leukotriene antagonist zafirlukast can be helpful in reducing capsule formation and preventing capsular contracture via myofibroblast suppression, modulation of fibroblastic cytokines, and anti-inflammatory effect.