Microencapsulating stem cells in injectable microbeads can enhance delivery and localization, but their ability to act as growth factor production sources is still unknown. To address this concern, growth factor mRNA levels and production from alginate microbeads with encapsulated human adipose stem cells (ASC microbeads) cultured in both growth and chondrogenic media (GM and CM) were measured over a two week period. Human ASCs in microbeads were either commercially purchased (Lonza) or isolated from six human donors and compared to human ASCs on tissue culture polystyrene (TCPS). The effects of crosslinking and alginate compositions on growth factor mRNA levels and production were also determined. Secretion profiles of IGF-I, TGF-β3 and VEGF-A from commercial human ASC microbeads were linear and at a significantly higher rate than TCPS cultures over two weeks. For human ASCs derived from different donors, microencapsulation increased pthlh and both IGF-I and TGF-β3 secretion. CM decreased fgf2 and VEGF-A secretion from ASC microbeads derived from the same donor population. Crosslinking microbeads in BaCl2 instead of CaCl2 did not eliminate microencapsulation's beneficial effects, but did decrease IGF-I production. Increasing the guluronate content of the alginate microbead increased IGF-I retention. Decreasing alginate molecular weight eliminated the effects microencapsulation had on increasing IGF-I secretion. This study demonstrated that microencapsulation can enhance chondrogenic growth factor production and that chondrogenic medium treatment can decrease angiogenic growth factor production from ASCs, making these cells a potential source for paracrine factors that can stimulate cartilage regeneration.

This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts. hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3. Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system. pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines. The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM) components was detected after transfection by real-time quantitative PCR, ELISA, immunochemistry and Western blotting, respectively. The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing. Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs. Real-time quantitative PCR, immunochemistry and Western blotting showed that the cartilage-specific ECM markers, i.e., cartilage oligomeric matrix protein (COMP), Aggrecan, collagen type X and II were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells. It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.

The transforming growth factor-β1 (TGF-β1) bioassay developed in this study monitors increased luciferase activity in MCF10A cells containing the matrix metalloproteinase-2 (MMP-2) promoter with a luciferase reporter and treated with increasing TGF-β1 concentrations. The response was linear in the concentration range from 75 to 2,500 pg/mL. The abilities of 3 types of TGF-β in inducing MMP-2 were different. The luciferase activity induced by TGF-β1 was about 2 times more than that by TGF-β2 and TGF-β3. The MMP-2 promoter bioassay showed greater reproducibility (coefficient of variation [CV] 10%) than the previously developed anticell proliferation assay of TF-1 cell (CV 16%) and the MMP-2 zymogram assay (CV 40%).

UNASSIGNED

Cleft lip and palate (CLP) is the most common human cranial and maxillofacial birth defect. The aim of this bibliometric analysis was to provide an overview of the development of CLP-related research.

UNASSIGNED

Cleft lip and palate-related studies published from 2000 to 2017 were retrieved from the Science Citation Index Expanded core database. Publication date, journal, authors, first authors, keywords, and citations were extracted and quantitatively analyzed using Bibliographic Item Co-Occurrence Matrix Builder software. The word matrix and co-occurrence matrix were established, and the co-citation analysis, keyword clustering, and social network analysis (SNA) of highly cited papers were completed.

UNASSIGNED

A total of 9040 articles were retrieved from the 18 years of publications that were searched. The number of documents steadily increased over the period of interest, with a slight decrease in 2016 and 2017. This article separately examined the top most cited papers and high-frequency keywords from 3 time periods: 2000 to 2005, 2006 to 2011, and 2011 to 2017. The strategy coordinates of citation reflect TGF-β3, MSX1 gene, technique for cleft lip repair, TTF2, P63, IRF6 gene, FGF signaling, PVRL1, TGFBR2, and BMP4 gene as areas of research interest in the field. Moreover, the SNA of keywords highlighted new research topics: meta-analysis, cone beam computed tomography, tooth agenesis, case-control study, association study, micrognathia, DiGeorge syndrome, NSCL/P, UCLP, GWAS, MTHFR, and CLPTM1L.

UNASSIGNED

We conducted bibliometric research of CLP across an 18-year span. The results help to define an overall command of the latest topics in CLP and provide insight for launching new projects.

Polymeric multilayered capsules (PMCs) have found great applicability in bioencapsulation, an evolving branch of tissue engineering and regenerative medicine. Here, we describe the production of hierarchical PMCs composed by an external multilayered membrane by layer-by-layer assembly of poly(L-lysine), alginate, and chitosan. The core of the PMCs is liquified and encapsulates human adipose stem cells and surface-functionalized collagen II-TGF-β3 poly(L-lactic acid) microparticles for cartilage tissue engineering.

BACKGROUND

Tracheal stenosis constitutes one of the most frequently seen problems in thoracic surgery. Although many treatment modalities to prevent fibroblast proliferation, angiogenesis, or inflammation that causes tracheal stenosis have been attempted, an effective method has not yet been found. In this study, a transforming growth factor beta3 (TGF-β3)/chitosan combination was used for this purpose.

METHODS

A slow-release preparation containing a thin layer of TGF-β3 with a chitosan base was made. Thirty albino Wistar rats were divided into 3 groups. A full-layer vertical incision was made in the anterior side of the trachea of each rat between the second and fifth tracheal rings. The tracheal incision was sutured. Group A was evaluated as the control group. In Group B, a chitosan-based film was placed on the incision line. In Group C, a slow-release TGF-β3/chitosan-coated substance was placed on the incision line. The rats were killed on day 30, and their tracheas were excised by cutting between the lower edge of the thyroid cartilage and the upper edge of the sixth tracheal ring together with the esophagus. Epithelialization, fibroblast proliferation, angiogenesis, inflammation, and collagen levels were evaluated histopathologically by the same histopathologist.

RESULTS

Statistically significant differences were not found among the 3 groups. Cold abscesses were observed at the incision sites in both the TGF-β/chitosan and chitosan groups. These were thought to have formed due to the chitosan.

CONCLUSIONS

As this was the first experiment in the literature to use this type of TGF-β3 formulation, we intend to change the formulation and perform this study again with a different TGF-β3/chitosan preparation.

OBJECTIVE

To investigate the effect of mechano-growth factor (MGF) on the differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro.

RESULTS

Flow cytometry assay identified the isolated cells were human bone marrow mesenchymal stem cells, which had differentiation ability when cultured with specific induction culture media. Alizarin Red S, Oil Red O and Alcian Blue staining showed osteogenic, adipogenic and chondrogenic differentiation were significantly increased after hBMSCs were treated with MGF E peptide. Collagen II expression was considerably increased after hBMSCs were induced with chondrogenic induction culture medium supplemented with TGF-β3 and MGF E peptide. Overexpression of MGF by an expression plasmid further confirmed the MGF could enhance tri-lineage differentiation of hBMSCs. Moreover, we found that hBMSCs proliferation rate was decreased and G1 phase of the cell cycle was lengthened after MGF treatment when compared to the control group.

CONCLUSIONS

MGF can enhance differentiation of hBMSCs during specific induction culture media induction by lengthening G1 phase of cell cycle.

Herein, we hypothesized that pro-osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR-29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT-PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR-29b and TGF-β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR-29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR-29b inhibition. TGF-β3 was markedly downregulated while Smad3, Runx2, wnt3, and β-catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR-29b overexpression while the inhibition of miR-29b showed the inverse trends. Moreover, TGF-β3 was a direct target of miR-29b. Inhibition of miR-29b hinders valvular calcification through the upregulation of the TGF-β3 via inhibition of wnt/β-catenin and RUNX2/Smad3 signaling pathways.

The deposition of extracellular matrix (ECM)-which is mainly composed of type I collagen-in anterior subcapsular cataracts (ASCs) during epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs) decreases visual function. Transforming growth factor (TGF)-β is a key factor in the induction of EMT in LECs. Although Rho kinase (ROCK) plays an important role in EMT induced by TGF-β, it is unknown whether ROCK inhibition affects type I collagen expression in TGF-β-stimulated LECs and ASC formation. This was investigated in the present study both in vitro using human lens epithelium (HLE)-B3 cells and in vivo using mice with ultraviolet radiation (UVR)-B-induced cataracts. We found that TGF-β2 increased type I collagen mRNA expression in HLE-B3 cells; this was inhibited in a dose-dependent manner by treatment with the ROCK inhibitor Y-27632. UVR-B exposure caused ASC formation in mice. A histopathological examination revealed that LECs in the anterior subcapsular area were flattened and multi-layered, and had a spindle shape in cross section. Immunohistochemical analysis revealed the presence of α-smooth muscle actin and type I collagen around these flattened LECs; these opacities were reduced by topical instillation of Y-27632. These findings suggest that suppression of TGF-β signaling in LECs by topical application of a ROCK inhibitor can prevent the formation of ASCs.

The mechanism of active eruption of molars was examined in 36 male adolescent Wistar rats. Histological, histochemical [tetracycline (TC) labelling and alkaline phosphatase (ALP) activity], and immunohistochemical [transforming growth factor (TGF)-β1, -β2, and -β3] investigations were conducted of the rat molar areas. Real-time reverse transcription-polymerase chain reaction (RT-PCR) for mRNA of TGF-β was performed on the periodontal ligament (PDL) dissected out by laser capture microdissection. TC labelling lines showed that a considerable amount of bone formation occurred in the alveolar crest region, apical region, and intraradicular septum, indicating that the maxillary molars had moved downward. However, the periodontal fibres revealed a regular arrangement (alveolar crest, horizontal and oblique fibres) during the experimental period. This suggests that new formation of alveolar crest fibres and rearrangement of the periodontal fibres occurred in the PDL. ALP activity was intense on the bone surface and in the PDL. TGF-β1 was also detected in osteoblasts and fibroblasts but less so in cementoblasts. Real-time RT-PCR also demonstrated significant expression of mRNA of TGF-β1 in the PDL, indicating that TGF-β1 was involved in active eruption. These results suggest that active eruption occurs in adolescent rats and can be managed by TGF-β1.

Due to very sluggish turnover at the molecular and cellular level, the healing of chondral damages has been considered difficult. In the current study, the effects of the Kartogenin, a small heterocyclic molecule on chondrogenic differentiation of stem cells was compared to TGF-β3.

Human Adipose-Derived Stem Cells were extracted during an elective surgery. Cell viability was estimated by MTT assay, differentiated cells evaluated by histological and immunohistochemical techniques. Expression of cartilage specific genes (SOX9, Aggrecan, type II and X collagens) assessed by real-time PCR.

The real-time PCR assay has revealed the expression of gene marker of chondrogenesis, SOX9, Aggrecan and type II collagen, both in Kartogenin and TGFβ3 groups compared to the control group, significantly (p < 0.05). A low expression level of collagen type X as a hypertrophic marker was seen in cartilage produced by using Kartogenin. Meanwhile, the level of type X collagen protein in Kartogenin group was significantly decreased (p>> 0.05) compared to TGF-β3 group.

Kartogenin was suitable for successful chondrogenic differentiation of human adipose- derived stem cells and a suppressor of the consequent hypertrophy (Tab. 1, Fig. 5, Ref. 31).

The loss of nigral dopaminergic (DA) neurons is the disease-defining pathological change responsible for progressive motor dysfunction in Parkinson's disease. In this study, we sought to establish a culture method for adult rat tyrosine hydroxylase (TH)-immunoreactive DA neurons. In this context, we investigated the role of fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), transforming growth factor-β3 (TGF-β3), glial-derived neurotrophic factor (GDNF) and dibutyryl-cyclic AMP (dbcAMP) in these cultures. Culturing in the presence of FGF2, BDNF and GDNF enhanced the survival of DA neurons by 15-fold and promoted neurite growth. In contrast, dbcAMP promoted neurite growth in all neurons but did not enhance DA cell survival. This study demonstrates that long-term cultures of DA neurons can be established from the mature rat brain and that survival and regeneration of DA neurons can be manipulated by epigenetic factors such as growth factors and intracellular cAMP pathways.

The decrease in the bone mass associated with osteoporosis caused by ovariectomy, aging, and other conditions is accompanied by an increase in bone marrow adipose tissue. The balance between osteoblasts and adipocytes is influenced by a reciprocal relationship. The development of modalities to promote local/systemic bone formation by inhibiting bone marrow adipose tissue is important in the treatment of fractures or metabolic bone diseases such as osteoporosis. In this study, we examined whether raspberry ketone [4-(4-hydroxyphenyl)butan-2-one; RK], which is one of the major aromatic compounds of red raspberry and exhibits anti-obesity action, could promote osteoblast differentiation in C3H10T1/2 stem cells. Confluent C3H10T1/2 stem cells were treated for 6 days with 10-100 μg/mL of RK in culture medium containing 10 nM all-trans-retinoic acid (ATRA) or 300 ng/mL recombinant human bone morphogenetic protein (rhBMP)-2 protein as an osteoblast-differentiating agent. RK in the presence of ATRA increased alkaline phosphatase (ALP) activity in a dose-dependent manner. RK in the presence of rhBMP-2 also increased ALP activity. RK in the presence of ATRA also increased the levels of mRNAs of osteocalcin, α1(I) collagen, and TGF-βs (TGF-β1, TGF-β2, and TGF-β3) compared with ATRA only. RK promoted the differentiation of C3H10T1/2 stem cells into osteoblasts. However, RK did not affect the inhibition of early-stage adipocyte differentiation. Our results suggest that RK enhances the differentiation of C3H10T1/2 stem cells into osteoblasts, and it may promote bone formation by an action unrelated to adipocyte differentiation.

Transforming growth factor β (TGF-β) is a multifunctional cytokine recognized as an important regulator of inflammatory responses. The effect of inositol hexaphosphate (IP6), a naturally occurring phytochemical, on the mRNA expression of TGF- β1, TGF-β2, TGF-β3 and TβRI, TβRII, and TβRIII receptors stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium) and IL-1β in intestinal cells Caco-2 for 3 and 12 h was investigated. Real-time qRT-PCR was used to validate mRNAs level of examined genes. Bacterial endotoxin promoted differential expression of TGF-βs and their receptors in a time-dependent manner. IL-1β upregulated mRNA levels of all TGF-βs and receptors at both 3 h and 12 h. IP6 elicited the opposed to LPS effect by increasing downregulated transcription of the examined genes and suppressing the expression of TGF- β1 at 12 h. IP6 counteracted the stimulatory effect of IL-1β on TGF-β1 and receptors expression by decreasing their mRNA levels. IP6 enhanced LPS- and IL-1β-stimulated mRNA expression of TGF-β2 and -β3. Based on these studies it may be concluded that IP6 present in the intestinal milieu may exert immunoregulatory effects and chemopreventive activity on colonic epithelium under inflammatory conditions or during microbe-induced infection/inflammation by modulating the expression of genes encoding TGF-βs and their receptors at transcriptional level.

OBJECTIVE

To investigate the effects of KIOM-79 in preventing the development of diabetic complications, such as cataracts.

METHODS

The inhibitory effects of KIOM-79 were assessed in a model of xylose-induced lens opacity and on changes mediated by high levels of glucose in human lens epithelial (HLE-B3) cells.

RESULTS

In lenses treated with KIOM-79, opacity was significantly improved and glutathione (GSH) was increased compared to controls. In HLE-B3 cells treated with KIOM-79, high glucose-mediated increases in TGF-beta2, alphaB-crystallin, and fibronectin were significantly inhibited in a dose-dependent manner. KIOM-79 decreased the phosphorylation of p-Smad2/3, pp38MAPK, pp44/42, and NF-kappaB signaling in cells grown under high glucose conditions.

CONCLUSIONS

KIOM-79 is protective against lens opacity and protects HLE-B3 cells from the toxic effects of high glucose. Therefore, KIOM-79 may provide a potential therapeutic approach for preventing diabetic complications, such as cataracts.

MK5 is a protein serine/threonine kinase activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. Although little is known of the physiological role of MK5 in the heart, both hypertrophic growth and the increase in collagen 1-α₁ mRNA induced by increased afterload are attenuated in hearts of MK5 haploinsufficient (MK5^+/-) mice. MK5 transcripts are detected at high levels in the left ventricular myocardium; however, MK5 immunoreactivity is detected in adult cardiac fibroblasts, but not myocytes. The present study was to determine if MK5 has a potential role in remodeling of the extracellular matrix. Ventricular fibroblasts were isolated from MK5^+/+, MK5^+/-, or MK5^-/- mice and maintained in culture on either compliant (8 kPa) or rigid substrates to obtain quiescent fibroblasts or activated myofibroblasts, respectively. In quiescent fibroblasts, reduced MK5 had little effect: BMP7 and TGF-β1 mRNA was increased in MK5^+/- and MK5^-/-.cells, respectively. Ang-II altered the abundance of numerous transcripts in an MK5-sensitive manner. Both collagen 1-α₁ mRNA and secreted type 1 collagen immunoreactivity were increased by Ang-II in wild type but not MK5-deficient fibroblasts. The effects of deleting MK5 were quite different in myofibroblasts: both the abundance of collagen 1-α₁ mRNA and secreted type 1 collagen immunoreactivity elevated in the absence of added Ang-II and addition of Ang-II failed to evoke a further increase in either. In addition, whereas type I collagen immunoreactivity was distributed throughout the cytosol of wild-type myofibroblasts, it was perinuclear in MK5^-/- myofibroblasts. Furthermore, in MK5-deficient myofibroblasts the abundance of collagen 3-α₂, Timp3, Smad 6, Smad 7, TGF-β3, and snail homolog 1 transcripts was increased whereas integrin β3, latent TGF-β binding protein 1, thrombospondin 1, hepatocyte growth factor, and interleukin 13 were decreased. Finally, fibroblast contraction was decreased upon knocking down MK5. These results indicate that MK5 may be involved in fibroblast-mediated regulation of extracellular matrix homeostasis.

The aim was to explore the pulmonary surfactant regulatory effect of TGF-β1 and TGF-β3 during pre- and post-natal porcine development. Pigs on embryonic day 99 (E94) (term = 114 days) and 1-h (D0) and 15-day (D20) neonates were killed to obtain whole lungs. DSPC (disaturated phosphatidylcholine) was separated from other phospholipids, and chemical methods were used to determine the amounts of DSPC, TPL (total phospholipids) and TP (total protein) in BALF (bronchoalveolar lavage fluid). TPL was elevated at E94. DSPC in TPL was significantly higher in the D20 group than in the E94 group. Reductions in TP correlated with developmental age. The levels of TGF-β1 and TGF-β3 mRNA were determined by RT (reverse transcription)-PCR and Northern blot. The expression of TGF-β1 mRNA was low at E94, increased at D0 and then decreased at D20. The expression of TGF-β3 was high at E94, reduced at D0, and then elevated at D20. We further examined the effect of exogenously administered TGF-1 on the expression of SPs (surfactant proteins) and cytidine triphosphorylate: CCT (phosphocholine cytidylyltransferase) activity in porcine fetal lung cells cultured for 5 days. The results indicated that TGF-β1 inhibited the expression of all three SPs (SP-A, SP-B and SP-C) and CCT activity, but did not alter the expression level of SP-D transcripts. We conclude that TGF-1 inhibits the expression of surfactant components. The alterations of TGF-β3 seem to partly explain the pulmonary surfactant changes observed in development, but this result needs further investigation.

Prior findings in vitro of a TGF-β3 dependent mechanism induced by low dose-rate irradiation and resulting in increased radioresistance and removal of low dose hyper-radiosensitivity (HRS) was tested in an in vivo model. DBA/2 mice were given whole-body irradiation for 1 h at low dose-rates (LDR) of 0.3 or 0.03 Gy/h. Serum was harvested and added to RPMI (4% mouse serum and 6% bovine serum).This medium was transferred to reporter cells (T-47D breast cancer cells or T98G glioblastoma cells). The response to subsequent challenge irradiation of the reporter cells was measured by the colony assay. While serum from unirradiated control mice had no effect on the radiosensitivity in the reporter cells, serum from mice given 0.3 Gy/h or 0.03 Gy/h for 1 h removed HRS and also increased survival in response to doses up to 5 Gy. The effect lasted for at least 15 months after irradiation. TGF-β3 neutralizer added to the medium containing mouse serum inhibited the effect. Serum from mice given irradiation of 0.3 Gy/h for 1 h and subsequently treated with iNOS inhibitor 1400W did not affect radiosensitivity in reporter cells; neither did serum from the unirradiated progeny of mice given 1h LDR whole-body irradiation.

A universal method for reproducibly directing stem cell differentiation remains a major challenge for clinical applications involving cell-based therapies. The standard approach for chondrogenic induction by micromass pellet culture is highly susceptible to interdonor variability. A novel method for the fabrication of condensation-like engineered microtissues (EMTs) that utilizes hydrophilic polysaccharides to induce cell aggregation is reported here. Chondrogenesis of mesenchymal stem cells (MSCs) in EMTs is significantly enhanced compared to micromass pellets made by centrifugation measured by type II collagen gene expression, dimethylmethylene blue assay, and histology. MSCs from aged donors that fail to differentiate in pellet culture are successfully induced to synthesize cartilage-specific matrix in EMTs under identical media conditions. Furthermore, the EMT polysaccharides support the loading and release of the chondroinduction factor transforming growth factor β3 (TGF-β3). TGF-β-loaded EMTs (EMT(+TGF) ) facilitate cartilaginous tissue formation during culture in media not supplemented with the growth factor. The clinical potential of this approach is demonstrated in an explant defect model where EMT(+TGF) from aged MSCs synthesize de novo tissue containing sulfated glycosaminoglycans and type II collagen in situ.

OBJECTIVE

Abundance of connexin 43 (Cx43), a transmembrane protein that forms hemichannels (HCs) and gap junctions (GJs), is dynamically regulated in human gingival fibroblasts (GFBLs) during wound healing. This may be important for fast and scarless gingival wound healing as Cx43 is involved in key cell functions important during this process. Our aim was to uncover the factors that regulate Cx43 expression and abundance in GFBLs. We hypothesized that cytokines and growth factors released during wound healing coordinately regulate Cx43 abundance in GFBLs.

RESULTS

TGF-β1, -β2, -β3, PGE2 and IL-1β significantly upregulated, while TNF-α and IFN-γ downregulated Cx43 in cultured GFBLs. TGF-β1, -β2, -β3, IL-1β and IFN-γ modulated Cx43 abundance at both mRNA and protein levels, while TNF-α and PGE2 regulated only Cx43 protein abundance, suggesting involvement of distinct transcriptional/post-transcriptional and translational/post-translational mechanisms, respectively. TGF-β1-induced upregulation of Cx43 was mediated by TGFβRI (ALK5) and SMAD2/3 signaling, and this was potently suppressed by PGE2, IL-1β, TNF-α and IFN-γ that inhibited SMAD2/3 phosphorylation.

CONCLUSIONS

Regulation of Cx43 abundance in GFBLs involves transcriptional/post-transcriptional and translational/post-translational mechanisms that are distinctly modulated by an interplay between TGF-β isoforms and PGE2, IL-1β, TNF-α and IFN-γ.

An ideal scaffold for bone tissue engineering should have chondroinductive, biodegradable, and biocompatible properties, as well as the ability to absorb and slowly release the biological molecules. In order to develop such a system to support bone tissue regeneration, in the present study, we developed a three-dimensional poly(L-lactic-co-glycolic acid) (PLGA)/Polycaprolactone (PCL) nanohybrid scaffold embedded with PLGA macroparticles (MPs) conjugated with TGF-β3 for the growth and chondrogenic differentiation of human mesenchymal stem cells (hMSCs). First, a microfluidic device was used to fabricate porous PLGA MPs with the sizes ranging from 10 to 50 µm. Next, the PLGA MPs were loaded with TGF-β3, mixed with PCL solution, and then electrospun to obtain PLGA-TGF-β3 MPs/PCL nanohybrid scaffold. Our results demonstrated that PLGA MPs fabricated using a microfluidic-based approach exhibited enhanced conjugation of TGF-β3 with over 80% loading efficiency and sustained release of TGF-β3. Furthermore, the results of glycosaminoglycan (GAG) content measurement and Safranin O staining revealed that the PLGA-TGF-β3 MPs and PLGA-TGF-β3 MPs/PCL nanohybrid scaffold can promote the proliferation and chondrogenic differentiation of hMSCs in vitro. Therefore, the PLGA-TGF-β3 MPs/PCL nanohybrid scaffold could pave the way for cartilage regeneration and have wide applications in regenerative medicine.

Salmonella Typhimurium (ST) infection in chickens inhibits their growth and can lead to food-borne diseases in humans. Probiotics are expected to enhance the function of host intestinal barrier against pathogen infection. The aim of our study was to determine the effect of viable Lactobacillus reuteri (LR) on the response of the mucosal barrier function to antigen stimulation in broiler chicks. Day-old male (n=8) and female (n=4) broiler chicks were orally administered either 1 × 10⁸ LR or a water-only control, every day for 7 days. After 7 days, either 1 × 10⁸ heat-killed ST (k-ST), or a buffer-only control, was administered via intra-cardiac injection. The ileum and cecum were collected 3 h post-injection, and paraffin sections were prepared for either mRNA extraction (males), or gut permeability tests (females). Villus and crypt lengths were determined via histological analysis. Real-time PCR was used to calculate expression levels of Toll-like receptors (TLRs), pro-inflammatory cytokines, anti-inflammatory cytokines, avian β-defensins, and tight-junction-associated molecules. Gut permeability was assessed using the inverted intestine method. We found that (1) expression of TLR2-1, TLR21, TGF-β2 and TGF-β3 were reduced following k-ST stimulation, but were unaffected by LR-treatment; (2) oral administration of LR led to increased Claudin1, Claudin5, ZO2, and JAM2 expression following k-ST stimulation; (3) cecal permeability was reduced by co-treatment with LR and k-ST, but not by treatment with LR or k-ST alone. These results suggest that LR, as used in this study, may enhance the intestinal mucosal physical barrier function, but not the expression of other immune-related factors in newly hatched chicks.

Keywords: Salmonella Typhimurium; broiler chick; cytokine; intestine; probiotics; tight junction.