BACKGROUND

Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-β1 and -β3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma.

METHODS

The potential role of αVβ6 integrin-mediated activation of latent TGF-β was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVβ6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVβ6 integrin activity.

RESULTS

Both KAT-4 and Capan-2 cells expressed the αVβ6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-β in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-β1 and -β3 reduced collagen fibril thickness in both tumor models.

CONCLUSIONS

Our data demonstrate that the αVβ6-directed activation of latent TGF-β plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVβ6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-β activation and the extent of desmoplasia.

Identification of a suitable cell source and bioactive agents guiding cell differentiation towards tenogenic phenotype represents a prerequisite for advancement of cell-based therapies for tendon repair. Human adipose-derived stem cells (hASCs) are a promising, yet intrinsically heterogenous population with diversified differentiation capacities. In this work we investigated antigenically-defined subsets of hASCs expressing markers related to tendon phenotype or associated with pluripotency that might be more prone to tenogenic differentiation, when compared to unsorted hASCs. Subpopulations positive for tenomodulin (TNMD+ hASCs) and stage specific early antigen 4 (SSEA-4+ hASCs), as well as unsorted ASCs were cultured up to 21 days in basic medium or media supplemented with TGF-β3 (10 ng/ml), or GDF-5 (50 ng/ml). Cell response was evaluated by analysis of expression of tendon-related markers at gene and protein levels by real time RT-PCR, western blot and immunocytochemistry. A significant upregulation of scleraxis was observed for both subpopulations and unsorted hASCs in the presence of TGF-β3. More prominent alterations in gene expression profile in response to TGF-β3 were observed for TNMD+ hASCs. Subpopulations evidenced an increased collagen III and TNC deposition in basal medium conditions in comparison to unsorted hASCs. In the particular case of TNMD+ hASCs GDF-5 seems to influence more the deposition of TNC. Within hASCs populations, discrete subsets could be distinguished that offer varied sensitivity to specific biochemical stimulation leading to differential expression of tenogenic components suggesting that cell subsets may have distinctive roles in the complex biological responses leading to tenogenic commitment to be further explored in cell based strategies for tendon tissues.

An eight-week feeding trial was conducted to investigate the effects of dietary andrographolide on the growth performance, antioxidant capacity in the liver, intestinal inflammatory response and microbiota of Monopterus albus. A total of 900 health fish (25.00 ± 0.15 g) were randomly divided into five groups: AD1 (the basal diet) as the control, and AD2, AD3, AD4 and AD5 groups, which were fed the basal diet supplemented with 75, 150, 225 and 300 mg/kg andrographolide, respectively. The results showed that compared with the control group, dietary andrographolide supplementation (1) significantly increased trypsin and lipase activities in the intestine, and increased the weight gain rate but not significantly; (2) significantly increased the levels of glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GPx) and the content of in the liver; significantly decreased the contents of reactive oxygen species (ROS) and malondialdehyde (MDA); remarkably upregulated the Nrf2, SOD1, GSTK and GSTO mRNA levels in the liver; downregulated the Keap1 mRNA level; (3) significantly increased the villi length and goblet cell numbers in the intestine, remarkably upregulated the Occludin mRNA level in the intestine, downregulated the Claudin-15 mRNA level; (4) remarkably upregulated the IL-10, TGF-β1 and TGF-β3 mRNA levels in the intestine; downregulated the IL-12β and TLR-3 mRNA levels; (5) significantly decreased the richness and diversity of the intestinal microbioma, increased the percentages of Fusobacteria and Firmicutes and significantly decreased the percentages of Cyanobacteria and Proteobacteria. In conclusion, these results showed that dietary low-dose andrographolide (75 and 150 mg/kg) promoted growth and antioxidant capacity, regulated the intestinal microbioma, enhanced intestinal physical and immune barrier function in rice field eel.

Keywords: Monopterus albus; andrographolide; antioxidant capacity; growth; intestinal immune; intestinal microbioma.

The aim of this study was to assess the activity of extracts from Platycodon grandiflorum A. DC (PG) in a model of chronic bronchitis in rats. The research was carried out on three water extracts: E1 - from roots of field cultivated PG; E2 - from biotransformed roots of PG; E3 - from callus of PG. The extracts differed in saponins and inulin levels-the highest was measured in E3 and the lowest in E1. Identification of secondary metabolites was performed using two complementary LC-MS systems. Chronic bronchitis was induced by sodium metabisulfite (a source of SO₂). Animals were treated with extracts for three weeks (100 mg/kg, intragastrically) and endothelial growth factor (VEGF), transforming growth factors (TGF-β1, -β2, -β3), and mucin 5AC (MUC5AC) levels were determined in bronchoalveolar lavage fluid, whereas C reactive protein (CRP) level was measured in serum. Moreover, mRNA expression were assessed in bronchi and lungs. In SO₂-exposed rats, an elevation of the CRP, TGF-β1, TGF-β2, VEGF, and mucin was found, but the extracts' administration mostly reversed this phenomenon, leading to control values. The results showed a strong anti-inflammatory effect of the extracts from PG.

Keywords: Platycodon grandiflorum; bronchitis; cytokines; extracts; mRNA expression; saponins.

Tranilast (N-3,4-dimethoxycinnamoyl anthranilic acid) is an antiallergic agent with inhibitory effects on cell proliferation and extracellular matrix production. Here we assess the effect of tranilast on the expression of miR-29c and genes functionally involved in cell proliferation, fibrosis, and epigenetic regulation in isolated leiomyoma smooth muscle cells (LSMC). Tranilast significantly inhibited the rate of LSMC proliferation, which was associated with downregulation of cell cycle progression genes cyclin D1 (CCND1) and cyclin-dependent kinase 2 (CDK2) expression at messenger RNA and protein levels ( P < .05). Tranilast also suppressed the expression of collagen type I (COL1), collagen type III alpha 1 chain (COL3A1), the profibrotic cytokine, transforming growth factor β-3 (TGF-β3), DNA (cytosine-5)-methyltransferase 1 (DNMT1), and enhancer of zeste homolog 2 (EZH2), which regulate epigenetic status of gene promoters ( P < .05). Tranilast also significantly induced the expression of cellular and secreted miR-29c through downregulation of methylation status of miR-29c promoter ( P < .05). In addition, tranilast suppressed the activity of luciferase reporter containing 3'UTR of COL3A1 and CDK2, which are downstream targets of miR-29c ( P < .05). Knockdown of miR-29c expression attenuated the inhibitory effects of tranilast on COL3A1 and CDK2 protein expression ( P < .05). Collectively, these findings suggest that tranilast could have therapeutic potential as an inhibitory agent for leiomyoma growth and its associated symptoms.

Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.

Disc disease is characterised by degeneration of the nucleus pulposus (NP), the central gelatinous tissue of the intervertebral disc (IVD). As degeneration progresses, the microenvironment of the IVD becomes more hostile (i.e. decrease in oxygen, glucose and pH), providing a significant challenge for regeneration using cell-based therapies. Tissue engineering strategies such as priming cells or micro tissues with growth factors prior to implantation may overcome some of these issues by providing a pre-formed protective niche composed of extracellular matrix. The present study investigated the effect of priming on bone-marrow-derived stem cells (BMSCs) and articular chondrocytes (ACs) using transforming growth factor β3 (TGF-β3), cultured at different pH levels (pH 7.1, 6.8 and 6.5) representative of the in vivo disc microenvironment. Low pH was found to have a detrimental effect on both cell viability and matrix accumulation, which could be mitigated by priming cells using TGF-β3. Investigating the activation of the transmembrane acid-sensing ion channels (ASIC-1 and -3) showed an increased expression of ASIC-1 in BMSCs and ASIC-3 in ACs at lower pH levels post-priming. Metabolic activity in terms of lactic acid production was also found to be affected significantly by priming, whereas oxygen and glucose consumptions did not change considerably. Overall, the study demonstrated that cells could be equipped to sustain the harsh environment of the IVD and promote accumulation of NP-like matrix through priming. Such an approach may open new avenues to engineer tissues capable of sustaining challenging microenvironments such as those found in the IVD.

This study investigated the changes in the mRNA expression of transforming growth factor beta (TGF-β), plasminogen activators (PAs), and interleukin (IL) caused by sperm, as well as the regulatory mechanism of PA activity through TGF-β, in porcine uterine epithelial cells. The cells were isolated from the uterine horn of pig and co-incubated with Percoll-separated boar sperm (45% or 90%), or TGF-β for 24 h. The mRNA expression of TGF-β isoforms (TGF-β1, 2 and 3) and their receptors (TGF-β R1 and R2), PAs (urokinase-type, uPA; tissue-type, tPA; uPA receptor, uPAR; type 1 PA inhibitor, PAI-1), IL-6 and IL-8 was analyzed using real-time PCR. Supernatant was used to measure PA activity. Co-incubation with sperm from the 90% Percoll layer increased TGF-β1 mRNA, whereas TGF-β2 and TGF-β3 were decreased (P < 0.05). However, both TGF-βRs were not changed by the presence of the spermatozoa. Expression of tPA, PAI-1, IL-6, and IL-8 mRNA was down-regulated by 90% Percoll-separated sperm (P < 0.05), and sperm from 45% Percoll increased uPA expression (P < 0.05). TGF-β decreased tPA and IL-8 mRNA expression, and increased uPAR and PAI-1 mRNA (P < 0.05). The suppressive effect of TGF-β on PA activity was blocked by Smad2/3 and JNK1/2 signaling inhibitors (P < 0.05). In conclusion, sperm separated in 90% in porcine uterus could suppressed inflammation via modulation of TGF-β and down-regulation of PAs and ILs. Therefore, the regulatory mechanism of inflammation by sperm in the porcine uterus could be associated with interactions between numerous cytokines including TGF-β.

Keywords: Inflammation; Plasminogen activators; Porcine uterine epithelial cells; Spermatozoa; Transforming growth factor-beta.

Objective: To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-β3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs).

Methods: The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-β3.

Results: The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-β3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-β3 was observed in PA-MSCs, UC-MSCs and DP-MSCs.

Conclusion: Up-regulated expression of the Twist1, SIRT1 and TGF-β3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-β3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-β3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.

A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs). The recombinant pIRES-EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups: experimental group (MMP group), negative control group (no MMP) and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen (ColII) was detected by using Alcian blue staining and immunohistochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.

In the original article text presenting and discussing results shown in Fig. 6 omitted to mention that quantification of TGF-β2 and TGF-β3 was not included in Fig. 6a, c, e.

The processes of flat bones growth, sutures formation and interdigitation in the human calvaria are controlled by a complex interaction between genetic, biochemical and environmental factors that regulate bone formation and resorption during prenatal development and infancy. Despite previous experimental evidence accounting for the role of the main biochemical factors acting on these processes, the underlying mechanisms controlling them are still unknown. Therefore, we propose a mathematical model of the processes of flat bone and suture formation, taking into account several biological events. First, we model the growth of the flat bones and the formation of sutures and fontanels as a reaction diffusion system between two proteins: TGF-β2 and TGF-β3. The former is expressed by osteoblasts and allows adjacent mesenchymal cells differentiation on the bone fronts of each flat bone. The latter is expressed by mesenchymal cells at the sutures and inhibits their differentiation into osteoblasts at the bone fronts. Suture interdigitation is modelled using a system of reaction diffusion equations that develops spatio-temporal patterns of bone formation and resorption by means of two molecules (Wnt and Sclerostin) which control mesenchymal cells differentiation into osteoblasts at these sites. The results of the computer simulations predict flat bone growth from ossification centers, sutures and fontanels formation as well as bone formation and resorption events along the sutures, giving rise to interdigitated patterns. These stages were modelled and solved by the finite elements method. The simulation results agree with the morphological characteristics of calvarial bones and sutures throughout human prenatal development and infancy.

OBJECTIVE

To study the role of tranilast in the pathogenesis of myocardiac fibrosis in viral myocarditis.

METHODS

Seventy-two BALB/C mice were randomly divided into control, model and intervention groups (n=24 each). Mice in the model and intervention groups were infected with Coxsackievirus B3 to induce viral myocarditis. The intervention group was given with tranilast (200 mg/kg) by gavage until sacrifice for sampling, while the other two groups were administered with the same volume of normal saline. Cardiac tissues were obtained from 8 mice on 7, 14 and 28 days after modeling. The mast cell number was observed by toluidine blue staining and thionine staining. The cardiac tissues were stained with hematoxylin and eosin as well as masson trichrome to observe the pathological changes in cardiac tissues. The mRNA and protein expression of osteopontin and transforming growth factor-β1 was measured by RT-PCR and immunohistochemistry respectively.

RESULTS

In the model group, the mRNA and protein expression of osteopontin reached the highest level on the 7th day, decreasing from the 14th day, and became to the least on the 28th day; while the expression of TGF-β1 increased from the 7th day, reaching a peak on the 14th day, and decreased slightly on the 28th day. The mRNA and protein expression of TGF-β1 and OPN was lower in the intervention group than the model group (P<0.05), but higher than the control group (P<0.05). The expression of OPN mRNA was positively correlated to the number of mast cells.

CONCLUSIONS

Tranilast can reduce myocardial fibrosis by decreasing the number of mast cells, inhibiting the expression of TGF-β1 and OPN.

The present study aimed to assess the effect of a combination of naringin and rabbit bone marrow mesenchymal stem cells (BMSCs) on the repair of cartilage defects in rabbit knee joints and to assess possible involvement of the transforming growth factor-β (TGF-β) signaling pathway in this process. After establishing an articular cartilage defect model in rabbit knees, 20 New Zealand rabbits were divided into a sham operation group (Sham), a model group (Mod), a naringin treatment group (Nar), a BMSC group (BMSCs) and a naringin + BMSC group (Nar/BMSCs). At 12 weeks after treatment, the cartilage was evaluated using the International Cartilage Repair Society (ICRS)'s macroscopic evaluation of cartilage repair scale, the ICRS's visual histological assessment scale, the Modified O'Driscoll grading system, histological staining (hematoxylin and eosin staining, toluidine blue staining and safranin O staining) and immunohistochemical staining (type-II collagen, TGF-β3 and SOX-9 immunostaining). Using the above grading systems to quantify the extent of repair, histological quantification and macro quantification of joint tissue repair showed that the Nar/BMSCs group displayed repair after treatment in comparison to the untreated Mod group. Among the injury model groups (Mod, Nar, BMSCs and Nar/BMSCs), the Nar/BMSCs group displayed the highest degree of morphological repair. The results of histological and immunohistochemical staining of the repaired region of the joint defect indicated that the BMSCs had a satisfactory effect on the repair of the joint structure but had a poor effect on the repair of cartilage quality. The Nar/BMSCs group displayed satisfactory therapeutic effects on both repair of the joint structure and cartilage quality. The expression level of type-II collagen was high in the Nar/BMSCs group. Additionally, staining of TGF-β3 and SOX-9 in the Nar/BMSCs group was the strongest compared with that of any other group in the present study. Naringin and/BMSCs together demonstrated a more efficient repair effect on articular cartilage defects in rabbit knees than the use of either treatment alone in terms of joint structure and cartilage quality. One potential mechanism of naringin action may be through activation and continuous regulation of the TGF-β superfamily signaling pathway, which can promote BMSCs to differentiate into chondrocytes.

Keywords: bone mesenchymal stem cells; cartilage defect; knee joint; naringin; rabbit; superfamily signaling pathway; transforming growth factor-β.

The objective of this study was to identify a combination of growth factors that could be used with hydrostatic pressure (HP) stimulation to enhance the functional development of cartilaginous grafts engineered using human infrapatellar fat pad derived stem cells (FPSCs) isolated from osteoarthritic patients. Agarose hydrogels were first seeded with FPSCs at different seeding densities and maintained in a chondrogenic media supplemented with TGF-β3. It was found that chondrogenesis of human FPSCs in hydrogel culture is dependent on the cell seeding density (10 versus 30 million cells per ml), with greater sulphated glycosaminoglycan (sGAG) and collagen synthesis (normalised to DNA content) observed at higher seeding densities. Additional supplementation with BMP-6 was found to augment cartilage-specific matrix synthesis, also in a cell seeding density dependent manner, increasing both cell proliferation and sGAG synthesis in constructs seeded at higher densities, but having no significant effect at lower cell seeding densities. The application of cyclic HP to FPSC seeded constructs cultured in the presence of both TGF-β3 and BMP-6 had no significant effect on DNA content or sGAG accumulation, however it did improve the dynamic modulus of the engineered tissue. These tissues stained strongly for both alcian blue and type II collagen and negatively for type X collagen. The results of this study point to the benefits of combining both biochemical and biophysical stimulation to engineer functional cartilage grafts using diseased human FPSCs.

Background and aims: A vulnerable plaque is an atherosclerotic plaque that is rupture-prone with a higher risk to cause cardiovascular symptoms such as myocardial infarction or stroke. Mimecan or osteoglycin is a small leucine-rich proteoglycan, important for collagen fibrillogenesis, that has been implicated in atherosclerotic disease, yet the role of mimecan in human atherosclerotic disease remains unknown.

Methods: 196 human atherosclerotic carotid plaques were immunostained for mimecan. Smooth muscle cells, macrophages and intraplaque haemorrhage were also measured with immunohistochemistry. Neutral lipids were stained with Oil Red O and calcium deposits were quantified. Plaque homogenate levels of MCP-1, IL-6 and MIP-1β were measured using a Proximity Extension Assay and MMP-9 levels were measured using Mesoscale. Glycosaminoglycans, collagen and elastin were assessed by colorimetric assays and TGF-β1, β2 and β3 were measured using a multiplex assay. Mimecan gene expression in THP-1 derived macrophages was quantified by qPCR and protein expression in vitro was visualized with immunofluorescence. Cardiovascular events were registered using medical charts and national registers during follow-up.

Results: Mimecan correlated positively with plaque area of lipids, macrophages, intraplaque haemorrhage and inversely with smooth muscle cell staining. Mimecan also correlated positively with plaque levels of MMP-9 and MCP-1. Mimecan was upregulated in THP-1 derived macrophages upon stimulation with MCP-1. Patients with high levels of mimecan (above median) had higher risk for cardiovascular death.

Conclusions: This study indicates that mimecan is associated with a vulnerable plaque phenotype, possibly regulated by plaque inflammation. In line, plaque levels of mimecan independently predict future cardiovascular death.

Keywords: Atherosclerosis; Carotid artery plaque; Extracellular matrix proteins; Inflammation; Proteoglycans.

Embryo manipulations may cause the misexpression of various genes, most of which play critical roles in the regulation of implantation. This study aimed to evaluate the effects of embryo biopsy on the expression of miR-Let-7a and its gene targets including ErbB4, Tgf-α, Itg-αv, Itg β3 on the implantation of mouse embryo. Embryos were produced by in vitro fertilization followed by blastomere biopsy at the eight-cell stage. The effects of blastomere removal on the expression of genes ErbB4, Tgf-α, Itg αv, Itg β3, and miR-Let-7a as well as the alteration of the blastocyst cell number were compared in both biopsied and non-biopsied groups. Finally, blastocyst attachment was assessed on culture dishes precoated with Fibronectin. The results revealed that there were no significant differences between the biopsied and non-biopsied embryos with reference to the blastocyst formation rates, the average inner cell mass, trophectoderm cell number, and percentage of attachment of blastocysts (P > 0.05). The expression of ErbB4, Itg-β3, Itg-αv, TGF-α transcripts, and miR-Let-7a in blastocysts biopsied embryos did not differ from the non-biopsied blastocysts (P > 0.05). The results demonstrated that the preimplantation embryo development and attachment of biopsied embryos in vitro is not adversely affected by one blastomere biopsy at the eight-cell stage embryo.

Biological factors such as TGF-β3 are possible supporters of the healing process in chronic rotator cuff tears. In the present study, electrospun chitosan coated polycaprolacton (CS-g-PCL) fibre scaffolds were loaded with TGF-β3 and their effect on tendon healing was compared biomechanically and histologically to unloaded fibre scaffolds in a chronic tendon defect rat model. The biomechanical analysis revealed that tendon-bone constructs with unloaded scaffolds had significantly lower values for maximum force compared to native tendons. Tendon-bone constructs with TGF-β3-loaded fibre scaffolds showed only slightly lower values. In histological evaluation minor differences could be observed. Both groups showed advanced fibre scaffold degradation driven partly by foreign body giant cell accumulation and high cellular numbers in the reconstructed area. Normal levels of neutrophils indicate that present mast cells mediated rather phagocytosis than inflammation. Fibrosis as sign of foreign body encapsulation and scar formation was only minorly present. In conclusion, TGF-β3-loading of electrospun PCL fibre scaffolds resulted in more robust constructs without causing significant advantages on a cellular level. A deeper investigation with special focus on macrophages and foreign body giant cells interactions is one of the major foci in further investigations.

One candidate preparation of human sequence recombinant transforming growth factor-β3 (TGF-β3) was formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for its suitability to serve as an international standard. The preparation was tested by 8 laboratories using in vitro bioassays and immunoassays. The candidate preparation 09/234 was judged suitable to serve as an international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/234 was established by the WHO Expert Committee on Biological Standardisation (ECBS) as the WHO 1st IS for human TGF-β3 with an assigned value for TGF-β3 activity of 19,000 IU/ampoule.

This study aimed at determining the role of the transforming growth factor-beta (TGF-β) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-βs and their solvent/carrier was investigated in 300 μm ± 10-30 μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-βs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-β3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-β1 (p < 0.001), TGF-β2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-β combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-β combinations would be greater than those healing rates following treatments with TGF-β isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-β(2 + 3) and TGF-β(1 + 3) (p < 0.001). Any cell treated with TGF-β1, TGF-β(1 + 2) and TGF-β(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2 + 3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-β3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and β1. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In conclusion, combining TGF-β3 with any other TGF-β isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-β1 in any TGF-β combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-β1 has an inhibitory effect on bone wound healing while TGF-β3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment.

In spite of showing high sequence similarity and forming structurally similar ternary complex in vitro, the in vivo role of TGF-β1 and TGF-β3 ligands suggests against their functional redundancy and necessitates the importance for the study of the specificity of these ligands. A comparative computational analysis of binary and ternary complexes of these two ligands shows that anchor residues of ligand and receptor at TGF-β:TβR2 interface are similar in both complexes. However, the potential anchor residues of TGF-β at TGF-β:TβR1 interface are different, Tyr50 and Lys51 in TGF-β3 complex and Lys60 and Tyr6 in TGF-β1 complex. Pro55 and Asp57 of TβRI may act as anchor residues in complexes of both ligands along with Ile54 for TGF-β3 complex and Val61 for TGF-β1 complex. Arg58 of TβR1 acts as a potential hot residue for TGF-β3 ternary complex but not for TGF-β1 ternary complex formation whereas Pro55 and Phe60 may act as hot residues for both complexes. The Delphi analysis of the pH dependence of the binding energy indicates that pH has a remarkable effect on the binding energy of TβR2 to the open form of TGF-β3. Lowering of pH from 7 to 4 favors binding of the open form of TGF-β3 to TβR2. Now, apart from the residues at pH 7, residues Arg25, Lys31 and Arg94 of TGF-β3 and Asp118 and Glu119 of TβR2 also contribute significantly to the binding energy. Contrary to the binding energy of TβR2 to TGF-β3/TGF-β1, TβR1 shows appreciable pH dependence for its binding in ternary complex of TGF-β3/TGF-β1. In TGF-β3 ternary complex, the TβR1 electrostatic interaction energy disfavors complex formation at pH 7 while it is favored at pH 4.

OBJECTIVE

To investigate the effects of electromagnetic pulses (EMP) on the apoptosis and transforming growth factor beta 3 (TGF-β3) expression of mouse testis tissue.

METHODS

Thirty-two male BALB/c mice were randomly and equally divided into one control group and three EMP treated groups, which were whole-body exposed to EMP at 200 kV/m with 100, 200, and 400 pulses, respectively. The control group received no treatment. The pathological changes and cell apoptosis in testis tissue were analyzed by TUNEL assay. The mRNA expression of TGF-β3 in testis tissue was determined by RT-PCR, and the protein expression of TGF-β3 was determined by immunohistochemistry and Western blot.

RESULTS

No obvious pathological changes were found in testis tissue after EMP exposure at 200 kV/m with 100 and 200 pulses. However, after EMP exposure with 400 pulses, degeneration and shedding of testis tissue, accompanied by significant increase in apoptosis rate (P < 0.05), was observed. The RT-PCR, immunohistochemistry, and Western blot showed that the expression of TGF-β3 mRNA and protein increased significantly after EMP exposure with 400 pulses as compared with that of the control group (P < 0.05).

CONCLUSIONS

EMP exposure at 200 kV/m with 400 pulses increases the incidence of apoptosis and expression of TGF-β3 in mouse testis tissue, which is potentially one of the mechanisms by which EMP increases blood-testis barrier permeability in mice.