Human Transforming Growth Factor-β1 Research Articles

Evaluation of the potential of rhTGF- β3 encapsulated P(LLA-CL)/collagen nanofibers for tracheal cartilage regeneration using mesenchymal stems cells derived from Wharton's jelly of human umbilical cord

Tracheal injuries are one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, core-shell nanofibrous scaffold was fabricated to encapsulate bovine serum albumin & rhTGF-β3 (recombinant human transforming growth factor-β3) into the core of the nanofibers for tracheal cartilage regeneration. Characterization of the core-shell nanofibrous scaffold was carried out by scanning electron microscope (SEM), transmission electron microscope (TEM), laser scanning confocal microscopy (LSCM), and tensile mechanical test. The rhTGF-β3 released from the scaffolds in a sustained and stable manner for about 2months. The bioactivity of released rhTGF-β3 was evaluated by its effect on the synthesis of type II collagen (COL2) and glycosaminoglycans (GAGs) by chondrocytes. The results suggested that its bioactivity was retained during release process. The proliferation and morphology analyses of mesenchymal stems cells derived from Wharton's jelly of human umbilical cord (WMSCs) indicated the good biocompatibility of the fabricated nanofibrous scaffold. Meanwhile, the chondrogenic differentiation of WMSCs cultured on core-shell nanofibrous scaffold was evaluated by real-time qPCR and histological staining. The results suggested that the core-shell nanofibrous scaffold with rhTGF-β3 could promote the chondrogenic differentiation ability of WMSCs. Therefore, WMSCs could be a promising seed cells in the construction of tissue-engineered tracheal cartilage. Overall, the core-shell nanofibrous scaffold could be an effective delivery system for rhTGF-β3 and served as a promising tissue engineered scaffold for tracheal cartilage regeneration.

The synergistic induction of bone formation by the osteogenic proteins of the TGF-β supergene family.

The momentum to compose this Leading Opinion on the synergistic induction of bone formation suddenly arose when a simple question was formulated during a discussion session on how to boost the often limited induction of bone formation seen in clinical contexts. Re-examination of morphological and molecular data available on the rapid induction of bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) shows that hTGF-β3 replicates the synergistic induction of bone formation as invocated by binary applications of hOP-1:hTGF-β1 at 20:1 by weight when implanted in heterotopic sites of the rectus abdominis muscle of the Chacma baboon, Papio ursinus. The rapid induction of bone formation in primates by hTGF-β3 may stem from bursts of cladistic evolution, now redundant in lower animal species but still activated in primates by relatively high doses of hTGF-β3. Contrary to rodents, lagomorphs and canines, the three mammalian TGF-β isoforms induce rapid and substantial bone formation when implanted in heterotopic rectus abdominis muscle sites of P.ursinus, with unprecedented regeneration of full thickness mandibular defects with rapid mineralization and corticalization. Provocatively, thus providing potential molecular and biological rationales for the apparent redundancy of osteogenic molecular signals in primates, binary applications of recombinant human osteogenic protein-1 (hOP-1) with low doses of hTGF-β1 and -β3, synergize to induce massive ossicles in heterotopic rectus abdominis, orthotopic calvarial and mandibular sites of P.ursinus. The synergistic binary application of homologous but molecularly different soluble molecular signals has indicated that per force several secreted molecular signals are required singly, synchronously and synergistically to induce optimal osteogenesis. The morphological hallmark of the synergistic induction of bone formation is the rapid differentiation of large osteoid seams enveloping haematopoietic bone marrow that forms by day 15 in heterotopic rectus abdominis sites. Synergistic binary applications also induce the morphogenesis of rudimentary embryonic growth plates indicating that the "memory" of developmental events in embryo can be redeployed postnatally by the application of morphogen combinations. Synergistic binary applications or single relatively high doses of hTGF-β3 have shown that hTGF-β3 induces bone by expressing a variety of inductive morphogenetic proteins that result in the rapid induction of bone formation. Tissue induction thus invocated singly by hTGF-β3 recapitulates the synergistic induction of bone formation by binary applications of hTGF-β1 and -β3 isoforms with hOP-1. Both synergistic strategies result in the rapid induction and expansion of the transformed mesenchymal tissue into large corticalized heterotopic ossicles with osteoblast-like cell differentiation at the periphery of the implanted reconstituted specimens with "tissue transfiguration" invivo. Molecularly, the rapid induction of bone formation by binary applications of hOP-1 and hTGF-β3 or by hTGF-β3 applied singly resides in the up-regulation of selected genes involved in tissue induction and morphogenesis, Osteocalcin, RUNX-2, OP-1, TGF-β1 and -β3 with however the noted lack of TGF-β2 up-regulation.

Effect of rAAV2-hTGFβ1 Gene Transfer on Matrix Synthesis in an In Vivo Rabbit Disk Degeneration Model.

In vivo gene transfer for disk regeneration. To evaluate the efficiency and effect of human transforming growth factor β1 (hTGFβ1) gene transfer mediated by adeno-associated virus (AAV) in a rabbit disk degeneration model induced by fibronectin fragment (Fn-f). Gene therapy for disk degeneration has been reported to be effective. Nevertheless, few investigations have targeted the degenerative nucleus pulposus (NP) cells in vivo. Fn-f-induced degeneration has been previously verified to be a useful model for the study of disk degeneration at the molecular level. AAV vector is well suited for gene transfer in the disk for its lower immunogenicity and higher safety. The early dedifferentiated NP cells were transfected with rAAV2-mediated enhanced green fluorescent protein (EGFP) gene in vitro. Fluorescence expression was observed 48 hours later. The rabbit disk degeneration model was established with a microinjection of Fn-f. Ninety-six degenerative disks of 24 rabbits were injected with rAAV2-hTGFβ1 (group A), rAAV2-EGFP (group B), or PBS (group C). Immunohistochemical staining for hTGFβ1 and fluorescence observation were performed at the 1- and 12-week time points, respectively. 35S-sulfate incorporation assay and Western blot analysis were used to measure the synthesis of proteoglycan and collagen type II at 4-, 8-, and 12-week time points. The dedifferentiated NP cells exhibited intensive fluorescence expression in vitro, with a transfection rate of 90%. In vivo, disks in group A showed enhanced positive hTGFβ1 immunostaining at the 1-week time point. At the 4-, 8-, and 12-week time points, disks in group A exhibited significantly increased proteoglycan and collagen type II synthesis compared with the other 2 groups (P<0.01). Abundant green fluorescence was observed in the disks in group B at the 12-week time point. Early degenerative NP cells are susceptible to AAV-mediated gene transfer in vitro and in vivo. The rapid and prolonged target protein expressions and increased matrix synthesis indicated that AAV-mediated therapeutic gene transfer can be a promising form of treatment for disk regeneration in vivo.

Tissue segregation restores the induction of bone formation by the mammalian transforming growth factor-β3 in calvarial defects of the non-human primate Papio ursinus

A diffusion molecular hypothesis from the dura and/or the leptomeninges below that would control the induction of calvarial membranous bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) was investigated. Coral-derived calcium carbonate-based macroporous constructs (25 mm diameter; 3.5/4 mm thickness) with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were inserted into forty calvarial defects created in 10 adult Chacma baboons Papio ursinus. In 20 defects, an impermeable nylon foil membrane (SupraFOIL®) was inserted between the cut endocranial bone and the underlying dura mater. Twenty of the macroporous constructs were preloaded with hTGF-β3 (125 μg in 1000 μl 20 mM sodium succinate, 4% mannitol pH4.0), 10 of which were implanted into defects segregated by the SupraFOIL® membrane, and 10 into non-segregated defects. Tissues were harvested on day 90, processed for decalcified and undecalcified histology and quantitative real-time polymerase chain reaction (qRT-PCR). Segregated untreated macroporous specimens showed a reduction of bone formation across the macroporous spaces compared to non-segregated constructs. qRT-PCR of segregated untreated specimens showed down regulation of osteogenic protein-1 (OP-1), osteocalcin (OC), bone morphogenetic protein-2 (BMP-2), RUNX-2 and inhibitor of DNA binding-2 and -3 (ID2,ID3) and up regulation of TGF-β3, a molecular signalling pathway inhibiting the induction of membranous bone formation. Non-segregated hTGF-β3/treated constructs also showed non-osteogenic expression profiles when compared to non-segregated untreated specimens. Segregated hTGF-β3/treated 7% HA/CC constructs showed significantly greater induction of bone formation across the macroporous spaces and, compared to non-segregated hTGF-β3/treated constructs, showed up regulation of OP-1, OC, BMP-2, RUNX-2, ID2 and ID3. Similar up-regulated expression profiles were seen for untreated non-segregated constructs. TGF-β signalling via ID genes creates permissive or refractory micro-environments that regulate the induction of calvarial bone formation which is controlled by the exogenous hTGF-β3 upon segregation of the calvarial defects. The dura is the common regulator of the induction of calvarial bone formation modulated by the presence or absence of the SupraFOIL® membrane with or without hTGF-β3.

Chondroinductive Hydrogel Pastes Composed of Naturally Derived Devitalized Cartilage.

Hydrogel precursors are liquid solutions that are prone to leaking from the defect site once implanted in vivo. Therefore, the objective of the current study was to create a hydrogel precursor that exhibited a yield stress. Additionally, devitalized cartilage extracellular matrix (DVC) was mixed with DVC that had been solubilized and methacrylated (MeSDVC) to create hydrogels that were chondroinductive. Precursors composed of 10% MeSDVC or 10% MeSDVC with 10% DVC were first evaluated rheologically, where non-Newtonian behavior was observed in all hydrogel precursors. Rat bone marrow stem cells (rBMSCs) were mixed in the precursor solutions, and the solutions were then crosslinked and cultured in vitro for 6weeks with and without exposure to human transforming growth factor β3 (TGF-β3). The compressive modulus, gene expression, biochemical content, swelling, and histology of the gels were analyzed. The DVC-containing gels consistently outperformed the MeSDVC-only group in chondrogenic gene expression, especially at 6weeks, where the relative collagen II expression of the DVC-containing groups with and without TGF-β3 exposure was 40- and 78-fold higher, respectively, than that of MeSDVC alone. Future work will test for chondrogenesis in vivo and overall, these two cartilage-derived components are promising materials for cartilage tissue engineering applications.

Modulation of the EMT/MET process by pyrrole–imidazole polyamide targeting human transforming growth factor-β1

Transforming growth factor-β1 (TGF-β1) is a potent induction factor for epithelial–mesenchymal transition (EMT). Mesenchymal–epithelial transition (MET), as the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole–imidazole (PI) polyamide, a novel gene regulator that targets human TGF-β1, and investigated its effects on the EMT/MET process. PI polyamide targeted to TGF-β1 significantly inhibited the mRNA expression of TGF-β1 and SNAI1 as an EMT marker and increased mRNA and protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-β1 was applied to human fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline phosphatase-positive colonies. The expression of undifferentiated markers was also observed in these colonies. These results suggest that PI polyamide targeted to human TGF-β is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.

Fractalkine–CX3CR1-dependent recruitment and retention of human CD1c+ myeloid dendritic cells by in vitro–activated proximal tubular epithelial cells

Chemokines play pivotal roles in tissue recruitment and retention of leukocytes, with CX3CR1 recently identified as a chemokine receptor that selectively targets mouse kidney dendritic cells (DCs). We have previously demonstrated increased tubulointerstitial recruitment of human transforming growth factor-β (TGF-β)-producing DCs in renal fibrosis and chronic kidney disease (CKD). However, little is known about the mechanism of human DC recruitment and retention within the renal interstitium. We identified CD1c+ DCs as the predominant source of profibrotic TGF-β and highest expressors of the fractalkine receptor CX3CR1 within the renal DC compartment. Immunohistochemical analysis of diseased human kidney biopsies showed colocalization of CD1c+ DCs with fractalkine-positive proximal tubular epithelial cells (PTECs). Human primary PTEC activation with interferon-γ and tumor necrosis factor-α induced both secreted and surface fractalkine expression. In line with this, we found fractalkine-dependent chemotaxis of CD1c+ DCs to supernatant from activated PTECs. Finally, in comparison with unactivated PTECs, we showed significantly increased adhesion of CD1c+ DCs to activated PTECs via a fractalkine-dependent mechanism. Thus, TGF-β-producing CD1c+ DCs are recruited and retained in the renal tubulointerstitium by PTEC-derived fractalkine. These cells are then positioned to play a role in the development of fibrosis and progression of chronic kidney disease.

Effects of Biejia Ruangan Tablet-containing serum on matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in cultured renal interstitial fibroblasts.

To investigate the effects of Biejia Ruangan Tablet ([symbol in text], BRT)-containing serum on the expression of matrix metalloproteinase (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) in cultured renal interstitial fibroblasts. Different BRT-containing sera were prepared by gastric gavages to rats with the high-dose (7 g/kg), mid-dose (3.5 g/kg), and low-dose (1.75 g/kg) BRT respectively. The expression of extracellular matrix in NRK-49F cells was induced by treatment with human transforming growth factor-β1 (recombined human TGF-β1), and BRT-containing serum. Western blotting and Northern blotting were used to measure type I and III procollagen, MMP-9, and TIMP-1. The high dose BRT-containing serum could decrease the type I and III procollagen gene expression which boosted by TGF-β1, at the same time cut down TIMP-1 protein and gene expression which increased by TGF-β1 (P <0.05). Treatment of cells with recombined human TGF-β1 had no significant effect on MMP-9 expression and BRT-containing serum also had no effect on MMP-9 expression. High dose BRT has anti-fibrosis effects in NRK-49F cells, as indicated by its inhibition of type I and III procollagen and TIMP-1 expression.

Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells.

During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated. A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-β3 (TGF-β3). The hMSC pellet cultures treated with TGF-β3 were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR. SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α1 chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio. SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.

Engineered nasal cartilage by cell homing: a model for augmentative and reconstructive rhinoplasty.

Current augmentative and reconstructive rhinoplasties use auto logous tissue grafts or synthetic bioinert materials to repair nasal trauma or attain an aesthetic shape. Autologous grafts are associated with donor-site trauma and morbidity. Synthetic materials are widely used but often yield an unnatural appearance and are prone to infection or dislocation. There is an acute clinical need for the generation of native tissues to serve as rhinoplasty grafts without the undesirable features that are associated with autologous grafts or current synthetic materials. Bioactive scaffolds were developed that not only recruited cells in the nasal dorsum in vivo, but also induced chondrogenesis of the recruited cells. Bilayered scaffolds were fabricated with alginate-containing gelatin microspheres encapsulating cytokines atop a porous poly(lactic-co-glycolic acid) base. Microspheres were fabricated to contain recombinant human transforming growth factor-β3 at doses of 200, 500, or 1000 ng, with phosphate-buffered saline-loaded microspheres used as a control. A rat model of augmentation rhinoplasty was created by implanting scaffolds atop the native nasal cartilage surface that was scored to induce cell migration. Tissue formation and chondrogenesis in the scaffolds were evaluated by image analysis and histologic staining with hematoxylin and eosin, toluidine blue, Verhoeff elastic-van Geison, and aggrecan immunohistochemistry. Sustained release of increasing doses of transforming growth factor-β3 for up to the tested 10 weeks promoted orthotopic cartilage-like tissue formation in a dose-dependent manner. These findings represent the first attempt to engineer cartilage tissue by cell homing for rhinoplasty, and could potentially serve as an alternative material for augmentative and reconstructive rhinoplasty.

Association between TGFB1 915G/C polymorphism and susceptibility to chronic hepatitis C virus infection: A meta-analysis

The human transforming growth factor-β1 (TGF-β1) gene, namely TGFB1, contains several single-nucleotide polymorphisms (SNPs) and some of the polymorphic variants were shown to affect the TGF-β1 protein levels. A number of studies reported the association between 915G/C polymorphism and susceptibility to chronic hepatitis C virus (HCV) infection. However, the results were inconsistent. This meta-analysis was conducted to assess the association of TGFB1 915G/C polymorphism with susceptibility to chronic HCV infection. PubMed, ISI Web of Knowledge, ScienceDirect and Google Scholar databases were systematically searched up to August, 2013 to identify relevant studies. The pooled odds ratios (ORs) with their corresponding 95% confidence intervals (95% CIs) were calculated in 5 genetic comparison models (C vs. G, CC vs. GG, GC vs. GG, CC vs. GG+GC and CC+GC vs. GG). The Galbraith plot and subgroup analyses based on ethnicity, genotyping methods, sample size and fibrosis were performed to investigate possible sources of heterogeneity. A sensitivity analysis and assessment of publication bias were also conducted. Finally, 8 eligible case-control studies on TGFB1 915G/C polymorphism, including a total of 910 cases and 632 controls, were included in this meta-analysis. Overall, there was no evidence of any gene-disease association obtained from the subgroup analyses. Therefore, this meta-analysis demonstrated that there is no association between TGFB1 915G/C polymorphisms and susceptibility to chronic HCV infection.

The induction of bone formation by the recombinant human transforming growth factor-β3

Implantation of recombinant human transforming growth factor-β3 (hTGF-β3) with coral-derived calcium carbonate-based macroporous bioreactors with limited conversion to hydroxyapatite (7% HA/CC) in the rectus abdominis muscle of the non-human primate Chacma baboon Papio ursinus induces endochondral bone formation. The exact mechanisms by which hTGF-β3 signalling induces bone in heterotopic sites of P. ursinus are not known. Coral-derived 7% HA/CC bioreactors with and without 125 μg hTGF-β3 were implanted in triplicate in the rectus abdominis muscle of 6 adult baboons. 7% HA/CC bioreactors either with or without hTGF-β3 were loaded with 125 μg of recombinant human Noggin (hNoggin), a bone morphogenetic proteins (BMPs) antagonist. Tissues on day 15, 60 and 90 were analysed by histomorphometry and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Down-regulation of BMP-2 characterized 7% HA/CC constructs preloaded with 125 μg hNoggin with Noggin down-regulated on day 60 and 90 together with lack of TGF-β3 expression. Down-regulation of BMP-2 correlated with minimal bone formation by induction. hTGF-β3/hNoggin pre-treated bioreactors up-regulated BMP-2 but only on day 90 together with a significant down-regulation of Noggin on day 60 and 90, correlating with the induction of bone formation, albeit limited, on day 90 at the periphery of the macroporous bioreactors only. hTGF-β3 treated bioreactors significantly down-regulated BMP-2 on day 15 whilst up-regulating BMP-2 on day 60 and 90, together with down-regulation of Noggin on day 60 and 90 correlating with the prominent induction of bone formation. hTGF-β3 significantly up-regulated RUNX-2 and Osteocalcin expression on day 15 controlling the differentiation of progenitor stem cells into the osteoblastic lineage. The induction of bone as initiated by hTGF-β3 in the rectus abdominis muscle of P. ursinus is via the BMPs pathway with hTGF-β3 controlling the induction of bone formation by regulating the expression of BMPs via Noggin expression. These results unequivocally demonstrate that hTGF-β3 elicits bone induction by up-regulation of endogenous BMP-2 and is blocked by hNoggin.

Activation ofCOL1A2promoter in human fibroblasts byEscherichia coli

The relationship between bacterial infection and collagen production was investigated using human fibroblasts transfected with the promoter of COL1A2 , which encodes the α1 chain of human type I collagen, linked to a luciferase reporter. The cells were used to assess the gene promoter activity of COL1A2 following bacterial stimulation. The COL1A2 promoter was activated by stimulation with fixed Escherichia coli in a dose-dependent manner, but not by fixed Staphylococcus aureus. Enhancement of collagen production was observed in the E.coli-stimulated fibroblasts compared to those without stimulation. Both anti-human Toll-like receptor (TLR) 4 antibody and polymyxin B clearly blocked the COL1A2 promoter activity stimulated by E.coli, while antibodies against human TLR2 and human transforming growth factor-β (TGF-β) receptor type II did not. These results indicate that E.coli can directly interact with TLR4 expressed on the surface of fibroblasts and can further induce human type I collagen gene expression and collagen production in these cells. These data also suggest that infection by gram-negative bacteria may cause fibrosis.

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-β1 (TGF-β1)

This study demonstrates that the modification of hyaluronan (hyaluronic acid; Hya) and chondroitin sulfate (CS) with sulfate groups leads to different binding affinities for recombinant human transforming growth factor-β1 (TGF-β1) for comparable average degrees of sulfation (DS). In general, Hya derivates showed higher binding strength than CS derivatives. In either case, a higher degree of sulfation leads to a stronger interaction. The high-sulfated hyaluronan sHya3 (average DS≈3) exhibited the tightest interaction with TGF-β1, as determined by surface plasmon resonance and enzyme-linked immunosorbent assay. The binding strength was significantly weakened by carboxymethylation. Unmodified Hya and low-sulfated, native CS showed weak or no binding affinity. The interaction characteristics of the different sulfated glycosaminoglycans are promising for incorporation into bioengineered coatings of biomaterials to modulate growth factor binding in medical applications.

Induced transgene expression for the treatment of solid tumors by hematopoietic stem cell-based gene therapy

Tumor microenvironment is composed of different cell types including immune cells. Far from acting to eradicate cancer cells, these bone marrow-derived components could be involved in carcinogenesis and/or tumor invasion and metastasis. Here, we describe an alternative approach to treat solid tumors based on the genetic modification of hematopoietic stem and progenitor cells with lentiviral vectors. To achieve transgene expression in derivative tumor infiltrating leukocytes and to try to decrease systemic toxicity, we used the stress inducible human HSP70B promoter. Functionality of the promoter was characterized in vitro using hyperthermia. Antitumor efficacy was assessed by ex vivo genetic modification of lineage-negative cells with lentiviral vectors encoding the dominant-negative mutant of the human transforming growth factor-β receptor II (TβRIIDN) driven by the HSP70B promoter, and reinfusion of cells into recipient mice. Subsequently, syngeneic GL261 glioma cells were subcutaneously injected into bone marrow-transplanted mice. As a result, a massive antitumor response was observed in mice harboring TβRIIDN under the HSP70B promoter, without the need of any external source of stress. In summary, this study shows that stem cell-based gene therapy in combination with spatial and temporal control of transgene expression in derivative tumor-infiltrating cells represents an alternative strategy for the development of novel antitumor therapies.

Negative androgen-response elements mediate androgen-dependent transcriptional inhibition of TGF-β1 and CDK2 promoters in the prostate gland.

The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgen functions by regulating the expression of genes it targets. In the present study, we mapped 2 regions in human transforming growth factor β1 and cyclin-dependent kinase 2 promoters that negatively regulated transcriptional activity in an androgen-dependent manner in close proximity to positive androgen-response elements (pARE). We identified 7 negative androgen-response elements (nARE) in the negative androgen-response region in the TGF-β1 promoter. Each nARE is composed of 15 nucleotides and mediates androgen-dependent inhibition of transcription in close proximity of pAREs. The 5' portions of nAREs are highly conserved and resemble core half sites in pAREs. The nAREs interacted weakly with AR DNA-binding domain (AR DBD) in gel shift assays. Mutations on the conserved nucleotides in the nARE abolished its interaction with the AR DBD and abolished its inhibition of androgen-driven transcription, suggesting that nARE/AR interaction is essential for nARE-mediated inhibition of transcription. Taken together, these findings indicate that the AR negatively regulates the expression of its target genes via a negative androgen-response region composed of multiple nAREs.

A Macroporous Bioreactor Super Activated by the Recombinant Human Transforming Growth Factor-β3

Macroporous single phase hydroxyapatite (HA) and biphasic HA/β-tricalcium phosphate with 33% post-sinter hydroxyapatite (HA/β-TCP) were combined with 25 or 125 μg recombinant human transforming growth factor-β3 (hTGF-β3) to engineer a super activated bioreactor implanted in orthotopic calvarial and heterotopic rectus abdominis muscle sites and harvested on day 30 and 90. Coral-derived calcium carbonate fully converted (100%) and partially converted to 5 and 13% hydroxyapatite/calcium carbonate (5 and 13% HA/CC) pre-loaded with 125 and 250 μg hTGF-β3, and 1:5 and 5:1 binary applications of hTGF-β3: hOP-1 by weight, were implanted in the rectus abdominis and harvested on day 20 and 30, respectively, to monitor spatial/temporal morphogenesis by high doses of hTGF-β3. Bone formation was assessed on decalcified paraffin-embedded sections by measuring the fractional volume of newly formed bone. On day 30 and 90, single phase HA implants showed greater amounts of bone when compared to biphasic specimens; 5 and 13% HA/CC pre-loaded with 125 and 250 μg hTGF-β3 showed substantial induction of bone formation; 250 μg hTGF-β3 induced as yet unreported massive induction of bone formation as early as 20 days prominently outside the profile of the macroporous constructs. The induction of bone formation is controlled by the implanted ratio of the recombinant morphogens, i.e., the 1:5 hTGF-β3:hOP-1 ratio by weight was greater than the inverse ratio. The unprecedented tissue induction by single doses of 250 μg hTGF-β3 resulting in rapid bone morphogenesis of vast mineralized ossicles with multiple trabeculations surfaced by contiguous secreting osteoblasts is the novel molecular and morphological frontier for the induction of bone formation in clinical contexts.

Construction of eukaryotic expression plasmid of hTGF-β3 and its inducing effect on differentiation of precartilaginous stem cells into chondroblasts

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.

Chondrogenesis by chemotactic homing of synovium, bone marrow, and adipose stem cellsin vitro

Cell transplantation has been well explored for cartilage regeneration. We recently showed that the entire articular surface of a synovial joint can regenerate by endogenous cell homing and without cell transplantation. However, the sources of endogenous cells that regenerate articular cartilage remain elusive. Here, we studied whether cytokines not only chemotactically recruit adipose stem cells (ASCs), mesenchymal stem cells (MSCs), and synovium stem cells (SSCs) but also induce chondrogenesis of the recruited cells. Recombinant human transforming growth factor-β3 (TGF-β3; 100 ng) and/or recombinant human stromal derived factor-1β (SDF-1β; 100 ng) was control released into an acellular collagen sponge cube with underlying ASCs, MSCs, or SSCs in monolayer culture. Although all cell types randomly migrated into the acellular collagen sponge cube, TGF-β3 and/or SDF-1β recruited significantly more cells than the cytokine-free control group. In 6 wk, TGF-β3 alone recruited substantial numbers of ASCs (558±65) and MSCs (302±52), whereas codelivery of TGF-β3 and SDF-1β was particularly chemotactic to SSCs (400±120). Proliferation of the recruited cells accounted for some, but far from all, of the observed cellularity. TGF-β3 and SDF-1β codelivery induced significantly higher aggrecan gene expression than the cytokine-free group for ASCs, MSCs, and SSCs. Type II collagen gene expression was also significantly higher for ASCs and SSCs by SDF-1 and TGF-β3 codelivery. Remarkably, the expression of aggrecan and type II collagen was detected among all cell types. Thus, homing of multiple stem/progenitor cell populations may potentially serve as an alternative or adjunctive approach to cell transplantation for cartilage regeneration.

Adipose-derived stem cells transfected by adenoviral vector mediated human transforming growth factor-β1 gene with β-tricalcium phosphate scaffold in vitro

Objective To observe the biological behaviors of cultured adipose-derived stem cells (ADSCs) transfected by adenoviral vector-mediated human transforming growth factor-β1 (Ad-hTGF-β1) combined with β-tricalcium phosphate (β-TCP) scaffold and investigate the feasibility of them for cartilage tissue engineering. Methods The adenoviral plasmid vector containing hTGF-β1 gene was transfected into ADSCs. The ADSCs were seeded onto the β-TCP scaffold and the effects of adhesion and morphological changes were observed under the phase-contrast microscopy and scanning electron microscopy (SEM). Reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were applied to detect the expression of collagen Ⅱ in the β-TCP scaffold. Results The SEM results showed the porous β-TCP scaffold had macro- and micro-porous strutures with the porosity being 65%. The ADSCs transfected by Ad-hTGF-β1 were successfully cultured in vitro. The induced cells adhered to the surface of the β-TCP scaffold and proliferated well. RT-PCR, Western blotting and immunohistochemistry revealed that Collagen Ⅱ was detected after mixed culture. Conclusion The porous β-TCP scaffold with excellent property may be a good matrix for ADSCs transfected by Ad-hTGF-β1, and can be used for cartilage engineering. Key words: Adipose-derived stem cell; β-tricalcium phosphate; Scaffold; Tissue engineering