Sustained expression of transforming growth factor-beta1 by distraction during distraction osteogenesis

Distraction osteogenesis is a well-established clinical treatment for limb length discrepancy and skeletal deformities. In our previous studies, we have shown that the tension at the distraction gap correlated with the plasma bone specific alkaline phosphatase activity during distraction. Transforming growth factor-beta1 (TGF-beta1) has been shown to have a regulatory role in alkaline phosphatase activity during fracture healing. This study is to investigate the expression of TGF-beta1 during distraction as a biological response to mechanically stimulated osteoblastic activity by immunohistochemistry. The expression of TGF-beta1 in the distraction callus was compared with that in the fracture callus. During the distraction phase, the osteoblasts and osteocytes expressed a high level of TGF-beta1. Moderate expression of TGF-beta1 was observed in fibroblast-like cells in the fibrous zone of the distraction callus. After the distraction stopped, the expression of TGF-beta1 in different cell types decreased. In fracture healing, the strong expression of TGF-beta1 declined after the first week. Our results showed that the mechanical force induced and sustained TGF-beta1 expression in osteoblasts and fibroblasts-like cells of the distraction callus. Transforming growth factor-beta1 may play a role in transducing mechanical stimulation to biological tissue during in distraction osteogenesis.     

Effects of transforming growth factor-beta on normal clonal bone cell populations.

Although transforming growth factor-beta (TGF-beta) has been implicated in the local regulation of bone growth and remodelling, its specific effects on different subpopulations of bone cells have not been elucidated. Cells derived from bone are known to be heterogeneous and include both cells of different lineages and osteoblastic populations with different levels of expression of osteoblast-associated properties. Consequently, we have isolated clonal populations of bone cells to examine more precisely the effects of TGF-beta on individual subpopulations. Several clonal populations were isolated by limiting dilution from cells derived from 21-day-old fetal rat calvaria. Two of these clones, RCA 11 and RCB 2, were used here. While the two clones responded similarly to parathyroid hormone (PTH) and isoproterenol (ISP) with increases in intracellular cAMP, prostaglandin E2 (PGE2) elicited a 10-fold higher response in RCB 2 cells compared with RCA 11. RCB 2 cells expressed a 10-fold higher alkaline phosphatase activity compared with RCA 11. Both clones synthesized a variety of bone matrix associated proteins, but only RCA 11 synthesized SPP-1 (osteopontin) constitutively. TGF-beta stimulated growth of RCB 2 cells after 24 and 48 h of treatment, but had no effect on growth of RCA 11. TGF-beta supported anchorage-independent growth of RCB 2 cells, but not that of RCA 11. A 24-h exposure to TGF-beta decreased cAMP responsiveness to PTH and ISP slightly in both clones, but had no effect on PGE2 responses. Significant reductions in alkaline phosphatase activity were seen in both clones after 24- and 48-h treatments with TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of immune cell function in vitro by recombinant human transforming growth factor-beta

The influence of recombinant human transforming growth factor-beta (rHuTGF-beta) on B-cell function and antigen-specific T-cell responses in vitro was investigated. The addition of 0.1 ng/ml of rHuTGF-beta to cultures of peripheral blood mononuclear cells (PBMC) stimulated with tetanus toxoid resulted in a 50% inhibition of the PBMC proliferative response as determined by [3H]thymidine incorporation. Further, rHuTGF-beta at 0.37 ng/ml caused a greater than 50% reduction in the number of immunoglobulin G-secreting cells among PBMC induced by pokeweed mitogen. rHuTGF-beta also inhibited the generation of allospecific cytotoxic T lymphocytes (CTL) in the mixed-lymphocyte reaction but had no effect on the cytolytic function of CTL generated in the absence of exogenous HuTGF-beta. The results indicate additional immunoregulatory activities for HuTGF-beta and suggest that this factor may play an important role in the regulation of the antigen-dependent immune response.     

Effects of bovine inhibin, transforming growth factor-beta and bovine Activin-A on granulosa cell differentiation

To determine whether inhibin and its related peptides might act locally to control granulosa cell function and differentiation, the dose- and time-dependent effects of bovine inhibin, the homo-dimer of the beta-chain of bovine inhibin (Activin-A) and porcine TGF beta on rat granulosa cell aromatase activity and progesterone synthesis were investigated in vitro. TGF beta enhanced FSH-induced aromatase activity and progesterone synthesis, and accelerated the peak response for progesterone synthesis. Activin-A on the other hand, augmented FSH-induced aromatase activity while arresting progesterone synthesis, and anti-luteinization effect. By contrast, exogenous inhibin had no detectable effect on the steroidogenic potential of these cells. Thus TGF beta and Activin, unlike their similar effects on the release of FSH by the pituitary, appear to affect ovarian granulosa cell function in different fashion, under conditions where inhibin itself has no effect.     

The role of transforming growth factor-beta on retarded osteoblastic differentiation in vitro

Various matrix growth factors play important roles in the development and growth of cartilage and bone. Among them transforming growth factor-beta superfamily and especially bone morphogenetic proteins are known to be important factors, since they induce bone and cartilage formation in ectopic sites in vivo. We have previously shown that the human osteosarcoma cell line Saos-2 expresses molecules that in vivo induce new bone formation with asymmetric bone maturation. In this study we examined the role of Saos-2-conditioned medium in prolonged cultures of mesenchymal C3H/10T1/2 cells. The C3H/10T1/2 cells were cultured with Saos-2-conditioned medium for 28 days. We show that Saos-2-treated C3H/10T1/2 cells performed retarded osteoblastic differentiation when compared to recombinant BMP-2 and -4 induced differentiation. We further show that this retardation is due to excessive amounts of transforming growth factor-beta in Saos-2-conditioned medium. Our results also suggest that this model can well be used to study additional cofactors involved in retarded osteogenesis.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

转化生长因子β1对大鼠心肌细胞肥大的影响

目的：研究转化生长因子β1（TGF-β1）及其下游Smad3信号蛋白在大鼠心肌细胞肥大中的作用。方法：TGF-β1干预培养新生大鼠心肌细胞,流式细胞仪检测心肌细胞总蛋白含量。结扎大鼠腹主动脉复制心肌肥厚模型,在不同时间点处死动物,检测左室质量指数（LVM1）,RT—PCR检测TGF-β1及Smad3的mRNA表达,Westernblot检测Smad3蛋白的表达。结果：不同剂量TGF-β1均能明显增加体外培养的心肌细胞总蛋白含量,TGF-β1（3ng／ml）还增加心肌细胞Smad3 mRNA和蛋白的表达,其表达量1h达高峰,持续至TGF-β1刺激后8h。大鼠腹主动脉结扎术后3d LVMI开始上升并持续至术后28d,心肌组织中TGF-β1、Smad3的mRNA表达水平以及Smad3蛋白表达术后3d也开始上升持续至术后28d,术后14d为表达高峰（P〈0．01）。结论：TGF-β1能诱导大鼠心肌细胞肥大,其信号蛋白Smad3参与了大鼠心肌肥大的病理过程。     

Transforming growth factor-beta modulates the expression of osteoblast and chondroblast phenotypes in vitro

Transforming growth factor beta (TGF-beta) has been shown to induce chondrogenesis by embryonic rat mesenchymal cells (Seyedin et al., J. Biol. Chem., 261: 5693, 1986). Here we report the effects of bovine TGF-beta on the phenotypic expression of differentiated primary rat osteoblastic and chondroblastic cells. Culture of rat calvarial osteoblasts with TGF-beta resulted in a dose and time-dependent decrease in alkaline phosphatase activity. Levels of alkaline phosphatase were reduced to less than 10% of control values by 0.4 nM TGF-beta. The decrease became apparent after 24 hours and reached a maximum by 72 hours. Similarly, treatment of chondroblasts with 0.4 nM TGF-beta resulted in decreased production of cartilage-specific macromolecules: type II collagen and cartilage proteoglycan. Both cell types exhibited dramatic changes in cell shape after treatment with TGF-beta. Modulation of these differentiated markers by TGF-beta could be mimicked, in part, by addition of fibronectin. Addition of dihydrocytochalasin B blocked the inhibition of phenotypic expression by TGF-beta. These results indicate that TGF-beta inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of TGF-beta may be mediated through interactions between the extracellular matrix and cytoskeletal elements.     

Choice of osteoblast model critical for studying the effects of electromagnetic stimulation on osteogenesis in vitro

ABSTRACT

The clinical benefits of electromagnetic field (EMF) therapy in enhancing osteogenesis have been acknowledged for decades, but agreement regarding the underlying mechanisms continues to be sought. Studies have shown EMFs to promote osteoblast-like cell proliferation, or contrarily, to induce differentiation and enhance mineralization. Typically these disparities have been attributed to methodological differences. The present paper argues the possibility that the chosen osteoblast model impacts stimulation outcome. Phenotypically immature cells, particularly at low seeding densities, appear to be prone to EMF-amplified proliferation. Conversely, mature cells at higher densities seem to be predisposed to earlier onset differentiation and mineralization. This suggests that EMFs augment ongoing processes in cell populations. To test this hypothesis, mature SaOS-2 cells and immature MC3T3-E1 cells at various densities, with or without osteo-induction, were exposed to sinusoidal 50 Hz EMF. The exposure stimulated the proliferation of MC3T3-E1 and inhibited the proliferation of SaOS-2 cells. Baseline alkaline phosphatase (ALP) expression of SaOS-2 cells was high and rapidly further increased with EMF exposure, whereas ALP effects in MC3T3-E1 cells were not seen until the second week. Thus both cell types responded differently to EMF stimulation, corroborating the hypothesis that the phenotypic maturity and culture stage of cells influence stimulation outcome.     

Prostaglandin E2 enhances transforming growth factor-beta 1 and TGF-beta receptors synthesis: an in vivo and in vitro study

The aims of this study were to determine how Prostaglandin E2 (PGE2) locally applied affected the immunodistribution of latent transforming growth factor-beta 1 (TGF-beta1), and how the eicosanoid modified TGF-beta1 release and TGF-beta receptors gene expression in cultured osteoblasts. PGE2 locally delivered on the rat mandible at doses of 0.1 and 0.05 mg/day, but not 0.025 mg/day, over 20 days significantly increased latent TGF-beta1 immunodistribution (P<0.001), comparing with a placebo-treated group. Cultured osteoblasts stimulated with 10(-5) or 10(-7)M PGE2 significantly varied the level of activated TGF-beta1 released into supernatants at different experimental periods compared with negative and positive controls. TGF-beta receptor type I gene expression was significantly increased in osteoblasts (P<0.01) after 10 days of treatment with 10(-5) and 10(-7)M PGE2, whereas 10(-3) M PGE2 produced the opposite effect. It is concluded that PGE2 may stimulate bone deposition by affecting TGF-beta pathway. This effect on the pathway appears to be dose-dependent.     

Chondrocytes inhibit endothelial sprout formation in vitro: evidence for involvement of a transforming growth factor-beta.

Using a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti-TGF-beta antibodies to the cocultures significantly reduced the inhibitory effect of chondrocytes on sprout formation. Chondrocyte-conditioned medium or exogenously added TGF-beta 1 have a similar albeit transient inhibitory effect. Depletion of TGF-beta from chondrocyte conditioned medium with anti-TGF-beta antibodies and solid-phase protein-A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte-derived TGF-beta-like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF-beta.     

Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.     

The diabetic rat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor

Two models of wound repair compared the effect of defined, recombinant growth factors on the rate of wound repair in both normal and streptozotocin-induced diabetic rats: subcutaneous implantation of polyvinyl alcohol sponges and incisional wounding. Transverse incisional wounds were made on the dorsal surface of rats and closed with steel sutures. Three days postwounding the rats received a single injection of either transforming growth factor-beta or vehicle alone directly into the wound site. Animals were sacrificed 7, 14, and 21 days postwounding, and fresh and formalin-fixed wound tensile strength were measured. Diabetic rats had expected defects in wound repair, including decreased granulation tissue and reduced amounts of collagen, protein, and DNA. Fresh tensile strength of the diabetic incisions was 53% of normal on Day 7 (p < or = .01) and 29% of normal on Day 21. Fixed tensile strength was 41% of normal on Day 7 (p < or = .01) and fell to 78% of normal by Day 21 (p < or = .01), suggesting that collagen concentrations of diabetic wounds increased towards normal but did not undergo maturation. TGF beta produced a moderate increase in tensile strength of fresh and fixed wounds of diabetic rats, but not to the levels of wounds in untreated normal rats. Sponges fill with granulation tissue, their reproducible rate of organization being measured by histological and biochemical methods. A single injection into sponges 3 days postimplantation of basic fibroblast growth factor, transforming growth factor-beta, or vehicle only, was evaluated at 7 and 9 days postimplantation. In the sponge model, bFGF and TGF beta were each able to induce significant increases in the accumulation of granulation tissue in both diabetic and normal rats. TGF beta increased the collagen content of sponges by 136% in sponges from diabetic animals (p < or = .001), thereby raising the collagen content to that of normal control wounds, while stimulating a 49% (p < or = .02) increase in sponges from normal animals on Day 9. By contrast, the response to bFGF was predominantly an increase in the protein and DNA content of the sponges. These results emphasize the differential effects of the two cytokines in accelerating healing under conditions of defective wound repair.