Extracellularly Detectable Electrochemical Signals of Living Cells Originate from Metabolic Reactions.

Direct detection of cellular redox signals has shown immense potential as a novel living cell analysis tool. However, the origin of such signals remains unknown, which hinders the widespread use of electrochemical methods for cellular research. In this study, the authors found that intracellular metabolic pathways that generate adenosine triphosphate (ATP) are the main contributors to extracellularly detectable electrochemical signals. This is achieved through the detection of living cells (4,706 cells/chip, linearity: 0.985) at a linear range of 7,466-48,866. Based on this discovery, the authors demonstrated that the cellular signals detected by differential pulse voltammetry (DPV) can be rapidly amplified with a developed medium containing metabolic activator cocktails (MACs). The DPV approach combined with MAC treatment shows a remarkable performance to detect the effects of the anticancer drug CPI-613 on cervical cancer both at a low drug concentration (2 µm) and an extremely short treatment time (1 hour). Furthermore, the senescence of mesenchymal stem cells could also be sensitively quantified using the DPV+MAC method even at a low passage number (P6). Collectively, their findings unveiled the origin of redox signals in living cells, which has important implications for the characterization of various cellular functions and behaviors using electrochemical approaches.

Recent Advances in Electrochemical Biosensors for Monitoring Animal Cell Function and Viability.

Redox reactions in live cells are generated by involving various redox biomolecules for maintaining cell viability and functions. These qualities have been exploited in the development of clinical monitoring, diagnostic approaches, and numerous types of biosensors. Particularly, electrochemical biosensor-based live-cell detection technologies, such as electric cell-substrate impedance (ECIS), field-effect transistors (FETs), and potentiometric-based biosensors, are used for the electrochemical-based sensing of extracellular changes, genetic alterations, and redox reactions. In addition to the electrochemical biosensors for live-cell detection, cancer and stem cells may be immobilized on an electrode surface and evaluated electrochemically. Various nanomaterials and cell-friendly ligands are used to enhance the sensitivity of electrochemical biosensors. Here, we discuss recent advances in the use of electrochemical sensors for determining cell viability and function, which are essential for the practical application of these sensors as tools for pharmaceutical analysis and toxicity testing. We believe that this review will motivate researchers to enhance their efforts devoted to accelerating the development of electrochemical biosensors for future applications in the pharmaceutical industry and stem cell therapeutics.

Electrically controlled mRNA delivery using a polypyrrole-graphene oxide hybrid film to promote osteogenic differentiation of human mesenchymal stem cells.

Direct messenger ribonucleic acid (mRNA) delivery to target cells or tissues has revolutionized the field of biotechnology. However, the applicability of regenerative medicine is limited by the technical difficulties of various mRNA-loaded nanocarriers. Herein, we report a new conductive hybrid film that could guide osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) via electrically controlled mRNA delivery. To find optimal electrical conductivity and mRNA-loading capacity, the polypyrrole-graphene oxide (PPy-GO) hybrid film was electropolymerized on indium tin oxide substrates. We found that the fluorescein sodium salt, a molecule partially mimicking the physical and chemical properties of mRNAs, can be effectively absorbed and released by electrical stimulation (ES). The hADMSCs cultivated on the PPy-GO hybrid film loaded with pre-osteogenic mRNAs showed the highest osteogenic differentiation under electrical stimulation. This platform can load various types of RNAs thus highly promising as a new nucleic acid delivery tool for the development of stem cell-based therapeutics. Electronic Supplementary Material: Supplementary material (electrochemical and FT-IR analysis on the film, additional SEM, AFM and C-AFM images of the film, optical and fluorescence images of cells, and the primers used for RT-qPCR analysis) is available in the online version of this article at 10.1007/s12274-022-4613-y.

Raman Spectroscopy-Based 3D Analysis of Odontogenic Differentiation of Human Dental Pulp Stem Cell Spheroids.

Numerous efforts have been made to establish three-dimensional (3D) cell cultures that mimic the structure, cell composition, and functions of actual tissues and organs in vitro; however, owing to its intrinsic complexity, precise characterization of 3D differentiation remains challenging and often results in high variations in the resulting differentiated spheroids. This study reports a 3D Raman mapping-based analytical method that helps us to identify the crucial factors responsible for inducing variability in differentiated stem cell spheroids. Human dental pulp stem cell spheroids were generated at various cell densities using the hanging drop (HD) and molded parafilm-based (MP) methods and were then further subjected to odontogenic differentiation. Thereafter, the 3D cellular differentiation in these spheroids was analyzed based on three different Raman peaks, namely, 960, 1156/1528, and 2935 cm-1, which correspond to hydroxyapatite (HA, odontogenic differentiation marker), ß-carotene (precursor of HA), and proteins/cellular components (cell reference). By correlating such cell differentiation-related peaks and water/medium peaks (3400 cm-1), we discovered that the diffusion of the medium containing various nutrients and differentiation factors was crucial in determining the variations in 3D differentiation of stem cell spheroids. Odontogenic differentiation was majorly induced at the outer shell of HD spheroids (up to ∼20 µm), whereas odontogenic differentiation was markedly induced in MP spheroids (up to 40-50 µm). Considering the challenges associated with high variations in spheroid and organoid differentiation, we conclude that the proposed Raman-based 3D analysis plays a pivotal role in stem cell-based regenerative therapy and drug screening.

Enhancing osteogenesis of adipose-derived mesenchymal stem cells using gold nanostructure/peptide-nanopatterned graphene oxide.

Graphene derivatives are highly promising materials for use in stem-cell-based regenerative therapies, particularly for bone regeneration. Herein, we report a graphene oxide (GO)-based hybrid platform (GOHP) that is highly effective for guiding the osteogenesis of human adipose-derived mesenchymal stem cells (hAMSCs). A GO-coated indium tin oxide (ITO) substrate was electrochemically modified with Au nanostructures (GNSs), following which a cysteine-modified quadruple-branched arginine-glycine-aspartic acid was self-assembled on the ITO-GO-GNS hybrid via Au-S bonds. The synthesized GOHP, with the highest density of GNSs (deposition time of 120 s), exhibited the highest osteogenic differentiation efficiency based on the osteogenic marker expression level, osteocalcin expression, and osteoblastic mineralisation. Remarkably, although GO is known to be less efficient than the high-quality pure graphene synthesised via chemical vapour deposition (CVD), the fabricated GOHP exhibited an efficiency similar to that of CVD-grown graphene in guiding the osteogenesis of hAMSCs. The total RNA sequencing results revealed that CVD graphene and GOHP induced the osteogenesis of hAMSCs by upregulating the transcription factors related to direct osteogenesis, Wnt activation, and extracellular matrix deposition. Considering that GO is easy to produce, cost-effective, and biocompatible, the developed GOHP is highly promising for treating various diseases/disorders, including osteoporosis, rickets, and osteogenesis imperfecta.

A Spheroid-Forming Hybrid Gold Nanostructure Platform That Electrochemically Detects Anticancer Effects of Curcumin in a Multicellular Brain Cancer Model.

In this study, a multifunctional platform that enables the highly efficient formation of 3D multicellular cancer spheroids and precise real-time assessments of the anticancer effects of curcumin in a brain tumor coculture model is reported. A highly conductive gold nanostructure (HCGN) is fabricated to facilitate cancer spheroid formation without using anti-cell adhesion molecules. A neuroblastoma (SH-SY5Y) and glioblastoma (U-87MG) coculture model is generated on HCGN with a specific cell-to-cell ratio (SH-SY5Y: U-87MG = 1:1), and their redox behaviors are successfully measured without destroying the distinct 3D structure of the multicellular spheroids. Using electrochemical signals as an indicator of spheroid viability, the effects of potential anticancer compounds on cocultured spheroids are further assessed. Remarkably, decreased cell viability in 3D spheroids caused by a low concentration of curcumin (30 µM) is detectable using the electrochemical method (29.4%) but not with a conventional colorimetric assay (CCK-8). The detection is repeated more than ten times for both short- (63 h) and long-term cultivation (144 h) without damaging the spheroids, enabling real-time, non-destructive pharmacokinetic analysis of various drug candidates. Therefore, it can be concluded that the hybrid platform is a highly promising, precise, and high-throughput drug screening tool based on 3D cell cultivation.

Recent developments of nano-structured materials as the catalysts for oxygen reduction reaction.

Developments of high efficient materials for electrocatalyst are significant topics of numerous researches since a few decades. Recent global interests related with energy conversion and storage lead to the expansion of efforts to find cost-effective catalysts that can substitute conventional catalytic materials. Especially, in the field of fuel cell, novel materials for oxygen reduction reaction (ORR) have been noticed to overcome disadvantages of conventional platinum-based catalysts. Various approaching methods have been attempted to achieve low cost and high electrochemical activity comparable with Pt-based catalysts, including reducing Pt consumption by the formation of hybrid materials, Pt-based alloys, and not-Pt metal or carbon based materials. To enhance catalytic performance and stability, numerous methods such as structural modifications and complex formations with other functional materials are proposed, and they are basically based on well-defined and well-ordered catalytic active sites by exquisite control at nanoscale. In this review, we highlight the development of nano-structured catalytic materials for ORR based on recent findings, and discuss about an outlook for the direction of future researches.

Preclinical Studies of Mesenchymal Stem Cell (MSC) Administration in Chronic Obstructive Pulmonary Disease (COPD): A Systematic Review and Meta-Analysis.

BACKGROUND: In the last two decades, mesenchymal stem cells (MSCs) have been pre-clinically utilized in the treatment of a variety of kinds of diseases including chronic obstructive pulmonary disease (COPD). The aim of the current study was to systematically review and conduct a meta-analysis on the published pre-clinical studies of MSC administration in the treatment of COPD in animal models. METHODS AND RESULTS: A systematic search of electronic databases was performed. Statistical analysis was performed using the Comprehensive Meta-Analysis software (Version 3). The pooled Hedges's g with 95% confidence intervals (95% CIs) was adopted to assess the effect size. Random effect model was used due to the heterogeneity between the studies. A total of 20 eligible studies were included in the current systematic review. The overall meta-analysis showed that MSC administration was significantly in favor of attenuating acute lung injury (Hedges's g = -2.325 ± 0.145 with 95% CI: -2.609 ~ -2.040, P < 0.001 for mean linear intercept, MLI; Hedges's g = -3.488 ± 0.504 with 95% CI: -4.476 ~ -2.501, P < 0.001 for TUNEL staining), stimulating lung tissue repair (Hedges's g = 3.249 ± 0.586 with 95% CI: 2.103~ 4.394, P < 0.001) and improving lung function (Hedges's g = 2.053 ± 0.408 with 95% CI: 1.253 ~ 2.854, P< 0.001). The mechanism of MSC therapy in COPD is through ameliorating airway inflammation (Hedges's g = -2.956 ± 0.371 with 95% CI: -3.683 ~ -2.229, P< 0.001) and stimulating cytokine synthesis that involves lung tissue repair (Hedges's g = 3.103 ± 0.734 with 95% CI: 1.664 ~ 4.541, P< 0.001). CONCLUSION: This systematic review and meta-analysis suggest a promising role for MSCs in COPD treatment. Although the COPD models may not truly mimic COPD patients, these pre-clinical studies demonstrate that MSC hold promise in the treatment of chronic lung diseases including COPD. The mechanisms of MSCs role in preclinical COPD treatment may be associated with attenuating airway inflammation as well as stimulating lung tissue repair.

Matrix metalloproteinase-9 activates TGF-ß and stimulates fibroblast contraction of collagen gels.

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-ß1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-ß1 and reduced several TGF-ß1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-ß1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-ß1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts.

Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation.

BACKGROUND: The balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. This study was designed to investigate the effect of budesonide on fibroblast-mediated tissue repair and remodeling. METHODS: Using human fetal lung fibroblasts in a three-dimensional collagen gel culture system, we investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of Inflammatory cytokines (interleukin-1ß and tumor necrosis factor α; 5 ng/mL each) and, in order to activate latent proteases, serine protease trypsin 0.25 µg/mL. The effects of budesonide on metalloproteinase production and activation were also investigated. RESULTS: Inflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, P < 0.01). Budesonide 100 nM almost completely inhibited the release and mRNA expression of metalloproteinase-1, metalloproteinase-3, and metalloproteinase-9 induced by the cytokines (P< 0.05). Exposure to the cytokines plus trypsin increased collagen degradation and conversion of the metalloproteinases to lower molecular weight forms corresponding to their active forms. Budesonide blocked both enhanced collagen degradation (P< 0.01) and suppressed trypsin-mediated conversion of cytokine-induced metalloproteinase-9 and metalloproteinase-3 to lower molecular weight forms. Similar effects were observed with dexamethasone 1 µM, suggesting a class effect. CONCLUSION: These findings demonstrate that budesonide directly modulates contraction of collagen gels and can decrease collagen degradation under Inflammatory conditions. The mechanism of this effect is through suppressing gene expression, release, and activation of metalloproteinases. By modulating the release and activity of metalloproteinases, inhaled budesonide may be able to modify airway tissue repair and remodeling.

Dermatomyositis, complicated with pneumomediastinum, successfully treated with cyclosporine A: a case report and review of literature.

We report a rare case of patient with dermatomyositis (DM) who developed spontaneous pneumomediastinum (PnM) and subcutaneous emphysema. She was successfully treated with oral prednisolone and cyclosporine A (CsA). We reviewed the cases of PnM in patients with DM treated with CsA. A review of four previously reported cases revealed that treatment with systemic glucocorticoid and CsA was effective for the DM and PnM. We indicate that initial and early treatment of the patients with DM and PnM with CsA enabled rapid tapering of the dose of glucocorticoid and improved the disease.

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract.

BACKGROUND: We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-kappaB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-kappaB in mediating cell survival in response to cigarette smoke exposure in HBECs. METHODS: Both the pharmacologic inhibitor of NF-kappaB, curcumin, and RNA interference targeting p65 were used to block NF-kappaB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay. RESULTS: Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-kappaB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium. CONCLUSION: The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

PKCdelta mediates thrombin-augmented fibroblast-mediated collagen gel contraction.

Fibroblast-mediated collagen gel contraction has been used as an in vitro model of tissue remodeling. Thrombin is one of the mediators present in the milieu of airway inflammation and may be involved in airway tissue remodeling. We have previously reported that thrombin stimulates fibroblast-mediated collagen gel contraction partially through the PAR1/PKCepsilon signaling pathway [Q. Fang, X. Liu, S. Abe, T. Kobayashi, X.Q. Wang, T. Kohyama, M. Hashimoto, T. Wyatt, S.I. Rennard, Thrombin induces collagen gel contraction partially through PAR1 activation and PKC-epsilon, Eur. Respir. J. 24 (2004) 918-924]. Here, we further report that the delta-isoform of PKC (PKCdelta) is also activated by thrombin and involved in the thrombin-mediated augmentation of collagen gel contraction. Thrombin (10nM) significantly increased PKCdelta activity (over 5-fold increase after 15-30min stimulation) and stimulated phosphorylation of PKCdelta. Rottlerin, a PKCdelta inhibitor, completely inhibited activation of PKCdelta and partially blocked collagen gel contraction stimulated by thrombin. Similarly, PKCdelta-specific siRNA significantly inhibited PKCdelta activation without affecting PKCepsilon expression and activation. Furthermore, suppression of PKCdelta by siRNA resulted in partial blockade of thrombin-augmented collagen gel contraction. These results suggest that thrombin contributes to the tissue remodeling in inflammatory airways and lung diseases at least partially through both PKCdelta and PKCepsilon signaling.

The CC chemokine ligand 2 (CCL2) mediates fibroblast survival through IL-6.

Apoptosis of lung structural cells is crucial in the process of normal tissue repair. Insufficient apoptosis of lung fibroblasts may contribute to the development of fibrosis. Since the CC chemokine ligand 2 (CCL2) is associated with fibrotic disease and the cytokine IL-6 blocks apoptosis in many cell types, we hypothesized that CCL2 may contribute to the development of lung fibrosis by inducing IL-6, which, in turn, inhibits fibroblast apoptosis. Fibroblasts were cultured in the presence of CCL2, which stimulated IL-6 production and mRNA expression in a concentration-dependent manner (250-1,000 ng/ml). This effect was mediated through the ERK1/2 signaling pathway. In addition, through a feedback loop, the secreted IL-6 activated the fibroblasts as evidenced by immunoblotting for phosphorylated STAT3. CCL2 reduced fibroblast apoptosis induced by staurosporin as detected by DNA content profiling (53.6 +/- 10.8%, P < 0.05) and apoptosis induced by serum starvation as detected by COMET assay (Tail moment: 36.6 +/- 9.9 of control versus 3.6 +/- 1.4 of CCL2, P < 0.01). In the presence of anti-IL-6 neutralizing antibody, however, this anti-apoptotic effect of CCL2 was eliminated. These data suggest that CCL2 mediates fibroblast survival by inhibiting apoptosis through IL-6/STAT3 signaling and provides a novel mechanism through which CCL2 may contribute to the development and maintenance of lung fibrosis.

Cigarette smoke extract induces DNA damage but not apoptosis in human bronchial epithelial cells.

Whether DNA damage caused by cigarette smoke leads to repair or apoptosis has not been fully elucidated. The current study demonstrates that cigarette smoke induces single-strand DNA damage in human bronchial epithelial cells. Cigarette smoke also stimulated caspase 3 precursors as well as intact poly (ADP-ribose) polymerase (PARP) production, but did not activate caspase 3 or cleave PARP, while the alkaloid camptothecin did so. Neither apoptosis nor necrosis was induced by cigarette smoke when the insult was removed within a designated time period. In contrast, DNA damage following cigarette smoke exposure was repaired as evidenced by decreasing terminal dUTP-biotin nick-end labeling positivity. The PARP inhibitor, 3-aminobenzamide blocked this repair. Furthermore, cells subjected to DNA damage were able to survive and proliferate clonogenically when changed to smoke-free conditions. These results suggest that cigarette smoke-induced DNA damage in bronchial epithelial cells is not necessarily lethal, and that PARP functions in the repair process. Our data also suggest that the potency of cigarettes as a carcinogen may result from their ability to induce DNA damage while failing to trigger the apoptotic progression permitting survival of cells harboring potentially oncogenic mutations.

Reversible cigarette smoke extract-induced DNA damage in human lung fibroblasts.

Cigarette smoke contains thousands of chemicals, many of which may contribute to cytotoxicity and carcinogenesis. Using assays detecting DNA strand breaks (terminal transferase dUTP nick end labeling [TUNEL]) and DNA content (flow cytometry), we evaluated the genotoxic effect of cigarette smoke extract (CSE) on human fetal lung fibroblasts (HFL-1) cultured in three-dimensional collagen gels as well as in monolayer culture. When HFL-1 cells were exposed to CSE, DNA strand breaks were detected in most, as determined by TUNEL. This effect was dependent on CSE concentration, duration of CSE exposure, and the density of HFL-1 cells cast into the collagen gels. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, significantly increased DNA damage induced by 1% CSE, and N-acetylcysteine, a glutathione precursor, blocked 5% CSE from inducing DNA damage. After CSE exposure, most cells were TUNEL-positive, but DNA quantification revealed no hypodiploid cells, indicating that apoptosis was not occurring during the CSE exposure. CSE-induced DNA damage was reversible, and cells proliferated when CSE was removed after 24 h exposure. These results demonstrate that cigarette smoke can induce DNA damage in HFL-1 cells cultured in both three-dimensional collagen gels and monolayer cultures, and that oxidants likely play a role in this damage. Moreover, this DNA damage is reversible, with cells surviving and TUNEL positivity reversing when CSE is removed within 24 h.

Cigarette smoke stimulates MMP-1 production by human lung fibroblasts through the ERK1/2 pathway.

An imbalance between proteases and anti-proteases is believed to play an important role in the pathogenesis of emphysema. In this study, we explored the hypothesis that cigarette smoke can alter tissue structure through an effect on the release of matrix metalloproteinase-1 (MMP-1) and type I tissue inhibitor of metalloproteinases (TIMP-1). Cigarette smoke extract (CSE) significantly stimulated pro-MMP-1 production (determined by ELISA and immunoblots) and mRNA expression (by real-time RT-PCR) by human fetal lung fibroblasts (HFL-1) in a concentration-dependent manner (2.5-10%). High concentrations of CSE (10%) could potentially activate the latent form of MMP-1 as the high molecular weight (52 kDa) form was converted into a low molecular weight (42 kDa) form consistent with active MMP-1. TIMP-1 production, however, was not significantly altered by the concentrations of CSE tested. After 30 min exposure, CSE significantly induced ERK1/2 phosphorylation, which then gradually decreased from 90 minutes to 3 hours. PD98059, a specific inhibitor of ERK-MAPK, significantly blocked the CSE effect on ERK1/2 phosphorylation. Furthermore, PD98059 significantly inhibited the CSE effect on MMP-1 production and mRNA expression by fibroblasts. These results suggest that cigarette smoke stimulates production and likely activates MMP-1 through activating ERK1/2 signal transduction pathway. By inducing MMP-1, cigarette smoke may result in excess tissue destruction and contribute to the development of emphysema.

Ultrafine carbon black particles inhibit human lung fibroblast-mediated collagen gel contraction.

Both acute and chronic exposure to particulates have been associated with increased mortality and morbidity from a number of causes, including chronic obstructive pulmonary disease and other chronic lung diseases. The current study evaluated the hypothesis that ultrafine carbon particles, a component of ambient particulates, could affect tissue repair. To assess this, the three-dimensional collagen gel contraction model was used. Ultrafine carbon black particles, but not fine carbon black, inhibited fibroblast-mediated collagen gel contraction. Although previous research has indicated that inflammatory effects of ultrafine carbon black particles are mediated by oxidant mechanisms, the current study suggests that ultrafine carbon black's inhibition of fibroblast gel contraction is mediated by the binding of both fibronectin and transforming growth factor (TGF)-beta to the ultrafine particles. Binding of TGF-beta was associated with a reduction in nuclear localization of Smads, indicative of inhibition of TGF-beta signal transduction. There was also a decrease in fibronectin mRNA, consistent with a decrease in TGF-beta-mediated response. Taken together, these results demonstrate the ability of ultrafine particles to contribute to altered tissue repair and extend the known mechanisms by which these biologically active particles exert their effects.