Physico-chemical and biological characterization of synthetic and eggshell derived nanohydroxyapatite/carboxymethyl chitosan composites for pulp-dentin tissue engineering.

Hybrid nanohydroxyapatite/carboxymethyl chitosan (nHAp-CMC) scaffolds have garnered significant attention in the field of regenerative engineering. The current study comparatively analyzed the physicochemical and biological properties of synthetic nanohydroxyapatite (SnHA)- and eggshell-sourced nanohydroxyapatite (EnHA)- based CMC biocomposites for pulp-dentin regeneration. EnHA and CMC were synthesized through a chemical process, whereas SnHA was commercially obtained. Composite scaffolds of SnHA-CMC and EnHA-CMC (1:5 w/w) were prepared using freeze-drying method. All biomaterials were characterized by FTIR, micro-Raman, XRD, HRSEM-EDX, and TEM analyses, and their in vitro bioactivity was assessed by immersing them in simulated body fluid for 21 days. The biological properties of the composite scaffolds were evaluated by assessing cytocompatibility using MTT assay and biomineralization potential by analyzing the odontogenic gene expressions (ALP, DSPP, DMP-1 and VEGF) in human dental pulp stem cells (DPSCs) using RT-qPCR method. Characterization studies revealed that EnHA displayed higher crystallinity and superior surface morphology compared to SnHA. The composite scaffolds showed a highly interconnected porous microstructure with pore sizes ranging between 60 and 220 µm, ideal for cell seeding. All tested materials, SnHA, EnHA, and their respective composites, displayed high cytocompatibility, increased ALP activity and degree of mineralization with significant upregulation of odontogenic-related genes on DPSCs (p < 0.05). Nevertheless, the odontogenic differentiation potential of EnHA-CMC on DPSCs was significantly higher when compared to SnHA-CMC. The findings from this study highlight the potential of EnHA-CMC as a promising candidate for pulp-dentin engineering.

Assessment of dental age estimation using dentinal translucency in ground sections of single rooted teeth: a digital image analysis.

Human dentition is unique to individuals and helps in identification of individuals in forensic odontology. This study proposes to study the manually ground sections of single rooted teeth using digital methods for dental age estimation. To assess the dentinal translucency from the scanned digital images of manually ground section of teeth using commercially available image edition software. Corroborating the root dentinal translucency length and region of interest (ROI) of translucency zone in pixels (as a marker of dental age) with the chronological age of the subject, as stratified by different age groups. Twenty single-rooted extracted teeth from 20 patients each from 6 groups divided as per age. Manual sectioning of the teeth followed by scanning the sections was done. Root area in pixels and ROI of translucency zone were measured. From the observed values, translucency length percentage (TLP) and percentage of ROI in pixels (TPP) was calculated and tabulated. Pearson's correlation coefficients were obtained for age with TLP and TPP. Positive correlation existed between age and TLP and also between age and TPP. With the obtained data, multilinear regression equations for specific age groups based on 10-year intervals were derived. By a step-down analysis method, age was estimated with an average error of around ±7.9 years. This study gives a novel method for age-estimation that can be applied in real-time forensic sciences.

The use of aesthetic paediatric full coverage restorations among paediatric dental practitioners: an international survey.

Despite the recent growing evidence in support of prefabricated zirconia crown use as a successful option in managing grossly carious anterior and posterior primary teeth, the use of such crowns remains controversial within the paediatric dental profession. This study aims to assess the use of aesthetic full coverage paediatric restorations among paediatric dentists worldwide, with emphasis on aesthetic prefabricated zirconia crowns. This was an online cross-sectional survey questionnaire study comprising of 38 multiple-choice questions disseminated worldwide through the contact lists of national, regional and international paediatric dental organisations and social media platforms. The survey was completed by 556 respondents thus achieving power, with 391 (70.3%) females and 165 (29.7%) males. The respondents were from 55 different countries spread across six continents. The use of aesthetic full coverage restorations was reported by 80% (n = 444) of the respondents. For restoring anterior teeth, participants mainly used either composite strip crowns (94.4%, n = 419) or zirconia crowns (73.6%, n = 327), while those who used aesthetic crowns for restoring posterior teeth mainly used zirconia crowns (68.2%, n = 303). Within the limitations of this study, the results have shown a wide use of full coverage aesthetic restorations, including the use of zirconia crowns, of primary teeth within this international sample of practicing dentists.

Clinical Outcome of Nonsurgical Root Canal Treatment Using a Matched Single-Cone Obturation Technique with a Calcium Hydroxide-based Sealer: A Retrospective Analysis.

INTRODUCTION: The aim of this retrospective study was to estimate the outcome of nonsurgical root canal treatment using matched single-cones with a calcium hydroxide-based sealer and to identify the various prognostic factors influencing the outcome. METHODS: This was a retrospective study that enrolled 272 patients who underwent routine endodontic therapy and were obturated with matched single-cones and Sealapex as sealer. This study involved 223 patients with 261 teeth meeting the selection criteria and recalled between 24 and 84 months. The outcome was categorized as success (healed/healing clinically and radiographically) or failure (not healed clinically and/or radiographically). Two calibrated examiners assessed the treatment outcomes. A binomial logistic regression model was performed to identify the effect of various prognostic factors. The χ2 test was used to find the association between sealer extrusion and the initial periapical index scores. RESULTS: The overall success rate was found to be 89.7% for a mean recall period of 39.18 (±11.05) months. An increase in age was associated with increased odds of success, whereas the success rates were reduced by an increase in the number of roots and negative pulp sensibility status. Initial periapical index scores, presence of preoperative sinus tract, number of visits, and sealer extrusion did not affect the outcome significantly (P > .05). CONCLUSION: Within the limitations of this study, it was found that teeth obturated with matched single-cone and Sealapex achieved substantial success rates. Sealer extrusion did not have any significant effect on the treatment outcome.

Efficacy of Platelet Rich Plasma and Platelet Rich Fibrin for Direct Pulp Capping in Adult Patients with Carious Pulp Exposure- A Randomised Controlled Trial.

OBJECTIVE: This study evaluated the effectiveness of Platelet Rich Plasma (PRP), Platelet Rich Fibrin (PRF) and Mineral Trioxide Aggregate (MTA) as direct pulp capping materials in patients with carious pulp exposure using cone beam computed tomography. METHODS: This parallel, triple blinded, randomised trial was done on 30 permanent posterior teeth chosen as candidates for direct pulp capping based on stringent inclusion and exclusion criteria. The samples were allocated based on sequentially numbered opaque sealed envelope method into three groups: MTA, PRP and PRF for direct pulp capping. The treated teeth were restored with Glass Ionomer cement liner and light-cured resin composite. Patients were followed up for a period of 12 months. Statistical analyses were performed with statistical software SPSS version 25. Data were analysed for normality using Shapiro Wilk's test. Dentine bridge volume analysis across the groups were done using Kruskal Wallis test with the significance level at P=0.05. As the test revealed a significant difference, post hoc analysis was done with Dunn test. RESULTS: CBCT analysis revealed that the volume of dentine bridge formed by PRP and PRF was significantly higher than the volume of dentine bridge formed by MTA (P<0.001; CI: 90%). There was no significant difference in the efficacy between the three direct pulp capping agents based on clinical assessment. A total of 27 teeth (90%) showed positive response to pulp sensibility testing and evidence of dentine bridge formation in radiograph at 12 months. No adverse events were encountered.

Neuro-protective effects of nano-formulated hesperetin in a traumatic brain injury model of Danio rerio.

Understanding the mechanism behind neuronal regeneration is critical for treating ischemic stroke and traumatic brain injury. The presence of neural stem cells in and around the sub-ventricular zone of human and also in zebrafish is evidenced. In this current study, the neuro-protective potential of nano-formulated hesperetin on injury-induced neurogenesis in zebrafish was assessed. Nanoformulation of hesperetin was prepared by anti-solvent precipitation technique using sodium dodecyl sulfate (SDS) as the stabilizing agent. The synthesized particles were characterized using SEM, DLS, XRD and FT-IR. Anti-oxidant capacity of nano hesperetin (nHST) in in vitro followed by in vivo studies in a traumatic brain injury (TBI) model of adult zebrafish (Danio rerio), catalase activity, histological analysis and gene expression studies for the genes Sox2, Nestin, Fabp7a and HuC were carried out. The synthesized particles were found to be in nanoscale and SDS had successfully integrated with hesperetin. Moreover, nHST had a significantly higher anti-oxidant capacity in vitro. Catalase levels in nHST treated group were significantly restored compared to other groups. Histological studies supported reduced tissue damage on oral administration of nano-hesperetin as compared to other groups. Gene expression studies showed that nano-hesperetin at a concentration of 10 µM when administered orally induced proliferation of neural stem cells without inducing cell death.

Prenatal exposure to hexavalent chromium disrupts testicular steroidogenic pathway in peripubertal F1 rats.

We have reported sub-fertility in F1 progeny rats with gestational exposure to hexavalent chromium [Cr(VI)], which had disrupted Sertoli cell (SC) structure and function, and decreased testosterone (T). However, the underlying mechanism for reduced T remains to be understood. We tested the hypothesis "transient prenatal exposure to Cr(VI) affects testicular steroidogenesis by altering hormone receptors and steroidogenic enzyme proteins in Leydig cells (LCs)." Pregnant Wistar rats were given drinking water containing 50, 100, and 200 mg/L potassium dichromate during gestational days 9-14, encompassing fetal differentiation window of the testis from the bipotential gonad. F1 male rats were euthanized on postnatal day 60 (peripubertal rats with adult-type LCs alone). Results showed that prenatal exposure to Cr(VI): (i) increased accumulation of Cr(III) in the testis of F1 rats; (ii) increased serum levels of luteinizing and follicle stimulating hormones (LH and FSH), and 17ß estradiol, and decreased prolactin and T; (iii) decreased steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A1, 3ß- and 17ß-hydroxysteroid dehydrogenases, cytochrome P450 aromatase and 5α reductase proteins, (iv) decreased specific activities of 3ß and 17ß hydroxysteroid dehydrogenases; (v) decreased receptors of LH, androgen and estrogen in LCs; (vi) decreased 5α reductase and receptor proteins of FSH, androgen, and estrogen in SCs. The current study concludes that prenatal exposure to Cr(VI) disrupts testicular steroidogenesis in F1 progeny by repressing hormone receptors and key proteins of the steroidogenic pathway in LCs and SCs.

Determination on the binding of thiadiazole derivative to human serum albumin: a spectroscopy and computational approach.

4-[3-acetyl-5-(acetylamino)-2,3-dihydro-1,3,4-thiadiazole-2-yl]phenyl benzoate from the family of thiadiazole derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiadiazole derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance, r between the donor (HSA) and acceptor (thiadiazole derivative) was estimated according to Forster's theory of non-radiative energy transfer. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes of temperature-dependent Kb was calculated, which explains that the reaction is spontaneous and exothermic. The microenvironment of HSA have also been studied using synchronous fluorescence spectroscopy, and the feature of thiadiazole derivative-induced structural changes of HSA have been carried using Fourier transform infrared spectroscopy and the Molecular modelling simulations explore the hydrophobic and hydrogen bonding interactions.

Cissus quadrangularis prevented the ovariectomy induced oxidative stress in the femur of adult albino rats.

UNLABELLED: The increasing evidence suggesting the role of free radicals in bone resorption and bone loss prompted us to explore whether the consumption of antioxidant rich medicinal plant C. quadrangularis modifies antioxidant status in ovariectomized rats. METHODS: Twenty four female adult rats, 90days old showing regular estrous cycles were used for the present study. The animals were divided into two groups. The Group-1 rats (n=6) were sham operated and Group-II rats were bilaterally ovariectomized (n=18) and treated with C. quadrangularis for sixty days (100mg/kg body weight and 250mg/kg body weight). After sixty days, the rats were killed, femora were dissected out, minced and homogenized in Tris-HCl buffer (pH 7.4) and the supernatant was collected and used for biochemical assays. RESULTS: Ovariectomy registered a decrease (p<0.05) in the activities of SOD, GPx, GST, ALP, collagen content and increased (p<0.05) the activities of TRAP and lipid peroxidation. Simultaneous administration of C. quadrangularis maintained the enzyme activities in ovariectomized rats. CONCLUSION: C. quadrangularis, a natural herb may be used to treat the estrogen deficiency/menopause onset and ovariectomy induced oxidative stress.

Insights into the binding of thiosemicarbazone derivatives with human serum albumin: spectroscopy and molecular modelling studies.

4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl acetate [Ace semi],4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl propanoate [Pro semi] from the family of thiosemicarbazones derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and it is also less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiosemicarbazone derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance (r) between donor (HSA) and acceptor (thiosemicarbazone derivative) was estimated according to Forster's theory of non-radiative energy transfer using fluorescence spectroscopy. The binding dynamics has been elaborated using synchronous fluorescence spectroscopy, and the feature of thiosemicarbazone derivative induced structural changes of HSA has been studied by circular dichorism, Fourier transform infrared spectroscopy. Molecular modelling simulations explore the hydrophobic interaction and hydrogen bonding which stabilizes the interaction.

Microshear bond strength evaluation of surface pretreated zirconia ceramics bonded to dentin.

OBJECTIVES: To comparatively assess the micro shear bond strength (MSBS) of dentin bonded surface pre-treated zirconia ceramics. MATERIALS AND METHODS: Zirconia blocks were sectioned into 50 cubical blocks. The blocks were further categorized into five groups (n = 10 each). Group I: No treatment was performed on zirconia samples; Group II: The zirconia samples were sand-blasted; Group III: Group II + etched with 9.8% of hydrofluoric (HF) acid for 60 s; Group IV: The sandblasted zirconia samples were selectively infiltrated with low fusing porcelain; and Group V: Group IV + etched using 9.8% HF acid gel. The zirconia specimens were then bonded to dentin samples, and the samples were tested for MSBS evaluation using universal testing machine. RESULTS: The MSBS of all the four experimental groups shows greater value than group I. Among the experimental groups, group V and group IV do not show any statistical significant difference, whereas the mean MSBS of groups IV and V were statistically greater than group III and group II. However, groups I, II, and III do not show any statistical significant difference in mean MSBS values between them. CONCLUSION: Selective infiltration etching of zirconia ceramics provides the highest bond strength with resin cement.

Enhanced tribological, corrosion, and microstructural properties of an ultrathin (<2 nm) silicon nitride/carbon bilayer overcoat for high density magnetic storage.

An ultrathin bilayer overcoat of silicon nitride and carbon (SiNx/C) providing low friction, high wear resistance, and high corrosion resistance is proposed for future generation hard disk media. The 16 Å thick SiNx/C overcoat consists of an atomically thin SiNx underlayer (4 Å) and a carbon layer (12 Å), fabricated by reactive magnetron sputtering and filtered cathodic vacuum arc (FCVA), respectively. When compared with monolithic overcoats of FCVA-deposited carbon (16 Å) and sputtered SiNx (16 Å), the SiNx/C bilayer overcoat demonstrated the best tribological performance with a coefficient of friction < 0.2. Despite showing marginally less electrochemical corrosion protection than monolithic SiNx, its ability to protect the magnetic media from corrosion/oxidation was better than that of an ∼27 Å thick commercial hard disk overcoat and 16 Å thick monolithic FCVA-deposited carbon. From X-ray photoelectron spectroscopy and Raman spectroscopy analyses, it was found that the introduction of the 4 Å SiNx underlayer facilitated higher sp(3) hybridization within the carbon layer by acting as a barrier and promoted the formation of strong bonds at the SiNx/C and the SiNx/media interfaces by acting as an adhesion layer. The higher sp(3) carbon content is expected to improve the thermal stability of the overcoat, which is extremely important for future hard disk drives employing heat assisted magnetic recording (HAMR).

Probing the role of an atomically thin SiNx interlayer on the structure of ultrathin carbon films.

Filtered cathodic vacuum arc (FCVA) processed carbon films are being considered as a promising protective media overcoat material for future hard disk drives (HDDs). However, at ultrathin film levels, FCVA-deposited carbon films show a dramatic change in their structure in terms of loss of sp3 bonding, density, wear resistance etc., compared to their bulk counterpart. We report for the first time how an atomically thin (0.4 nm) silicon nitride (SiNx) interlayer helps in maintaining/improving the sp3 carbon bonding, enhancing interfacial strength/bonding, improving oxidation/corrosion resistance, and strengthening the tribological properties of FCVA-deposited carbon films, even at ultrathin levels (1.2 nm). We propose the role of the SiNx interlayer in preventing the catalytic activity of Co and Pt in media, leading to enhanced sp3C bonding (relative enhancement~40%). These findings are extremely important in view of the atomic level understanding of structural modification and the development of high density HDDs.

Age-related changes in serum levels of insulin-like growth factor-II and its binding proteins correlate with calcaneal bone mineral density among post-menopausal South-Indian women.

BACKGROUND: Insulin-like growth factor (IGF) system components are important regulators of bone metabolism, which have a predominant role in determining bone mineral density (BMD). While the serum levels of IGF-I are regulated by various systemic hormones and growth factors, IGF-II levels reflect the skeletal production relative to physical activity, mechanical loading, aging, race etc. Though various studies have been carried out among women of different ethnic groups to understand the relationship between serum levels of IGF-II and BMD, the results seem to be quite inconclusive. METHODS: We evaluated the same, recruiting South-Indian women who engage themselves in a wide variety of physical activities pertaining to their profession and life style. RESULTS: Serum levels of IGF-II and IGF binding protein (IGFBP)-3 showed positive correlation with calcaneal BMD, whereas IGFBP-4 showed negative correlation. These IGF system components exhibited similar correlations with serum bone formation markers and opposite trend with bone resorption marker. While both IGF-II and IGFBP-3 levels were observed to be decreased with aging and menopause, IGFBP-4 levels increased. CONCLUSIONS: The alterations in serum levels of IGF-II and its binding proteins due to aging and menopause could be some of the major contributors of decreased calcaneal BMD observed among elderly women.

Comparative evaluation of propolis and triantibiotic mixture as an intracanal medicament against Enterococcus faecalis.

INTRODUCTION: The aim of this study was to evaluate and compare the antimicrobial activity of calcium hydroxide, triantibiotic mixture (TAM), and an ethanol extract of propolis as intracanal medicaments on Enterococcus faecalis-infected root canals. METHODS: One hundred twenty extracted intact human permanent incisors were decoronated, and chemomechanical preparation of the root canal was performed. After sterilization of the samples, they were inoculated with pure culture of E. faecalis and incubated. After incubation, colony-forming units were recorded before medication. Then, samples were divided randomly into five groups (n = 24). Each group was then exposed to various intracanal medicaments, namely calcium hydroxide (group 1), TAM (group 2), propolis (group 3), ethanol (group 4), and saline as the control group (group V). The antibacterial effectiveness of the different intracanal medicament was recorded by determining the percentage reduction in colony counts (%RCC) at the end of days 1, 2, and 7. The data were statistically analyzed using one-way analysis of variance and Tukey Honestly Significant Difference (HSD) post hoc test. RESULTS: The %RCC was highest for propolis showing 100% reduction on day 2 followed by TAM showing 82.5%, 92.2%, and 98.4% of reduction on days 1, 2, and 3, respectively. Calcium hydroxide showed a gradual increase in antibacterial activity with a maximum of 59.4% on day 7. CONCLUSIONS: Propolis was more effective than TAM against E. faecalis at a 2-day time period, and both were equally effective at 7 days.

Effects of Cissus quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells.

Osteoporosis is a public health problem which is associated with significant morbidity and mortality. The repair of bone defect is still a big challenge for orthopedic surgeons. Traditional use of Cissus quadrangularis (C. quadrangularis) in the treatment of bone disorders has been documented. The present study was employed to delineate the effects of ethanolic extract of C. quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells. Lactate dehydrogenase assayed in the conditioned medium of control and C. quadrangularis treated cells did not differ significantly indicating that ethanolic extract of C. quadrangularis is nontoxic to osteoblastic cells. [(3)H] Thymidine incorporation assay revealed that C. quadrangularis treatment has increased the DNA synthesis of human osteoblastic SaOS-2 cells indicating increased proliferation of these cells. The data on alizarin red and ALP staining revealed increased matrix mineralization of human osteoblast like SaOS-2 cells. The study also revealed that the anabolic actions of ethanolic extract of C. quadrangularis in human osteoblast like cells are mediated through increased mRNA and protein expression of Runx2, a key transcription factor involved in the regulation of bone matrix proteins. Chromatin immunoprecipitation analysis revealed increased transcriptional activity of Runx2 on the promoter of osteocalcin after C. quadrangularis treatment. These results indicate positive regulation of C. quadrangularis on the proliferation, differentiation, and matrix mineralization of human osteoblast like SaOS-2 cells.

Biocomposites containing natural polymers and hydroxyapatite for bone tissue engineering.

Bone tissue engineering is an alternative strategy to generate bone utilizing a combination of biomaterials and cells. Biomaterials that mimic the structure and composition of bone tissues at nanoscale are important for the development of bone tissue engineering applications. Natural or biopolymer-based composites containing chitin, chitosan, or collagen have advantages such as biocompatibility, biodegradability that are essential for bone tissue engineering. The inclusion of nanoparticles of hydroxyapatite (one of the most widely used bioceramic materials) into the biopolymer matrix improves the mechanical properties and incorporates the nanotopographic features that mimic the nanostructure of bone. This review summarizes the recent work on the development of biocomposites containing natural polymers with hydroxyapatite particles suitable for use in bone defects/bone regeneration.

Polymeric composites containing carbon nanotubes for bone tissue engineering.

Several natural and synthetic polymers are now available for bone tissue engineering applications but they may lack mechanical integrity. In recent years, there are reports emphasizing the importance of carbon nanotubes (CNTs) in supporting bone growth. CNTs possess exceptional mechanical, thermal, and electrical properties, facilitating their use as reinforcements or additives in various materials to improve the properties of the materials. Biomaterials containing polymers often are placed adjacent to bone. The use of CNTs is anticipated in these biomaterials applied to bone mainly to improve their overall mechanical properties and expected to act as scaffolds to promote and guide bone tissue regeneration. This review paper provides a current state of knowledge available examining the use of the polymeric composites containing CNTs for promoting bone growth.

Dihydrotestosterone is a determinant of calcaneal bone mineral density in men.

Male osteoporosis is an increasingly important health problem worldwide. Though androgen deficiency leads to bone loss in men, information on the relative contribution of aromatizable and non-aromatizable androgens in maintaining bone mineral density (BMD) and the mechanisms involved are unclear. This cross-sectional study was designed to explore the same. Hundred osteoporotic men with age matched normal were studied for serum levels of sex steroids, PTH, IGF system components, cytokines and bone turnover markers. Our findings show that serum DHT, IGF-I, IGF-II and IGFBP-3 levels were significantly decreased while IL-1beta and bone turnover markers were significantly increased in osteoporotic men compared to normal. Pearson correlation analysis revealed that serum DHT, IGF-I, IGF-II and IGFBP-3 levels were positively and strongly correlated with BMD, while serum IL-1beta levels were negatively correlated with BMD. Serum PTH, testosterone, estradiol, IGFBP-4, TNF-alpha, IL-4 and IFN-gamma levels were similar between the two groups. We observed that DHT levels significantly declined with age. However, the significant difference in DHT between the osteoporotic and normal groups is the same regardless of age. A multiple regression model adjusted for age demonstrated that DHT/BMD association is fairly stronger among those with osteoporosis than the normal. Our findings for the first time point out that DHT is an important determinant of BMD in men. Most importantly, the strong positive correlation of serum DHT with BMD offers new perspectives in understanding the role of non-aromatizable androgen in regulating bone metabolism in men, and might serve as a potential clinical marker in the diagnosis of male osteoporosis.

Transforming growth factor-beta1 regulation of ATF-3 and identification of ATF-3 target genes in breast cancer cells.

Transforming growth factor-beta1 (TGF-beta1) is a crucial molecule for stimulation of breast cancer invasion and formation of bone metastases. The molecular mechanisms of how TGF-beta1 mediates these effects have yet to be completely determined. We have found that activating transcription factor-3 (ATF-3) is strongly stimulated and its level is sustained by TGF-beta1 in highly invasive and metastatic human breast cancer (MDA-MB231) and in mouse mammary pad tumor cells (r3T). ATF-3 is also overexpressed in human primary breast cancer tissue. Overexpression of ATF-3 increased normal human mammary epithelial cell number and DNA synthesis suggesting a role for ATF-3 in cell proliferation. The functional role of ATF-3 in breast cancer progression was determined by the RNA interference technique. Knockdown of ATF-3 by ATF-3 shRNA in MDA-MB231 cells decreased expression of cell cycle gene, cyclin A1 in MDA-MB231 cells. ATF-3 shRNA also decreased expression of an invasive and metastatic gene, matrix metalloproteinase-13 (MMP-13; collagenase-3) in these cells. Chromatin immunoprecipitation experiments identified the direct physical interaction of ATF-3 protein on the human MMP-13 promoter. Thus, the dysregulation of ATF-3 by TGF-beta1 is likely to activate cyclin A1 and MMP-13 genes in breast cancer cells and that would be key to the subsequent cancer cell invasion and metastasis.