An in vivo comparison of wound healing characteristics of two commercial acellular dermal matrices.

OBJECTIVES: Many acellular dermal matrices (ADMs) are available for use in periodontal surgical procedures. However, few studies exist evaluating their in vivo healing properties. The objectives of this study were to compare the wound healing and remodeling of two ADMs used for gingival augmentation procedures in the rat model. MATERIALS AND METHODS: This was a nonrandomized controlled split-mouth study. Envelope flaps were surgically created in the maxillary quadrants of 24 Sprague Dawley rats. Each received either (a) AlloDerm Regenerative Tissue Matrix, or (b) OrACELL. Gingival tissue from one mandibular quadrant served as the untreated control. Six male and six female rats were treated for 7 or 21 days. Biopsies were processed for histologic analysis (H&E, Picro-sirius red, Verhoeff's solution) or RNA analysis (RT-PCR) to analyze the expression of type I collagen (Col1a1), fibronectin (Fn-1) and VEGF-A (Vegf-A). RESULTS: There was a greater density of fibroblasts in OrACELL compared to AlloDerm at both timepoints. There was a greater density of elastin present in AlloDerm compared to OrACELL at 7 days but no differences at 21 days. There were no differences between test groups in the percentage of birefringent collagen or in the expression of Vegf-A or Fn-1. At 7 days, there were significantly more fibroblasts for males in the OrACELL group compared to females. At 21 days, there was a significantly greater expression of Col1a1 for males in the OrACELL group compared to females. CONCLUSIONS: Early wound healing and remodeling of OrACELL appeared to occur more rapidly than AlloDerm and was accelerated in male rats. Whether these results have clinical implications for soft tissue grafting procedures in humans remains to be determined.

SM1997 downregulates mustard-induced enzymes that disrupt corneal epithelial attachment.

Amino-Plex (SM1997) is a spray or liquid cosmeceutical that has been used for skin dryness, aging, or sun exposure. Its formulation includes electrolytes, trace minerals, amino acids, peptides, nucleosides and nucleotides, all substances that are <10 kDa. It is designed to increase oxygen levels in cells, improve glucose transport, stimulate ATP synthesis, and stimulate collagen formation, actions that can help facilitate repair of damaged cells. It also supports collagen synthesis and formation of healthy granulation tissue, accelerating reepithelization of damaged skin. Here, SM1997 has been tested as an agent to improve the healing of mustard injury to the cornea. The results indicate that SM1997 facilitates the retention of corneal epithelial attachment when applied to corneal organ cultures after nitrogen mustard exposure. In addition, it reduces the activation of enzymes that lead to epithelial-stromal separation, namely, ADAM17 and MMP-9. Therefore, SM1997 should be further investigated as a potential therapy sulfur mustard and nitrogen mustard exposure.

Comparing dentinal tubule penetration of conventional and 'HiFlow' bioceramic sealers with resin-based sealer: An in vitro study.

This study compared the depth and percentage of dentinal tubule penetration for single-cone (SC) and warm vertical (WV) obturation techniques with two different bioceramic sealers (BC Sealer [BCS], BC Sealer HiFlow [BCSHF]) and an epoxy resin-based sealer (2Seal easymiX). Fifty canals were filled with BCS, BCSHF or resin-based sealer (RBS). Teeth in BCS and BCSHF groups were filled with SC or WV techniques, and teeth in the control group (RBS) filled with WV technique only. The roots were sectioned at 3 mm and 6 mm levels from the apex and evaluated with a confocal laser microscope. There was significantly greater depth and percentage of sealer penetration at the 6 mm section compared to 3 mm (P < 0.05). No statistically significant difference was found in sealer type or obturation technique at the examined levels (P > 0.05). In conclusion, dentinal tubule penetration was similar comparing BC Sealer, BC Sealer HiFlow and RBS using SC and WV techniques.

Extracellular matrix: The proteins that function throughout the body.

The idea and meetings that planned this issue focused on extracellular matrix (ECM) started over 4 years ago. The invitations were sent to investigators over 2 years ago and manuscripts have been submitted, reviewed, and edited since the summer and fall of 2018. Most of the manuscripts were published in early view in 2019, and we are thrilled to share the final collection. This volume contains 6 reviews, 13 original research papers, and 4 remembrances. Marion (Emmy) Gordon and I organized the articles into seven topic areas, including ECM structure, genetics, and development; cancer; vascular structures and development; inflammation and wound healing; collagen in special structures; cornea and other ocular tissues; and extracellular vesicles. Anat Rec, 2020. © 2020 American Association for Anatomy.

Expression of Collagen Types I, II, IX, and X in the Mineralizing Turkey Gastrocnemius Tendon.

The turkey gastrocnemius tendon mineralizes by intramembranous ossification with a transient chondrogenic phase. The mineralizing zone has hypertrophic chondrocytes similar to endochondral bone formation. These similarities prompted the evaluation of this tendon for the presence of type X collagen in the mineralizing zone. Tendons were removed, radiographed, decalcified, and embedded for frozen sections. Seral sections were H&E stained and immunostained individually with antibodies specific collagens (types I, II, IX, and X). Type I collagen was distributed widely throughout the mineralized tendon extracellular matrix. Types II and IX collagen were at the mineralized/non-mineralized junction. Type X collagen was in the pericellular matrix of hypertrophic chondrocytes and in some calcified matrix. These data support the theory that the gastrocnemius tendon has fibrocartilage characteristics and that type X collagen has a role in the tissue's mineralization. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Extracellular Matrix in Secondary Palate Development.

The secondary palate arises from outgrowths of epithelia-covered embryonic mesenchyme that grow from the maxillary prominence, remodel to meet over the tongue, and fuse at the midline. These events require the coordination of cell proliferation, migration, and gene expression, all of which take place in the context of the extracellular matrix (ECM). Palatal cells generate their ECM, and then stiffen, degrade, or otherwise modify its properties to achieve the required cell movement and organization during palatogenesis. The ECM, in turn, acts on the cells through their matrix receptors to change their gene expression and thus their phenotype. The number of ECM-related gene mutations that cause cleft palate in mice and humans is a testament to the crucial role the matrix plays in palate development and a reminder that understanding that role is vital to our progress in treating palate deformities. This article will review the known ECM constituents at each stage of palatogenesis, the mechanisms of tissue reorganization and cell migration through the palatal ECM, the reciprocal relationship between the ECM and gene expression, and human syndromes with cleft palate that arise from mutations of ECM proteins and their regulators. Anat Rec, 2019. © 2019 American Association for Anatomy.

Cell polarization: From epithelial cells to odontoblasts.

Cell polarity identifies the asymmetry of a cell. Various types of cells, including odontoblasts and epithelial cells, polarize to fulfil their destined functions. Odontoblast polarization is a prerequisite and fundamental step for tooth development and tubular dentin formation. Current knowledge of odontoblast polarization, however, is very limited, which greatly impedes the development of novel approaches for regenerative endodontics. Compared to odontoblasts, epithelial cell polarization has been extensively studied over the last several decades. The knowledge obtained from epithelia polarization has been found applicable to other cell types, which is particularly useful considering the remarkable similarities of the morphological and compositional features between polarized odontoblasts and epithelia. In this review, we first discuss the characteristics, the key regulatory factors, and the process of epithelial polarity. Next, we compare the known facts of odontoblast polarization with epithelial cells. Lastly, we clarify knowledge gaps in odontoblast polarization and propose the directions for future research to fill the gaps, leading to the advancement of regenerative endodontics.

Histology of NeoMTA Plus and Quick-Set2 in Contact with Pulp and Periradicular Tissues in a Canine Model.

INTRODUCTION: NeoMTA Plus (Avalon Biomed Inc, Bradenton, FL) is a tricalcium silicate material similar to the first mineral trioxide aggregate product, ProRoot MTA (Dentsply Sirona, York, PA), but with improvements such as decreased setting time, increased ion release, increased water sorption, and nonstaining radiopacifiers. Quick-Set2 (Avalon Biomed Inc) is a newly formulated calcium aluminosilicate material that has a faster setting time and increased acid resistance and is nonstaining. The purpose of this study was to compare the healing of pulpal and periapical tissues in dogs after exposure to NeoMTA Plus and Quick-Set2 after pulpotomy and root-end surgery procedures. METHODS: Seventy-two teeth (36 for each procedure) in 6 beagle dogs received pulpotomy or root-end surgery using either NeoMTA Plus or Quick-Set2. The dogs were sacrificed at 90 days, and the teeth and surrounding tissues were prepared for histologic evaluation. Sixty teeth were evaluated and scored histologically (29 with pulpotomies and 31 with root-end resections). Specimens were scored for inflammation, quality and thickness of dentin bridging, pulp tissue response, cementum and periodontal ligament formation, and apical bone healing. RESULTS: Both materials displayed favorable healing at 90 days. The only significant difference was the quality of dentin bridge formation in pulpotomies using NeoMTA Plus compared with Quick-Set2. CONCLUSIONS: Quick-Set2 and NeoMTA Plus had similar effects on inflammation, pulp response, periodontal ligament and cementum formation, and apical tissue healing in dogs. NeoMTA Plus had superior dentin bridge quality compared with Quick-Set2.

The molecules in the corneal basement membrane zone affected by mustard exposure suggest potential therapies.

Mustard exposures result in epithelial-stromal separations in the cornea and epidermal-dermal separations in the skin. Large blisters often manifest in skin, while the cornea develops microblisters, and, when enough form, the epithelium sloughs. If the exposure is severe, healing can be imperfect and can result in long-term adverse consequences. For the cornea, this could manifest as recurrent corneal erosions. Since the corneal epithelial-stromal separations are in the region identified by electron microscopy as the lamina lucida, the same region affected by the blistering disease junctional epidermolysis bullosa (JEB), we postulated that the molecules that are defective in JEB would be the same ones cleaved by mustard compounds. These molecules are α6ß4 integrin and collagen XVII, which can be cleaved by matrix metalloproteinase-9 (MMP-9) and ADAM17, respectively. Therefore, our laboratory has tested MMP-9 and ADAM17 inhibitors as potential therapies to attenuate corneal mustard injury. Our results demonstrated that inhibiting MMP-9 and ADAM17 resulted in less epithelial-stromal separation in the corneas at 24 h postexposure, as compared with using only medium as a therapy.

ADAM17 Inhibitors Attenuate Corneal Epithelial Detachment Induced by Mustard Exposure.

PURPOSE: Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS: Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 µL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS: Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS: Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.

The biological function of type I receptors of bone morphogenetic protein in bone.

Bone morphogenetic proteins (BMPs) have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors (BMPRI and BMPRII). In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 (ALK2, also called type IA activin receptor), activin-like kinase 3 (ALK3, also called BMPRIA), and activin-like kinase 6 (ALK6, also called BMPRIB). The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future.

Activation of Receptor Activator of Nuclear Factor-κB Ligand and Matrix Metalloproteinase Production in Periodontal Fibroblasts by Endothelin Signaling.

BACKGROUND: Periodontitis is a group of inflammatory diseases affecting the tissues supporting the teeth that will progressively cause the loss of alveolar bone and periodontal ligaments and eventually the dentition. Activation of osteoclast activity by receptor activator of nuclear factor-κB ligand (RANKL) and released enzymes such as matrix metalloproteinases (MMPs) are among the factors involved in the breakdown of the periodontium. However, the mechanisms regulating their production in periodontitis are poorly understood. Endothelin signaling via the activation of the endothelin-A receptor (EDNRA) by endothelin-1 may play a role in the disease because the expression of the receptor and ligand is elevated in the periodontal tissues of patients with periodontitis. METHODS: Cultured primary human periodontal fibroblasts were treated with 20 and 100 nM endothelin-1 for 6 and 24 hours and then collected to assess MMP and RANKL production by immunoblotting. Inhibitors were used to identify the molecular pathways activated by EDNRA in these cells. RESULTS: Endothelin-1 stimulated the production of MMP1, MMP8, and RANKL in a dose- and time-dependent manner; blocking EDNRA function with the antagonist TBC3214 inhibited the response, although EDNRA activation had no effects on osteoprotegerin production. These mechanistic studies indicate that EDNRA activates phospholipase C, which then 1) increases the MMP1 protein levels through activation of the extracellular signal-regulated kinase mitogen-activated protein kinase-dependent pathway and 2) upregulates RANKL by a different pathway. CONCLUSION: These results suggest that EDNRA may function in the breakdown of the periodontal tissues associated with periodontitis by promoting the protein expression of MMPs and RANKL via the phospholipase C pathway.

Method of Studying Palatal Fusion using Static Organ Culture.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate (1). Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Ephrin reverse signaling mediates palatal fusion and epithelial-to-mesenchymal transition independently of Tgfß3.

The mammalian secondary palate forms from shelves of epithelia-covered mesenchyme that meet at midline and fuse. The midline epithelial seam (MES) is thought to degrade by apoptosis, epithelial-to-mesenchymal transition (EMT), or both. Failure to degrade the MES blocks fusion and causes cleft palate. It was previously thought that transforming growth factor ß3 (Tgfß3) is required to initiate fusion. Members of the Eph tyrosine kinase receptor family and their membrane-bound ephrin ligands are expressed on the MES. We demonstrated that treatment of mouse palates with recombinant EphB2/Fc to activate ephrin reverse signaling (where the ephrin acts as a receptor and transduces signals from its cytodomain) was sufficient to cause mouse palatal fusion when Tgfß3 signaling was blocked by an antibody against Tgfß3 or by an inhibitor of the TgfßrI serine/threonine receptor kinase. Cultured palatal epithelial cells traded their expression of epithelial cell markers for that of mesenchymal cells and became motile after treatment with EphB2/Fc. They concurrently increased their expression of the EMT-associated transcription factors Snail, Sip1, and Twist1. EphB2/Fc did not cause apoptosis in these cells. These data reveal that ephrin reverse signaling directs palatal fusion in mammals through a mechanism that involves EMT but not apoptosis and activates a gene expression program not previously associated with ephrin reverse signaling.

Anti-inflammatory protein tumor necrosis factor-α-stimulated protein 6 (TSG-6) promotes early gingival wound healing: an in vivo study.

BACKGROUND: Human multipotent mesenchymal stromal cells (hMSCs) produce tumor necrosis factor (TNF)-α-stimulated protein 6 (TSG-6). TSG-6 modulates proinflammatory cytokine cascades and enhances tissue repair. This study tests the effects of recombinant human TSG-6 (rhTSG-6) on gingival wound healing within the first 2 days post-surgery. METHODS: After gingival resection in 120 Sprague-Dawley rats, 2 µg rhTSG-6 in 5-µL phosphate-buffered saline (PBS) or the same volume of only PBS solution was injected into gingival tissue approximating the surgical wound. Control animals did not receive injections. Tissue biopsies and blood were collected at 1 to 2, 6 to 8, 24, and 48 hours post-surgery (n = 10 per group). Specimens were analyzed via histologic analysis and enzyme-linked immunosorbent assay (ELISA) for quantification and comparison of inflammatory markers interleukin (IL)-1ß, IL-6, TNF-α, and myeloperoxidase (MPO). Wound photographs were taken for a double-masked clinical assessment at each time period. Weights were recorded for all animals pre- and post-surgery. RESULTS: Animals injected with rhTSG-6 had significantly less severe clinical inflammation at 6 to 8 (P = 0.01228), 24 (P = 0.01675), and 48 (P = 0.0186) hours. Sham and control animals had more weight loss at 24 and 48 hours. Sham and control animals had more pronounced cellular infiltrate. rhTSG-6-treated animals had significantly less MPO (P = 0.027) at 24 hours and IL-1ß (P = 0.027) at 24 and 48 hours. IL-6 showed a marginal significant difference at 6 to 8 hours, but there was no significant difference for TNF-α. CONCLUSION: rhTSG-6 reduced postoperative gingival inflammation by reducing levels of proinflammatory cytokines and cellular infiltrate and may offer significant promise as an anti-inflammatory agent for gingival surgery.

Cell-matrix and cell-cell interactions of human gingival fibroblasts on three-dimensional nanofibrous gelatin scaffolds.

An in-depth understanding of the interactions between cells and three-dimensional (3D) matrices (scaffolds) is pivotal to the development of novel biomaterials for tissue regeneration. However, it remains a challenge to find suitable biomimetic substrates and tools to observe cell-material and cell-cell interactions on 3D matrices. In the present study, we developed biomimetic nanofibrous 3D gelatin scaffolds (3D-NF-GS) and utilized confocal microscopy combined with a quantitative analysis approach to explore cell-matrix and cell-cell interactions on the 3D-NF-GS. Human gingival fibroblasts (HGFs) migrated throughout the 3D-NF-GS by 5 days and formed stable focal adhesions by 14 days. The focal adhesions were detected using integrin-ß1, phospho-paxillin and vinculin expression, which were quantified from specific wavelength photon data generated using a spectral separation confocal microscope. As the cells became more confluent after 14 days of culture, cell-cell communication via gap junctions increased significantly. Collagen I matrix production by HGFs on 3D-NF-GS was visualized and quantified using a novel approach incorporating TRITC label in the scaffolds. Based on confocal microscopy, this study has developed qualitative and quantitative methods to study cell-matrix and cell-cell interactions on biomimetic 3D matrices, which provides valuable insights for the development of appropriate scaffolds for tissue regeneration.

Comparison of Vibringe, EndoActivator, and needle irrigation on sealer penetration in extracted human teeth.

INTRODUCTION: Vibringe is a new device that allows continuous sonic irrigation of the canal system during endodontic treatment. The aim of this study was to compare the effect of different irrigation systems on sealer penetration into dentinal tubules of extracted single-rooted teeth. METHODS: Fifty single-rooted human teeth were instrumented and randomly divided into 4 groups: group 1 (control), saline; group 2 (conventional irrigation), 17% EDTA followed by 6% NaOCl; group 3 (EndoActivator), same irrigants as group 2; group 4 (Vibringe), same irrigants as group 2. Obturation of all teeth was done with gutta-percha and SimpliSeal labeled with fluorescent dye. Transverse sections at 1 mm and 5 mm from the root apex were examined by using confocal laser scanning microscopy. Percentage and maximum depth of sealer penetration were measured by using NIS-Elements Br 3.0 imaging software. RESULTS: Groups 3 and 4 had a significantly greater percentage of the canal wall penetrated by sealer at the 5-mm level than group 1 (P < .0125), but not group 2. No other differences were found between the groups at either section level for both the percentage of sealer penetration and maximum depth. The 5-mm sections in each experimental group had a significantly higher percentage and maximum depth of sealer penetration than did the 1-mm sections (P < .0125). CONCLUSIONS: The use of sonic activation with either the EndoActivator or Vibringe did not significantly improve the sealer penetration when compared with conventional irrigation.

Analysis of Snail1 function and regulation by Twist1 in palatal fusion.

Palatal fusion is a tightly controlled process which comprises multiple cellular events, including cell movement and differentiation. Midline epithelial seam (MES) degradation is essential to palatal fusion. In this study, we analyzed the function of Snail1 during the degradation of the MES. We also analyzed the mechanism regulating the expression of the Snail1 gene in palatal shelves. Palatal explants treated with Snail1 siRNA did not degrade the MES and E-cadherin was not repressed leading to failure of palatal fusion. Transforming growth factor beta 3 (Tgfß3) regulated Snail1 mRNA, as Snail1 expression decreased in response to Tgfß3 neutralizing antibody and a PI-3 kinase (PI3K) inhibitor. Twist1, in collaboration with E2A factors, regulated the expression of Snail1. Twist1/E47 dimers bond to the Snail1 promoter to activate expression. Without E47, Twist1 repressed Snail1 expression. These results support the hypothesis that Tgfß3 may signal through Twist1 and then Snail1 to downregulate E-cadherin expression during palatal fusion.

Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model.

The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing from 24h to 168h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury.

Antioxidant combinations protect oral fibroblasts against metal-induced toxicity.

OBJECTIVE: In dentistry, the use of metals in fillings, braces, implants, bridges and other prosthodontic restorations is a common practice. Previous studies revealed that zinc (Zn) and copper (Cu) released from gold alloys, and nickel (Ni) released from nickel-chromium alloys, have a highly cytotoxic effect on fibroblast cell cultures. Our working hypothesis is that oral fibroblasts are susceptible to damage from metals because they elevate reaction oxygen species (ROS). In this study, we investigated specific antioxidant (AO) combinations to determine if they counteract the effects of Cu, Ni and Zn on cultured oral fibroblast proliferation and oxidative damage. METHODS: Oral fibroblasts were pretreated with Cu, Ni and Zn for 60min. Thereafter, cells were treated with 10(-5)M combinations of bioactive AO resveratrol (R), ferulic acid (F), phloretin (P) and tetrahydrocurcuminoids (T) (RFT, PFR, PFT) for 24h. Cell viability and DNA synthesis were monitored by 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS) and 5-bromo-2-deoxyuridine (BrDU) assays. ROS was measured using the fluorescence response of dichlorodihydrofluorescein diacetate (DCF). RESULTS: AO compounds increased recovery of cells exposed to Cu and Zn. Moreover, AO treatment induced DNA synthesis in the presence of the metal stressors. Cu and Ni stimulated production of ROS. PFR treatment decreased ROS in the presence of Cu, Ni and Zn. SIGNIFICANCE: These data indicate that pure AOs counteracted the detrimental effects of Cu, Ni, Zn on oral fibroblasts in vitro by increasing cell viability, and DNA synthesis and decreasing ROS activity.