“In vitro” osteogenic and angiogenic potential evaluation of a coculture of dental pulp stem and endothelial cells grown on the BisGMA/TEGDMA Chitlac coated thermosets

Securing an adequate blood supply for survival of cell transplants is critical for a successful outcome in tissue engineering. Moreover during regeneration of weaken teeth, which is susceptible to reinfection, fracture and loss, the teeth apical canal is open and a limited blood supply is allowed. Thus the interactions between endothelial and dental pulp progenitor stem cells are important for vascularization of regenerating tissue cells. In particular, the interplay of dental pulp stem cells and endothelial cells can enhance “in vitro” osteo/odontogenic and angiogenic potential (Dissanayaka J Endod 2012, 38,454-463) and “in vivo” ensure angiogenesis and pulp regeneration (Dissanayaka Tissue Engin Part A 2015, 3-4, 550-563). Since dental pulp microenvironment supports HUVEC survival and capillary network formation in the absence of scaffolding material and external angiogenic stimulation , “in vitro” osteogenic and angiogenic potential of dental pulp stemcells cocultured with endothelial cells grown on BisGMA/TEGDMA Chitlac coated thermosets was evaluated. Results: DPSCs were grown on BisGMA/TEGDMA Chitlac coated thermosets, a composite material used in dental restoration, in the presence of two different concentrations of endothelial cells (1:1 e 1:5) for 28 days and their metabolic activity and cytotoxic response were evaluated. MTT analysis discloses that cell metabolic activity significantly increases in the presence of endothelial cells, mainly at 21 days of culture, along with cytotoxic response, while at 28 days of culture a light cytotoxic response occurs. An increasing ALP activity is evidenced in the coculture systems up to 28 days, both in the presence and in absence of Chitlac thermosets and this evidence is further supported by Alizarin red staining, which does not detect mineralization in the early stages of differentiation, but is significantly increased at 28 days of culture in both the conditions (1:1 e 1:5). Even though the positive effect on DPSC differentiation, Chitlac thermosets could induce an inflammatory response in the system and thus an ELISA IL6 assay reveals an increased inflammatory response in 1:1 coculture system after 28 days of culture, furtherly increased in 1:5 coculture system. In parallel an increased PGE2 release is evidenced in 1:1 coculture system in the presence of thermosets, reduced in 1:5 coculture system, suggesting the potential occurrence of neoangiogenesis, furtherly supported by a tubular network formation when DPSC are grown on matrigel. These results evidencing that endothelial cells enhance “in vitro” osteo/odontogenic differentiation of DPSCs and angiogenesis, and that this response is revealed in the presence of Chitlac, usually used in dental restorative practice, indicate a coculture of DPSC and endothelial cells as a promising source for regenerative endodontics

pPKC ε mediated metalloproteinase 2 and 9 levels upon ibuprofen and lipoic acid coniugate (codrug 1) treatment in an Alzheimer disease rat brain model

Alzheimer’s Disease (AD) begins with loss of recent memory and is associated to pathological and histological hallmarks such as β amyloid plaques, neural tangles (NFT), cholinergic deficit, extensive neuronal loss and synaptic changes in the cerebral cortex and hippocampus. The amyloid cascade hypothesis implies the activity of β, γ secretases which mediate the cleavage of Amyloid Precursor Protein (APP), the formation of amyloidogenic Aβ fragment (1-42), which compacts into amyloid plaques, while the cleavage by ? secretase of APP, within the Aβ segment forms sAPP and prevents the formation of Aβ (Zetterberg et al. 2010 Exp Gerontol 45, 23-29). Among the proteases which have Aβ-degrading activity, Metalloproteinase (MMP) 2, disclosing β secretase-like activity, is included, while MMP9 seems to contribute to neuronal death (Yan et al. 2006 J Biol Chem 281, 24566-74). In addition, intracellular signalling protein Protein Kinase C (PKC) can control ? secretase, preventing the formation of β amyloid, and prolonging the life span of AD (de Barry et al. 2010 Exp Gerontol 45, 64-69). A lipophilic molecular combination (codrug 1), obtained by joining an antioxidant molecule, lipoic acid, with an anti-inflammatory compound, ibuprofen (IBU) has been synthetized in our lab and administered in a chronic treatment during intracerebroventricular infusion of Aβ (1-40) peptide in rat brains as a model of AD (Sozio et al. 2010 Arch Pharm 343,133-42). Here we show the effects exerted by codrug 1 on PKC ε-mediated MMP2 and MMP9 levels regulation in Aβ (1-40) infused rat cerebral cortex. Interestingly codrug 1, lowering Metalloproteinases expression via PKC ε down-modulation, seems to control Alzheimer’s disease induced cerebral amyloid deposits, neuronal death and, lastly, behavioural deterioration. Moreover the cognitive test evidences that codrug 1 treatment decreases the number of reference memory errors and the time spent to perform the test suggesting this compound as an useful therapeutical tool against AD

Tumor necrosis factor-alpha - mediated 2-hydroxyethyl methacrylate cytotoxic and inflammatory effect on human gingival fibroblasts

2-Hydroxyethyl methacrylate (HEMA), deriving from polymerized dental resinous biomaterials, can diffuse throughout the dentin organic matrix, preventing collagen collapse, but also at gingival and tooth pulp level [1]. HEMA could induce toxic effects, such as tissue inflammation, also at relatively low concentrations. Our study aimed to investigate the cytotoxic and inflammatory effect exerted on human gingival fibroblasts (HGFs) by a low HEMA concentration evaluating cell viability by Trypan blue dye exclusion test, early apoptosis and reactive oxygen species (ROS) production by flow cytometry and gene expression of specific proteins involved in the inflammatory process, such as tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2), by real-time reverse transcription polymerase chain reaction (real-time RT PCR). Cultured HGFs, obtained from fragments of gingival tissue, were exposed to 3 mM HEMA in Dulbecco’s modified Eagle’s medium for 0, 24 or 96 hours. In our experimental model, both 24- and 96-hour HEMA treatment decreased cell viability of about 20%. In parallel Annexin-V/PI assay, which detects apoptosis, indicated a 18% of Annexin-V positive cells after 24- and 96-hour HEMA incubation. After 24-hour HEMA treatment we observed an increase of ROS persisting up to 96 hours. Interestingly, 24-hour HEMA treatment increased TNF-α gene expression of about 80% and COX-2 mRNA levels of about 70% compared to control. After 96-hour HEMA incubation, TNF-α gene expression was about sixfold and COX-2 mRNA levels were about fivefold compared to control. Increase of TNF-α and COX-2 gene expression was hence HEMA exposure time-dependent. Since TNF-α - induced inflammation has been shown to be mediated by the activation of COX-2 transcription in HGFs [2], we can hypothesize that, in our experimental model, 24- or 96-hour HEMA treatment in HGFs induces a ROS-mediated cytotoxicity and an inflammatory process modulated by increase of TNF-α gene expression, which could rapidly produce the observed up-regulation of COX-2 transcription. Thus, the knowledge of molecular mechanisms underlying cellular response to dental resinous biomaterials, identifying threshold over which these compounds become toxic, could allow to set up protocols for a more effective clinical practice and for a better performance of tested materials. [1] Schweikl H, Spagnuolo G, Schmalz G. J Dent Res 2006; 85: 870-7. [2] Nakao S, Ogata Y, Shimizu E, Yamazaki M, Furuyama S, Sugiya H. Mol Cell Biochem 2002; 238: 11-8

Effects of methacrilyc thermosets coated with Silver-polysaccharide nanocomposite on HGFs adhesion in a S. mitis co-culture system

Silver based medical products have been proven to be effective in retarding and preventing bacterial growth, being silver reported to control infections since ancient times (1). In the field of dentistry, the use of silver ions/nanoparticles has been explored to counteract bacteria in resins and implants, as silver can destroy bacterial cell walls by reacting with the thiol groups (–SH) of proteins exposed to the extracellular portion of the bacterial membrane. Conversely, eukaryotic cells lack these exterior binding sites, so nanoparticles are supposed to interact with them only upon metal internalization (2). To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized silver nanoparticles with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a co-culture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rised up to 20%. Moreover, the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKC α translocated into nuclei. These data confirm that Chitlac-nAg thermosets have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.This work was supported by grants from MIUR FIRB 2010 and MIUR PRIN-2009

Effect of Chitlac-nAg on Streptococcus mitis internalization into human gingival fibroblasts

The surfaces of the oral cavity are always exposed to a broad variety of microor- ganisms able to form biofilms (Filoche et al, 2010) characterized by microbial com- munities that are organized as a network of cell-to-cell interactions. Streptococci are the predominant bacterial population of the oral environment and S.mitis in particular is the first colonizer of the oral biofilm (Di Giulio et al, 2013). Silver-based medical products have been proven to be effective in retarding and preventing bacterial growth. In order to prevent silver nanoparticles aggregation, a lactose-modified chitosan has been set up and resulted effective in stabilizing colloidal solution of nanoparticles (Chitlac-nAg) (Travan et al, 2009). Since many bacteria are able to internalize into eukaryotic cells, in our study we have investigated both the intracellular signaling governing S. mitis internalization into HGFs and the biological effect of ChitlacnAg on eukaryotic and prokaryotic cells in a co-culture model system. The internalization of S. mitis into HGFs is due to F-actin cytoskeleton reorganization and reduced expression within the cell. Immunofluorescence shows actin polymerization at invasion sites along with vinculin increased expression and spot organization. Vinculin is an adaptor protein that regulates the adhesion of integrin receptors to actin cytoskeleton. In presence of S. mitis an increment of integrin β1 and FAK expression, responsible for the entrance of the microorganism in HGFs is consistent, as revealed by electron microscopy analysis. This adhesion and uptake proteins profile is the same in the presence of saliva as well as bacteria uptake. When Chitlac-nAg is administred to cell culture the expression of all four proteins decreases and Ag nano- particles are recognized within the cells. Further, in presence of Ag nanoparticles the low amount of FAK is almost localized at nuclear level. In presence of Ag and S.mitis, the expression of all four proteins is increased, with respect to control, and F-actin cytoskeleton rearranged, while a raised number of bacteria is shown. This effect is mit- igated by the presence of saliva in cell culture, which probably prevents bacteria entry into the cell. These results let us hypothesize that Chitlac-nAg, developing its bacteri- cidal action could represent a good component of tooth paste and mouthwash

Escape from cell death through authophagy in Human Gingival Fibroblast/Streptococcus mitis co-culture treated with Chitlac n-Ag

Since ancient times, silver has been extensively used to control infections. Silver based medical products have been proved to be effective in retarding and preventing bacterial infections (Chen et al., 2007). In order to prevent silver nanoparticles aggregation a lactose-modified chitosan was shown to be effective in stabilizing colloidal solutions of silver nanoparticles: “Chitlac-nAg” (Travan et al., 2009). Silver ions and nanoparticle are capable to destroy the bacterial cell wall by reacting with sulfhydryl groups on membrane proteins (Kruszewski et al., 2003). Since the cells are capable of internalizing nanoparticles there is the risk of a massive uptake by eukaryotic cells, which eventually leads to their death through oxidative DNA damage (Li et al. 2013) In the present work we investigated the effects of Chitlac-nAg on primary human gingival fibroblast (HGFs) co-cultured with Streptococcus mitis in the presence of saliva. HGFs were obtained from fragments of healty marginal gingival tissue, co-cultured with the clinical strain of S. mitis and treated for 24-48h with Chitlac or Chitlac-nAg. Cytotoxicity evaluated by LDH assay showed an increment in LDH release in co-culture in the presence of Chitlac n-Ag and saliva. Oxidative stress detected by means of Reactive Oxygen Species formation highlighted an early ROS presence in samples with Chitlac-nAg and saliva, but this value was similar to control after 48h; apoptotic and necrotic cells were detected by means of Annexin V/PI showing an increase in cell death in HGFs treated with Ag and saliva after 24h, and returned to basal levels after 48h; the uptake of nanoparticles by cells was determined by optical and electronic microscopy revealing the Ag uptake in vesicles. The presence of lysosomes and autophagosomes was verified by Lysotracker and by LC3 respectively. In vitro results showed that in our co-culture model, which mimics the microenvironment of the oral cavity, chitlac n-Ag does not exert cytotoxic effect towards HGFs that are able to execute a homeostasis mechanism through autophagy promoting cell survival