Bisphosfonate matrix metalloproteinase inhibitors for the treatment of periodontitis: An in vitro study

Periodontitis is an inflammatory disease caused by anaerobic bacteria, including Porphyromonas gingivalis. Lipopolysaccharide (LPS)-stimulated persistent inflammation is responsible for an increase in matrix metalloproteinase (MMP) expression, resulting in periodontal tissue destruction. The aim of the present study was to investigate synthesized bisphosphonic MMP inhibitors, in an in vitro model consisting of human gingival fibroblasts exposed to LPS, and to compare the biological responses to those induced by zoledronate (ZA), a commercial bisphosphonate. MTT and lactate dehydrogenase (LDH) assays were used to measure cell viability and cytotoxicity, respectively. ELISA was performed to evaluate prostaglandin E2 (PGE2), interleukin (IL)6 and collagen secretion, while western blotting was used to analyze MMP expression. No effect on viability and low cytotoxicity were observed following treatment with bisphosphonate compounds. In the present study, treatment with compound 1 did not increase the release of PGE2and IL6. Increased levels of collagen I secretion were reported when compound 3 and ZA were administered. An increase of MMP8 was observed following ZA treatment, while a decrease of MMP9 and MMP14 following treatment with compounds 1, 2 and ZA were reported. The performance of compound 1 was optimal in terms of cell viability. Compound 1 also did not induce inflammation, and had the ability to counteract LPS-induced increases in MMP expression. These data suggested that compound 1 was the most suitable treatment to progress to an in vivo animal study, with the aim to confirm its use for the treatment of periodontitis

“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

Ibuprofen and Lipoic Acid Diamide as Co-Drug with Neuroprotective Activity: Pharmacological Properties and Effects in β-Amyloid (1–40) Infused Alzheimer's Disease Rat Model

Both oxidative stress and inflammation are elevated in brains of Alzheimer's disease patients, but their pathogenic significance still remains unclear. Current evidence support the hypothesis that non-steroidal anti-inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimer's disease, and ibuprofen has the strongest epidemiological support. In the present work our attention was focused on (R)-α-lipoic acid considered as a potential neuroprotective agent in Alzheimer's disease therapy. In particular, we investigated a new co-drug (1) obtained by joining (R)-α-lipoic acid and ibuprofen via a diamide bond, for evaluating its potential to antagonize the deleterious structural and cognitive effects of β-amyloid (1–40) in an infused Alzheimer's disease rat model. Our results indicated that infusion of β-amyloid (1–40) impairs memory performance through a progressive cognitive deterioration; however, ibuprofen and co-drug 1 seemed to protect against behavioural detriment induced by simultaneous administration of β-amyloid (1–40) protein. The obtained data were supported by the histochemical findings of the present study: β-amyloid protein was less expressed in 1-treated than in ibuprofen and (R)-α-lipoic acid alone-treated cerebral cortex. Taken together, the present findings suggest that co-drug 1 treatment may protect against the cognitive dysfunction induced by intracerebroventricular infusion of β-amyloid (1–40) in rats. Thus, co-drug 1 could prove useful as a tool for controlling Alzheimer's disease-induced cerebral amyloid deposits and behavioural deterioration

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

Dual Acting Carbon Monoxide Releasing Molecules and Carbonic Anhydrase Inhibitors Differentially Modulate Inflammation in Human Tenocytes

Sustained oxidative stress and inflammation have been reported as the major factors responsible for the failure of tendon healing during rotator cuff tears (RCTs) and rotator cuff disease (RCD). Although, their therapeutic management remains still challenging. Carbonic anhydrases (CAs) are involved in many pathological conditions, and the overexpression of both CA9 and 12 in inflamed joints has been recently reported. Consequently, a selective CA9/12 inhibition could be a feasible strategy for improving tendon recovery after injury. In addition, since carbon monoxide (CO) has been proven to have an important role in modulating inflammation, CO releasing molecules (CORMs) can be also potentially suitable compounds. The present study aims at evaluating five newly synthesized dual-mode acting CA inhibitors (CAIs)-CORMs compounds, belonging to two chemical scaffolds, on tendon-derived human primary cells under H2O2 stimulation in comparison with Meloxicam. Our results show that compounds 2 and 7 are the most promising of the series in counteracting oxidative stress-induced cytotoxicity and display a better profile in terms of enhanced viability, decreased LDH release, and augmented tenocyte proliferation compared to Meloxicam. Moreover, compound 7, as a potent superoxide scavenger, exerts its action inhibiting NF-kB translocation and downregulating iNOS, whereas compound 2 is more effective in increasing collagen I deposition. Taken together, our data highlight a potential role of CA in RCTs and RCD and the prospective effectiveness of compounds acting as CAI-CORM during inflammation

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

In vitro comparison of new bisphosphonic acids and zoledronate effects on human gingival fibroblasts viability, inflammation and matrix turnover

Bisphosphonates (BPs) are well known clinically used drugs, commonly applied to treat osteoclast-mediated bone resorption. Some clinically used BPs were demonstrated to be able to inhibit the activity of matrix metalloproteinases (MMPs) (1), a protease family required to fully degrade all the components of the extracellular matrix during connective tissue remodelling (2). Combining the arylsulfonamide function with the bisphosphonic group, several compounds were synthesized to obtain selective inhibitors of MMPs. The aim of the present work is to compare the effects on cell adhesion, cytotoxicity, inflammatory response occurrence and matrix turnover process in an in vitro model of primary human gingival fibroblasts (HGFs) treated with newly synthesized sulfonamide BPs and with zoledronic acid (ZA), a clinically used drug. Western blot was used to measure Procollagen I, β1 integrin MMP-8 and MMP-9, phase contrast and MTT for cell viability, LDH was performed for toxicity evaluation, ELISA for Prostaglandin E2 (PGE2) secretion assessment. When compared with ZA, the treatment with the newly synthetized compounds shows increasing viability, Procollagen I expression and decreased expression of β1 integrin in HGFs. Higher levels of released LDH, PGE2 and MMP-9 expression are recorded in ZA-treated HGFs. Increased levels of MMP-8 are recorded in newly synthetized compounds-treated samples.These findings imply that new BPs could accelerate the physiological matrix turnover, they are more able to preserve the soft tissue surrounding bone as they have neither inflammatory effects nor toxicity, along with reduced effects on the cell viability, which are instead typical side effects of ZA administration. We can conclude that the newly synthesized compounds are better tolerated, leading to the hypothesis that their use leads to connective tissues side effects reduction compared to clinically used drugs, even though several studies are required to deeply investigate the signaling cascades involved in the mechanism of action of these new BPs