K. Zeka

Development of a new protocol: a macroscopic study of the tongue dorsal surface

The presence on the dorsal tongue of additional irregularities such as fissures, grooves and the distribution of papillae constitutes a retention area for harbouring bacteria, particularly those that produce sulphur compounds. A protocol to study the morphology of the tongue in a macroscopic way was developed, aimed at better investigating on the relationship between the dorsal surface of the tongue, considered as a microbial ecosystem, and the oral halitosis (1). A patient affected by oral malodour was chosen and included in the study. A picture of his lingual dorsum was taken to show the areas where the coating was visible. Then, an impression with alginate was taken obtaining a replication in plaster and, on the base of this one, a sort of impression tray was modelled utilizing an impression material made of silicone putty. After this, a second impression was taken combining the tray in silicone putty with a silicone material having a very low-light density (using the 2-step double-mix impression technique). The impression obtained was divided and cut with a blade in six parts, according to Winkel Tongue Coated Index (2), and their contour was observed with the stereo-microscope. The images were analysed with the Image J programme, and the parameter considered was the depth among the papillae. The mean measure of this parameter, in the parts where the coating was visible, resulted in a range between 0,25 + 0,019 mm and 0,55 + 0,11 mm. The same measures in the parts where the coating was not visible swung instead in a range between 0,14 + 0,08 mm and 0,23 + 0,07 mm. This new protocol can be considered clinically relevant for the suitable diagnosis and for the personalized treatment of halitosis.

In vitro biocompatibility of a new hydrogel with Crocin, powerful antioxidant found in Crocus Sativus L. flowers

Recently, attention has been paid to the identification of natural antioxidants from the petals of Crocus S. flowers that are normally considered waste[1]. The antioxidant activities are mainly attributed to carotenoid compounds, like crocin. Scientific evidences demonstrate that this kind of compounds are among the most important natural plant sources of antioxidant activity in the human diet, protecting the body against damages caused by reactive oxygen species (ROS). This antioxidant was extracted using methanol and ethanol. In this experiment, it was used a new hydrogel consisting of three polymers - Polyvinyl pyrrolidone (PVP), Agar and Polyethylene glycol (PEG) – and Crocin, mixed, reticulated and then sterilized by gamma irradiation at 25 kGy. For the in vitro experimental protocol, it was used a primary culture of fibroblasts taken from the subcutaneous tissue of a newborn mice, seeding the cells on a little square (1cm2 area) of both kinds of hydrogel. As the ISO protocol prescribes, the experiments were repeated 3 times for each kind of hydrogel, stopping the culture at the 3rd, 7th and 14th day after the seeding. For all steps, 3 Petri dishes were used as controls without biomaterials. At fixed deadline, all Petri dishes were stained, using the Wright method for cell counting and morphological evaluations. The microscopic analysis revealed the complete biocompatibility of the hydrogel. The Petri dishes with this kind of new hydrogel has an overproduction of collagen from the fibroblasts. The presence of this natural important compound in saffron petals, now offers new possibilities for the best use of the hole flower. This study is still in progress.

Kaempferol, a powerful antioxidant from Crocus sativus L. flowers: an in vitro study

Recently, it has been demonstrated the presence of Kaempferol in the petals of Crocus S. flowers that are normally discarded [1]. Kaempferol is an important antioxidant of the flavonoid family. Numerous reports have shown that kaempferol and/or its glycosides induce cell death in a variety of cancer cells from different tissues, also it can slow skin aging by contrasting enzymes that degrade the extracellular matrix. This antioxidant has been extracted from Crocus S. petals with different alcohols. Isolation and purification was done by flash column chromatography. Fractions were analysed by Thin Layer Chromatography (TLC). Kaempferol was further characterised by infrared (IR), mass spectroscopy (MS) and nuclear magnetic resonance (1H & 13C NMR) spectroscopy. To be sure of the antioxidant property of the extracted Kaempferol, a DPPH test was performed. In this experiment, it was used a new hydrogel consisting of three polymers - Polyvinyl pyrrolidone (PVP), Agar, Polyethylene glycol (PEG) – and Kaempferol, mixed, reticulated and then sterilized by gamma irradiation at 25 kGy. [2]. For the in vitro experimental protocol, it was used a primary culture of fibroblasts taken from the subcutaneous tissue of a newborn mice, seeding the cells on a little square (1cm2 area) of the hydrogel with Kaempferol. As the ISO protocol prescribes, the experiments were repeated 3 times, stopping the culture at the 3rd, 7th and 14th day after the seeding. For all steps, 3 Petri dishes were used as controls without the biomaterial. At fixed deadline, all Petri dishes were stained, using the Wright method for cell counting and morphological evaluations. The microscopic analysis revealed the complete biocompatibility of the hydrogel. The Petri dishes with this kind of new hydrogel showed a production of collagen fibres similar of the Petri dishes without the hydrogel. The presence of this bioactive compound in saffron petals, paves the way of new possibilities for the best use of the Crocus S. hole flower.

New Hydrogels Enriched with Antioxidants from Saffron Crocus Can Find Applications in Wound Treatment and/or Beautification

Saffron extracts have a long history of application as skin protectant, possibly due to their ability to scavenge free radicals. In this work, the performance of a hydrogel enriched with antioxidant compounds isolated from saffron crocus (Crocus sativus L.) petals was tested. These hydrogels could be considered as new drug delivery system. Hydrogels are crosslinked polymer networks that absorb large quantities of water but retain the properties of a solid, thus making ideal dressings for sensitive skin. We tested antioxidant-enriched hydrogels on primary mouse fibroblasts. Hydrogels enriched with kaempferol and crocin extracted from saffron petals showed good biocompatibility with in vitro cultured fibroblasts. These new types of hydrogels may find applications in wound treatment and/or beautification.

The synthesis of chalcones as anticancer prodrugs and their bioactivation in CYP1 expressing breast cancer cells

BACKGROUND Although the expression levels of many P450s differ between tumour and corresponding normal tissue, CYP1B1 is one of the few CYP subfamilies which is significantly and consistently overexpressed in tumours. CYP1B1 has been shown to be active within tumours and is capable of metabolising a structurally diverse range of anticancer drugs. Because of this, and its role in the activation of procarcinogens, CYP1B1 is seen as an important target for anticancer drug development. OBJECTIVE To synthesise a series of chalcone derivatives based on the chemopreventative agent DMU-135 and investigate their antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1. METHOD A series of chalcones were synthesised in yields of 43-94% using the Claisen-Schmidt condensation reaction. These were screened using a MTT assay against a panel of breast cancer cell lines which have been characterised for CYP1 expression. RESULT A number of derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing significantly lower toxicity towards a non-tumour breast cell line with no CYP expression. Experiments using the CYP1 inhibitors acacetin and α-naphthoflavone provided supporting evidence for the involvement of CYP1 enzymes in the bioactivation of these compounds. CONCLUSION Chalcones show promise as anticancer agents with evidence suggesting that CYP1 activation of these compounds may be involved.

Flavonoids and Their Metabolites: Prevention in Cardiovascular Diseases and Diabetes

The occurrence of atherosclerosis and diabetes is expanding rapidly worldwide. These two metabolic disorders often co-occur, and are part of what is often referred to as the metabolic syndrome. In order to determine future therapies, we propose that molecular mechanisms should be investigated. Once the aetiology of the metabolic syndrome is clear, a nutritional intervention should be assessed. Here we focus on the protective effects of some dietary flavonoids, and their metabolites. Further studies may also pave the way for development of novel drug candidates.

Detection of hidden vertical root fracture by stereomicroscope

The vertical root fractures resulting as from trauma, as from the “cracked tooth syndrome”, are spotted within the “iatrogenic periodontal lesions” according to the Al-Fouzan (1) classification. The stereo microscope, an optical microscope variant typically exploiting the light reflected from the surface of an object rather than that transmitted through it (2), in the reported case has revealed the best tool to perform the resolving three-dimensional examination of the tooth. Aim of this paper is to describe a vertical root fracture, not otherwise detectable. A second upper molar, has been endodontically treated, because of the presence of an endo-periodontal lesion. Even though the treatment was performed according the standard protocol, after two years the lesion persisted and the tooth was extracted. After the extraction, the tooth was diaphanized to be observed with the stereomicroscope (Leica LED2000). The investigation clearly showed a vertical fracture, starting directly from the cervical line and a small hole on the distal surface of the root palatal cone. Mean values of measurements taken in 15 randomly points at 1x, 4x and 8x magnification were made. The fracture resulted to be long 9,79 mm and to have a mean width of 0,37 + 0,07 mm externally, and 0,15 + 0,02 mm internally. The mean area of the hole resulted of 0,85 mm2. All the analytical procedure was validated by t-student test, showing a p value < 0,0001. This vertical root fracture represented the unexpected pathway between the pulp and periodontal tissues and sure it caused the endodontic treatment failure, with tooth loss. It appears clear that a good diagnosis, following exactly the proto- col stated in the literature, together with the patient’s compliance, are crucial basis to face such clinical challenge.

Hydrogels with natural or chemical compounds: in vitro biocompatibility

Biomaterials are very popular topics in advanced medical applications. Our group performed different types of hydrogels. Two of the most successful concern hydro- gels with natural or chemical compounds inside. The first step of experiments has been the PVP-Hydrogel production. This new hydrogel consists of three polymers – Polyvinyl pyrrolidone (PVP), Agar and Polyethylene glycol (PEG) – mixed, reticulated and together sterilized by gamma irradiation at 25 kGy. Simultaneously, a different kind of hydrogel embedding Crocus sativus L. petals compounds was prepared. This hydrogel consists of PVP, Agar, PEG and Kaempferol and/or Crocin. Same sterilization process as the PVP-hydrogel. The target clinical applications of this new biomaterial are the “difficult wounds” and cosmetic sector. These hydrogels were compared with Neoheal. For the in vitro experimental protocol, it was used a primary culture of fibroblasts, from the subcutaneous tissue of a newborn mice, seeding the cells on 1cm2 area of both kinds of hydrogel. As the ISO protocol prescribes, the experiments were repeated 3 times for each kind of hydrogel, stopping the culture at the 3rd, 7th and 14th day after the seeding. For all steps, three Petri dishes were used as controls without biomaterials. At each fixed deadline, all Petri dishes were colored using the Wright method for cell counting and other morphological evaluations. The microscopic analysis revealed the total biocompatibility of both hydrogels at each step of the experiment. The new PVP-Hydrogel was found to be more adsorbent, increasing its dimensions day by day and free floating in the medium. The hydrogel with the compounds extracted from Crocus sativus L. had the cells that growth faster. The Neoheal, indeed, revealed more sticky properties, adhering to the dish floor but neither swelling or increasing its volume. These results showed that both the new hydrogels are biocompatible and the different chemical properties caused a very different behavior in cells growth comparing with Neoheal. It is hoped that this study will stimulate further investigations in this field.

The Antral Artery Anastomosis: An In Vivo Investigation

The arterial vascular supply of maxillary sinus has to be considered in all the surgical procedures where it is involved. In particular, the intraosseous anastomosis between the posterior superior alveolar artery and the infraorbital artery branches in the bony canal can be tricky to a not well aware clinician (1). The aim of this study is to investigate the arterial blood supply of the maxillary sinus to give clinicians the basis for a better understanding of vascular complications that can derive from surgical procedures at this level. 50 Cone Beam Computed Tomography (CBCT) were analysed by the Imaging software for 3D images, i-Dixel 2.0. The parameters considered have been the presence (i), the calibre (ii), the dorso-ventral length (iii) and the cranio-caudal distance (iv). The data have been processed with Means, Standard Deviations and verified by T-Student test. The statistical outputs showed that less than half of samples presented the intraosseous anastomosis. Those anastomosis resulted both in dorso-ventral way (13,59 + 0,04 mm) and in cranio-caudal way (15,38 + 0,94 mm) long. The calibre measurements resulted quite big as well: 1,63 + 0,03 mm. The differences between the right and left sides were found not statistically significant. This in vivo investigation shows how a knowledge of the maxillary sinus vascularization is essential during the programming surgical phase in order to prevent blood complications during the operations involving this region.

In vitro biocompatibility of a new PVP-Hydrogel

These experiments show the in vitro biocompatibility of a new PVP-Hydrogel comparing it with Neoheal, an already experimented biomaterial completely biocompatible. The new hydrogel consists of three polymers - Polyvinyl pyrrolidone (PVP), Agar and Polyethylene glycol (PEG) - mixed, reticulated and at the same time sterilized by gamma irradiation at 25 kGy. The target clinical applications of this new biomaterial are the “difficult wounds”. For the in vitro experimental protocol, it was used a primary culture of fibroblasts taken from the subcutaneous tissue of a newborn mice, seeding the cells on a little square (1cm2 area) of both kinds of hydrogel. As the ISO protocol prescribes, the experiments were repeated 3 times for each kind of hydrogel, stopping the culture at the 3rd, 7th and 14th day after the seeding. For all steps, three Petri dishes were used as controls without biomaterials. At each fixed deadline, all Petri dishes were colored using the Wright method for cell counting and other morphological evaluations. The microscopic analysis revealed the complete biocompatibility of both hydrogels at every step of the experiment and the test produced very interesting results. The new PVP-Hydrogel was found to be more adsorbent, increasing its dimensions day by day and free floating in the medium. The Neoheal, indeed, revealed more sticky properties, adhering to the dish floor but neither swelling nor increasing its volume. These different chemical properties caused a very different behavior of the cells that could grow very well on and under the PVP-Hydrogel, but not on or under the Neoheal. These results showed that the new PVP-hydrogel is biocompatible with the cells just like the Neoheal, even if in a very different way. Other experiments have to complete the protocol to test the biodegradation and the biocompatibility in vivo of both biomaterials, when they enter in contact with the tissues.