Three dimensional oral mucosa models: development and applications

Animal experimentation has been extensively and for a long time applied in several research fields, but since 2011 it has been substantially limited by the Commission of the European Parliament to ensure people/animals safety and reduce research costs. To respond to these directives, many attempts have been focused on the development and validation of new in vitro 3D systems, bypassing the traditional 2D cell cultures. In this regard, diverse approaches to tissue-engineered bone and oral mucosa have been developed. Despite the promising premises and the cutting-edge results, the used 3D in vitro bone-oral mucosal models still lack interaction between the mucosal and the bone components. Therefore, this project aimed to create 3D models, entirely made with primary human cells (keratinocytes, fibroblasts, and osteoblasts), able to mimic the natural structure and interaction of bone and oral mucosa. In the present work, the regulatory role of the mesenchymal tissue onto epithelia was evaluated. The main results showed that that during the differentiation hMSC produce and secrete factors that induce the keratinization and the expression of the marker of differentiation CK10; in particular in the middle stage of differentiation (OB14). The proteomic analysis revealed that this effect can be ascribable to KGF secretion. This finding may impact the design of new implantable devices able to induce, alone, the epithelial growth and keratinization to improve implant graft avoiding epithelial graft linked to the morbidity of another zone. Moreover, we also showed that OM might have a pro-innervation effect, at least during the last stages of keratinocytes stratification. Finally, we obtained and characterized an innervated mucoperiosteal model that could open new in vitro frontiers for oral biomaterials validation as well as improve knowledge regarding the mesenchymal stem cells roles onto oral mucosa development

Functional health literacy of asylum seekers and refugees. A pilot study in italy

Literature shows how some groups of populations, among which are people seeking international protection and refugees, find it difficult to access services in national health systems. Usually, asylum seekers have limited Health Literacy (HL), which makes understanding the appropriate health information difficult. The objective of this research is to consider the relationship between how people requesting international protection and refugees approach the Italian Health System to request health services and their level of Functional Health Literacy (FHL). These relationships are examined through mixed methods. Data were obtained using several tools: a self-administered questionnaire in which the subjects revealed social and demographic data and a face-to-face interview together with the S-FHL Scale fulfilment in order to identify the functional level of HL. Twenty-one subjects were interviewed in two Centers of Protection System for Asylum Seekers and Refugees (SPRAR). Results show a picture of the actual situation. Data report a problematic or insufficient FHL level. Some factors, such as gender, age and health perceptions, play a role in the FHL levels. Some racial prejudices were reported. Language barriers had the most impact on the communication gap. Nevertheless, none of the subjects were denied health services. In conclusion, although this study is a pilot, we have experienced difficulty in obtaining asylum seekers’ trust to be open about their experience. This explains the number of the sample that should be more indicated for a qualitative study. Our results are in accordance with literature for inadequate level of FHL and lack of knowledge of the Italian Health System. This study highlighted several other issues to be taken into consideration for future research on the subject

High-risk HPVs, microbiota and epithelial carcinogenesis: state of the art and research contribution of in vitro 3D models

Persistent high-risk human papillomavirus (HR-HPV) infection is associated with anogenital and head & neck squamous epithelial (HNSCC) tumors, which altogether cause about 550,000 new cases every year. Several evidences suggest that the microbiota could have a role on the inflammatory, epithelial mesenchymal transition and tumorigenesis processes promoted by HR-HPV infection, yet the mechanisms involved remain to be clarified. In this review we report the state of the art on this topic and on the most promising in vitro developed models for studying the host-pathogen interactions. Using MEDLINE, several terms were searched and combined to select the most pertinent papers. The investigation was limited to the international indexed articles published in PubMed in the last 10 years. This review reports the latest knowledge in the field of the microbial-associated anogenital tumors and HNSCC. In addition, we also discuss the in vitro epithelial culture systems that reproduce the pathophysiological features of the tumoral microenvironment and the in vivo response to microbial agents, thus representing a useful tool for analyzing at cellular and molecular levels the role played by infective agents in tumorigenesis

EVALUATION OF THE INTERACTION AMONG HPV16 E6 AND E7 ONCOPROTEINS AND THE DNA DAMAGE SENSOR 53BP1 IN 2D AND 3D EPITHELIAL CULTURES BY THE PROXIMITY LIGATION ASSAY

INTRODUCTION. Human papillomaviruses (HPV) group several viruses able to infect squamous stratified epithelia and cause benign papillomas, warts and anogenital lesions. They also correlate to oropharyngeal and anogenital malignancies, mainly promoted by the high risk (HR) \u3b1-HPV16 E6E7 oncoproteins. Despite scientific progresses and the development of vaccines, these tumors are still common and represent one of the major causes of women\u2019s death. Host\u2019s cell replication fidelity depends by the DNA Damage Response (DDR). Unlike from other DNA viruses, HR-HPVs encourage cells proliferation without inactivating the DDR: the mechanisms at the basis haven\u2019t been clarified yet. During HPV16 infection, E6 binds and degrades p53 through the E6AP LXXLL domain. Similarly, E7 competes with E2F1-pRb interaction, thus inactivating pRb, and promoting the linking the pRb-like proteins CBP/p300 and p107, that also harbor a LXXLL sequence. Unfortunately, E6 E7 role in the DDR activation is not elucidated yet. EXPERIMENTAL MODEL. To gain new information, we reproduced an in vitro 3D HPV16-E6E7 infected epithelium, already characterized for HPVs studies, and checked for cellular and viral markers, among them HPV16E6E7 oncoproteins and the double strand breaks (DSB) sensor 53BP1; we then made a Co-IF for E6 and E7 with 53BP1. Since E6 and E7 both interact with LXXLL containing proteins, we analyzed 53BP1 BRCT2 domain and we explored the binding hypothesis via the in situ PLA technique in 2D in CaSki and E6E7HPV16 keratinocytes and in the 3D model. RESULTS. The in vitro infected epithelium resembles the in vivo tissue. E6E7HPV16, both in basal and differentiated strata, induce a 53BP1 increase in nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the binding in all the employed cellular models. CONCLUSION. Our results add new information on HPV16 oncoproteins capability in overcome cellular defense strategies

In Vitro Reconstructed Human Epithelial Models for Microbial Infection Research: Why Do We Need them?

In the last 50 years, the Replacement, Reduction and Refinement principles have become a framework for conducting high quality academic, pre-clinical, clinical and industrial research experimentation studies, in order to respond to the European Union legislative demand of alternatives to animal-based experimentation, often difficult to translate to human applications, expensive and not ethically approved. Thanks to the improvement of cellular isolation protocols, culture and co-culture conditions, together with the increased clinical demand, several novel in vitro three-dimensional tissue engineered human epithelial models, able to create sophisticate pre-clinical tests and produce results more reliable than the traditional bi-dimensional flat cell culture systems, have been developing also for microbial infection research purposes

Bioactive glasses functionalized with polyphenols: in vitro interactions with healthy and cancerous osteoblast cells

Bioactive glasses are widely studied as biomaterials for bone contact applications. In this research work, the opportunity to modify the surface of a bioactive glass with polyphenols (gallic acid, and natural polyphenols extracted from red grape skin and green tea leaves) has been investigated in order to induce a selective anti-tumor activity in vitro. The presence of surface grafted molecules has been optically proved by fluorescence microscopy exploiting their autofluorescence. Direct and indirect cytotoxicity assays have been performed with human bone osteosarcoma cells (U2OS) and human fetal pre-osteoblasts (hFOB), as well as the quantification of oxygen and nitrogen reactive species (RONS) engendered from cells in response to the materials. Finally, the DNA damage of U2OS cells upon contact with the bioactive glass has been evaluated in order to verify any selective cytotoxic activity of functionalized materials against cancer cells. Results showed a selective cytotoxic activity of functionalized bioactive glasses toward osteosarcoma cells that was particularly evident when cells were cultivated directly onto glasses surface. Moreover, the presence of grafted polyphenols increased the RONS production and induced a permanent DNA damage on the U2SOS cells while they promote a certain anti-inflammatory action toward hFOB. These preliminary results suggest polyphenols grafted bioactive glasses as promising material for bone substitution in cancer treatment

Exploring sexual dimorphism of the modern human talus through geometric morphometric methods

Sex determination is a pivotal step in forensic and bioarchaeological fields. Generally, scholars focus on metric or qualitative morphological features, but in the last few years several contributions have applied geometric-morphometric (GM) techniques to overcome limitations of traditional approaches. In this study, we explore sexual dimorphism in modern human tali from three early 20th century populations (Sassari and Bologna, Italy; New York, USA) at intra- and interspecific population levels using geometric morphometric (GM) methods. Statistical analyses were performed using shape, form, and size variables. Our results do not show significant differences in shape between males and females, either considering the pooled sample or the individual populations. Differences in talar morphology due to sexual dimorphism are mainly related to allometry, i.e. size-related changes of morphological traits. Discriminant function analysis using form space Principal Components and centroid size correctly classify between 87.7% and 97.2% of the individuals. The result is similar using the pooled sample or the individual population, except for a diminished outcome for the New York group (from 73.9% to 78.2%). Finally, a talus from the Bologna sample (not included in the previous analysis) with known sex was selected to run a virtual resection, followed by two digital reconstructions based on the mean shape of both the pooled sample and the Bologna sample, respectively. The reconstructed talus was correctly classified with a Ppost between 99.9% and 100%, demonstrating that GM is a valuable tool to cope with fragmentary tali, which is a common occurrence in forensic and bioarchaeological contexts