Dottorato in TECNOLOGIE INNOVATIVE NELLE MALATTIE DELLO SCHELETRO,
DELLA CUTE E DEL DISTRETTO ORO-CRANIO-FACCIALE

Titolo della tesi: Positional differences in the gene expression profile of human alveolar mucosa, buccal attached gingiva and palatal tissue-derived fibroblasts in the early phases of wound healing and the role of chlorhexidine digluconate and hyaluronic acid as modifiers of oral gingival-derived cells behaviour. A clinical, histological, immunohistochemical and biomolecular analysis

ABSTRACT Wound healing is a complex process orchestrated by a variety of known and unknown factors. Oral wound healing presents peculiar characteristics as accelerated wound closure and reduced scar formation when compared with cutaneous wound repair, and it has been recognized the key role of fibroblasts in this concern. In fact, oral-derived fibroblasts represent a resource of great interest for regenerative approaches due to their self-renewal capacity and plasticity. The biomolecular basis of the differences between oral and skin repair have been described by several studies, showing the main differences in transcriptional changes between the first 12-24 hours after injury. Nevertheless, variations in wound healing also exists inside the oral environment. The evidence we have nowadays is that wounding in the alveolar mucosa leads to scarring, while in the gingival tissue does not, but the literature lacks clear evidence concerning comprehensive in vivo biomolecular data on the differences between alveolar mucosa, buccal attached gingiva and palatal tissue-derived fibroblasts behaviour. The course of wound repair process can be modified by different factors. It is well know how the presence of biofilm jeopardizes the healing process. To counteract this during the post-surgical period, chlorhexidine digluconate (CHX) mouthrinse is commonly indicated. Although several in vitro studies have reported cytotoxicity effects on fibroblasts, , in vitro assays cannot fully represent the oral environment as a whole and this could be a limitation. Nevertheless, there are no in vivo data on the effect of post-surgical CHX use in oral cells behaviour in the early phases of wound healing. Improving wound healing through the use of bioactive substances that can influence cells behaviour, supporting tissue repair/regeneration, is of major clinical interest. Hyaluronic acid (HA) is the major endogenous component of ECM, involved in cell proliferation, migration and tissue remodeling. In vitro and animal studies have demonstrated the ability of exogenous HA to improve wound healing, enhancing proliferative and migratory ability of oral-derived fibroblasts. However, in vivo human studies evaluating its specific mechanisms on cellular activation and gene expression modulation in the early phases after oral surgical wounding are lacking. The present thesis was organized and divided in the following sections: Chapter I The first chapter is intended to give evidence on the peculiar characteristics of oral soft tissues in the early phases of wound healing process from a clinical and biomolecular point of view. In addition, evidence available concerning the role of post-surgical chlorhexidine digluconate (CHX) mouthrinse and exogenous hyaluronic acid (HA) in oral wound repair is presented. The aim of this chapter is to provide current knowledge on these concerns, pointing out the missing evidences that lead the research topic during the course of the doctorate and presenting the rationale of the topic that allowed to conclude in the three main branches of this research: - Differences in the gene expression profiles between human alveolar mucosa, buccal attached gingiva and palatal tissue-derived fibroblasts in the early phases of oral wound repair; - Effect of post-surgical CHX mouthrinse on gingival tissue features and oral gingival-derived cells behaviour in the early phases of oral wound repair; - Effect of intra-surgical HA application on gingival tissue features and oral gingival-derived cells behaviour in the early phases of oral wound repair. Chapter II The second chapter is intended to provide evidence on the behaviour of fibroblasts derived from different oral soft tissues in the early phases of the wound healing process. The main aim was to analyse and compare the gene expression profiling of fibroblasts from human alveolar mucosa (M), gingival (G) and palatal (P) tissues in the early phases following surgical wounding, correlating it with the clinical response, autophagy activation and fibrotic markers expression. M, G and P biopsies were harvested from six patients at baseline and twenty-four hours after surgical procedure. Clinical response was evaluated through Early wound Healing Score (EHS). Fibrotic markers expression and autophagy activation were assessed on fibroblasts isolated from those tissues by Western blot and quantitative real time PCR analysis (qRT-PCR). Fibroblasts from two patients were subjected to RT2 profiler array, followed by network analysis of the differentially expressed genes. The expression of key genes was validated with qRT-PCR on all patients. At twenty-four hours after surgery, EHS was higher in P and G than in M. In line with the clinical results, no autophagy and myofibroblast differentiation were observed in G and P. Significant variations in mRNA expression of key genes were observed: RAC1, SERPINE1 and TIMP1, involved in scar formation; CDH1, ITGA4 and ITGB5, contributing to myofibroblast differentiation; and IL6 and CXCL1, involved in inflammation. Some genes involved in the oral soft tissue differential clinical wound healing outcome were identified, providing novel insights into the molecular mechanisms of oral repair and allowing to develop new approaches of essential impact in periodontal surgery. Chapter III The present chapter focuses on the study of the in vivo effect of post-surgical chlorhexidine digluconate (CHX) mouthrinse on the gingival tissue features and oral gingival-derived cells behaviour in the early phases of wound repair. G biopsies were obtained in three patients twenty-four hours after surgery with the indication of post-surgical 0.12% CHX use and were compared with those obtained from the same patients without any antiseptic use. Each gingival biopsy was divided in two parts: one for histological-immunohistochemical (IHC) analysis and one for gene expression analysis in order to carry out a morphological and molecular analysis. For the first one, epithelial tissue/chorion features and collagen fibers organization/content were evaluated through Hematoxylin-Eosin and Masson trichrome staining, respectively. The expressions of proteins related to cell proliferation (ki67) and apoptosis (p53) were examined by IHC analysis. Fibrotic markers expression (Vimetin, Col1a1 and αSMA) were also analysed by IHC in order to evaluate collagen deposition and myofibroblasts differentiation. For the molecular analysis, qRT-PCR was carried out: fibrotic markers expression (Col1a1 and αSMA) and proapoptotic protein (BAX) were analysed in all the patients, and to evaluate the re-epithelialization and collagen turnover, RAC1, SERPINE1 and TIMP1 gene expression were analysing in two patients. Twenty-four hours after surgery, CHX was able to reduce cellular proliferation and to increase collagen deposition, proapoptotic protein and fibrotic markers expression, and myofibroblast differentiation. In addition, a reduction in the expression of RAC1 and triggering in the expression of SERPINE1 and TIMP1 were observed, showing a “scar wound healing response” pattern. The demonstration of CHX-induced fibrotic transformation, leading to scar repair, could support the need for new post-surgical clinical protocols based on a strategic and personalised use of CHX. Chapter IV The present chapter focuses on the study of the in vivo effect of exogenous hyaluronic acid (HA) on the gingival tissue features and oral gingival-derived cells behaviour in the early phases of wound repair. G biopsies were obtained in eight patients twenty-four hours after surgery with intra-surgical application of HA and were compared with those obtained from the same patients without HA application (no treatment group - NT). Clinical response was evaluated through EHS. Each gingival biopsy was divided in three parts: one for histological-IHC analysis, one for protein analysis and one for gene expression analysis in order to carry out a morphological and molecular analysis. For the first one, tissue structure and inflammatory infiltrate, extracellular matrix (ECM) organization and microvascular density (MVD), and collagen fibers organization/content were evaluated through Hematoxylin-Eosin, Sinus red and Masson trichrome staining, respectively; whereas cellular proliferation was evaluated by immunohistochemical detection of Ki67. For the molecular analysis, collagen turnover was evaluated through MMP-1, MMP-2 and MMP-9 protein analysis by Western Blot and LOX, MMP-1, TIMP-1, TGF-β1 genes expression by real time PCR. Since ECM remodeling is also influenced by mechanical stimuli, and fibroblasts are mechanoresponsive cells, the expression of key mechanosensors paxillin (PAX), focal adhesion kinase (FAK) and vinculin (VNC) were also analysed. Twenty-four hours after surgery, EHS was significantly higher in HA than in NT group. In line with the clinical results, gene expression analysis showed that mRNA levels of LOX-involved in collagen maturation-, resulted significantly up-regulated in HA-treated gingiva compared to NT group but this was independent of TGF-β1 since no difference was found its expression. MMP-1/TIMP-1 balance was modified: significant increase of MMP-1 protein and TIMP-1 gene expression in HA compared to NT group were revealed. No significant differences were observed in MVD, key mechanosensors expression, collagen content and cell proliferation. Intra-surgical HA application enhance wound healing properties such as ECM remodeling and collagen maturation, and improve the clinical repair response in human in vivo gingival wounds 24 hrs after injury. HA might be an important component in future regimens aiming to accelerate and improve the wound healing after periodontal surgery. Chapter V The last chapter aims to provide general conclusions concerning the three branches of the research performed during the course of doctorate, integrating the results obtained and pointing out some key points and recommendations. In addition, future perspectives in the wound healing research field are mentioned.