Bioactive Membranes and Nanocoatings for Guided Tissue Regeneration in Periodontal Diseases

Periodontal diseases are highly prevalent in population of all ages. Initiated by bacterial accumulation at the interface of bone and soft tissue, they lead to the loss of gingival tissue adherent to the root surface and, eventually, to tooth loss. Regenerative approaches to treat periodontitis offer exciting possibilities; guided tissue/bone regeneration (GTR/GBR) approaches are promising because, through the insertion of a physical barrier, they can exclude unwanted epithelial and gingival connective tissue cells from the healing area and allow bone tissue cells to repopulate the bony defect. Different resorbable and non-resorbable membranes have been developed. Expanded polytetrafluoroethylene (ePTFE) membranes are the "gold standard" for GTR/GBR applications but they are non-resorbable and they need a second surgical operation to repair dehiscence. Biodegradable synthetic membranes avoid a second surgical operation but they show drawbacks concerning the capacity of space maintenance, early/late absorption, mechanical properties and bacterial infection during degradation. Collagen membranes have advantages related to collagen biological properties but are characterized by low mechanical strength. The "ideal" membrane for use in periodontal regenerative therapy has yet to be developed. The main purpose of this thesis was the design of biologically active products, with improved osteoconductive and antimicrobial properties, for GTR/GBR applications in periodontal diseases. In a more traditional approach, a commercially available membrane (based on PTFE) was surface modified by environmentally friendly technique to allow rapid bone re-growth and exert antimicrobial action. Binding ability of 3,4-dihydroxy-DL-phenylalanine (DOPA) to samples of any type, size and shape was exploited to improve PTFE surface properties. In particular, a hydroxyapatite nanoparticles (HAp) coating was applied by DOPA self-polymerization on PTFE surface in the presence of HAp nanoparticles, to promote the bone re-growth properties of PTFE films. Chemical composition analysis demonstrated the successful deposition of polyDOPA and HAp on coated films. Morphological and topographical characterizations further confirmed the total surface coverage causing an increase in surface roughness (39.8±5.2 nm for PTFE films vs 236.5±12.0 nm for polyDOPA/HAp coated films) and wettability (110.8±2.8° for PTFE films vs 46.1±12.4° for polyDOPA/HAp coated samples). A discontinuous HAp coating was still present after 14 days of incubation of coated PTFE films in phosphate buffered saline. Pre-osteoblastic MC3T3-E1 cells cultured on polyDOPA/HAp coated films showed a pronounced increase of cell proliferation and adhesion. Regarding the antimicrobial action, silver nanoparticles (AgNPs) have been selected due to their good antimicrobial efficacy against bacteria, viruses and other eukaryotic micro-organisms. The successful deposition of AgNPs on PTFE surface, through the functional groups of DOPA, has been demonstrated by physico-chemical and morphological analyses. Nanoparticles exhibited a diameter around 68 nm and were homogeneously distributed on the surface. In vitro cell tests with fibroblast NIH 3T3 cells showed an inhibition of cells proliferation on AgNPs functionalized films after 3 days of culture, while good cell adhesion was observed with cells randomly distributed on sample surface and extensively spread. The antimicrobial efficiency was demonstrated against S. aureus and Ag release was sustained for at least 14 days. The mussel-inspired coated PTFE membrane could find potential application as GTR/GBR strategy for the treatment of periodontal diseases. In a highly innovative approach, a bi-layered bioabsorbable membrane was developed, by the assembly of a compact and a porous layer. GTR/GBR membranes can be considered an interface-implant between gingival connective tissue/epithelium and alveolar bone tissue. Developing a multi-component structure membrane with compositional and structural gradients that meet the local functional requirements could represent a challenge. Binary blends of poly(DL-lactide-co-ε-caprolactone) (PLCL) and poly(DL-lactide-co-glycolide) (PLGA) with various compositions (100/0, 75/25, 50/50, 25/75, 0/100 wt/wt) were prepared by solvent casting technique as compact layer of the bi-layered membrane. Morphological analysis did not evidence phase separation between PLCL and PLGA and the behavior of blend glass transition temperatures as a function of composition suggested some degree of blend compatibility. However, blends elastic modulus showed a negative deviation from the additive law of mixture. In vitro cell tests with fibroblast NIH 3T3 cells showed improved cell adhesion and growth on PLCL/PLGA 25/75 blend. Due to its biocompatibility, its superior mechanical properties (E=10.2±0.6 MPa, σmax=0.8±0.0 MPa, and εmax=548.8±57.9%) and compatibility between the components, PLCL/PLGA 25/75 blend was selected for this application. Compact films were then surface modified via layer-by-layer (LbL) technique to enhance fibroblast cell response and confer antibacterial efficacy. A surface priming treatment (aminolysis) was optimized before depositing LbL coating. The following parameters were used: C=0.08 g/mL, t=8 min and T=37 °C. Then, multilayered chondroitin sulfate/chitosan (CHS/CH) coatings were deposited on the aminolysed films. The feasibility of multilayer coating was confirmed by QCM-D analysis. Further confirmations derived from water contact angle measurements (contact angle jumped alternatively between 45° and 65° depending on the outmost layer component) and FTIR-ATR analysis (appearance of absorbance peaks characteristics of CHS and CH). FTIC-labelled CH was also employed to follow LbL built up by fluorescence microscopy analysis. In vitro cell tests demonstrated the ability of coated samples to improve NIH 3T3 fibroblast adhesion and proliferation. Biocompatibility properties increased with increasing the layer number and were superior in the case of CH-terminating layers but no antibacterial activity was observed for films coated with 16 layers. Three dimensional sponge-like composite membranes fabricated by freeze-drying, with a composition similar to natural bone, and based on β-tricalcium phosphate (TCP) dispersed in a chitosan/gelatin (CH/G) network cross-linked with genipin (GP) and disodium phosphate salt (DSP) were developed as porous layer of the bi-layered device. Three membranes were developed (CH/G, CH/G+GP-DSP and CH/G/TCP+GP-DSP) and characterized. Successful double cross-linking of CH/G network was confirmed by Kaiser test, chemical and thermal analysis. All membranes showed a typical foam-like morphology with interconnected pores having an average diameter of 100-200 μm. Both cross-linking and TCP presence caused a marked increase of membrane stability in water solution, as well as of tensile modulus and maximum tensile strength (respectively, 14.9±5.1 MPa and 0.6±0.0 MPa for CH/G, and 29.4±2.7 MPa and 0.8±0.1 MPa for CH/G/TCP+GP-DSP.). Compared to CH/G samples, CH/G+GP-DSP and CH/G/TCP+GP-DSP membranes showed improved MG-63 human osteoblast-like cells response, in terms of cell viability and morphology. The assembly process of the compact and porous layer was developed based on the insertion of an intermediate adhesive layer composed by a polyvinylpyrrolidone/polyethylene glycol 70/30 wt/wt blend. Preliminary characterizations were carried out. Morphological analysis did not show changes in compact and porous layer structure due to the presence of the adhesive. The final device showed an elastic modulus of about 61 MPa in dry condition that markedly decreased in wet state (to about 5 MPa). Qualitative analysis of membrane manageability revealed its ability to adapt to mandible conformation after immersion in physiological solution. Despite the need for additional tests, the bi-layered membrane appeared promising for GTR/GBR application