Novel nanostructured biomaterials for biomedical applications

The research activity reported in this thesis was focused on bottom-up design, production and characterization of biomaterials in the form of medical devices for biomedical applications. More specifically, both a biomaterial for anastomosis after colorectal cancer resection, and a biomaterial for chronic non-healing wounds have been designed and developed, by exploiting the same manufacturing strategy. In the first part of the work, the research has been mainly aimed to obtain a device for preventing the leakage of the anastomosis following surgical treatment of colorectal cancer. The anastomotic leakage is a defect of the intestinal wall at the anastomotic site, which leads to a communication between the intra- and extra-luminal compartments. This study was part of the scientific activity forecasted by the European project AnastomoSEAL (FP7, c.n.280929). The project aim was to develop a patch to be wrapped around the anastomosis, capable to promote the healing of the wound. Specific biomolecules were chosen as biomaterial components. Alginate was selected for its ability to form gels thus providing the physical matrix; Hyaluronic Acid (HA) was chosen for its ability to stimulate wound healing; Butyric acid (But) was chosen since recent data demonstrated its beneficial effect on colorectal anastomosis in animal models. The last two components have been also chemically combined in the Hyaluronic Acid Butyric ester (HABut) molecule. Patches with alginate and HA were produced by using various polymer concentrations, different alginate types (algal sources), and HA with different molecular weights, in order to fine-tune the composition and the performances for the final application. Moreover, in vitro biological tests were performed on the patch components (raw materials): the effects on cell viability, proliferation and extracellular matrix production were studied on primary human fibroblasts and on a normal-derived colonocyte cell line. Additional biological in vitro tests were conducted in order to study more in depth the effect of But on colonocytes. The obtained in vitro data enabled the selection of the best performing formulation and lead to the decision to exclude the use of HABut and But from the medical device. The second part of the work was focused on a biomaterial for chronic non-healing wounds treatment. Chronic non\u2013healing wounds are defined as wounds that do not heal in eight weeks and are characterized by a prolonged inflammation, excess of proteolytic enzymes, reduced cell proliferation and migration, and infections which further sustains these deregulations. For the development of the medical device for chronic non-healing wounds application alginate and HA were also chosen. Moreover, silver nanoparticles (nAgs) were added for their antibacterial and anti-inflammatory activity, and for their ability to inhibit proteolytic enzymes. nAgs are produced in wet conditions from silver nitrate and reducing compounds in the presence of the biopolymer Chitlac as dispersion agent. A foamed biomaterial was prepared in order to increase the ratio surface/volume and therefore to enhance the bacterial exposure to nAg. The foam has been obtained by using hydroxy-methyl-2-propyl cellulose (HPMC), a cellulose water soluble derivative already employed for this purpose in many biomedical and pharmaceutical applications. The HPMC-foamed patch was characterized by structural, mechanical and biological analysis: scanning electron microscopy (SEM) and tensile strength measurement were performed. The overall data confirmed that the biomaterial obtained is a promising material for the chronic non-healing wound application

Alginate membranes loaded with hyaluronic acid and silver nanoparticles to foster tissue healing and to control bacterial contamination of non-healing wounds

Chronic non-healing wounds are a clinically important problem in terms of number of patients and costs. Wound dressings such as hydrogels, hydrocolloids, polyurethane films and foams are commonly used to manage these wounds since they tend to maintain a moist environment which is shown to accelerate re-epithelialization. The use of antibacterial compounds is important in the management of wound infections. A novel wound-dressing material based on a blended matrix of the polysaccharides alginate, hyaluronic acid and Chitlac-silver nanoparticles is here proposed and its application for wound healing is examined. The manufacturing approach to obtain membranes is based on gelling, foaming and freeze-casting of alginate, hyaluronic acid and Chitlac-silver nanoparticles mixtures using calcium ions as the cross-linking agent. Comprehensive evaluations of the morphology, swelling kinetics, permeability, mechanical characteristics, cytotoxicity, capability to inhibit metalloproteinases and of antibacterial property were conducted. Biological in vitro studies demonstrated that hyaluronic acid released by the membrane is able to stimulate the wound healing meanwhile the metal silver exploits an efficient antibacterial activity against both planktonic bacteria and biofilms. Overall, the experimental data evidence that the studied material could be used as antibacterial wound dressing for wound healing promotion

Hyaluronan delivery by polymer demixing in polysaccharide-based hydrogels and membranes for biomedical applications

Alginate-based membranes containing hyaluronic acid (HA) were manufactured by freeze-drying calcium-reticulated hydrogels. The study of the distribution of the two macromolecules within the hydrogel enabled to highlight a polymer demixing mechanism that tends to segregate HA in the external parts of the constructs. Resistance and pliability of the membranes were tuned, while release and degradation studies enabled to quantify the diffusion of both polysaccharides in physiological solution and to measure the viable lifetime of the membranes. Biological studies in vitro proved that the liquid extracts from the HA-containing membranes stimulate wound healing and that fibroblasts are able to colonize the membranes. Overall, such novel alginate-HA membranes represent a promising solution for several medical needs, in particular for wound treatment, giving the possibility to provide an in situ administration of HA from a resorbable device

Biological Responses of Human Gingival Fibroblasts (HGFs) in an Innovative Co-Culture Model with Streptococcus mitis to Thermosets Coated with a Silver Polysaccharide Antimicrobial System

This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs) co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA) thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL- 6) and prostaglandin E2 (PGE2) secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with ChitlacnAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE2 production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE2 secretion. Data obtained in the present work suggest that Chitlac n- Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs

Biological responses of human gingival fibroblasts (HGFs) in an innovative co-culture model with Streptococcus mitis to thermosets coated with a silver polysaccharide antimicrobial system.

This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs) co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA) thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6) and prostaglandin E2 (PGE2) secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE2 production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE2 secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs

Effect of Chitlac and Chitlac-nAg thermosets on cell migration in HGFs/<i>Streptococcus mitis</i> co-culture model.

<p>The graph represents the mean fold increase of cell number ± SD of three experiments. <b>U</b>: HGFs; <b>Th</b>: HGFs with Chitlac thermoset; <b>Ag</b>: HGFs with Chitlac-nAg thermoset; <b>MTh</b>: HGFs with Chitlac thermoset and <i>S. mitis</i>; <b>MAg</b>: HGFs with Chitlac-nAg thermoset and <i>S. mitis</i>; <b>MSTh</b>: HGFs with Chitlac thermoset, <i>S. mitis</i> and saliva; <b>MSAg</b>: HGFs with Chitlac-nAg thermoset, <i>S. mitis</i> and saliva; <b>M bottom</b>: <i>S. mitis</i> in the bottom chamber: <b>S bottom</b>: Saliva in the bottom chamber; <b>MS bottom</b>: <i>S. mitis</i> and saliva in the bottom chamber. <b>*</b>MTh, M bottom, S bottom vs U, p = 0.0421, p = 0.0397, p = 0.0041; § MSTh vs MTh, p = 0.0461; £ MS bottom vs M bottom, p = 0.0037; °MS bottom vs S bottom, p = 0.0234.</p

Effect of Chitlac and Chitlac-nAg thermosets on IL-6 and PGE₂ release in HGFs/<i>Streptococcus mitis</i> co-culture model.

<p>Graph represents the mean concentration (pg/ml) ± SD of three different consistent experiments. <b>U</b>: HGFs; <b>Th</b>: HGFs with Chitlac thermoset; <b>Ag</b>: HGFs with Chitlac-nAg thermoset; <b>MSTh</b>: HGFs with Chitlac thermoset, <i>S. mitis</i> and saliva; <b>MSAg</b>: HGFs with Chitlac-nAg thermoset, <i>S. mitis</i> and saliva; *Th, Ag, MSTh and MSAg vs U IL-6, p = 0.0391, p = 0.0299, p = 0.0325, p = 0.0408; § Th, Ag, MSTh and MSAg vs U PGE₂, p = 0.0300, p = 0.0262, p = 0.0472, p = 0.0362.</p

Effect of BisGMA/TEGDMA thermosets on LDH release.

<p><b>A</b>: in HGFs. The graph represents the mean percentage ± SD of three different consistent experiments. <b>*</b>BT 24 h and BT 48 h vs U, p = 0.0392 and p = 0.0019. <b>B</b>: in HGFs/<i>Streptococcus mitis</i> co-culture model. The graph represents the mean percentage ± SD of three different consistent experiments. <b>U</b>: HGFs; <b>BT</b>: HGFs with uncoated thermoset; <b>Th</b>: HGFs with Chitlac thermoset; <b>Ag</b>: HGFs with Chitlac-nAg thermoset; <b>MTh</b>: HGFs with Chitlac thermoset and <i>S. mitis</i>; <b>MAg</b>: HGFs with Chitlac-nAg thermoset and <i>S. mitis</i>; <b>MSTh</b>: HGFs with Chitlac thermoset, <i>S. mitis</i> and saliva; <b>MSAg</b>: HGFs with Chitlac-nAg thermoset, <i>S. mitis</i> and saliva. <b>*</b>Th and Ag 24 h vs U 24 h, p = 0.0335 and p = 0.0018; § Th and Ag 48 h vs U 48 h, p = 0.0257 and p = 0.0181; £ MTh 48 h vs Th 48 h, p = 0.0304; °MSTh 48 h vs MTh 48 h, p = 0.0272.</p