Liliana Grenho

Antibacterial activity and biocompatibility of three-dimensional nanostructured porous granules of hydroxyapatite and zinc oxide nanoparticles—an in vitro and in vivo study

Ceramic scaffolds are widely studied in the bone tissue engineering field due to their potential in regenerative medicine. However, adhesion of microorganisms on biomaterials with subsequent formation of antibiotic-resistant biofilms is a critical factor in implant-related infections. Therefore, new strategies are needed to address this problem. In the present study, three-dimensional and interconnected porous granules of nanostructured hydroxyapatite (nanoHA) incorporated with different amounts of zinc oxide (ZnO) nanoparticles were produced using a simple polymer sponge replication method. As in vitro experiments, granules were exposed to Staphylococcus aureus and Staphylococcus epidermidis and, after 24 h, the planktonic and sessile populations were assessed. Cytocompatibility towards osteoblast-like cells (MG63 cell line) was also evaluated for a period of 1 and 3 days, through resazurin assay and imaging flow cytometry analysis. As in vivo experiments, nanoHA porous granules with and without ZnO nanoparticles were implanted into the subcutaneous tissue in rats and their inflammatory response after 3, 7 and 30 days was examined, as well as their antibacterial activity after 1 and 3 days of S. aureus inoculation. The developed composites proved to be especially effective at reducing bacterial activity in vitro and in vivo for a weight percentage of 2% ZnO, with a low cell growth inhibition in vitro and no differences in the connective tissue growth and inflammatory response in vivo. Altogether, these results suggest that nanoHA-ZnO porous granules have a great potential to be used in orthopaedic and dental applications as a template for bone regeneration and, simultaneously, to restrain biomaterial-associated infections.

Micropatterned silica thin films with nanohydroxyapatite micro-aggregates for guided tissue regeneration

UNLABELLED Surface modification of biomaterials has been shown to improve the biological response to dental implants. The ability to create a controlled micro-texture on the implant via additive surface modification techniques with bioactive nanohydroxyapatite (nanoHA) may positively influence guided tissue regeneration. OBJECTIVE The main goal of this study was to produce micro-fabricated SiO(2) surfaces modified with nanohydroxyapatite particles and to characterize their influence on the biological response of Human Dental-Pulp Mesenchymal Stem Cells (hDP-MSCs) and Streptococcus mutans. MATERIALS AND METHODS A combined methodology of sol-gel and soft-lithography was used to produce micropatterned SiO(2) thin films with different percentages of nanoHA micro-aggregates. The surfaces were characterized by SEM/EDS, FT-IR/ATR, AFM, XPS quantitative elemental percentage and contact angle measurements. Biological characterization was performed using hDP-MSCs cultures, while Streptococcus mutans was the selected microorganism to evaluate the bacterial adhesion on the thin films. RESULTS Micropatterned SiO(2) surfaces with 0%, 1% and 5% of nanoHA micro-aggregates were successfully produced using a combination of sol-gel and soft-lithography. These surfaces controlled the biological response, triggering alignment and oriented proliferation of hDP-MSCs and significant differences in the adhesion of S. mutans to the different surfaces. SIGNIFICANCE The micropatterned surfaces exhibited biocompatible behavior that induced an oriented adhesion and proliferation of hDP-MSCs while SiO(2) presented low bacterial adhesion. These results show that the combination of sol-gel with soft-lithography is a good approach to create micropatterned surfaces with bioactive nanoparticles for guided tissue regeneration.

Biodegradation, biocompatibility, and osteoconduction evaluation of collagen‐nanohydroxyapatite cryogels for bone tissue regeneration

Designing biomimetic biomaterials inspired by the natural complex structure of bone and other hard tissues is still a challenge nowadays. The control of the biomineralization process onto biomaterials should be evaluated before clinical application. Aiming at bone regeneration applications, this work evaluated the in vitro biodegradation and interaction between human bone marrow stromal cells (HBMSC) cultured on different collagen/nanohydroxyapatite cryogels. Cell proliferation, differentiation, morphology, and metabolic activity were assessed through different protocols. All the biocomposite materials allowed physiologic apatite deposition after incubation in simulated body fluid and the cryogel with the highest nanoHA content showed to have the highest mechanical strength (DMA). The study clearly showed that the highest concentration of nanoHA granules on the cryogels were able to support cell types survival, proliferation, and individual functionality in a monoculture system, for 21 days. In fact, the biocomposites were also able to differentiate HBMSCs into osteoblastic phenotype. The composites behavior was also assessed in vivo through subcutaneous and bone implantation in rats to evaluate its tissue-forming ability and degradation rate. The cryogels Coll/nanoHA (30 : 70) promoted tissue regeneration and adverse reactions were not observed on subcutaneous and bone implants. The results achieved suggest that scaffolds of Coll/nanoHA (30 : 70) should be considered promising implants for bone defects that present a grotto like appearance with a relatively small access but a wider hollow inside. This material could adjust to small dimensions and when entering into the defect, it could expand inside and remain in close contact with the defect walls, thus ensuring adequate osteoconductivity.

In vitro analysis of the antibacterial effect of nanohydroxyapatite–ZnO composites

Hydroxyapatite (HA) is a biocompatible and bioactive synthetic material for biomedical applications as it binds to bone and enhances bone tissue formation. Particularly, nanophased HA can mimic the dimensions of constituent components of natural tissues; can modulate enhanced osteoblast adhesion and resorption with long-term functionality of tissue engineered implants. However, HA does not inhibit bacteria from adhering onto its surface, and this has implications in the bone healing process required for patient recovery, since infection can lead to the implant failure. In the present work a composite that combines the favorable biological characteristics of nanohydroxyapatite (nanoHA) and, simultaneously, possesses antimicrobial activity as expressed by ZnO was synthesized. To determine whether the size of ZnO particles was playing an important role in inhibiting bacterial growth, ZnO particle of different sizes (from the microscale down to the nanoscale) and concentration were incorporated into nanoHA and tested. The composite samples were characterized by SEM, FT-IR, XRD, XPS and zeta potential. The antibacterial activity of the composites was investigated, as well as the biofilm formation, using both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) microorganisms. The characterization revealed that ZnO particles were dispersed homogeneously within the nanoHA matrix. The composites antibacterial activity increased with decreasing ZnO particle size and increasing concentration. Biofilm formation tests revealed that the nanoHA-ZnO composites exhibit a strong effect against the common pathogens S. aureus and E. coli.

Influence of nanohydroxyapatite surface properties on Staphylococcus epidermidis biofilm formation

Nanohydroxyapatite (nanoHA), due to its chemical properties, has appeared as an exceptionally promising bioceramic to be used as bone regeneration material. Staphylococcus epidermidis have emerged as major nosocomial pathogens associated with infections of implanted medical devices. In this work, the purpose was to study the influence of the nanoHA surface characteristics on S. epidermidis RP62A biofilm formation. Therefore, two different initial inoculum concentrations (Ci) were used in order to check if these would affect the biofilm formed on the nanoHA surfaces. Biofilm formation was followed by the enumeration of cultivable cells and by scanning electron microscopy. Surface topography, contact angle, total surface area and porosimetry of the biomaterials were studied and correlated with the biofilm data. The surface of nanoHA sintered at 830℃ (nanoHA830) showed to be more resistant to S. epidermidis attachment and accumulation than that of nanoHA sintered at 1000℃ (nanoHA1000). The biofilm formed on nanoHA830 presented differences in terms of structure, surface coverage and EPS production when compared to the one formed on nanoHA1000 surface. It was observed that topography and surface area of nanoHA surfaces had influence on the bacterial attachment and accumulation. Ci influenced bacteria attachment and accumulation on nanoHA surfaces over time. The choice of the initial inoculum concentration was relevant proving to have an effect on the extent of adherence thus being a critical point for human health if these materials are used in implantable devices. This study showed that the initial inoculum concentration and surface material properties determine the rate of microbial attachment to substrata and consequently are related to biofilm-associated infections in biomaterials.

Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa onto nanohydroxyapatite as a bone regeneration material.

In orthopedics due to the enormous number of surgical procedures involving invasive implant biomaterials, infections have a huge impact in terms of morbidity, mortality, and medical costs. In this study the initial adhesion of several strains namely Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa, to nanohydroxyapatite, previously heat-treated at 725 °C and 1000 °C was assessed. Adherent cells were evaluated by scanning electron microscopy and quantified by confocal laser scanning microscopy and as colony forming units after being released by sonication. The wettability and roughness of samples surfaces were assessed by contact angle measurements and atomic force microscopy, respectively. Nanohydroxyapatite heat-treated at 1000 °C appeared to be more resistant to bacterial adhesion, over time, in five of the six tested strains while the clinical strains isolated from orthopedic infections presented superior ability to adhere, as well as better capacity to produce slime. The increase in materials sintering temperature resulted in increased hydrophobicity and roughness; however, other surface features such as the decrease in surface area and on porosity as well as the decrease on zeta potential may be the aspects that contributed to a lower bacterial adhesion on the materials sintered at 1000 °C.

A modular reactor to simulate biofilm development in orthopedic materials

Surfaces of medical implants are generally designed to encourage soft- and/or hard-tissue adherence, eventually leading to tissue- or osseo-integration. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation. To understand the mechanisms of bone tissue infection associated with contaminated biomaterials, a detailed understanding of bacterial adhesion and subsequent biofilm formation on biomaterial surfaces is needed. In this study, a continuous-flow modular reactor composed of several modular units placed in parallel was designed to evaluate the activity of circulating bacterial suspensions and thus their predilection for biofilm formation during 72 h of incubation. Hydroxyapatite discs were placed in each modular unit and then removed at fixed times to quantify biofilm accumulation. Biofilm formation on each replicate of material, unchanged in structure, morphology, or cell density, was reproducibly observed. The modular reactor therefore proved to be a useful tool for following mature biofilm formation on different surfaces and under conditions similar to those prevailing near human-bone implants.

Anti-sessile bacterial and cytocompatibility properties of CHX-loaded nanohydroxyapatite.

Nanohydroxyapatite possesses exceptional biocompatibility and bioactivity regarding bone cells and tissues, justifying its use as a coating material or as a bone substitute. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation. Surface functionalization with antimicrobials is a promising strategy to reduce the likelihood of bacterial infestation and colonization on medical devices. Chlorhexidine digluconate is a common and effective antimicrobial agent used for a wide range of medical applications. The purpose of this work was the development of a nanoHA biomaterial loaded with CHX to prevent surface bacterial accumulation and, simultaneously, with good cytocompatibility, for application in the medical field. CHX (5-1500 mg/L) was loaded onto nanoHA discs and the materials were evaluated for CHX adsorption and release profile, physic-chemical features, antibacterial activity against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, and cytocompatibility toward L929 fibroblasts. Results showed that the adsorption of CHX on nanoHA surface occurred by electrostatic interactions between the cationic group of CHX and the phosphate group of nanoHA. The release of CHX from CHX-loaded nanoHA showed a fast initial rate followed by a slower kinetics release, due to constraints caused by dilution and diffusion-limiting processes. NanoHA.50 to nanoHA.1500 showed strong anti-sessile activity, inhibiting bacterial adhesion and the biofilm formation. CHX-nanoHA caused a dose- and time-dependent inhibitory effect on the proliferation of fibroblasts for nanoHA.100 to nanoHA.1500. Cellular behavior on nanoHA.5 and nanoHA.50 was similar to control. Therefore, CHX-loaded nanoHA surfaces appear as a promising alternative to prevention of devices-related infections.

Levofloxacin-loaded bone cement delivery system : highly effective against intracellular bacteria and Staphylococcus aureus biofilms

Staphylococcus aureus is a major pathogen in bone associated infections due to its ability to adhere and form biofilms on bone and/or implants. Moreover, recrudescent and chronic infections have been associated with S. aureus capacity to invade and persist within osteoblast cells. With the growing need of novel therapeutic tools, this research aimed to evaluate some important key biological properties of a novel carrier system composed of acrylic bone cement (polymethylmethacrylate - PMMA), loaded with a release modulator (lactose) and an antibiotic (levofloxacin). Levofloxacin-loaded bone cement (BC) exhibited antimicrobial effects against planktonic and biofilm forms of S. aureus (evaluated by a flow chamber system). Moreover, novel BC formulation showed high anti-bacterial intraosteoblast activity. This fact led to the conclusion that levofloxacin released from BC matrices could penetrate the cell membrane of osteoblasts and be active against S. aureus strains in the intracellular environment. Furthermore, levofloxacin-BC formulations showed no significant in vitro cytotoxicity and no allergic potential (measured by the in vivo chorioallantoic membrane assay). Our results indicate that levofloxacin-loaded BC has potential as a local antibiotic delivery system for treating S. aureus associated bone infections.

Micropatterned Coatings for Guided Tissue Regeneration in Dental Implantology

Dento-alveolar trauma and congenital absences are the most important causes of edentulism that are not associated with bacteria. However, the World Health Organization reports show that dental caries and periodontitis, two conditions of bacterial origin, are the most frequent oral diseases in humans [1]. These conditions might be avoided if an adequate oral preventive health policy is implemented, including preventive and educational measures that, regardless of the population s socioeconomic factors, have shown their effectiveness. Despite these facts, tooth extraction1, defined as the surgical removal of a tooth, is currently the most frequent surgical procedure in the world [1].