Poly(DL-lactic acid) scaffolds as a bone targeting platform for the co-delivery of antimicrobial agents against S. aureus-C.albicans mixed biofilms.

New strategies for the treatment of polymicrobial bone infections are required. In this study, the co-delivery of two antimicrobials by poly(D,L-lactic acid) (PDLLA) scaffolds was investigated in a polymicrobial biofilm model. PDLLA scaffolds were prepared by solvent casting/particulate leaching methodology, incorporating minocycline and voriconazole as clinically relevant antimicrobial agents. The scaffolds presented a sponge-like appearance, suitable to support cell proliferation and drug release. Single- and dual-species biofilm models of Staphylococcus aureus and Candida albicans were developed and characterized. S. aureus presented a higher ability to form single-species biofilms, compared to C. albicans. Minocycline and voriconazole-loaded PDLLA scaffolds showed activity against S. aureus and C. albicans single- and dual-biofilms. Ultimately, the cytocompatibility/functional activity of PDLLA scaffolds observed in human MG-63 osteosarcoma cells unveil their potential as a next-generation co-delivery system for antimicrobial therapy in bone infections.

Inhibitory Effect of 5-Aminoimidazole-4-Carbohydrazonamides Derivatives Against Candida spp. Biofilm on Nanohydroxyapatite Substrate.

Candida can adhere and form biofilm on biomaterials commonly used in medical devices which is a key attribute that enhances its ability to cause infections in humans. Furthermore, biomaterial-related infections represent a major therapeutic challenge since Candida biofilms are implicated in antifungal therapies failure. The goals of the present work were to investigate the effect of three 5-aminoimidazole-4-carbohydrazonamides, namely (Z)-5-amino-1-methyl-N'-aryl-1H-imidazole-4-carbohydrazonamides [aryl = phenyl (1a), 4-fluorophenyl (1b), 3-fluorophenyl (1c)], on Candida albicans and Candida krusei biofilm on nanohydroxyapatite substrate, a well-known bioactive ceramic material. To address these goals, both quantitative methods (by cultivable cell numbers) and qualitative evaluation (by scanning electron microscopy) were used. Compounds cytocompatibility towards osteoblast-like cells was also evaluated after 24 h of exposure, through resazurin assay. The three tested compounds displayed a strong inhibitory effect on biofilm development of both Candida species as potent in vitro activity against C. albicans sessile cells. Regarding cytocompatibility, a concentration-dependent effect was observed. Together, these findings indicated that the potent activity of imidazole derivatives on Candida spp. biofilms on nanohydroxyapatite substrate, in particular compound 1c, is worth further investigating.

Alginate-nanohydroxyapatite hydrogel system: Optimizing the formulation for enhanced bone regeneration.

Ceramic/polymer-based biocomposites have emerged as potential biomaterials to fill, replace, repair or regenerate injured or diseased bone, due to their outstanding features in terms of biocompatibility, bioactivity, injectability, and biodegradability. However, these properties can be dependent on the amount of ceramic component present in the polymer-based composite. Therefore, in the present study, the influence of nanohydroxyapatite content (30 to 70 wt%) on alginate-based hydrogels was studied in order to evaluate the best formulation for maximizing bone tissue regeneration. The composite system was characterized in terms of physic-chemical properties and biological response, with in vitro cytocompatibility assessment with human osteoblastic cells and ex vivo functional evaluation in embryonic chick segmental bone defects. The main morphological characteristics of the alginate network were not affected by the addition of nanohydroxyapatite. However, physic-chemical features, like water-swelling rate, stability at extreme pH values, apatite formation, and Ca2+ release were nanoHA dose-dependent. Within in vitro cytocompatibility assays it was observed that hydrogels with nanoHA 30% content enhanced osteoblastic cells proliferation and expression of osteogenic transcription factors, while those with higher concentrations (50 and 70%) decreased the osteogenic cell response. Ex vivo data underlined the in vitro findings, revealing an enhanced collagenous deposition, trabecular bone formation and matrix mineralization with Alg-nanoHA30 composition, while compositions with higher nanoHA content induced a diminished bone tissue response. The outcomes of this study indicate that nanohydroxyapatite concentration plays a major role in physic-chemical properties and biological response of the composite system and the optimization of the components ratio must be met to maximize bone tissue regeneration.

Novel cerium doped glass-reinforced hydroxyapatite with antibacterial and osteoconductive properties for bone tissue regeneration.

The aim of this work was to develop a bioactive bone substitute with an effective antibacterial ability based on a cerium (Ce) doped glass-reinforced hydroxyapatite (GR-HA) composite. Developed composites were physicochemically characterized, using x-ray diffraction (XRD) analysis, SEM, energy dispersive x-ray spectroscopy (EDS), and flexural bending strength (FBS) tests. X-ray photoelectron spectroscopy (XPS) analysis was performed to analyze the oxidation state of Ce in the prepared doped glass. The antimicrobial activity of the composites was evaluated against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa; whether the cytocompatibility profile was assayed with human osteoblastic-like cells (Mg-63 cell line). The results revealed that the Ce inclusion in the GR-HA matrix induced the antimicrobial ability of the composite. In addition, Ce-doped materials reported an adequate biological behavior following seeding of osteoblastic populations, by inducing cell adhesion and proliferation. Developed materials were also found to enhance the expression of osteoblastic-related genes. Overall, the developed GR-HA_Ce composite is a prospective candidate to be used within the clinical scenario with a successful performance due to the effective antibacterial properties and capability of enhancing the osteoblastic cell response.

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.

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.

In vitro antimicrobial activity and biocompatibility of propolis containing nanohydroxyapatite.

The high number of biomaterial associated infections demands new strategies to prevent this problem. In this study the suitability of nanohydroxyapatite (nanoHA)-based surfaces containing two Brazilian extracts of propolis (green and red ones) to prevent bacterial growth and biofilm formation, as well as its non-cytotoxic nature, was investigated. Optical density, colony forming units and MTT reduction assay were used to assess the materials' antibacterial activity against planktonic and sessile growth of Staphylococcus aureus. NanoHA matrix was able to absorb both types of propolis and the obtained results revealed the antibacterial effectiveness of the novel materials expressed as the reduction of bacterial growth and biofilm formation ability. Additionally, cell culture tests showed the growth of fibroblasts with high metabolic activity and without membrane damage. Therefore, these nanoHA-based surfaces containing natural products deriving from bees may be a promising bioactive biomaterial to be further studied with the aim of application to orthopaedic or dental devices.

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.

Biological evaluation of alginate-based hydrogels, with antimicrobial features by Ce(III) incorporation, as vehicles for a bone substitute.

A novel hydrogel, based on an alginate/hyaluronate mixture and Ce(III) ions, with effective bioactive and antimicrobial ability was developed to be used as vehicle of a synthetic bone substitute producing an injectable substitute (IBS). Firstly, three different IBSs were prepared using three developed alginate-based hydrogels, the hydrogel Alg composed by alginate, the hydrogel Alg/Ch composed by an alginate/chitosan mixture and the hydrogel Alg/HA composed by an alginate/hyaluronate mixture. MG63 cells viability on the IBSs was evaluated, being observed a significantly higher cell viability on the Alg/HA_IBS at all time points, which indicates a better cell adaptation to the material, increasing their predisposition to produce extracellular matrix and thus allowing a better bone regeneration. Moreover, SEM analysis showed evident filopodia and a spreader shape of MG63 cells when seeded on Alg/HA_IBS. This way, based upon the in vitro results, the hydrogel Alg/HA was chosen to the in vivo study by subcutaneous implantation in an animal model, promoting a slight irritating tissue response and visible tissue repairing. The next step was to grant antimicrobial properties to the hydrogel that showed the best biological behavior by incorporation of Ce(III) ions into the Alg/HA, producing the hydrogel Alg/HA2. The antimicrobial activity of these hyaluronate-based hydrogels was evaluated against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans. Results showed that Ce(III) ions can significantly enhance the hydrogel antimicrobial ability without compromising the osteoconductivity improvement promoted by the vehicle association to the synthetic bone substitute.

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.

Equisetum arvense hydromethanolic extracts in bone tissue regeneration: in vitro osteoblastic modulation and antibacterial activity.

OBJECTIVES: Equisetum arvense preparations have long been used to promote bone healing. The aim of this work was to evaluate osteogenic and antibacterial effects of E. arvense hydromethanolic extracts. MATERIALS AND METHODS: Dried aerial components of E. arvense were extracted using a mixture of methanol:water (1:1), for 26 days, yielding three extracts that were tested (10-1000 µg/ml) in human osteoblastic cells: E1, E2 and EM (a mixture of E1 and E2, 1:1). Cell cultures, performed on cell culture plates or over hydroxyapatite (HA) substrates, were assessed for osteoblastic markers. In addition, effects of the extracts on Staphylococcus aureus were addressed. RESULTS: Solution E1 caused increased viability/proliferation and ALP activity at 50-500 µg/ml, and deleterious effects at levels ≥1000 µg/ml. E2 inhibited cell proliferation at levels ≥500 µg/ml. EM presented a profile between those observed with E1 and E2. In addition, E1, E2 and EM, 10-1000 µg/ml, inhibited expansion of S. aureus. Furthermore, E1, tested in HA substrates colonized with osteoblastic cells, causing increase in cell population growth (10-100 µg/ml). E1 also exhibited antibacterial activity against S. aureus cultured over HA. CONCLUSIONS: Results showed that E. arvense extracts elicited inductive effects on human osteoblasts while inhibiting activity of S. aureus, suggesting a potentially interesting profile regarding bone regeneration strategies.

Staphylococcus aureus and Staphylococcus epidermidis adhesion to nanohydroxyapatite in the presence of model proteins.

Bacterial infections can have adverse effects on the efficacy, lifetime, and safety of an implanted device. The aim of this study was to investigate the initial adhesion of several strains, namely S. aureus and S. epidermidis, on two distinct types of nanohydroxyapatite (nanoHA), sintered at 725 °C and 1000 °C. A comparison was also made with nanohydroxyapatite having adsorbed fetal bovine serum (FBS), human fibronectin (FN) and human serum albumin (HSA). Adhered bacterial cells were examined by scanning electron microscopy and quantified as colony forming units after being released by sonication. The wettability of the sample surface with and without adsorbed protein was assessed by contact-angle measurements. NanoHA sintered at 1000 °C showed lower bacterial adhesion than this heat-treated at 725 °C. Adsorption of FBS onto the nanoHA surface caused a decrease in the adhesion of all strains on both materials. The bacterial adhesion patterns in the presence of FN were different for both nanoHA substrates; the adherence of the bacterial strains, except for the clinical strain of S. epidermidis, was significantly higher on nanoHA 1000 in comparison to nanoHA 1000 without protein and the bacterial adhesion on the FN-coated nanoHA 725 was lower in comparison to the bare nanoHA 725. The effect of HSA on bacterial adhesion was concentration and bacterial strain dependent.

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.

Supplementation of collagen scaffolds with SPARC to facilitate mineralization.

The extracellular matrix-associated protein, SPARC (Secreted Protein Acidic and Rich in Cysteine) is known to play a role in the mineralization of collagen in bone formation. The objectives of this study were to determine: 1) if SPARC supplementation of type 1 collagen scaffolds in vitro facilitated the binding of pre-formed HA nanoparticles added to the scaffolds; 2) if SPARC supplementation of the scaffolds enhanced the uptake of calcium and phosphorus from calcium phosphate solutions; and 3) if pretreatment in a calcium phosphate solution enhanced the subsequent binding of the nanoparticles. A related objective was to begin to determine the behavior of mesenchymal stem cells in the scaffolds when the constructs were grown in osteogenic medium. The calcium and phosphorus contents of the scaffolds were evaluated by inductively coupled plasma analysis, and the elastic modulus of the scaffolds determined by unconfined compression testing. Scaffolds were seeded with goat bone marrow-derived mesenchymal stem cells and the cell-seeded constructs grown in osteogenic medium. Supplementation of the scaffolds with as little as 0.008 % SPARC (by wt. of collagen) resulted in an increase in the binding of hydroxyapatite nanoparticles to the scaffold, but had no effect on incorporation of calcium or phosphorus from a calcium phosphate solution. The incorporation of hydroxyapatite nanoparticles into the scaffolds did not result in an increase in modulus. Supplementation of the scaffolds with SPARC and the increase in the binding of hydroxyapatite nanoparticles did not affect the proliferation of mesenchymal stem cells.

Proliferation and mineralization of bone marrow cells cultured on macroporous hydroxyapatite scaffolds functionalized with collagen type I for bone tissue regeneration.

This study concerns the preparation and in vitro characterization of functionalized hydroxyapatite (HA) porous scaffolds, which are intended to be used as drug-delivery systems and bone-regeneration matrices. Hydroxyapatite scaffolds were prepared using the polymer replication method, and, after being submitted to a specific sintering cycle, collagen Type I was incorporated on the surface. After the coating procedure, collagen was crosslinked using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) conjugation method. In this study, hydroxyapatite scaffolds with uncrosslinked and crosslinked Type I collagen were evaluated. Cell morphology and deposition of extracellular matrix were assessed by scanning electron microscopy, whereas cell distribution was visualized by means of methylene blue staining. MTS and total DNA quantification assays were used to evaluate the viability and proliferation of human bone marrow cells cultured on all the materials for 28 days. Results showed that the cells were able to adhere, proliferate, and form a mineralized matrix on the surface of all the materials. Furthermore, the cells were able to spread from one pore to another and form cell clusters. The results show that these scaffolds are good candidates to serve as drug delivery vehicles and for tissue engineering purposes.

Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering.

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.

In vivo evaluation of highly macroporous ceramic scaffolds for bone tissue engineering.

During the last decades, different materials of both natural and synthetic origin have been developed with the aim of inducing and controlling osteogenic differentiation of mesenchymal stem cells (MSCs). In order for that to happen, it is necessary that the material to be implanted obey a series of requirements, namely: osteoconduction, biocompatibility, and biodegradability. Additionally, they must be low-priced, easy to produce, shape, and store. Hydroxyapatite (HA) is a well known ceramic with a composition similar to the mineral component of bone and is highly biocompatible and easy to obtain and/or process. On the other hand, collagen is the main structural protein present in the human body and bone. In this study, a polymer replication method was applied and a highly porous HA scaffold was produced. Collagen was later incorporated to improve the biological properties of the scaffold while resembling the bone composition. The scaffolds were characterized by means of scanning electron microscopy, Fourier transform infrared spectroscopy and energy dispersive spectroscopy. In vitro and in vivo testing was performed in all scaffolds produced. The goal of this study was to evaluate the in vivo osteogenic potential of MSCs from two different species seeded on the different HA basedporous scaffolds with collagen type I. The resultsindicate that all scaffolds exhibit relevant bone formation, being more prominent in the case of the HA scaffolds.

Cationic liposome-DNA complexes as gene delivery vectors: Development and behaviour towards bone-like cells.

Modulation of the biological pathways responsible for fracture repair and osteogenisis may accelerate regeneration. Gene therapy is an alternative method for the release of osteogenisis-stimulating proteins into tissues. The development of vectors for gene release is still a problem in terms of ethics and techniques. In this work we evaluated whether cationic liposomes constitute a valuable strategy for the release of genetic material into bone tissue cells as non-viral vectors. Liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-2-dioleoyl-sn-glycero-3-phosphatidylethanolamine and DOTAP-cholesterol, and characterized according to their size, zeta potential, DNA protection capacity and cytotoxicity. Transfection studies were also carried out using pCMVbeta-gal plasmid in two osteoblastic cell lines (MG63 and MC3T3-E1) and in the 294T line, varying the charge ratio and the applied DNA dose. Inclusion of transferrin to increase the expression was also tested. The results suggest that there is great dependency between the transfection activity and the lipid formulation, the charge ratios of the complexes, the applied DNA dose and the cell type. There were even some differences concerning both osteoblastic lines under study. The cells of the MC3T3-E1 line present greater expression levels than the cells of the MG-63 line. The conjugation of the transferrin with the complexes contributes to the increase in transfection levels, possibly due to an increase in internalization of complexes. It is thus a good strategy for inducing the expression of specific genes in osteoblast-like cells.

Laser surface treatment of hydroxyapatite for enhanced tissue integration: surface characterization and osteoblastic interaction studies.

Biocompatibility has long been associated with surface microtopography, microtexture, and microchemistry. The surface topography eventually affects the nature and the intensity of the interactions that occur at biomaterial-biological interface (cell adhesion, mobility, spreading, and proliferation). Therefore, it is necessary to produce and work with controlled microtopographical surfaces that present reproducible microdomains of a dimension similar to that of the biological elements of interest (in this case, osteoblasts). There are a number of substrates that already have been studied in terms of surface topography; however, few studies are related to hydroxyapatite (HA) substrates. As it is well established, HA is a well-known ceramic that is extremely used in medical applications, namely implants and coatings. In this work, the surface topography of dense HA substrates was altered by using KFr excimer laser. The surface was characterized by atomic force microscopy and contact angle measurements, while the cell distribution and morphology was assessed by scanning electron microscopy and confocal laser scanning microscopy. Results revealed that the surface is characterized by a homogeneous columnar structure with high specific area. Moreover, cells were able to attach and spread on the surface of the samples, and gradually grow into nearly confluent monolayers.