M.P. Ferraz

Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions.

Staphylococcus comprises up to two-thirds of all pathogens in orthopedic implant infections and they are the principal causative agents of two major types of infection affecting bone: septic arthritis and osteomyelitis, which involve the inflammatory destruction of joint and bone. Bacterial adhesion is the first and most important step in implant infection. It is a complex process influenced by environmental factors, bacterial properties, material surface properties and by the presence of serum or tissue proteins. Properties of the substrate, such as chemical composition of the material, surface charge, hydrophobicity, surface roughness and the presence of specific proteins at the surface, are all thought to be important in the initial cell attachment process. The biofilm mode of growth of infecting bacteria on an implant surface protects the organisms from the host immune system and antibiotic therapy. The research for novel therapeutic strategies is incited by the emergence of antibiotic-resistant bacteria. This work will provide an overview of the mechanisms and factors involved in bacterial adhesion, the techniques that are currently being used studying bacterial-material interactions as well as provide insight into future directions in the field.

CaO-P2O5 glass hydroxyapatite double-layer plasma-sprayed coating: In vitro bioactivity evaluation

Double-layer composite coatings composed of a P2O5-based glass/Ca10(PO4)6(OH)2 (HA) mixture top layer and a simple HA underlayer, on Ti-6Al-4V substrates, were prepared using a plasma-spraying technique. The in vitro bioactivity of these coatings was assessed by immersion testing in simulated body fluid. Both scanning electron microscopy (SEM) analysis and the ionic solution changes followed by atomic absorption spectroscopy and the molybdenum blue method demonstrated that these composite coatings induce a faster surface Ca-P layer formation than the simple HA coatings used as a control. X-ray photoelectron spectroscopy (XPS) analysis demonstrated that the Ca-P layer formed was apatite. The combination of SEM and XPS analyses showed that the apatite layer was a calcium-deficient hydroxyapatite with a Ca/P ranging from 1.3 to 1.4 with CO3(2-) groups contained in the structure.

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.

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.

Effect of chemical composition on hydrophobicity and zeta potential of plasma sprayed HA/CaO-P2O5 glass coatings.

Multilayered plasma sprayed coatings on the surface of Ti-6Al-4V alloys have been prepared, which were composed of an underlayer of HA and a surface layer of a CaO-P2O5 glass-HA composite, with 2 or 4wt% of glass. Contact angle and surface tension variation with time, for both water and a protein solution, were determined by the sessile and pendent drop methods respectively using the ADSA-P software. Wettability studies showed that hydrophobicity of the coatings increase with the glass addition. The work of adhesion of albumin was also altered in a controlled manner by the addition of the CaO-P2O5 glass, being lower on the composite coatings than on HA. Zeta potential (ZP) results showed that composite coatings presented a higher net negative charge than HA coatings and that ZP values were also influenced by the content of the glass. This study demonstrated that the surface properties of those coatings may be modified by the addition of CaO-P2O5 glass.

Flow cytometry analysis of the effects of pre-immersion on the biocompatibility of glass-reinforced hydroxyapatite plasma-sprayed coatings.

Multilayered coatings composed of mixtures of HA and P2O5-based bioactive glasses are of potential clinical benefit in orthopaedic and dental surgery. Pre-immersion of these materials has been reported to further enhance their efficacy in vivo, although the precise biological effects of this treatment are not yet known. In this study we have therefore prepared double-layer plasma-sprayed coatings and evaluated the effects of pre-immersion on the growth and function of human osteosarcoma cells in vitro, using the MTT assay and flow cytometry analysis, respectively. The results showed that the increase in numbers of viable cells was the same or elevated following incubation on the pre-immersed HA and glass-reinforced HA coatings compared with the non-immersed materials. In addition, the expression of bone sialoprotein and fibronectin, two key connective tissue antigens, was up-regulated in cultures grown on the pre-immersed surfaces compared with the non-treated materials. Moreover, cell numbers and antigen expression both improved as the proportion of glass increased, particularly in the pre-immersed samples. Our findings thus suggest that the immersion treatment of these materials appeared to improve the response of these bone-like cells.

In vitro growth and differentiation of osteoblast- like human bone marrow cells on glass reinforced hydroxyapatite plasma-sprayed coatings

Human osteoblastic bone marrow cells were cultured for periods of up to 28 days in control conditions and on the surface of a glass reinforced hydroxyapatite composite (HA/G1) and commercial hydroxyapatite (HA) plasma-sprayed coatings, in the “as-received” condition and after immersion treatment in culture medium for 21 days. Cultures were characterized for total protein content and alkaline phosphatase activity. Scanning electron microscope analyses were performed on control cultures, seeded materials and materials incubated in the absence of cells. Culture media were analyzed for total and ionized calcium and phosphorus concentrations throughout the incubation period. Immersion of HA/G1 and HA coatings in culture medium resulted in significant alterations to the levels of calcium and phosphorus in the medium, leading to surface modifications. However, seeded material samples showed significant differences in the pattern of variation of the levels of these species. Cell proliferation was observed in the “as-received” HA/G1 composite, but cell mediated formation of mineral deposits was not proved. In contrast, “as-received” HA hardly supported cell growth. Previously immersed material samples showed cell proliferation and evidence of biological formation of mineral deposits. However, the HA/G1 composite presented better surface characteristics for cell growth as the behavior of bone marrow cells was closer to that observed in control cultures.

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.

Flow cytometry analysis of effects of glass on response of osteosarcoma cells to plasma-sprayed hydroxyapatite/CaO-P2O5 coatings

Multilayered coatings composed of mixtures of hydroxyapatite (HA) and P2O5-based bioactive glasses offer potential clinical benefits in orthopedic and dental surgery. In this study double-layer plasma-sprayed coatings were prepared and the biological response evaluated in tissue cul- ture using two human osteosarcoma cell lines, MG63 and HOS TE85 (HOS). The cells were cultured on the materials and the effects on cell growth were determined using a spec- trophometric assay of a mitochondrial enzyme that is active in viable cells. While none of the materials influenced the growth of the MG63 cells, the HOS cells appeared to un- dergo less proliferation on all the HA materials. Flow cy- tometry analysis was carried out using rabbit antibodies against osteonectin, osteopontin, bone sialoprotein, fibro- nectin, and collagen type I to measure the effects of the materials on key cellular functions. The results showed that the materials downregulated the expression of these extra- cellular matrix antigens by MG63 cells whereas they had less effect on the HOS cells compared to the same cells incubated on a plastic surface. Notably, with both cell lines the com- posite with the higher percentage of glass restored the pro- duction of connective tissue proteins to levels that were more similar to those present in the control cells.

HA and double-layer HA-P2O5/CaO glass coatings: influence of chemical composition on human bone marrow cells osteoblastic behavior

Human osteoblastic bone marrow derived cells were cultured for 28 days onto the surface of a glass reinforced hydroxyapatite (HA) composite and a commercial type HA plasma sprayed coatings, both in the “as-received” condition and after an immersion treatment with culture medium during 21 days. Cell proliferation and differentiation were analyzed as a function of the chemical composition of the coatings and the immersion treatment.Cell attachment, growth and differentiation of osteoblastic bone marrow cells seeded onto “as-received” plasma sprayed coatings were strongly affected by the time-dependent variation of the surface structure occurring during the first hours of culture. Initial interactions leading to higher amounts of adsorbed protein and zeta potential shifts towards negative charges appeared to result in surface structures with better biological performance. Cultures grown onto the pretreated coatings showed higher rate of cell proliferation and increased functional activity, as compared to those grown onto the corresponding “as-received” materials. However, the cell behavior was similar in the glass composite and HA coatings.The results showed that the glass composites present better characteristics for bone cell growth and function than HA. In addition, this work also provide evidence that the biological performance of the glass composites can be modulated and improved by manipulations in the chemical composition, namely in the content of glass added to HA.