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Plasma sprayed wollastonite/TiO2 composite coatings on titanium alloys.

Wollastonite/TiO2 composite coatings were prepared using plasma spraying technology onto Ti-6Al-4V substrate. The composite coatings exhibit obvious lamellar structure with alternating wollastonite coating and TiO2 coating. No obvious cracks exist on the interface between coatings and substrate. In the case of composite coatings, the primarily crystalline phases of the coatings are wollastonite and rutile, indicating wollastonite and TiO2 did not react during plasma spraying process. Some of rutile in the powders transforms into anatase due to plasma spraying. The mean bond strength of the composite coatings is higher than 30 MPa. The Vickers microhardness of coatings increase with the increase in the content of TiO2. Wollastonite/TiO2 composite coatings were soaked in simulated body fluid to examine their bioactivity. Carbonate-containing hydroxyapatite (CHA) layer was formed on the surface of the wollastonite and W7T3 coatings soaked in simulated body fluid, while was not formed on the surface of the TiO2 and W3T7 coatings after immersion. In addition, a rich-silica layer appeared at the interface of CHA and wollastonite and W7T3 coatings. In order to investigate the cytocompatibility of the coatings, osteoblast was seeded onto the surface of the coatings. The scanning electron microscopy observation showed that the addition of wollastonite promote the proliferation of osteoblast. It is enough to prove that the wollastonite and wollastonite/TiO2 composite coatings possess excellent cytocompatibility.

Alloys↗

Chondrocyte behaviors on poly-L-lactic acid (PLLA) membranes containing hydroxyl, amide or carboxyl groups.

Hydrophilic groups, i.e. hydroxyl (-OH), carboxyl (-COOH) or amide (-CONH(2)) were introduced onto the poly-L-lactic acid (PLLA) membrane surfaces via the photo-induced grafting copolymerization of the corresponding monomers, i.e. hydroxyethyl methacrylate, methacrylic acid or acrylamide, respectively. Chondrocyte culture was used to study the correlation between the cell behaviors and the hydrophilic functional groups. The results showed that the cytocompatibility of the PLLA membranes with hydroxyl or amide groups on the surface was greatly improved compared to that of the original PLLA membrane. However, the PLLA membrane with carboxyl groups on the surface had even worse cytocompatibility though possessed a similar hydrophilicity.

Animals↗

Biocompatibility and enzymatic degradation studies on sulphated hyaluronic acid derivatives.

The biocompatibility and susceptibility to enzymatic degradation of new heparin-like polysaccharides obtained by hyaluronic acid sulphation (HyalSx) were evaluated. In particular, HyalSx cytotoxicity and cytocompatibility were assessed by the direct contact method using fibroblasts L929 and human endothelial cells. The results showed that hyaluronic acid derivatives are devoid of any cytotoxic effects on mouse fibroblasts and they are cytocompatible. The haemolysis test showed that the sulphated polysaccharides are not haemolytic. HyalSx susceptibility to enzymatic degradation was tested in the presence of both hyaluronidase and chondroitinase ABC. It was demonstrated that the introduction of sulphate groups along the hyaluronic acid chain makes the macromolecules resistant to enzymatic digestion.

Animals↗

Improved cell adhesion to ion beam-irradiated polymer surfaces.

A very strong improvement of the cell adhesion, spreading and proliferation was observed for ion beam irradiated surfaces of polyethersulphones and polyurethane. The improvement shows a characteristic ion fluence-dependence with a threshold around 1 x 10(15) ions cm-2. We have compared this improvement of surface cytocompatibility with the various ion-induced chemical and physical modifications of the polymeric surfaces, taking into account their characteristic fluence-dependence. The irradiation induced a severe compositional and chemical modification of the polymeric surfaces as detected by X-ray photoelectron spectroscopy. Contact angle measurements showed that the surface free energy was strongly modified by ion irradiation. The fluence-dependent formation of a hydrogenated amorphous carbon phase was demonstrated by Raman spectroscopy. Our findings indicate that neither the simple modification of the functional groups nor the mere elemental composition nor the modification of the surface free energy can explain the observed fluence-dependence of the cell adhesion enhancement. On the contrary, we show that this enhancement correlates with the formation of a highly specific ion-induced 'unsaturated' a-C:H phase. According to these findings, we suggest that the formation of a substantial amount of hydrogenated amorphous carbon phases is the major factor promoting the cytocompatibility of ion irradiated polymer surfaces.

Astrocytoma↗

In vitro and in vivo behaviour of Ca- and P-enriched anodized titanium.

The influence of different surface preparations on titanium biocompatibility and bone integration was evaluated. Commercially grade 2 titanium rods (diameter 2 mm, length: 3 mm), vacuum annealed and hydrofluoric acid etched was selected for its promising surface characteristics to achieve good direct osseointegration. Some rods were surface modified by Anodic Spark Discharge anodization and a thin layer (approximately 5 microm) of amorphous TiO2 containing Ca and P (Ti/AM) was obtained. Some of the Ti/AM specimens underwent a further hydrothermal treatment to produce a thin outermost layer (approximately 1 microm) of hydroxyapatite (Ti/AM/HA). Cytotoxicity tests (direct contact: ISO 10993-5) showed good cytocompatibility for all tested samples. Ti and tissue culture substrate + DMEM control, respectively, were associated with a significant higher proportion of attached cells than Ti/AM and Ti/AM/HA (P < 0.0005), but this was in the normal range of 10-20% of unattached cells for cytocompatible materials. Histomorphometric analysis conducted on samples inserted in the cancellous bone of distal femoral epiphysis of Sprague-Dawley rats gave the following results at 4 and 8 weeks: Affinity index (AI%) data proving the surface osteconductive properties of non-anodized acid etched Ti (AI-4 weeks: 67.1 +/- 17.0%; AI-8 weeks: 74.8 +/- 11.5%). Ti/AM samples showed the lowest values (AI-4 weeks: 45.8 +/- 15.9%; AI-8 weeks: 68.5 +/- 13.6%) while the best performances of the Ti/AM/HA samples (AI-4 weeks: 60.4 +/- 21.8%; AI-8 weeks: 79.5 + 9.37%) indicated that hydroxyapatite allowed a higher bone to implant contact respect to Ti only. Further investigations should be performed in order to better understand the mechanism of observed in vitro behaviour and to achieve information on long-term osseointegration process.

Analysis of Variance↗

Improvement of titanium alloy for biomedical applications by nitriding and carbonitriding processes under glow discharge conditions.

Although titanium alloys are used in medicine, they present low wear resistance. In this paper we present the results of studies on surface layers produced by nitriding at three different temperatures, and by carbonitriding under glow discharge conditions in order to improve wear resistance, hardness, and to modulate microstructure and chemical composition of surface layers. A cell culture model using human fibroblasts was chosen to study the effect of such treatments on the cytocompatibility of these materials. The results showed that nitrided and carbonitrided surface layers were cytocompatible. Modulation of surface microstructure by temperature in the nitriding process and chemical composition of surface layers by carbonitriding led to differences in cellular behaviour. Cell proliferation appeared to be slightly reduced from the 6th day of culture on nitrided surfaces produced at 730 degrees C and 1000 degrees C, however after 12 days of culture, the best growth was on surface layers produced at 850 degrees C. The best viability was observed on the carbonitrided layer. The orientation and shape of the cells corresponded to surface topography. Nitriding and carbonitriding under glow discharge conditions may constitute interesting techniques allowing the formation of surface layers on parts with sophisticated shapes. They may also permit modulating surface topography in a way improving the features of titanium alloys for various applications in medicine.

Journal Article↗

The influence of polymeric membrane surface free energy on cell metabolic functions.

In membrane bioartificial organs using isolated cells, polymeric semipermeable membranes are used as immunoselective barriers, means for cell oxygenation and also as substrata for adhesion of anchorage-dependent cells. The selection of cytocompatible membranes that promote in vitro cell adhesion and function could be dependent on its membrane properties. In this study we investigated the physicochemical aspects of the interaction between the membrane and mammalian cells in order to provide guidelines to the selection of cytocompatible membranes. We evaluated the metabolic behavior of isolated liver cells cultured on various polymeric membranes such as the ones modified by protein adsorption. The physico-chemical properties of the membranes were characterized by contact angle measurements. The surface free energy of membranes and their different parameters acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) were calculated according to Good-van Oss's model. The adsorption of protein modified markedly both contact angle and membrane surface tension. In particular, membrane surface free energy decreased drastically with increased water contact angle. For each investigated membrane we observed that liver specific functions of cells improve on hydrophilic membrane surfaces. For all investigated membranes the rate of ammonia elimination increased with increasing of membrane surface free energy.

Journal Article↗

Evaluation of cellular affinity and compatibility to biodegradable polyesters and Type-II collagen-modified scaffolds using immortalized rat chondrocytes.

Immortalized rat chondrocytes (IRCs) were employed to evaluate the cytocompatibility of different biodegradable polyester scaffolds for chondrocyte seeding and cartilage tissue engineering in vitro due to the limitation of using freshly harvested chondroctyes. Cells were seeded onto the films and the porous substrates as well as into the three-dimensional scaffolds made of the biodegradable polyesters including poly(l-lactide) (PLLA) and two poly(lactide-co-glycolide)s (PLGAs). The materials were characterized by water contact angle, electron spectroscopy for chemical analysis (ESCA), and microscopy. PLGA50/50, one of the PLGAs, had the largest cell numbers at 24 h and 96 h (close to the tissue culture polystyrene control), possibly due to its lower contact angle, higher oxygen/carbon (O/C) atomic ratio, and larger degradation rate. When the surface was further modified by cross-linked Type-II collagen, cell population was significantly enhanced (two- to fourfold). The adhesion and proliferation behavior of IRCs on different materials was parallel to that of rabbit chondrocytes, but was more reproducible in general. IRCs are thus suitable for evaluation of different polymer scaffolds. Despite the favorable cytocompatibility of PLGA50/50, blending with a small portion of PLLA is required for easy fabrication and collagen modification. Scaffolds made of blended materials by freeze-drying procedure with the surface modified by cross-linked Type-II collagen were demonstrated as the ideal templates for chondrocyte seeding in our study.

Animals↗

Poly-L-lactide surfaces subjected to long-term cell cultures: cell proliferation and polymer degradation.

Short term cell cultures are usually grown in contact with biomaterials to assess cytocompatibility. Depending on the rate of material degradation or corrosion, the time of culture can be a key-point in the method which, if too short, may not show any effect of the released material on the cells. A long term culture was therefore carried out with L929 fibroblast cells in contact with PLLA/PDLA samples for up to eight months. The degradation was measured in terms of shear-strength properties, intrinsic viscosity of the material and its cristallinity. The effect of the material on the cells was evaluated by measuring the growth rate of the cells. A significant decrease in the shear strength of the material was measured after three months. The rate of modification of the intrinsic viscosity was regular and decreased progressively throughout the culture period. Differential scanning calorimetry showed that the samples were initially essentially amorphous and that contact with the cell culture and its medium did not change its crystallinity level. The growth rate of the cells was not modified by the presence of the material when compared to the control. This study showed this material to be cytocompatible for a long period of time, even after detection of modifications of its physico-chemical properties.

Animals↗

In vitro and in vivo evaluation of a small caliber vascular prosthesis fixed with a polyepoxy compound.

A small caliber vascular prosthesis obtained from an ovine internal thoracic artery (3.8-4.5 mm ID) fixed with a polyepoxy compound and treated with heparin has been evaluated. Cytocompatibility was evaluated in vitro using human endothelial cells (HEC). HEC were obtained from human saphenous vein and cultivated in culture medium supplemented with 25% human serum. Graft segments were rinsed using a standard protocol proposed by the manufacturer. Tissue reaction was tested on a rabbit model of subcutaneous implantation. The patency rate and healing patterns were evaluated comparatively with polytetrafluorethylene (PTFE) 4 mm ID prosthesis in a canine model of carotid interposition. Cytocompatibility assay showed that there was low adhesion on vascular grafts (20 +/- 2% of endothelial cells seeded) and no growth of HEC on the graft surface. The graft patency rate was 55% in both groups, and actuarial freedom from occlusion was not different at 3 months (37.7 +/- 15% in Denacol-fixed grafts versus 38.1 +/- 14% in PTFE). Histological studies on the biological grafts shows a frequent neointimal hyperplasia at the anastomosis (5/12), a lack of endothelial cells lining the graft surface, a good preservation of the media, and a moderate inflammatory response in the adventicia. The Denacol-fixed graft has presented excellent surgical properties and preservation of the histological structure. Nevertheless, the patency rate was not improved when compared with the PTFE control graft.

Animals↗

Evaluation of chitosan-alginate-hyaluronate complexes modified by an RGD-containing protein as tissue-engineering scaffolds for cartilage regeneration.

In this study, a series of natural biodegradable materials in the form of chitosan (C)-alginate (A)-hyaluronate (H) complexes are evaluated as tissue-engineering scaffolds. The weight ratio of C/A is 1 : 1 or 1 : 2. Sodium hyaluronate is mixed in 2%. The complexes can be cast into films or fabricated as scaffolds. Their surface can be further modified by an Arg-Gly-Asp (RGD)-containing protein, a cellulose-binding domain-RGD (R). Cytocompatibility tests of the films are conducted using immortalized rat chondrocyte (IRC) as well as primary articular chondrocytes harvested from rabbits. The neocartilage formation in cell-seeded scaffolds is examined in vitro as well as in rabbits, where the scaffolds are implanted into the defect-containing joints. The results from cytocompatibility tests demonstrate that R enhances cell attachment and proliferation on C-A and C-A-H complex films. Complex C1A1HR (C : A = 1 : 1 with H and R) has better performance than the other formulation. Cells retain their spherical morphology on all C-A and C-A-H complexes. The in vitro evaluation of the seeded scaffolds indicates that the C1A1HR complex is the most appropriate for 3-D culture, manifested by the better cell growth as well as higher glycosaminoglycan and collagen contents. When the chondrocyte scaffolds are implanted into rabbit knee cartilage defects, partial repair is observed after 1 month in C1A1HR as well as in C1A1 (C : A = 1 : 1 without H and R) scaffolds. The defects are completely repaired in 6 months when C1A1HR constructs are implanted. It is concluded that C1A1HR is a potential tissue-engineering scaffold for cartilage regeneration.

Alginates↗

Aptamer-based capture molecules as a novel coating strategy to promote cell adhesion.

The improvement of the cytocompatibility of medical implants is a major goal in biomaterials research. During the last years many researchers worked on the fascinating approach to seed the respective cell types on various artificial substrates before implantation. For instance, cell-seeded implants are supposed to be better candidates for transplantable bone substitutes than conventional artificial bone grafts. To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for medical implants. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). Aptamers do have high binding affinity and selectivity to their target. In our study, human osteoblasts from osteosarcoma tissue were used as a target to create the aptamer. Single aptamer mediated cell sorting assays showed the binding affinity with osteoblasts. Additionally, the aptamers immobilized on tissue culture plates could capture osteoblasts directly and rapidly from the cell solution. This model proves that aptamer coated artificial surfaces can greatly enhance cell adhesion. We assume that this strategy is capable to improve the clinical application of tissue engineered implants.

Base Sequence↗

Photografting of poly(hydroxylethyl acrylate) onto porous polyurethane scaffolds to improve their endothelial cell compatibility.

In order to improve the cytocompatibility of polyurethane (PU) porous scaffolds obtained by thermally-induced phase separation, poly(hydroxylethyl acrylate) (PHEA) was covalently immobilized by grafting copolymerization of HEA by photo-oxidation of the scaffolds and initiation of UV light. FT-IR-ATR spectroscopy and X-ray photoelectron spectroscopy (XPS) characterizations confirmed the occurrence of the grafting co-polymerization of HEA on the porous PU scaffolds. The measurement of water absorption demonstrated the improvement of the hydrophilicity after grafting with PHEA. The results obtained in a human umbilical vein endothelial cell (HUVEC) culture proved that the porous PU scaffold modified with the hydrophilic PHEA had better cytocompatibility than the control. The influence of surface pore size on the HUVEC growth behavior was assessed regarding cell anchorage, proliferation and viability, as well as morphology. An overall increase of cell number and viability with the decrease of the surface pore size was found.

Absorption↗

Properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films modified with polyvinylpyrrolidone and behavior of MC3T3-E1 osteoblasts cultured on the blended films.

A series of composite films of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) modified with polyvinylpyrrolidone (PVP) was prepared by varying the ratio of constituents, and their properties and cytocompatibility were evaluated. The hydrophilicity of the blended materials surfaces increased and the amounts of fibronectin and laminin adsorbed on the materials surface increased remarkably compared with PHBHHx. FT-IR spectra of the blended films showed a new band, implying that a surface physical interpenetrating network structure had formed. Scanning electron microscopy showed that there were dense pits and holes on the blended films surface. For the films of PHBHHx with 20 wt% and 40 wt% PVP, MTT assay indicated that PVP enhanced cell adhesion and proliferation, but that the effects were impaired by excessive PVP. The results suggested that proper addition of PVP increased the cytocompatibility of PHBHHx because the material surface had increased hydrophilicity and presented an appropriate morphology.

3-Hydroxybutyric Acid↗

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue.

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.

Alkaline Phosphatase↗

The effects of crosslinking density on cartilage formation in photocrosslinkable hydrogels.

Photoencapsulation of chondrocytes to produce tissue engineered cartilage provides many benefits including rapid polymerization times, the ability to fabricate complex architectures in vivo, and spatial and temporal control during polymerization. Recently, we have examined the cytocompatibility of several photoinitiation schemes and found that low photoinitiator concentrations and light intensities in the ultraviolet and visible range are cytocompatible. In this work, we are currently investigating photocrosslinkable hydrogels based on poly(vinyl alcohol) (PVA) and poly(ethylene oxide) (PEO) as scaffolds for tissue engineering cartilage. In particular, the influence of the network crosslinking density, swelling ratio, and chemical composition on the ability of encapsulated chondrocytes to form extracellular matrix is examined. The cartilage produced in these hydrogels will be quantified using biochemical assays that measure DNA content and the amount of sulfated glycosaminoglycans and total collagen in the extracellular matrix. We have demonstrated that chondrocytes encapsulated in a polymer scaffold made from a 20 wt% solution of PEODM/PEO (40 wt% dimethacrylated PEO (MW 3400)/60 wt% PEO (MW 100 K)) form cartilage, and after four weeks the results based on the wet weight of cartilage were approximately 0.03 million cells/mg cartilage, approximately 1.5% glycosaminoglycans and approximately 4.5% total collagen.

Biomedical Engineering↗

A new porous titanium-nickel alloy: Part 1. Cytotoxicity and genotoxicity evaluation.

Porous titanium-nickel (PTN) alloys represent new biomaterials for long-term implantation. Their porosity properties might confer them the capacity to trigger fluid capillarity, tissue ingrowth, as well as good tissue-implant apposition and fixation. Before PTN materials are used as long-term implants, their biocompatibility level must be assessed. In this study, porous titanium-nickel was therefore extracted in a saline semi-physiological solution and materials were evaluated for potential cytotoxicity and genotoxicity reactions. The cytocompatibility elution test was performed in order to determine PTN toxic potential at the in vitro cellular level: no reactivity was detected in cell layers exposed to PTN extracts or the negative controls. In parallel, the genocompatibility of porous titanium-nickel was evaluated using three different assays in order to assess potential damage at the DNA level: the test for chemical induction of chromosome aberrations, the Salmonella typhimurium and Escherichia coli reverse mutation assay, and the mouse micronucleus test. No significant increase in the number of chromosomal aberrations, bacterian revertant colonies, or micronuclei was observed in presence of PTN extracts when compared to negative control exposition. Based on the above results, porous titanium-nickel can be considered completely cytocompatible and genocompatible, and therefore represents a good candidate for long-term implantation.

Animals↗

[Non-biodegradable drug-sustained capsular ring for prevention of secondary cataract. Part I: In vitro evaluation].

PURPOSE: The appearance of secondary cataract constitutes the most common complication of cataract surgery since the advent of phacoemulsification. Prevention is today one of the most crucial challenges of research in ophthalmology. We have developed a drug-sustained capsular ring releasing 5-fluorouracil (5-FU). This system aims to combine mechanical and pharmacological properties with the ability to interfere on the lens epithelial cellular processes of secondary cataract. This first article will report on the in vitro evaluation of this system. MATERIALS AND METHOD: Development of a hydrogel that can serve as a mechanical support and as a release of an active agent requires a comparative trial of multiple hydrophobic products providing rigidity to the ring and of hydrophilic products providing the ability to release a drug. The choice of 5-FU as a non-proliferate agent was based on its well-known pharmacological properties and its wide use in ophthalmology. Evaluation comprised adjustment of the adequate copolymer, then of the loading phase, and finally assessment of 5-FU release. The mechanical characteristics and cytocompatibility of the ring were also assessed. RESULTS: The characteristics of drug release differed depending on the water content and resulting volume of the copolymer. The pharmaceutical and mechanical testing led to selecting the HPMA-MMA combination (75:25). However, a burst effect within the first 2 hours was observed in all cases. That limit was eliminated by creating a biodegradable surface coating of Poly Lactid-co-Glycolid (PLG 50:50) that could provide a positive barrier effect against migration and a controlled continual release of 0.25 microg/h extended to 9 days. Mechanical tests of tension, compression, and traction showed the crucial influence of the ring's hydration state. The cytocompatibility study showed no evidence of cellular toxicity. DISCUSSION/CONCLUSIONS: The synthesized copolymer is able to release a constant, regular and safe rate of 5-FU, higher than CI50 over a period of 9 days for a cell proliferation phase of 3-5 days. We adjusted the ring associating the mechanical properties and the ability to release 5-FU, allowing further evaluation on animal models, the subject of the second part of this report.

Biocompatible Materials↗