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In vitro and ex vivo intestinal tissue models to measure mucoadhesion of poly (methacrylate) and N-trimethylated chitosan polymers
Keely, S., A. Rullay, et al. (2005), Pharm Res 22(1): 38-49.
Abstract: PURPOSE: The adhesion of a range of polymers based on poly(2-(dimethylamino-ethyl) methacrylate (pDMAEMA) was assessed using human mucus-secreting and non mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12) and HT29 monolayers, as well as excised non-everted intestinal sacs from rats. Differentiation of mucoadhesion from bioadhesion was achieved by pre-treatment with the mucolytic agent, N-acetyl cysteine (NAC). Adherence of pDMAEMA polymers was compared to that obtained with the mucoadhesive, N-trimethylated chitosan (TMC). METHODS: The quantity of adherent coumarin 343-conjugated polymers to HT29, E12, and intestinal sacs was measured by fluorescence. Confocal laser scanning microscopy (CLSM), light microscopy, and fluorescent microscopy were used to provide direct evidence. Measurements of transepithelial electrical resistance (TEER), permeability to FITC-dextran 4000 (FD-4), and the release of lactate dehydrogenase (LDH) were used to assess potential cytotoxicity of polymers. RESULTS: Adherence of unquaternized and of 10%, 24%, and 32% methyl iodide-quaternized pDMAEMA polymers was measured in E12, HT29, and sacs. All pDMAEMA polymers showed significantly higher levels of adhesion to mucus (mucoadhesion) than to epithelium (bioadhesion). Colocalization of pDMAEMA with mucus was confirmed in E12 by microscopy. TMC showed equally high levels of mucoadhesion as unquaternized and 24% quaternized pDMAEMA, but displayed higher levels of bioadhesion. pDMAEMA-based polymers demonstrated lower levels of adherence to E12 and rat sacs in the presence of NAC, whereas adherence of TMC was unchanged. pDMAEMA significantly decreased the permeability of FD-4 across E12 monolayers and sacs and was less cytotoxic in E12 than in HT29. In contrast, TMC increased the permeability of FD-4 across E12 and sacs and was less cytotoxic in E12 than in HT29. CONCLUSIONS: Human mucus-producing E12 monolayers can be used to assess polymer mucoadhesion and give similar data to isolated rat intestinal sacs. pDMAEMA displayed similar levels of mucoadhesion and lower levels of bioadhesion than a chitosan derivative and it was not cytotoxic. pDMAEMA decreased FD-4 flux in the presence of mucus, whereas TMC increased it. The combination of mucus and methacrylate polymers appears to increase barrier function of the apical membrane.

In vitro and in vivo antitumorigenic activity of a mixture of lysine, proline, ascorbic acid, and green tea extract on human breast cancer lines MDA-MB-231 and MCF-7
Roomi, M. W., V. Ivanov, et al. (2005), Med Oncol 22(2): 129-38.
Abstract: Current treatments are generally ineffective once breast cancer has metastasized; median survival is reduced to 2-3 yr. Previous research studies demonstrating potent synergistic antitumor activity of lysine, proline, ascorbic acid, and epigallocatechin gallate prompted us to investigate the in vivo inhibitory effect of a nutrient mixture containing lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate (NM) on the growth of human cancer xenografts in female athymic nude mice. Five to six week old female mice were inoculated with 3x106 breast cancer cells MDA-MB-231. After injection, the mice were randomly divided into two groups A and B; group A was fed a regular diet and group B with the regular diet supplemented with 0.5% of the nutrient mixture (NM). Four weeks later, the mice were sacrificed, and their tumors were excised, weighed, and processed for histology. We also tested the effect of NM in vitro on estrogen-receptor positive (ER+) MCF-7 and estrogen-receptor negative (ER-) MDA-MB-231 breast cancer cell lines by measuring: cell proliferation by MTT assay, expression of MMPs by gelatinase zymography, invasion through Matrigel, and VEGF by ELISA. MCF-7 cells were also treated with estradiol to study enhanced invasion and expression of MMPs and VEGF. Results showed that NM inhibited the growth and reduced the size of tumors in female nude mice by 27%. Furthermore, histological evaluation revealed increased mitotic index, MMP-9 and VEGF secretion, and PAS material (mucin) in the control group tissues. In vitro studies showed NM inhibited MDA-MB-231 cell growth by 34% at 500 microg/mL and MCF-7 cell growth by 18% at 1000 microg/mL. Invasion of MDA-MB-231 through Matrigel was inhibited by 50%, 60%, and 95% by 10, 50, and 100 microg/mL of NM, respectively. The results of this study demonstrated that the nutrient mixture tested significantly suppressed tumor growth of breast cancer cells in female athymic nude mice and significantly inhibited MMP expression, angiogenesis, and invasion in breast cancer cells, in vitro, offering promise for therapeutic use in the treatment of breast cancer.

In vitro and in vivo bactericidal activities of vancomycin dispersed in porous biodegradable poly(epsilon-caprolactone) microparticles
Le Ray, A. M., H. Gautier, et al. (2005), Antimicrob Agents Chemother 49(7): 3025-7.
Abstract: Treatment of methicillin-resistant Staphylococcus aureus osteomyelitis requires a prolonged antibiotic therapy with vancomycin. Because of its weak diffusion, the in situ implantation of vancomycin could be interesting. The activity of vancomycin encapsulated in microparticles was evaluated in vitro and in vivo on rabbit osteomyelitis and showed a good activity compared to intravenous administration.

In vitro and in vivo characterization of a novel biocompatible polymer-lipid implant system for the sustained delivery of paclitaxel
Ho, E. A., V. Vassileva, et al. (2005), J Control Release 104(1): 181-91.
Abstract: Recently, a novel chitosan-based implantable formulation (chitosan-ePC) was developed to provide controlled, local release of paclitaxel (PTX) for the treatment of ovarian tumors. Hence, the objective of this study was to evaluate this delivery system in vitro in human ovarian SKOV-3 cells and in vivo in mice with intraperitoneal implants of drug-free or 14C-PTX-chitosan-ePC films. In vitro, 14C-PTX-chitosan-ePC implants (10 mg) provided zero-order constant release of 0.92+/-0.03 pg/day PTX over 5 days. Released PTX retained dose-dependent activity; effectively inhibiting SKOV-3 proliferation with an ED50 of 211 ng/ml of released PTX. Drug-free implants did not affect cell viability or cell morphology of SKOV-3 cells. A sustained, zero-order release of PTX was also seen in vivo over a 2 week period in mice implanted with 14C-PTX-chitosan-ePC films. Correlations between the in vitro and in vivo release of PTX was highly significant (R2 = 0.975). After 2-4 weeks, mice with chitosan-ePC implants did not demonstrate any signs of encapsulation, inflammation or infection. Overall, our in vitro and in vivo results demonstrated zero-order drug release and biocompatibility of the novel chitosan-ePC film. This indicates potential usefulness of chitosan-ePC implants in the sustained and local delivery of anti-neoplastic agents.

In vitro and in vivo evaluation of Adacolumn cytapheresis in healthy subjects
Ramlow, W., J. Emmrich, et al. (2005), J Clin Apher 20(2): 72-80.
Abstract: Adacolumn is a medical device for adsorptive cytapheresis. It has been developed for selective adsorption of granulocytes and monocytes from peripheral blood of patients with immune disorders, such as autoimmune diseases and chronic inflammatory diseases. A double blind sham-controlled crossover study design was used in order to evaluate in vivo biological responses of leukocytes as well as biocompatibility during and after Adacolumn cytapheresis in healthy volunteers. In addition, experiments were undertaken to further evaluate leukocyte reactions to Adacolumn carrier (G-1: cellulose diacetate) beads in vitro. Six healthy volunteers, 4 males and 2 females, with a mean age of 26.7 years were randomly assigned to one of the two treatment arms in a crossover fashion. Three subjects received a single Adacolumn treatment, followed by a single sham treatment at an interval of 7 days. The other three subjects received the two treatments in reverse order. All subjects were followed up 7 days after the last treatment. Additionally, in vitro investigations were carried out using blood from the healthy donors to examine the effect of G-1 beads on granulocyte functions. In vitro exposure of human peripheral blood to G-1 beads caused downregulation of L-selectin expression and upregulation of Mac-1 expression on granulocytes, leading to a marked reduction of adhesive capacity of granulocytes to endothelial cells. The exposure also led to decreased granulocyte chemotactic activity to IL-8. The number of granulocytes and monocytes clearly decreased during Adacolumn cytapheresis. Granulocytes showed marked phenotypic changes of L-selectin(Low) and Mac-1(Hi) after passing through Adacolumn in vivo. Expression of TNF-alpha and chemokine receptors was downregulated. In addition, TNF-alpha and IL-1beta producing capacity of peripheral blood leukocytes was decreased after Adacolumn cytapheresis and these changes lasted even one week after the cytapheresis. The level of complement fragments, C3a and C5a, increased, while bradykinin concentration did not change during Adacolumn cytapheresis. Exposure of human peripheral blood to G-1 beads, both in vitro and in vivo, caused a significant reduction of adhesive capacity and proinflammatory cytokine producing capacity of peripheral blood leukocytes. Such changes were not observed after sham apheresis. Despite complement activation, tolerability of Adacolum cytapheresis was not influenced. These findings may at least partly explain the beneficial effect of Adacolumn cytapheresis in the treatment of autoimmune diseases.

In vitro and in vivo interactions of cells with biomaterials
Ziats, N. P., K. M. Miller, et al. (1988), Biomaterials 9(1): 5-13.
Abstract: The biocompatibility of materials at an implant site involves a complex interaction of cells and tissues with the biomaterial. This cell-cell and cell-polymer interaction evokes the release of mediators such as chemotactic and growth factors that elicit and sustain inflammatory responses at the implant site. In this review, we summarize the interaction of cells with biomaterials in vitro and in vivo.

In vitro and in vivo methods to determine the interactions of osteogenic cells with biomaterials
Oreffo, R. O. and J. T. Triffitt (1999), J Mater Sci Mater Med 10(10/11): 607-11.
Abstract: To assess new biomaterials for possible use as bone graft substitutes, a number of techniques allow interactions with osteoblastic cells to be studied, with respect to effects on proliferation and differentiation of osteoprogenitors. In vitro models include the use of bone explant cultures, fetal rat calvarial-derived osteoblast cells, primary stromal populations, transformed and non-transformed cell lines and immortalized osteoblast cell lines. However, these assessments are limited by the extent of osteogenic differentiation and bone formation that can be observed in vitro, species differences and phenotypic drift of cells cultured in vitro. The use of in vivo experimental systems such as the segmental/calvarial bone defect model, the subcutaneous implant model and the diffusion chamber implantation model circumvent some of these issues and, in the appropriate model, provide data on efficacy, biocompatibility and osteointegration of a biomaterial. The combination of in vitro and in vivo approaches together with the development of new cell labeling techniques, in particular the ability to genetically mark and select specific human bone cell populations provides new avenues for their potential evaluation in combination with appropriate biomaterials for clinical use. These in vitro and in vivo techniques are reviewed and those recently developed for assessment of human osteogenic cells should be applicable to many other cell systems where knowledge of specific human tissue or cell interactions with biomaterials is required.

In vitro and in vivo studies of cyclosporin A-loaded microspheres based on copolymers of lactide and epsilon-caprolactone: comparison with conventional PLGA microspheres
Li, Y., K. J. Zhu, et al. (2005), Int J Pharm 295(1-2): 67-76.
Abstract: A hydrophobic peptide, cyclosporin A (CyA), was incorporated in microspheres based on poly(lactide-b-epsilon-caprolactone) (P(LA-b-CL), LA/CL (in molar ratio): 78.7/21.3 and 48.1/51.9) and poly(lactide-co-glycolide) (PLGA, LA/GA: 80/20) using oil-in-water (O/W) emulsion solvent evaporation method. The microspheres were characterized by SEM, DSC and X-ray diffraction, and CyA release rate was determined by HPLC. It was revealed that CyA can be efficiently loaded into all the microspheres (exceed 96%). Compared to PLGA microspheres, P(LA-b-CL) microspheres liberated CyA more rapidly. Within the first day, about 75, 50 and 12% of CyA released from P(LA-b-CL) (48.1/51.9), P(LA-b-CL) (78.7/21.3) and PLGA microspheres, respectively, which can be attributed to the partial crystallization occurring in P(LA-b-CL) microspheres. CyA levels in whole blood were also tested. In comparison with PLGA microspheres, P(LA-b-CL) microspheres provided a higher blood level of CyA. The maximum CyA concentration in whole blood (approximately 520, 450 and 400 ng ml(-1) for P(LA-b-CL) (48.1/51.9) P(LA-b-CL) (78.7/21.3) and PLGA microspheres, respectively) was reached at the second day post administration. And then P(LA-b-CL) microspheres showed a constant CyA level (about 100-200 ng ml(-1)) for extended periods of time (several weeks). Such CyA-loaded P(LA-b-CL) microspheres displaying higher CyA concentration during the first few days and similar constant blood CyA level thereafter showed more advantages than those prepared with PLGA and could meet clinical needs more efficiently.

In vitro and in vivo studies of ibuprofen-loaded biodegradable alginate beads
Arica, B., S. Calis, et al. (2005), J Microencapsul 22(2): 153-65.
Abstract: The irritation effects of ibuprofen, a widely used non-steroidal anti-inflammatory drug (NSAID), were evaluated on mouse gastric and duodenal mucosa when suspended in 0.5% (w/v) sodiumcarboxymethylcellulose (NaCMC) solution and loaded in alginate beads. The ionotropic gelation method was used to prepare controlled release alginate beads of ibuprofen. The influence of various formulation factors on the encapsulation efficiency, as in vitro drug release and micromeritic properties, was investigated. Other variables included the alginate concentration, percentage drug loading and stirring speed during the microencapsulation process. Scanning electron micrographs of alginate beads loaded with ibuprofen showed rough surface morphology and particle sizes in the range of 1.15 +/- 0.4 - 3.15 +/- 0.6 mm. The yield of microspheres, as collected after drying, was generally 80-90%. Formulation code H showing t50% value of 3.5 h was chosen for in vivo trials because of the appropriate drug release properties. For in vivo trials, free ibuprofen (100 mg kg(-1)), blank and ibuprofen (100 mg kg(-1)) loaded alginate beads (formulation code H) were suspended in 0.5% (w/v) NaCMC solution and each group was given to six mice orally by gavage. NaCMC solution was used as a control in experimental studies. In vivo data showed that the administration of ibuprofen in alginate beads prevented the gastric lesions.

In vitro and in vivo studies on blends of isotactic and atactic poly (3-hydroxybutyrate) for development of a dura substitute material
Kunze, C., H. Edgar Bernd, et al. (2006), Biomaterials 27(2): 192-201.
Abstract: Blends of semicrystalline isotactic poly(3-hydroxybutyrate) (PHB) with amorphous atactic PHB (at-PHB) were prepared by solution-casting using 30%, 50% and 70% at-PHB, and were studied for medical applications. The mechanical properties of the blends including the elastic modulus and elongation at break are strongly affected by the blend composition. The elastic modulus decreases with increasing fraction of at-PHB in the blend from 3350 MPa in the case of PHB to 170 MPa of PHB/at-PHB (30/70). In contrast, the elongation at break increases from 2% in pure PHB up to 50% in the case of the blend with 70% at-PHB. The in vitro degradation is changed as well. The molecular weight of PHB/at-PHB (30/70) is reduced to 5% after 2 years storage in phosphate buffer compared to 35% for pure PHB stored at identical conditions. The in vitro cell vitality is slightly reduced depending on the composition. PHB/at-PHB blends with 30% and 50% at-PHB were selected as dura substitute in minipigs based on the results of the in vitro investigation and the mechanical testing. Patch films with a structured surface on one side were fabricated by a dipping-leaching method. Dura defects were clinically and histologically examined 3, 6, and 9 months after implantation, confirming defect closure, prevention of adhesions to brain tissue, and no signs of inflammation or malignant degeneration. The PHB-based patch materials fulfill the requirements which are necessary for a dura substitute.

In vitro antimycotic activity and nail permeation models of a piroctone olamine (octopirox) containing transungual water soluble technology
Dubini, F., M. G. Bellotti, et al. (2005), Arzneimittelforschung 55(8): 478-83.
Abstract: Several in vitro studies with a new medical device (Myfungar) containing 0.5% of piroctone olamine (CAS 68890-66-4, octopirox) in a hydroxypropyl chitosan hydroalcoholic solution were performed. The chemical name of the active ingredient is 1-hydroxy-4-methyl-6 (2,4,4-trimethylpentyl)-2(1H)-pyridone; combination with 2-amino-ethanol (1:1). The antimycotic activity was determined in the most common fungi responsible of nail infections such as Candida parapsilosis, Scopulariopsis brevicaulis or Trichophyton rubrum. The minimum inhibitory concentration (MIC), found by means of the broth dilution susceptibility method, ranged between 0.0003% and 0.006% for all pathogens considered. The in vitro permeation study was performed by using bovine hoof membranes inserted in a modified Gummer vertical permeation cell. The experiment showed, at 30 h, a retention of the product in the nail membranes by 11.1% of the applied dose. No piroctone permeation through bovine hoof membranes could be detected by HPLC due to the limit of quantitation of this method. On the other hand, permeation of nail membranes has been demonstrated by a biological assay: the study of in vitro permeation through bovine hoof membranes, performed by biological assay, showed dose-dependent inhibition rings of T. rubrum growth by the tested device, placed either on disks for antibiogram or on nail fragments. The placebo did not show any inhibition. In vitro experimental infection by T. rubrum showed a preventive activity of the tested device on fungal growth as well as a curative activity, as the pathogen was eradicated by the tested solution in previously infected nails.

In vitro anti-staphylococcal activity of heparinized biomaterials bonded with combinations of rifampicin
Fallgren, C., M. Utt, et al. (1998), Zentralbl Bakteriol 287(1-2): 19-31.
Abstract: Biomaterial implants in various human body tissues are highly susceptible to bacterial colonization. We report here on the coating of heparinized biomaterials with heparin binding extracellular matrix proteins giving special regard to the efficient adsorption and slow release of antibiotics. Heparin was partially degraded and the resulting fragments were covalently end-point attached to 0.5 cm long silicone biomaterial surface. Collagen type I was immobilized on the heparinized biomaterials and then cross-linked with acyl-azide or carbodiimide. Finally, the resulting biosurfaces were exposed to antibiotics, i.e. rifampicin in combination with cefuroxime, fusidic acid, ofloxacin or vancomycin, respectively. The antibiotic bonded biomaterials were evaluated for their anti-staphylococcal activity after elution in NaCl, serum or blood by measuring the zones of inhibition for S. epidermidis strain RP12. Furthermore, we examined the in-vitro colonization resistance to S. epidermidis RP12 for these combinations of rifampicin-bonded biomaterials by an ATP bioluminescence assay. The ATP measurements showed that initially adherent bacteria were eradicated from the polymer surface, for at least 24 or 48 h (fusidic acid > cefuroxime > vancomycin > ofloxacin). The anti-staphylococcal activity of rifampicin-fusidic acid bonded heparinized biomaterials seems of sufficient duration and efficacy to merit testing in an animal model.

In vitro assessment of corrosive properties of titanium as a biomaterial
Koike, M. and H. Fujii (2001), J Oral Rehabil 28(6): 540-8.
Abstract: Titanium (Ti) is thought to be a highly biocompatible material, and its clinical applications are becoming increasingly frequent. However, there have recently been some clinical papers reporting hypersensitivity and allergic reactions to Ti. The purpose of this study was to assess the corrosive properties of Ti in the intra-oral environment in vitro. Cast pure Ti specimens were immersed in artificial saliva, physiological saline solution, and 128 mmol x L(-1) of lactic, formic and acetic acids for 3 weeks at 37 degrees C with shaking. The colour, weight, surface morphologies and chemical binding state of specimens were observed before and after immersion. Marked discoloration was recognized on the surface of specimens immersed in formic acid, and a significant difference was found between the immersion solutions. Weight changes also varied with solutions; a tendency to increase in formic acid and to decrease in lactic acid. A slight loss was observed in specimens immersed in lactic acid and artificial saliva. The oxide layer composed mainly of Ti-oxide on the surface of the immersed specimens was thinnest in lactic acid, and thickest in formic acid. The present study indicates that both hydrogen evolution type and oxygen diffusion type corrosion on Ti surfaces are possible in the living body. (Our results also confirm some clinical studies reporting Ti accumulation in surrounding tissues and Ti causing allergic reactions.)

In vitro assessment of the enzymatic degradation of several starch based biomaterials
Azevedo, H. S., F. M. Gama, et al. (2003), Biomacromolecules 4(6): 1703-12.
Abstract: The susceptibility of starch-based biomaterials to enzymatic degradation by amylolytic enzymes (glucoamylase and alpha-amylase) was investigated by means of incubating the materials with a buffer solution, containing enzymes at different concentrations and combinations, at 37 degrees C for 6 weeks. Two polymeric blends of corn starch with poly(ethylene-vinyl alcohol) copolymer and poly(epsilon-caprolactone), designated by SEVA-C and SPCL, respectively, were studied. The material degradation was characterized by gravimetry measurements, tensile mechanical testing, scanning electron microscopy (SEM), and Fourrier transform infrared-attenuated total reflectance (FTIR-ATR). The degradation liquors were analyzed for determination of reducing sugars, as a result of enzyme activity, and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used to identify the degradation products. All of the analysis performed showed that starch polymeric blends are susceptible to enzymatic degradation, as detected by increased weight loss and reducing sugars in solution. alpha-Amylase caused significant changes on the overall mechanical properties of the materials, with a decrease of about 65% and 58% being observed in the moduli for SEVA-C and SPCL, respectively, when compared with the control (samples incubated in buffer only). SEM analysis detected the presence of fractures and pores at the material's surface as a result of starch degradation by amylolytic enzymes. FTIR spectra confirmed a decrease on the band corresponding to glycosidic linkage (-C-O-C-) of starch after incubation of the materials with alpha-amylase. In contrast, the incubation of the polymers in buffer only, did not cause significant changes on the material's properties and morphology. Comparing the two materials, SEVA-C exhibited a higher degradability, which is related to the physicochemical structure of the materials and also to the fact that the starch concentration is higher in SEVA-C. The identification of the degradation products by HPAEC-PAD revealed that glucose was the major product of the enzymatic degradation of starch-based polymers. alpha-Amylase, as expected, is the key enzyme involved in the starch degradation, contributing to major changes on the physicochemical properties of the materials. Nevertheless, it was also found that starch-based polymers can also be degraded by other amylolytic enzymes but in a smaller extent.

In vitro assessment of the osteointegrative potential of a novel multiphase anodic spark deposition coating for orthopaedic and dental implants
Sandrini, E., C. Morris, et al. (2005), J Biomed Mater Res B Appl Biomater 73(2): 392-9.
Abstract: Hydroxyapatite coatings have been proven to improve the osteointegration of metal implants through a tight binding to the bone mineral phase as well as through favorable osteoblast adhesion and proliferation onto the implant surface. However, hydroxyapatite coatings are not stable and they tend to delaminate from the metal surface when challenged by the mechanical stresses experienced by the implant. Recently, a new multiphase anodic spark deposition (ASD) method has been optimized where the formation of a thick oxide film is followed by the deposition of a calcium phosphate mineral phase and its etching by alkali. The data in this paper demonstrate that this novel type of coating, BioSpark, improves the material osteointegration potential when compared to conventional ASD while offering more mechanical stability. A faster mineralization was obtained by incubation in simulated body fluids and osteoblasts showed better adhesion, proliferation, differentiation, and collagen production. These performances were related to the surface morphology, to the film calcium/phosphate ratio and its surface oxygen content, as well as to a preferential binding of structural proteins such as fibronectin.

In vitro bacterial adherence to teicoplanin and calcium sulfate-soaked bone cement
Sencan, I., I. Sahin, et al. (2005), J Chemother 17(2): 174-8.
Abstract: The aim of this study was to assess in vitro the improvement in release kinetics for teicoplanin and the inhibition of bacterial adhesion on calcium sulfate-soaked PMMA discs. Calcium sulfate has been used in vivo and shown to be biocompatible, and prevention of bacterial adhesion may be expected with calcium sulfate-soaked polymethylmethacrylate (PMMA). Discs were made by adding teicoplanin and calcium sulfate in powder form to PMMA powder. The antibiotic concentration eluted from PMMA discs was assayed by agar diffusion assay. Nonadherent bacteria were removed by washing and adherent bacteria were detached by sonication. The suspension including nonadherent bacteria was seeded on sheep blood agar plate and incubated for 24 h at 37 degrees C for the growth of microorganisms. The teicoplanin released from discs containing calcium sulfate was higher than that released from discs which had not been soaked with calcium sulfate. The count of bacteria adhering to the calcium sulfate-soaked discs was lower than that from the discs without calcium sulfate. In conclusion, the addition of calcium sulfate to teicoplanin-loaded PMMA bone cement may provide local antibiotic concentrations higher than MIC values due to increased antibiotic release. Furthermore, calcium sulfate was found to be effective in reducing bacterial adherence to treated discs.

In vitro behavior of osteoblast-like cells on fluoridated hydroxyapatite coatings
Cheng, K., W. Weng, et al. (2005), Biomaterials 26(32): 6288-95.
Abstract: In this work, fluoridated hydroxyapatite (Ca10(PO4)6Fx(OH)(2-x) or FHA) coatings are prepared by sol-gel method for study of the influence of F content on the behavior of osteoblast-like cells. The results show that the cells well attach and proliferate on the FHA coatings studied (Ca10(PO4)6F(0.67-2.00)(OH)(0-1.33)). With increasing F content in the FHA coatings, percentage of cell in S period increases, indicating F in the coating favors the proliferation process of the cells. On the other hand, the proliferation rate increases inversely with zeta potential of the coating surface. As tested from the MTT of the cells cultured in the leaching out solution, increase of F content in the FHA coatings results in a slight decrease in cell proliferation rate, which is most probably due to reduction in release of Ca2+ ions. As a compromise among cell attachment, cell proliferation, apatite deposition and ability to resist dissolution, it is suggested that FHA coatings (Ca10(PO4)6Fx(OH)(2-x)) with x in the range of 0.67-1.48, from the results of this study, may be most suitable for real case implantation.

In vitro behaviour of osteoblasts cultured on orthopaedic biomaterials with different surface roughness, uncoated and fluorohydroxyapatite-coated, relative to the in vivo osteointegration rate
Fini, M., R. Giardino, et al. (2003), Int J Artif Organs 26(6): 520-8.
Abstract: The effects of two surfaces with different roughness (Low Roughness, LR: Ra: 5.6-5.9 microm; High Roughness, HR: Ra: 21.5-22.5 microm), uncoated and fluorohydroxyapatite(FHA)-coated, were investigated in MG-63 osteoblasts. At 72 hours, cells proliferated on biomaterials more slowly than in the control group (p < 0.0001), the proliferation rate was higher on FHA-coated LR than uncoated HR (p = 0.037). Collagen-I production was positively affected by the LR surface (p = 0.001) as compared to controls, while it was significantly lower (p = 0.0001) in the HR surfaces. Compared to controls, LR and HR surfaces led to enhanced production of TGF-beta1, further improved by FHA (FHA-coated LR: p = 0.007; FHA-coated HR p < 0.0001 respectively). ALP, OC, IL-6 and TNF-alpha levels were not significantly different from the controls. Results suggest that collagen-I production could be useful in predicting the in vivo osteointegration rate of biocompatible biomaterials observed in previous studies.

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications
Suzuki, S., L. Grondahl, et al. (2005), Biomaterials 26(26): 5303-12.
Abstract: The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of CaP minerals, whereas all the grafted membranes revealed the growth of CaP minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity (theta(adv) = 120 degrees, theta(rec) = 80 degrees) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces (theta(adv) = 60 degrees and 80 degrees, theta(rec) = 25 degrees and 15 degrees, respectively) exhibited irregular growth of non-apatitic CaP minerals. Irreversibly adsorbed protein measured after a 1h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3-6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample. The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro.

In vitro bioactivity of starch thermoplastic/hydroxyapatite composite biomaterials: an in situ study using atomic force microscopy
Leonor, I. B., A. Ito, et al. (2003), Biomaterials 24(4): 579-85.
Abstract: The in vitro bioactivity of a composite composed by a biodegradable starch-based polymeric matrix and hydroxyapatite fillers was investigated, in situ, as a function of immersion time in a simulated body fluid (SBF) using atomic force microscopy (AFM). The surface roughness of the composite started to increase after the initial 8h because of both the degradation of the polymer matrix and the nucleation of calcium phosphate. After 24h of immersion the surface of the composite was fully covered with calcium phosphate nuclei with diameters around 126 nm. As the immersion time increased, the nuclei increased both in number and size, and coalesced leading to the formation of a dense and uniform calcium phosphate layer on the surface of the composite only after 126 h of SBF immersion. The results of in situ AFM observation agreed with those of standard in vitro bioactivity tests in combination with scanning electron microscopy observations. Thin-film X-ray diffraction demonstrated that the ratio of apatite to the polymer matrix was higher within the surface layer (40 microm deep from the surface) than that in the bulk after the immersion for 7 days. The water-uptake capability of the polymer contributes to the nucleation and growth of the calcium phosphate layer. These results suggest the great potential of the composite for a range of temporary applications in which bone-bonding ability is a desired property.

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