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In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery
Trindade, M. C., M. Lind, et al. (2001), Biomaterials 22(3): 253-9.
Abstract: Periprosthetic tissues observed at sites of loose total joint implants exhibit abundant macrophages, lymphocytes, fibroblasts and particulate debris. Macrophages phagocytose orthopaedic debris and release proinflammatory cytokines, chemokines, matrix metalloproteinases and other substances. In addition, other cell types present in tissues harvested from the bone-implant interface are thought to influence periprosthetic bone resorption. The present study examined the effects of polymethylmethacrylate (PMMA), cobalt chrome molybdenum alloy (CoCr), and titanium-alloy particle challenge on macrophages co-cultured with lymphocytes in vitro. Potential synergistic effects of lymphocytes on macrophage activation were determined by measuring interleukin-6 and tumor necrosis factor-alpha release following exposure to orthopaedic biomaterial particles. Exposure of macrophages or macrophages co-cultured with lymphocytes to all three types of particles resulted in increased release of interleukin-6 and tumor necrosis factor-alpha at 48 h, when compared to macrophages or macrophages co-cultured with lymphocytes, respectively, cultured in the absence of particles. Lymphocytes isolated from periprosthetic tissues secreted increased basal levels of cytokines relative to peripheral blood lymphocytes. Higher doses of PMMA and titanium-alloy particles stimulated increased levels of cytokine release in the macrophage and macrophage/lymphocyte groups. In contrast, a higher dose of CoCr particles (0.075% v/v) was not as effective as the 0.015% v/v dose, indicating probable CoCr toxicity. The macrophage/lymphocyte co-culture did not show synergism between the two types of cells with respect to cytokine release. T-cells at the bone-implant interface may alter the biological response to particulate debris.

In vitro reconstruction of an endothelialized skin substitute provided with a microcapillary network using biopolymer scaffolds
Tonello, C., V. Vindigni, et al. (2005), Faseb J 19(11): 1546-8.
Abstract: Successful in vitro reconstruction of skin requires the inclusion of several cell types that give rise in coculture to the specific elements present in native skin, and the appropriate scaffolding structure to house and support these cells. In addition to the two main structural components, epidermis and dermis, one critical apparatus of the skin is a capillary network that guarantees adequate perfusion of nutrients and oxygen. The aim of the present study was to develop an in vitro coculture system that assumed the human dermal-epidermal architecture and included a microcapillary network in a three-dimensional biomaterial that guaranteed ease of handling in a clinical setting. Endothelialized skin (ES) was prepared by coculturing three human cell types: keratinocytes, fibroblasts, and endothelial cells, obtained from human full-thickness skin samples, in scaffolds produced from modified hyaluronic acid. Results were evaluated by histological and immunohistochemical analyses at different time points. In vitro, engineered skin obtained with this composite culture developed into a well-differentiated upper layer of stratified keratinocytes lining a dermal-like structure, in which fibroblasts, extracellular matrix and a microvascular network were present. Furthermore, the biodegradable fabric produced from hyaluronic acid and used as the scaffolding support for this in vitro constructed skin graft greatly facilitated handling in the perioperative period.

In vitro release of levonorgestrel from phase sensitive and thermosensitive smart polymer delivery systems
Chen, S. and J. Singh (2005), Pharm Dev Technol 10(2): 319-25.
Abstract: The objective of this research is to develop injectable controlled delivery systems for the contraceptive hormone, levonorgestrel (LNG), using phase sensitive and thermosensitive polymers. A combination of poly (lactide) (PLA) and a solvent mixture of benzyl benzoate (BB) and benzyl alcohol (BA) was used in the phase-sensitive polymer delivery systems. The effects of solvent systems and polymer concentrations on the in vitro LNG release were evaluated. In the case of thermosensitive polymer delivery systems, a series of low-molecular-weight poly (lactide-co-glycolide)-poly (ethylene glycol)-poly (lactide-co-glycolide) (PLGA-PEG-PLGA) triblock copolymers with varying ratios of lactide/glycolide (LA/GA, 2.0-3.5) were used. The effects of varying block length of copolymers 1, 2, 3, and 4 on the in vitro LNG release were evaluated. Phosphate buffer saline (pH 7.4) containing 0.5% w/v Tween-80 was used as in vitro release medium. The amount of the released LNG was determined by an high pressure liquid chromatography (HPLC) method. A controlled (zero-order) in vitro release of LNG was observed from both phase-sensitive and thermosensitive-polymer delivery systems. Increasing the concentration of the phase-sensitive polymer from 5% to 30% significantly (p < 0.05) decreased the release rate of LNG from 38.32 microg/day to 31.45 microg/day; and increasing the hydrophilic fraction of the solvents mixture (i.e., BA) significantly (p < 0.05) increased the release rate of LNG. In the case of the thermosensitive polymer delivery system, increasing the hydrophobic PLGA block length of copolymers significantly (p < 0.05) decreased the release rate of LNG (98.65 microg/day to 67.60 microg/day). It is evident from this study that both the phase sensitive and thermosensitive polymers are suitable for developing prolonged-release injectable delivery systems for the contraceptive hormone.

In vitro release of plasmid DNA from oligo(poly(ethylene glycol) fumarate) hydrogels
Kasper, F. K., S. K. Seidlits, et al. (2005), J Control Release 104(3): 521-39.
Abstract: This research investigates the release of plasmid DNA in vitro from novel, injectable hydrogels based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF). These biodegradable hydrogels can be crosslinked under physiological conditions to physically entrap plasmid DNA. The DNA release kinetics were characterized fluorescently with the PicoGreen and OliGreen Reagents as well as through the use of radiolabeled plasmid. Further, the ability of the released DNA to be expressed was assessed through bacterial transformations. It was found that plasmid DNA can be released in a sustained, linear fashion over the course of 45-62 days, with the release kinetics depending upon the molecular weight of the poly(ethylene glycol) from which the OPF was synthesized. Two formulations of OPF were synthesized from poly(ethylene glycol) of a nominal molecular weight of either 3.35K (termed OPF 3K) or 10K (termed OPF 10K). By the time the gels had completely degraded, 97.8+/-0.3% of the initially loaded DNA was recovered from OPF 3K hydrogels, with 80.8+/-1.9% of the initial DNA retaining its double-stranded form. Likewise, for OPF 10K gels, 92.1+/-4.3% of the initially loaded DNA was recovered upon complete degradation of the gels, with 81.6+/-3.8% of the initial DNA retaining double-stranded form. Experiments suggest that the release of plasmid DNA from OPF hydrogels is dominated by the degradation of the gels. Bacterial transformation results indicated that the DNA retained bioactivity over the course of 42 days of release. Thus, these studies demonstrate the potential of OPF hydrogels in controlled gene delivery applications.

In vitro response of encapsulated somatic embryos of Lagerstroemia indica L
Niranjan, M. H. and M. S. Sudarshana (2005), Indian J Exp Biol 43(6): 552-4.
Abstract: A method to produce encapsulatable units for synthetic seeds was developed in L. indica. Somatic embryos were harvested from leaf derived embryogenic callus on Murashige and Skoog's basal medium supplemented with 2, 4-dichlorophenoxy acetic acid (2, 4-D, 0.5 mg/l), 6-benzyl amino purine (BAP, 1 mg/l) and ascorbic acid (AA, 50 mg/l). The embryos were encapsulated in alginate beads and dehydrated. Germination ability of the artificial seeds were investigated. The frequency of regeneration from the encapsulated embryos was significantly affected by (i) the concentration of alginate (ii) the duration of storage, and (iii) the effect of different types of media. A 2% sodium alginate concentration on MS salts resulted in significantly higher germination frequencies than at other concentrations. L. indica showed maximum germination on MS medium (93.84%) after 6 weeks of culture. The germinated synthetic seeds with well developed roots and shoots were transferred successfully to green house. This is the first report on artificial seeds in Lagerstroemnia indica.

In vitro screening studies of the toxicological testing of synthetic biomaterials. Introduction and comparative evaluation of a new method of testing applying breeding bull semen
Paluch, D., M. Borzemska-Szymonowicz, et al. (1980), Polim Med 10(4): 193-204.

In vitro studies of elastin-fibrin biomaterial degradation: preservative effects of protease inhibitors and antibiotics
Collet, D., F. Lefebvre, et al. (1991), Biomaterials 12(8): 763-6.
Abstract: The degradability of a new elastin-fibrin material was tested in vitro versus human pancreatic elastase (HPE) and plasmin (PL) activities. It is shown that aprotinine Iniprol, a well-known protease inhibitor and Eglin C, a new potent inhibitor of HPE, especially when used in synergy, efficiently protected the material. A small amount of specific antibiotics was incorporated into the material. The two products will allow the material to be used in digestive surgery with improved safety.

In vitro studies on some probiotic properties of immobilised lactic acid bacteria after freezing and freeze-drying
Nazzaro, F., A. Sorrentino, et al. (2001), Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet 66(3a): 307-12.

In vitro studies on the effect of sodium tripolyphosphate on the interactions of stain and salivary protein with hydroxyapatite
Shellis, R. P., M. Addy, et al. (2005), J Dent 33(4): 313-24.
Abstract: OBJECTIVES: To study properties of sodium tripolyphosphate (STP) relevant to inhibition or removal of dental stain in vitro. METHODS: The effects of STP and other phosphates on adsorption of a dietary chromogen (black tea polyphenol) and salivary protein to hydroxyapatite (HA) powder were studied by analysing loss of protein or tea stain from solutions mixed with HA or HA pre-treated with the test agents. The effects on desorption of protein and stain from HA were studied by analysis of water or solutions of test agents mixed with HA or HA pre-treated with saliva or tea solution. RESULTS: At concentrations and pH representative of those likely to occur in the mouth, STP inhibited adsorption of salivary protein and black tea polyphenol to, and desorbed these substances from, HA surfaces. Adsorption and desorption of protein and stain were not influenced by pH of the STP solutions but adsorption varied with concentration. STP showed equivalent effectiveness with respect to salivary protein adsorption and desorption as a longer-chain condensed phosphate. The inhibitory activity of HA-bound STP on adsorption of salivary protein and stain resisted extensive washing. CONCLUSIONS: STP is likely to be an effective agent for inhibiting and removing dental stain, whether bound directly to mineralised surfaces or indirectly via salivary pellicle.

In vitro study of alginate-chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure
Haque, T., H. Chen, et al. (2005), Biotechnol Lett 27(5): 317-22.
Abstract: The application of alginate-chitosan (AC) microcapsules to liver cell transplantation has not been previously investigated. In the current in vitro study, we have investigated the potential of AC microcapsules for the encapsulation of liver cells and show that the AC membrane supports the survival, proliferation and protein secretion by entrapped hepatocytes. The AC membrane provides cell immuno-isolation and has the potential for cell cryopreservation. The AC microcapsule has several advantages compared to more widely used alginate-poly-L-lysine (APA) microcapsules for the application of cell therapy.

In vitro study of dentin hypersensitivity treated by Nd:YAP laser and bioglass
Lee, B. S., C. W. Chang, et al. (2005), Dent Mater 21(6): 511-9.
Abstract: OBJECTIVES: An ideal material has yet to be discovered that can completely treat dentin hypersensitivity. However, if a highly biocompatible material such as bioglass, could be melted by laser irradiation to achieve better sealing depth for dentinal tubules, it may subsequently bond to dentin structures under a physiological environment and offer a prolonged therapeutic effect. METHODS: The authors used four types of energy parameters to melt the composition-modified bioglass. These four types were 30 Hz, 330 mJ/pulse (G+ mode), 30 Hz, 160 mJ/pulse (G- mode), 10 Hz, 400 mJ/pulse (D+ mode), and 10 Hz, 200 mJ/pulse (D- mode). The temperature elevation, occlusive depth of bioglass, and phase changes in the bioglass after laser irradiation were evaluated by means of scanning electron microscope (SEM), thermometer, and X-ray diffractometer (XRD). RESULTS: The occlusive depths of 2 and 10 microm in the dentinal tubules were achieved when the bioglass underwent 30 Hz, 160 mJ/pulse (G- mode) and 30 Hz, 330 mJ/pulse (G+ mode) of laser treatments, respectively. The bioglass experienced a temperature increase of less than 600 degrees C, and no phase transformation was observed after Nd:YAP laser irradiation. SIGNIFICANCE: The melting point of a composition-modified bioglass could be reduced and its use plus Nd:YAP laser have the potential in clinical use to treat dentin hypersensitivity.

In vitro study of intradentinal calcium diffusion induced by two endodontic biomaterials
Guigand, M., J. M. Vulcain, et al. (1997), J Endod 23(6): 387-90.
Abstract: The aim of this in vitro study was to assess intratubular calcium penetration induced by two root canal restoration materials, one calcium oxide based, and the other calcium hydroxide based. Pig teeth were restored with no preliminary root canal preparation. The filing materials were left in place for 8, 15, or 21 days. The samples were then examined using various microanalytical techniques and, in parallel, by backscattered electron image (BEI) scanning electron microscopy. The Ca/P ratios obtained by microanalysis were higher for samples restored with calcium oxide. In addition, the distances over which the ratios increased were also greater than those obtained using calcium hydroxide. BEI photographs confirm these results and show corresponding retrodiffusion fringes.

In vitro test of new biomaterials for the development of a bioartificial pancreas
Lembert, N., P. Petersen, et al. (2001), Ann N Y Acad Sci 944: 271-6.
Abstract: The implantation of macroencapsulated islets has the potential to restore endogenous insulin secretion in type 1 diabetics, with no need for lifetime immunosuppression. To match the physiological fluctuations of blood glucose concentrations with appropriate insulin release, the macroencapsulation material must combine immunoprotection with optimal diffusion properties for glucose and insulin. The impact of chemical modifications of polysulphone (PSU) capillary polymers with a cutoff of 50 kD on glucose-induced insulin secretion of macroencapsulated rat islets was studied in perifusion experiments. The insulin release of free-floating islets showed the typical rapid response to glucose stimulation. Total insulin release (AUC between minute 30 and 120 of perifusion) reached 117+/-22 ng/ml. Blending PSU with polyvinylpyrrolidone or sodium-dodecyl-sulfate was not suitable for islet macroencapsulation, since glucose-induced insulin release was absent or disturbed. Hydroxy-methylation (CH2OH) of PSU improved the secretory behavior of macroencapsulated islets depending on the degree of PSU substitution (DS 0.8, AUC 62+/-15 ng/ml; DS 1.8, 111+/-24 ng/ml). In highly substituted PSU-capillaries the kinetics of glucose-induced insulin release was very similar to that observed in free-floating islets. Two consecutive glucose stimulations potentiated insulin release of free-floating islets during the second period of stimulation. Furthermore, freshly isolated macroencapsulated islets responded with more efficient insulin secretion after the initial priming. In conclusion, in vitro membrane screening identified highly substituted hydroxy-methylated PSU as the material of choice for islet encapsulation in a bioartificial pancreas.

In vitro testing of surface-modified biomaterials
Leitao, E., M. A. Barbosa, et al. (1998), J Mater Sci Mater Med 9(9): 543-8.
Abstract: The influence of surface modification treatments such as ion implantation and sputter coating on an in vitro rat bone-marrow cell culture was studied by scanning electron microscopy and X-ray microanalysis. 316 L stainless steel, Ti-6Al-4V and Ti-5Al-2.5Fe were nitrogen ion-implanted with three fluences: 10(15), 10(16) and 10(17) ion cm-2 with an energy beam of 40 keV. Both nitrogen and carbon sputter-coated 316 L stainless steel samples were also studied. Polished 316 L stainless steel, Ti-6Al-4V, Ti-5Al-2.5Fe and Thermanox were also studied, in order to give comparative information. The materials were inoculated with a droplet of cell suspension and were maintained for 3 wk. A mineralized extracellular matrix was formed on all materials except on nitrogen sputter-coated 316 L stainless steel. The morphology of the cell cultures obtained on nitrogen-ion implanted materials was similar to those obtained on the untreated materials and Thermanox. The observation of the interface between the cell layer and the substrata showed the presence of calcium- and phosphorus-rich globular deposits associated with collagen fibres. A higher density of these globular deposits was observed on the ion-implanted materials.

In vitro thrombogenicity investigation of new water-dispersible polyurethane anionomers bearing carboxylate groups
Poussard, L., F. Burel, et al. (2005), J Biomater Sci Polym Ed 16(3): 335-51.
Abstract: New segmented polyurethane (PU) anionomers based on hydroxytelechelic polybutadiene were synthesized via an aqueous dispersion process. Incorporation of carboxylic groups was achieved using thioacids of different length. Surface properties were investigated by mean of water absorption analysis and static contact-angle measurements using water, diiodomethane, formamide and ethylene glycol. Blood compatibility of the PUs was evaluated by in vitro adhesion assays using 111In-radiolabeled platelet-rich plasma and [125I]fibrinogen. Morphology of the adhered platelets was examined by scanning electron microscopy (SEM). Results were compared to two biomedical-grade PUs, namely Pellethane and Tecoflex. Insertion of carboxylic groups increased surface hydrophilicity and limited water uptake (< 8% for an ion content of 5% by weight). Surface energy of all synthesized PUs was between 40 and 45 mJ/m2. Platelet adhesion and fibrinogen adsorption on the PU anionomer surfaces were affected as a function to the increase of graft length; thiopropionic was the most haemocompatible, followed by thiosuccinic and then thioglycolic acid. SEM analyses of all ionic PU samples exhibited low platelet adhesion to surfaces with no morphological modification. In conclusion, increased hydrophily, dynamic mobility and charge repulsion are synergistic key factors for enhanced haemocompatibility.

In vitro toxicity of biomaterials determined with cell density, total protein, cell cycle distribution and adenine nucleotides
Wieslander, A. P., M. K. Nordin, et al. (1993), Biomater Artif Cells Immobilization Biotechnol 21(1): 63-70.
Abstract: Inhibition of cell growth is the most commonly used endpoint for in vitro toxicity of biomaterials. The use of several different endpoints might however generate more information concerning the nature of the toxicity. Thus, we examined the toxicity of two biomaterials, Polyvinylchloride (PVC) and Polyoximethene (POM), with different selected endpoints. The influence of cell growth on these endpoints was also investigated. Water extracts from the polymeric materials were tested on the continuous cell line L-929. Cell density, total protein, total protein per cell, fraction of cells in G0/G1- or S-phase, the concentration of ATP, ADP and AMP were used as endpoints. The PVC material did not significantly influence any of these endpoints until after 72 hours of exposure and the main part of the toxicity at 72 hours was related to higher proliferation rate in control cultures. After the cells had been incubated for 8 hour with POM the main toxic effect was on the energy parameters. In conclusion the PVC material was less toxic than the POM material. Our results also implies that the choice of endpoint will influence the evaluation of cytotoxicity.

In vitro toxicity test of ethyl 2-cyanoacrylate, a tissue adhesive used in cardiovascular surgery, by fibroblast cell culture method
Kaplan, M. and K. Baysal (2005), Heart Surg Forum 8(3): E169-72.
Abstract: BACKGROUND: The aim of this study was to evaluate the cytotoxicity of cyanoacrylate polymers (ethyl 2-cyanoacrylate) by an elution test system. In such systems, the material is extracted with a cell culture medium, which is subsequently added onto cultured cells, resulting in an indirect contact between the biomaterial and cells. METHODS: A cell line commonly utilized for cytotoxicity experiments; L929 mouse fibroblasts were used in this study. The effects of extract dilutions on cells were evaluated by two experiments: (a) The cells were suspended and seeded in a medium containing the extract, followed by a short incubation to observe the effects on cell attachment; (b) cells were seeded in a normal medium. Following cell attachment, this was replaced with a medium containing the extract and long-term effects on cell proliferation were measured. The cytotoxicity was quantified using a cell viability assay, well established for use in the evaluation of cell-biomaterial interactions. RESULTS: These results indicate that, in the test system utilized, a tenfold dilution of the extract results in an approximately 10% decrease in cells; this increases to between 30% and 45% in a 1:1 dilution. When a large number of cells (3000/well) were used, proliferation of cells overcame the cytotoxic effect and consistent results could not be observed. CONCLUSIONS: In this study, the observed outcomes follow a similar trend on cell attachment and proliferation with acute effects (4 hours incubation) of the extracts on the cells, producing slightly higher toxicity. Our findings are parallel with the literature findings.

In vivo and ex vivo evaluation of the antithrombogenicity of human thrombomodulin immobilized biomaterials
Kishida, A., Y. Akatsuka, et al. (1995), Asaio J 41(3): M369-74.
Abstract: Human thrombomodulin (hTM) is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. Focusing on the establishment of the practical evaluation of hTM immobilized materials, the activity of immobilized hTM was evaluated by in vivo and ex vivo blood contacting tests. As the basis for immobilization, regenerated cellulose films and hollow fibers were used. For the in vivo test, hTM immobilized cellular hollow fibers were implanted into dog blood vessels. Using hTM immobilized cellulose hollow fibers, a small scale dialyzer was assembled and its antithrombogenic activity was studied using human blood. As a result, it was revealed that the immobilized hTM still has co-enzymatic activity for activation of Protein C and anticoagulant activity. The coagulation time of the human blood passed through the hTM immobilized small dialyzer was effectively prolonged. It is expected that hTM immobilized cellulose should be a useful antithrombogenic biomaterial.

In vivo assessment of hydroxyapatite and silicate-substituted hydroxyapatite granules using an ovine defect model
Patel, N., R. A. Brooks, et al. (2005), J Mater Sci Mater Med 16(5): 429-40.
Abstract: Phase pure hydroxyapatite (HA) and two silicate-substituted hydroxyapatites (0.8 and 1.5 wt% Si, or 2.6 and 4.9 wt% SiO4) were prepared by aqueous precipitation methods. The filter-cakes of HA and silicate-substituted hydroxyapatite (SiHA) compositions were processed into granules 1.0-2.0 mm in diameter and sintered at 1200 degrees C for 2 h. The sintered granules underwent full structural characterisation, prior to assessment in an ovine defect model by implantation for a period of 6 and 12 weeks. The results indicate that HA and SiHA implants were well accepted by the host tissue, with no evidence of inflammation. New bone formation was observed directly on the surfaces and in the spaces between the granular implants. Quantitative histomorphometry as determined by the percentage of bone ingrowth and bone coverage for both SiHA implant compositions was significantly greater than that for phase pure HA. These findings indicate that the in vivo bioactivity of hydroxyapatite was significantly improved by the incorporation of silicate ions into the HA structure, making SiHA ceramics attractive alternatives to conventional HA materials for use as bone graft substitute ceramics.

In vivo behavior of acrylic bone cement in total hip arthroplasty
Ries, M. D., E. Young, et al. (2006), Biomaterials 27(2): 256-61.
Abstract: Polymethylmethacrylate (PMMA) bone cement serves as the primary fixation material between bone and the prosthetic component in cemented total hip arthroplasty. In vivo degradation of bone cement may lead to a decrease in mechanical properties of PMMA and result in aseptic loosening. However, other factors such as porosity and location of the cement relative to the bone implant interface may also contribute to mechanical behavior in vivo. This study investigated the mechanical properties of Simplex cement retrieved from 43 patients undergoing revision total hip arthroplasty. The time in vivo was between 1 month and 27 years. The variables studied included fracture toughness (KIC), porosity, molecular weight, time in vivo of the cement, and relative in vivo location of the cement with respect to the implant and bone. KIC did not correlate with time in vivo of the samples or with molecular weight. This suggests that time in vivo may not be the limiting factor in the mechanical integrity of the bone cement, A significant and inverse relationship was found between porosity and KIC. This implies that porosity is the most important factor in the mechanical behavior of bone cement during in vivo use.

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Last Modified: 8 February 2006