In vitro assessment of the biocompatibility of chemically modified GaAs surfaces

GaAs has excellent optical, electrical, and mechanical properties and shows promise to be used in the fabrication of novel devices. However, the unprotected GaAs surface can release heavy metal compounds such as AsOx, which are toxic to living cells. A promising approach to reduce or eliminate this release relies on the passivation of the GaAs surface using different chemical approaches. In this work, we compared three different passivation methods aimed at enhancing the viability of cells on GaAs. Protective layers composed of self-assembled alkyl thiols, polypeptides, thick polymer layers, and shells of polyelectrolytes were tested. We confirmed that the GaAs surface can be made biocompatible for several days based on in vitro tests with HeLa and KB cells. In addition, we compared the cell spreading behavior on the GaAs substrates modified by different chemical approaches. Our results suggest that when the toxicity of the GaAs surface is reduced or eliminated, the cells’viability and spreading depend on the chemical and topographical nature of the surface.     

Anin vitro model for the biological evaluation of dental materials using human periodontal ligament cells

Anin vitro system is described which uses human diploid cells derived from the periodontal ligament. It simulates the clinical situation and is suitable for rapid cytotoxicity screening of dental materials. It can also be adapted to study various factors influencing biocompatibility.     

Biocompatibility testing of an experimental fluoride releasing resin using human gingival epithelial cells in vitro

Summary Cell culture is a valuable method of evaluating the biocompatibility of new dental materials. The purpose of this study was to compare the in vitro biocompatibility of an experimental fluoride composite resin with fluoride and non-fluoride-releasing materials currently available. The dental materials tested were: MQ Silicate (silicate cement), KETAC-CEM and FUJI (type II glass ionomer cements), VISIO DISPERS (a light-cured, nonfluoridated, microfilled composite resin), and FR-17 (an experimental fluoride-releasing composite resin). The Smulow-Glickman (S-G) human, gingival epithelial cell line, which exhibits semidifferentiated characteristics, was used in the study as a test system. Biocompatibility was quantified by counting the viable cells per unit area remaining after 24 and 48 h at two radial distances from cured specimens immersed in the cell culture medium. The test materials were observed to be most toxic to cells nearest the materials. A Time-Distance Cytotoxicity Index (TDCI) was calculated to relate the percentage of dead cells to viable cells at each diffusion distance for each exposure time compared to a nontoxic control. The relative toxicity ranking of the materials tested based on the TDCI was VISIO DISPERS (91%), FUJI (82%), FR-17 (30%), MQ Silicate (23%), and KETAC-CEM (10%), which exhibited the least toxicity. The cytotoxicity of the experimental resin FR-17 was within the range of cytotoxicity of currently accepted restorative materials. this study was supported in part by grant R01-DE04749 from the National Institutes of Health, Bethesda, MD, to H. R. R., and by grant no. S07-RR05704-13 from the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health, awarded to the Louisiana State University School of Dentistry.     

In vitro antiseptic properties of an ammonium compound combined with denture base acrylic resin

Background: Denture base acrylic resin is easily colonised by oral endogenous bacteria and Candida spp., and eventually by extra‐oral species such as Staphylococcus spp., Pseudomonadaceae or members of Enterobacteriaceae. This microbial reservoir can be responsive for denture related stomatitis and aspiration pneumonia, a life‐threatening infection especially in geriatric patients. However, the oral and denture hygiene of dependant elderly individuals is extremely poor. Objective: This in vitro study aimed to determine the per cent of a quaternary ammonium compound heat‐polymerised in acrylic resin necessary to obtain denture base displaying antiseptic properties. Design: Acrylic resin discs containing 2–50% ammonium polymer (Poly 202063A; 0% control) were soaked in artificial saliva for 4 weeks. Resin discs were incubated for 24 hours with Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa [37°C, brain–heart infusion (BHI) broth and phosphate‐buffered saline (PBS) buffer] and Candida albicans (30°C, Schaedler broth), in 15 ml (168 discs) and 600 μl (168 discs) of inoculum. Microbial growth was verified at t 0 hours and t 24 hours. Data were recorded as the mean of three colony forming unit (CFU) numerations. The borderline of antimicrobial effect was determined at 0.1% viable cells. Results: In 600 μl of PBS inoculum, resin specimens had a bactericidal effect (E. coli and S. aureus: 2%; P. aeruginosa: 10%) and a fungicidal effect (C. albicans: 50%). Long‐term stability and toxicity in vivo studies are now required. Conclusion: A 2% quaternary ammonium compound polymerised with a denture acrylic resin displayed antiseptic properties after a 4‐week soaking period in artificial saliva. Such antiseptic denture base could help geriatric patients to improve their oral health.     

Weight loss and changes in surface roughness of denture base and reline materials after simulated toothbrushing in vitro

doi: 10.1111/j.1741‐2358.2010.00422.x
Weight loss and changes in surface roughness of denture base and reline materials after simulated toothbrushing in vitro Objective: To evaluate the weight loss and the surface roughness of acrylic resins after simulated brushing tests. Material and methods: Ten specimens of each material (Tokuyama Rebase II‐TR, New Truliner‐NT, Ufi Gel Hard‐UH and Lucitone 550‐L) were made. The wear loss (mg) by weight and the surface roughness (Ra μm) of each specimen was determined before and after brushing. The specimens were placed on the brushing machine and a total of 20 000 brushing cycles was performed. The results of weight loss and roughness values were submitted to the anova followed by the Tukey’s test (p = 0.05). Results: The mean weight loss of material L was statistically higher (p < 0.001) than that of the relines TR, UH and NT. No significant differences were found among the roughness values of resins TR, UH and L (p > 0.05). Only for L, toothbrushing increased the surface roughness. After toothbrushing, there was no significant difference between the roughness values of materials L and NT. The highest mean weight loss during the simulated toothbrushing tests was observed for L. Before the toothbrushing tests, the NT exhibited the highest mean roughness. Conclusion: Brushing resulted in increase in roughness only for resin L.     

Biocompatibility of denture base acrylic resins evaluated in culture of L929 cells. Effect of polymerisation cycle and post-polymerisation treatments

Objective: The purpose of this study was to evaluate the effect of two post‐polymerisation treatments and different cycles of polymerisation on the cytotoxicity of two denture base resins. Materials and methods: The resins tested were Lucitone 550 and QC 20. Discs of resins were fabricated following the manufacturer's instructions. Lucitone 550 was processed by long cycle or short cycle. The resin QC 20 was processed by reverse cycle or normal cycle. The specimens were divided into groups: (i) post‐polymerised in microwave for 3 min at 500 W; (ii) post‐polymerised in water‐bath at 55°C for 60 min and (iii) without post‐polymerisation. Eluates were prepared by placing three discs into a sterile glass vial with 9 ml of Eagle's medium and incubated at 37°C for 24 hours. L929 cells were seeded into 96 well culture plates and DNA synthesis was assessed by ³H‐thymidine incorporation assay. Results: The results were submitted to two‐way anova and Tukey HSD test. QC 20 specimens polymerised by the normal cycle and submitted to microwave post‐polymerisation were graded as moderately cytotoxic. Similar results were observed for Lucitone 550 processed by long cycle without post‐polymerisation. The other experimental groups were graded as not cytotoxic. After water‐bath post‐polymerisation, specimens of Lucitone 550 processed by long cycle produced significantly lower inhibition of DNA synthesis than the other groups. Conclusion: The long cycle increased the cytotoxicity of Lucitone 550 and water‐bath post‐polymerisation reduced the cytotoxicity of Lucitone 550 processed by long cycle.     

In situ evaluation of surface roughness and micromorphology of temporary soft denture liner materials at different time intervals

Objective

To perform an in situ evaluation of surface roughness and micromorphology of two soft liner materials for dentures at different time intervals.

Background

The surface roughness of materials may influence the adhesion of micro‐organisms and inflammation of the mucosal tissues. The in situ evaluation of surface roughness and the micromorphology of soft liner materials over the course of time may present results different from those of in vitro studies, considering the constant presence of saliva and food, the changes in temperature and the pH level in the oral cavity.

Materials and methods

Forty‐eight rectangular specimens of each of the two soft liner materials were fabricated: a silicone‐based material (Mucopren Soft) and an acrylic resin‐based material (Trusoft). The specimens were placed in the dentures of 12 participants (n = 12), and the materials were evaluated for surface roughness and micromorphology at different time intervals: 0, 7, 30 and 60 days. Roughness (Ra) was evaluated by means of a roughness tester. Surface micromorphology was evaluated by scanning electron microscopy.

Results

Analysis of variance for randomised block design and Tukey's test showed that surface roughness values were lower in the groups using the silicone‐based material at all the time intervals (P < .0001). The average surface roughness was higher at time interval 0 than at the other intervals, for both materials (P < .0001). The surface micromorphology showed that the silicone material presented a more regular and smoother surface than the acrylic resin‐based material.

Conclusion

The surface roughness of acrylic resin‐based and silicone‐based denture soft liner materials decreased after 7 days of evaluation, leading to a smoother surface over time. The silicone‐based material showed lower roughness values and a smoother surface than the acrylic resin‐based material, thereby making it preferred when selecting more appropriate material, due its tendency to promote less biofilm build‐up.     

造血细胞体外悬浮培养和生物反应器开发

为解决造血细胞的静态培养中由浓度梯度引起的培养不稳定、环境不均一、难放大等问题,首先采用转瓶对脐血单个核细胞进行了悬浮培养研究,结果表明,悬浮培养中总细胞、集落和CD34^＋细胞的扩增都高于静态的方瓶培养。在测试了所用材料生物相容性的基础上,开发了可以控制溶氧和pH的生物反应器,并将其应用到造血细胞的批培养中,结果表明反应器的培养环境均一,可实现较高密度的培养,而且总细胞、集落和CD34^＋细胞的扩增都优于静态培养。大规模的反应器培养有利于解决临床应用中细胞数量不足的问题。     

In vitro culture of blood cells from the colonial protochordateBotryllus schlosseri

Summary Primary cultures of circulatory blood cells from the colonial tunicateBotryllus schlosseri were cultivated in 96-well plates for up to 3 mo. in a medium based on Dulbecco’s modified Eagle’s medium, supplemented with salts to the botryllid ascidian hemolymph osmolarity, HEPES buffer,l-glutamin, fetal bovine serum, and antibiotics. Intercellular bridges between granular pigment cells were established within 24 h. The viability of these cells decreased slowly, and most died within 1 mo. without any sign of cell proliferation. Other cell types remained in an arrested state and were subjected to a weekly medium exchange. Spontaneous cell proliferation was randomly recorded in 6 to 10% of the wells from 2 wk to 1 mo. This proliferation was followed by the formation of masses of cell clumps, from which uniform hemocytes (5 μm, lymphocytelike cells) migrated peripherally. Stress conditions, which included longer intervals between medium exchanges and partial medium replacement, increased the probability of cell proliferation. From each proliferating primary culture, we successfully performed up to 10 plating cycles over a period of 15 wk, during which the cells differentiate in size but are uniformly structured. This produced the firstBotryllus lymphocytelike cell line. From this stage, cell numbers remained constant for up to 6 mo. without increase in cell number. Several mitogenic factors were employed on primary cultures.Botryllus and sea cucumber hemolymphs and mixed interleukins were found to augment significantly proliferation of at least one specific cell size, whereas cells were not markedly responsive to lectins (Concavalin A, wheat germ agglutinin,Ulex europaeus agglutinin), insulin, and retinoic acid. The results are discussed with respect to future efforts in the development of tunicate blood cell cultures.     

In vitro models of differentiated sertoli cell structure and function

Summary Primary cultures of Sertoli cells maintained in conventional cultures on plastic culture vessels do not retain many of the structural and functional properties of their in vivo counterparts. Sertoli cell phenotype is better maintained by incorporating certain environmental parameters, intrinsic to the testic, into the Sertoli cell culture system. These environmental parameters include a) high cell density, b) a unique extracellular matrix, c) a semipermeable support between the basal plasma membrane of the cells and blood-derived nutrients in the interstitium, d) chemically distinct microenvironments at the apical and basal surfaces of the cells, and e) cell-to-cell interactions among Sertoli cells and other testicular cell types. Using three variations of Sertoli cell culture we have demonstrated the importance of each of these environmental parameters in obtaining a better Sertoli cell culture model. Paper presented a the 38th Annual Meeting of the Tissue Culture Association in Arlington, Virginia, in May 1987. The session was chaired by Dr. Carlton H. Nadolney, member of the TCA Committee on Toxicity, Carcinogenesis and Mutagenesis Evaluation. This work was supported by grant HD-16260 from the National Institutes of Health, Bethesda, MD, and a grant from the Mellon Foundation.     

In vitro models of intestinal epithelial cell differentiation

The intestinal epithelium is a particularly interesting tissue as (1) it is in a constant cell renewal from a stem cell pool located in the crypts which form, with the underlying fibroblasts, a stem cell niche and (2) the pluripotent stem cells give rise to four main cell types: enterocytes, mucus, endocrine, and Paneth cells. The mechanisms leading to the determination of phenotype commitment and cell-specific expressions are still poorly understood. Although transgenic mouse models are powerful tools for elucidating the molecular cascades implicated in these processes, cell culture approaches bring easy and elegant ways to study cellular behavior, cell interactions, and cell signaling pathways for example. In the present review, we will describe the major tissue culture technologies that allow differentiation of epithelial cells from undifferentiated embryonic or crypt cells. We will point to the necessity of the re-creation of a complex microenvironment that allows full differentiation process to occur. We will also summarize the characteristics and interesting properties of the cell lines established from human colorectal tumors.     

Metallic dental material biocompatibility in osteoblastlike cells

Ions released from metallic dental materials used in orthodontic appliances could induce undesirable effects on cells and tissues. This study evaluates the biocompatibility of two of the most labile components of metallic dental alloys on osteoblastlike cells. The influence of protein and ions on metal dissolution properties is also investigated using different electrolyte solutions. Morphological alterations, cell growth, and differentiation of osteoblasts were assessed after exposure to pure metals (Ag, Cu, Pd, Au) and Ni−Ti alloy and correlated with the kinetics of elements released into the culture media. Results showed that Cu and Ag were the most cytotoxic elements and the other metals were biocompatible with the osteoblasts. The parameters of biocompatibility were correlated with the levels of ions detected into the culture media. Metal ions induced cell death through early mitosis arrest, apoptotic phenomena, and necrotic processes. Voltammograms showed that anions and proteins interfered in the corrosion process. Fetal bovine serum (FBS) strongly affected the electrochemical process, decreasing the oxidation rate of the metals. In conclusion, copper and silver ions showed a time-dependent low biocompatibility, which correlated with the concentration of released ions. The dissolution of the metallic materials was dependent on the composition of the simulated biological media.     

In vitro cytotoxicity test for estimating non-ocular irritation dose of ophthalmic solutions

The in vitro cytotoxicity test for estimating the non-ocular irritation dose of ophthalmic solutions was investigated. In the in vitro test, normal human epidermal keratinocytes (NHEK) in a confluent monolayer were incubated for 48hr in a medium with test compounds. The concentration of a test compound which causes a 50% reduction in NHEK viability was determined as IC₅₀ by MTT colorimetric assay. For comparison, the in vivo rabbit ocular irritation tests were carried out by the standard Draize method. The maximum concentration, which did not show any ocular irritation, was determined as DS₀. The results showed the correlation coefficient between the IC₅₀ values and the DS₀ values for 19 test compounds to be 0.82. However, the correlation coefficients for 10 compounds, which have IC₅₀ values of less than 300g/ml, and for 7 alcohols were 0.99. The IC₅₀-DS₀ correlation curves obtained could be utilized as the critical concentrations for ocular irritation. These results suggest that our in vitrolin vivo test can estimate non-ocular irritation dose of the ophthalmicpreparations in advance of the in vivo tests.Abbreviations DS₀ Draize Score 0 - KGM keratinocyte growth medium - NHEK normal human epidermal keratinocytes     

人脐静脉血管内皮细胞的分离、培养、鉴定及试验研究

血管内皮细胞体外培养,在血管再生、新血管生成机理、内皮细胞在心血管系统疾病中的作用、内皮细胞与造血的关系等方面的研究中有很高的应用价值。本研究借鉴了前人的工作,建立了胶原酶灌流消化获得人脐静脉血管内皮细胞并进行体外培养的方法,探讨了影响人脐静脉血管内皮细胞（HUVEC）分离培养的影响因素。同时应用建立的HUVEC体外培养进行了内皮抑素的抑制活性检测,证明了内皮抑素对于激活增殖的HUVEC的抑制作用,为今后的试验研究打下了基础。     

In vitro electrical impedance spectroscopy of human dentine: the effect of restorative materials

The influence of different restorative materials on in vitro dielectric properties of sound dentine was investigated. The studied samples were three-layer materials consisting of successive disks of dentine and silver amalgam or nanohybrid composite resin. Before being tested, the samples were maintained in physiological solution never more than 48 h from the extraction. Also, sections of intact dentine were similarly prepared for electrical measurements. Complex dielectric permittivity of these specimens was determined in a wide frequency range using the parallel-plate capacitor technique. Very similar dielectric responses of intact dentine and amalgam-dentine material were observed. This is explained on the basis of high dc conductivity exhibited by both samples. In contrast, resin-dentine specimen revealed a much more insulating behavior. A simple theoretical model for heterogeneous systems could be applied to these dental three-layer materials. The dielectric properties of restored dentine are strongly dependent on the kind of restorative material employed in each case. This suggests that electrical data should be used carefully in caries diagnosis on restored teeth.     

In vitro behaviour of human osteoblasts on dentin and bone

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culture polystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material. Cellular attachment, viability and growth were monitored in parallel cultures of human osteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells (HOBs). Culture plastic was either left untreated or roughened with abrasive paper of various grit sizes (4000-1200 grit) in order to obtain a level of roughness comparable to that of the dentin slices. Cell counting and intracellular BCECF staining showed that after an initial incubation of 2 h, the primary cells attached and spread out more quickly on the different substrates than the three cell lines. The primary cells also showed a stronger mitochondrial staining and viability on dentin. During subsequent culture morphological differences appeared with the cells on dentin displaying more cellular extensions. All three cell lines proliferated more slowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affected in their growth by the different substrates. Total and specific alkaline phosphatase (AP) activity of the cell lines was not significantly affected by the different substrates after short-term adhesion, but it was increased for the primary cells on the dentin. However, after 2-3 days of culture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasma treatment of the roughened plastic did not alter cellular viability or AP activity, suggesting that grinding of the surface did not affect the property of the culture plastic to support cell attachment and growth. In conclusion, the results show that not only do osteoblastic cells behave differently on a natural calcified substrate surface than on standard culture plastic, but also that differences were evident between the various cell types, in particular the primary HOB versus the continuous cell lines.     

In vitro correlation of ultrastructural morphology and creatine phosphokinase release in L6 skeletal muscle cells after exposure to parenteral antibiotics

Summary Morphologic changes in a rat skeletal muscle cell line (L6) exposed for 1 h to the parenteral antibiotics amphotericin B (AMP), tetracycline-HCl (TET), erythromycin lactobionate (ERY), and cephaloridine (CEP) were characterized by transmission and scanning electron microscopy and compared to cellular release of creatine phosphokinase (CRK). AMP (0.05, 0.1, 0.5 mg/ml) caused a concentration-related swelling of nuclei, endoplasmic reticulum, and mitochondria. Loss of membrane integrity associated with AMP exposure was evident at the middle concentration and extensive at the high concentration, which correlated well with the 43 and 90% depletion of CPK from the muscle cells, respectively. TET (0.25, 1.0, 2.5 mg/ml) caused dilation of endoplasmic reticulum and cytoplasmic blebbing at the low concentration but had no effect on the cytoplasmic membrane or CPK. Cells exposed to the high concentration of TET had extensive damage to the cytoplasmic membrane, and CPK was completely depleted. ERY (2.5, 5.0, 25 mg/ml) caused a pattern of morphologic changes and CPK depletion similar to TET. CEP (4.0, 20, 50 mg/ml) had no effect on membrane integrity or CPK; however, membranous whorls were prominent in the cytoplasm. A good correlation between CPK release and cytoplasmic membrane integrity was evident and the ability of these agents to release CPK from muscle cells in culture correlated with the known irritancy potential of these parenteral antibiotics. Furthermore, CPK depletion seems to be a reliable indicator of muscle cell damage after cytoplasmic membrane perturbation and is therefore an appropriate index of toxicity in this in vitro muscle irritation model.     

Irritation and cytotoxic potential of denture adhesives

Objective: The present study aimed to examine the in vitro biocompatibility of denture adhesives. Background: Denture adhesives absorb water to become viscous and sticky, and by this process, other constituents like colouring, flavouring, wetting and preserving agents may be released. Some of these constituents may induce adverse reactions among users of denture adhesives. Materials and methods: Five commercially available denture adhesives; three different creams, a powder, and a cushion were included in the study. The irritation and cytotoxic potential was evaluated using the Hen's Egg Test Chorioallantoic Membrane (HET‐CAM) method and three cell culture methods; filter diffusion, dimethylthiazol diphenyltetrazolium bromide (MTT) assay and agar diffusion. Results: None of the tested denture adhesives showed a noteworthy acute irritation as evaluated by the HET‐CAM method. None of the tested denture adhesives induced cytotoxicity in the filter diffusion test. One of the denture adhesives induced a severe cytotoxic reaction in both the MTT and agar diffusion assays. These tests employ longer exposure times than in both the filter diffusion and the HET‐CAM test. Conclusion: Denture adhesives are commonly used throughout the day, and our results raise the concern that denture adhesives may contribute to mucosal inflammation in denture wearers.