Culture Polystyrene Plates Research Articles

Rifampicin induced virulence determinants increase Candida albicans biofilm formation

Increased intravenous catheter use has been paralleled by increased bacterial and yeast bloodstream infection. Biofilm formation, which is associated with the cell surface hydrophobicity (CSH) phenotype, represents a major pathogenicity strategy ofCandida albicans, becoming especially important in the colonization of intravascular medical devices. Increasing evidence shows the induction of virulence factors inC. albicansby diverse substances. Therefore, we investigated whether rifampicin, an antibiotic shown to be capable of inducing MDR1 expression inC. albicansmay also promote the formation of a pathogenic biofilm. In response to 40 µg/mL rifampicin, an enhanced retention ofC. albicansSC5314 cells on polystyrene culture plates was observed by measuring increased metabolic activity by XTT assay, indicating induction of biofilm formation. Rifampicin treatment also induced fibronectin binding, cell hydrophobicity and germ tube formation. Furthermore, increased RNA and protein expression of CSH1p, a major mediator of CSH, was demonstrated. We conclude that exposure to rifampicin may result in upregulation of keyCandidavirulence determinants, potentially boosting pathogenicity and supporting biofilm formation. This finding gains clinical significance from the increasing popularity of rifampicin-coated catheters, which might provide an advantageous gateway forCandidabloodstream infections.

Effects of irradiation on osteoblast‐like cells on different titanium surfaces in vitro

The aim of this study was to investigate the effects of irradiation on adhesion ability, proliferation, and differentiation of MC3T3-E1 cells on microarc oxidation (MAO) titanium surfaces and polished titanium (PT) surfaces. MC3T3-E1 cells were exposed to a single dose at 2, 4, 6, 8, or 10 Gy using a (60) Co source, with tissue culture polystyrene plates chosen as controls. On all surfaces, irradiation resulted in a dose-dependent decrease in cellular proliferation. At 4 Gy dose, the cell proliferation of cells decreased by 17.8% on MAO and 18.6% on PT surfaces, respectively, compared with nonirradiated controls. Cells exposed to 8 Gy dose showed significant inhibition in collagen secretion and osteogenesis-related genes expression (OSX, COL-Iα1, and OCN). In contrast, irradiation increased cell adhesion to three surfaces dose dependently. It was also demonstrated that cells on MAO surface showed higher adhesion and collagen secretion than on PT surface at different radiation doses. This study revealed the effects of irradiation on osteoblasts in vitro on two titanium surfaces. MAO surface could be used in dental implants in irradiated bone due to enhanced adhesion ability and collagen secretion in osteoblasts.

Cell cycle arrest and apoptosis of OVCAR-3 and MCF-7 cells induced by co-immobilized TNF-α plus IFN-γ on polystyrene and the role of p53 activation

The aim of this study is to reveal the biological mechanism for high anti-cancer efficiency of co-immobilized TNF-α plus IFN-γ polymeric drug (co-immobilized drug) in mediating two gynecologic cancer cell lines: MCF-7 and OVCAR-3. The co-immobilized drug is prepared by mixing 10 ng/ml TNF-α plus 10 ng/ml IFN-γ which are then photo-immobilized onto cell culture polystyrene plates. The drug compositions and microstructures are characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The MCF-7 and OVCAR-3 cell cycle arrest and programmed cell death are checked by flow cytometry, and the expression of p53 is probed by immunofluorescence staining. The phosphorylation sites of the p53 regulation and the apoptosis key protein expressions of caspase3, 8 and 9 are detected by western blot assay. Our data show that, in case of short treatment time (48 h) at low cytokine concentrations (20 ng/ml), the co-immobilized drug demonstrates visible effects in comparison with the treatment using TNF-α plus IFN-γ freely attached on the polymeric plate (free drug). It is revealed that the co-immobilized drug leads to significant cell arrest in the S phase or G1 and G2 phase and offer high efficiency in mediating a caspase-dependent apoptosis via p53 transcriptional regulation. Moreover, upon the treatment by the co-immobilized drug, the two gynecologic cancer cell lines show different phosphorylation sites of p53 and then different caspase-dependent apoptosis pathways. The present work sheds deep insights into the p53 regulation mechanism responsible for the high anti-cancer efficiency of the co-immobilized TNF-α plus IFN-γ polymeric drug against MCF-7 and OVCAR-3.

Application of anodized titanium for enhanced recruitment of endothelial progenitor cells

ObjectivesTo study the efficacy of an effective anodized titanium surface with enhanced attachment of endothelial progenitor cell (EPC).BackgroundIn-stent restenosis is a major obstacle for vascular patency after catheter-based intravascular interventions. Recently, stents that capture EPCs have been paid attention in order to make a functional endothelialized layer at the site of stent-induced endothelial denudation. Anodized titanium has been shown to enhance stem cell attachment. Anodization is a quick and inexpensive method, which can provide suitable stent surface.MethodsSurface topography was examined by high-resolution scanning electron microscopy (SEM). Substrates were co-cultured with EPCs at second passage in 24-well culture plates. Evaluation of cell growth, proliferation, viability, surface cytotoxicity and cell adhesion was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and 4,6-diamidino-2-phenylindole dihydrochloride staining. For platelet attachment, platelets added to substrates were evaluated under SEM.ResultsThe average MTT values for tissue culture polystyrene plate, unanodized and anodized titanium with nanostructure were equal to 0.49, 0.16 and 0.72, respectively (P < 0.05). The surface had no cytotoxic effects on cells. The average cell attachment results showed that 9,955 ± 461.18, 3,300 ± 197.98 and 11,359 ± 458.10 EPCs were attached per well of tissue culture polystyrene plate, unanodized and anodized titanium surfaces, respectively (P < 0.05).ConclusionsAnodized titanium surfaces can be potentially applied for devices that need enhanced recruitment of EPCs. This unique property makes these anodized surfaces good and cheap candidates for designing cardiovascular medical devices as endovascular stents.

Comparison of several attachment methods for human iPS, embryonic and adipose-derived stem cells for tissue engineering

As actual stem cell application quickly approaches tissue engineering and regenerative medicine, aspects such as cell attachment to scaffolds and biomaterials become important and are often overlooked. Here, we compare the effects of several attachment proteins on the adhesion, proliferation and stem cell identity of three promising human stem cell types: human adipose-derived stem cells (hASCs), human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Traditional tissue culture polystyrene plates (TCPS), Matrigel (Mat), laminin (Lam), fibronectin (FN) and poly-L-lysine (PLL) were investigated as attachment protein surfaces. For hASCs typically cultured on TCPS, laminin resulted in the greatest cell attachment and proliferation with largest cell areas, indicating favourability by cell spreading. However, mesenchymal stem cell markers indicative of hASCs were slightly more expressed on surfaces with lowest cell attachment, corresponding to increased cell roundness, a newly observed attribute in hASCs possibly indicating a more stem cell-like character. hESCs preferred Matrigel as a feeder-free culture surface. Interestingly, hiPSCs favoured laminin over Matrigel for colony expansion, shown by larger cell colony area and perimeter lengths, although cell numbers and stem cell marker expression level remained highest on Matrigel. These data provide a practical reference guide for selecting a suitable attachment method for using human induced pluripotent, embryonic or adipose stem cells in tissue engineering and regenerative medicine applications.

Descemet's membrane substrate from human donor lens anterior capsule

To study the potential use of human donor anterior lens capsule as a Descemet's membrane substrate. Anterior lens capsules were recovered from the lenses of 30 cornea donors. Human corneal endothelial cells were recovered from the remaining corneal sclera rims of 15 donor corneas used for penetrating keratoplasty. Samples were sorted into three groups. Group 1 consisted of 10 samples in which the endothelial cells were allowed to grow on anterior lens capsules. In Group 2 human corneal endothelial cells grew on a collagen membrane and in Group 3 on polystyrene culture plates. Cell density, morphology and adherence of the cell-capsule complex were evaluated at 1, 4, 7 and 14days with a phase-contrast microscope, a scanning electron microscope and by histology. Cell viability was quantified by a microscopic live-dead assay. Expression of zonula occludens-1, Na(+) /K(+) -adenosine triphosphatase, tissue transglutaminase and vimentin were investigated by immunohistochemistry. A mean diameter of 10.05±0.13mm of anterior capsule was obtained as a substrate for cell culture. Endothelial cell density of Group 1 was measured at 2455.4±283.8cells/mm(2) , which was also comparable with the cell density of the control group. Cell viability was 95% or superior in all groups and multiple cellular interconnections developed between growing cells. Immunohistochemical analysis demonstrated strongly positive staining for all investigated proteins. Electron microscopy confirmed the adherence and monolayer growth of the endothelial cells. Human donor anterior lens capsule might therefore be a potential scaffold for the ex vivo expansion of human corneal endothelial cells.

Evaluation of Hemocompatibility of Titanium after Various Surface Treatments: An in vitro Study

ABSTRACT Objective To evaluate the hemocompatibility of titanium after various surface treatments. Materials and methods A total of 27 disk-shaped specimens (3 ⨯ 10.0 mm) were prepared from a cylindrical rod of medical grade titanium. The disks were divided into three groups, of which one was considered as the control (mechanical surface polished surface). The other groups being sandblasted disks and anodized disks. Surface evaluation was done for sandblasted and anodized disks with scanning electron microscope. The specimens were placed in polystyrene culture plates and agitated with phosphate buffered saline for 5 minutes before they were exposed to blood taken from human volunteer. The materials were under 30 minutes agitation at 75 ± 5 rpm using an Environ shaker thermostated at 35 ± 20°C. The total hemoglobin from the initial sample was measured using automatic hematology analyzer. Percentage hemolysis, thrombin time, platelet adhesion and activation were assessed. Significant differences between different treated titanium materials were determined using Minitab® Version 15.1.1.0. A two-sample t-test was performed to find the p-values for different groups of data. Results After 30 minutes of agitation, cells began to spread on the test surfaces. There was a clear reduction in the number of platelets before and after exposure to titanium samples. Reduction of leukocytes was seen to a least extent on the anodized surface. Rough surface induced higher hemolysis than other groups. Platelet reduction and leukocyte reduction in all the three surfaces was quite higher than that obtained for reference plate. Surface variation has no significance on the thrombogenic capabilities of medical grade titanium (p &lt; 0.05). Significance The hemocompatibility of medical grade titanium did not vary with different surface modifications. How to cite this article BhavanChand Y, Ranzani R, Annapoorani H. Evaluation of Hemocompatibility of Titanium after Various Surface Treatments: An in vitro Study. Int J Prosthodont Restor Dent 2012;2(4):136-142.

Effect of nanocoating with rhamnogalacturonan‐I on surface properties and osteoblasts response

Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants.

Adhesion, phenotypic expression, and biosynthetic capacity of corneal keratocytes on surfaces coated with hyaluronic acid of different molecular weights

In ophthalmology, hyaluronic acid (HA) is an important extracellular matrix (ECM) component and is appropriate for use in generating a microenvironment for cell cultivation. The aim of this work was to evaluate the rabbit corneal keratocyte (RCK) growth in response to HA coatings under serum-free conditions. After modification with HA of varying molecular weights (MWs: 35–1500kDa), the surfaces were characterized by atomic force microscopy and contact angle measurements, and were used for cell culture studies. Our data indicated that the substrates coated with higher negatively charged HA become rougher and are more hydrophilic, resulting in the decrease of cell adhesion and cell–matrix interaction. This early cellular event was likely responsible for the determination of keratocyte configuration. Additionally, for the growth of RCKs on dry HA coatings with surface roughness of 1.1–1.7nm, a strong cell–cell interaction was observed, which may facilitate the formation of multicellular spheroid aggregates and maintenance of mitotically quiescent state. At each culture time point from 1 to 5days, a better biosynthetic capacity associated with a higher prevalence of elevated ECM production was found for the cells in a spherical configuration. Irrespective of polysaccharide MW of surface coatings, the RCKs presented good viability without hypoxia-induced death. As compared with a monolayer of adherent keratocytes on tissue culture polystyrene plates and low MW HA-modified samples, the cell spheroids (76–110μm in diameter) showed significantly higher expressions of keratocan and lumican and lower expressions of biglycan, similar to those of keratocytes in vivo. Moreover, the expression levels of corneal crystallin aldehyde dehydrogenase (7–9-fold increase) and nestin (10–16-fold increase) were greater in larger-sized spheroids, indicating higher ability to maintain cellular transparency and self-renewal potential. It is concluded that the cultured RCKs on surfaces coated with HA of different MWs can sense ECM cues, and the multicellular spheroids may potentially be used for corneal stromal tissue engineering applications.

The Role of Nanofibrous Structure in Osteogenic Differentiation of Human Mesenchymal Stem Cells with Serial Passage

Using scaffolds with autologous stem cells is a golden strategy for the treatment of bone defects. In this strategy, human mesenchymal stem cells (hMSCs) have often been isolated and expanded in vitro on a plastic surface to obtain a sufficient cell number before seeding on a suitable scaffold. Investigating the influence of serial passages (from passage two to passage eight) on the abilities of proliferation and osteogenic differentiation of hMSCs on 24-well tissue culture polystyrene plates and poly L-lactic acid electrospun nanofibrous scaffolds was performed to determine how prolonged culture affected these cellular abilities and how the nanofibrous scaffolds supported the osteogenic differentiation potential of hMSCs. Serial passage caused adverse changes in hMSCs characteristics, which were indicated by the decline in both proliferation and osteogenic differentiation abilities. Interestingly, the poly L-lactic acid nanofibrous scaffolds showed a significant support in recovering the osteogenic abilities of hMSCs, which had been severely affected by prolonged culture.

Dynamic Behavior and Spontaneous Differentiation of Mouse Embryoid Bodies on Hydrogel Substrates of Different Surface Charge and Chemical Structures

Differentiation of embryoid bodies (EBs) into particular cell lineages has been extensively studied. There is an increasing interest in the effect of soft hydrogel scaffolds on the behavior of EBs, such as the initial adhesion, dynamic morphology change, and differentiation. In this study, without adding any other bioactive factors in the serum-containing medium, dynamic behaviors of mouse EBs loaded on the surface of hydrogels with different surface charge and chemical structures are investigated. EBs adhered quickly to negatively charged poly(sodium p-styrene sulfonate) (PNaSS) hydrogels, which facilitates EBs spreading, migration, and differentiation into three germ layers with high efficiency of cardiomyocytes differentiation, similar to that on gelatin coated polystyrene (PS) culture plate. While on neutral poly(acrylamide) (PAAm) hydrogels, EBs maintained the initial spherical morphology with high expression of pluripotency-related markers in the short culture periods, and then showed the significantly greater levels of selected endoderm markers after long-time culture. EBs cultured on negatively charged poly(2-acrylamido-2-methyl-propane sulfonic acid sodium salt) (PNaAMPS) gels demonstrated the analogous behaviors with that of neutral PAAm gels at early differentiation phase (day 4+1). Then, their adhesion, spreading and differentiation were quite similar to that on negatively charged PNaSS gels. The correlation between surface properties of hydrogels and EBs differentiation was discussed.

Supercritical carbon dioxide processed resorbable polymer nanocomposite bone graft substitutes

The development of synthetic bone graft substitutes is an intense area of research due to the complications associated with the harvest of autogenous bone and concerns about the supply of allogenic bone. Porous resorbable polymers have been used extensively in hard tissue engineering applications, but currently lack load-bearing capacity. Supercritical carbon dioxide (scCO 2) processing is used as a novel method to simultaneously impart a porous structure and disperse a nano-clay in a resorbable polymer matrix suitable for load-bearing applications. Porous resorbable polylactic acid (PLA)/cloisite clay nanocomposite constructs prepared using scCO 2 processing exhibit a 2.5-fold increase in compressive strength compared with pure polymer constructs. The resulting mechanical properties are comparable with human cancellous and cortico-cancellous bone. In addition to the significant improvements in mechanical properties, the nanocomposite constructs display a biocompatibility greater than that of polystyrene culture plate controls. Furthermore, calcium phosphate-rich deposits could clearly be seen on the surface of the constructs, as well as at the center of the cultured constructs, indicating that osteoblasts are able to penetrate the porous network of the nanocomposite constructs. Cellular infiltration of these constructs is important for their in vivo use as bone graft substitutes. The diameter of the pores suggests that these constructs would also support neovascularization, which is integral for nutrient transport.

Using 96-well tissue culture polystyrene plates and a fluorescence plate reader as tools to study the survival and inactivation of viruses on surfaces

A method for studying the behavior of viruses on surfaces has been developed and is illustrated by determining the temperatures that inactivate adsorbed viral hemorrhagic septicemia virus (VHSV) and the concentration of 1-propanol that disinfected surfaces with adsorbed VHSV and chum salmon virus (CSV). VHSV is a rhabdovirus; CSV, a reovirus, and they were detected with two fish cell lines, EPC and CHSE-214, respectively. When polystyrene tissue culture surfaces were incubated with virus, rinsed, and left to dry, they still supported the attachment and spreading of cell lines and after 7days these cells showed the characteristic CPE of the viruses. Thus cells appeared to be infected directly from surfaces on which viruses had been adsorbed. Applying this property to 96-well plates allowed duplicate surfaces to be examined for their infectiousness or support of CPE. For each treatment 80 replicate surfaces in a 96-well plate were tested at one time and the results expressed as the number of wells showing CPE. VHSV adsorbed to polystyrene was inactivated by drying in the dark at temperatures above 14°C, but remained infectious for at least 15days of drying at 4°C. For chemical sterilization of polystyrene surfaces with adsorbed virus, disinfection was achieved with 1-propanol at 40% for VHSV and at 60% for CSV. As CPE can be conveniently monitored in 96-well plates with a fluorescence plate reader, this method can be used to rapidly evaluate a variety of treatments for their ability to inactivate surface-bound viruses.

High‐efficiency cell seeding method by relatively hydrophobic culture strategy

Cell adhesion efficiency is one of the key factors affecting the results of manufacturing tissue engineering constructs. High efficiency is required for seeding low proliferation cells onto scaffolds. In this study, we designed a strategy to improve the efficiency of cell adhesion using hydrophobic cell culture environment to enhance cells adhering to a scaffold. Cells have lower affinity to the surface of polydimethylsiloxane (PDMS) than tissue culture polystyrene (TCPS) plates. When cells were cultured with gelatin microspheres or chitosan films in a PDMS-coated plate instead of a normal TCPS plate, there was a significant increase in cell attachment efficiency. Cells cultured in the PDMS-coated system tended to selectively attach onto the gelatin microspheres or chitosan films, which are relatively more hydrophilic than the PDMS surface. However, minimal cell attachment on gelatin microspheres or chitosan films was observed when gelatin microspheres or chitosan films were placed in normal TCPS plate. Cell counting experiments with gelatin microspheres in the PDMS-coated system resulted in a cell attachment efficiency of 89.8% after 1 day of cultivation, whereas the cell attachment efficiency was less than 1% in normal TCPS plate. The results demonstrate that the method is easy to use and could be useful for fast cultivation of cell-scaffold constructs.

幹細胞の機能を制御する高分子表面グラフト化材料

The surface properties of biomaterials, including chemical composition, nano- or microstructured morphology, wettability, elasticity and electrostatic property, are very important for manipulation of stem cell functions such as adhesion, proliferation and differentiation. Photoreactive polyallylamine, poly （acrylic acid）, poly （ethylene glycol） and poly （vinyl alcohol） were grafted or micro-patterned onto cell culture polystyrene plates by UV irradiation. Human mesenchymal stem cells （MSC）, a promising cell source for regenerative medicine were cultured on the polymer-grafted or micropatterned plates to investigate the effect of functional groups on their chondrogenic, osteogenic, and adipogenic differentiation. The adhesion, proliferation, and differentiation of MSC could be manipulated by the polymer-grafted or micropatterned surfaces.

Inhibited cell spreading on polystyrene nanopillars fabricated by nanoimprinting and in situelongation

Polymer nanopillars (40–80 nm in diameter and 100 nm in pitch) were fabricated at highdensity over large areas directly on bulk tissue culture polystyrene plates using nanoimprintlithography. Nanoporous Si molds for imprinting were generated by transfer from an anodicalumina membrane. Ultrahigh aspect ratio polymer nanopillars were formed in a novelprocedure using controlled elongation of the imprinted pillars during mold release. Theresulting nanopillar arrays show significant changes in surface wettability upon briefO2 plasma treatment. Human dermal fibroblasts were cultured on the nanopillarsurfaces in order to study cell–substrate interaction at the nanoscale. The nanopillartopography shows strong effects on the cell morphology, with pillars of widelyvarying aspect ratios and surface energies resisting cell spreading. This effecton cell behavior can be rationalized in terms of the cells’ requirement to formmicron-scale focal adhesions. The study indicates that at the nanoscale, physical factorscan supersede the effects of chemical factors on the cell–substratum interaction.

Large-scale cultivation of transplantable dermal papilla cellular aggregates using microfabricated PDMS arrays

In this work we have developed a strategy for cultivating dermal papilla (DP) cells to form multiple arrayed spheroidal microtissues for transplantation on a micropatterned polydimethylsiloxane (PDMS)-based tissue culture polystyrene (TCPS) plate system. We also describe the behavior of dermal papilla cells on this platform and the spontaneous formation of spheroidal microtissues by DP cells. We used a hydrophobic PDMS arrayed chip as a master to separate the seeded cells in the TCPS culture plate. By controlling the cell seeding densities, a microwell with arrayed DP spheroidal microtissues was easily formed. Formation of DP microtissues was associated with overlapping multilayered cells on the microwells and low cell–substrate adhesivity on the PDMS film. The microwell environment enhanced the aggregation of DP cells into spheroidal microtissues on the TCPS culture plate. The spheroidal microtissues preserved their hair induction potential in vitro and in vivo. A large quantity of DP spheroidal microtissues could be obtained rapidly and simply using this platform. We could harvest hundreds of DP microtissues (352 microtissues) with a cell seeding density of 1 × 10 6 cells well −1 after 3 days cultivation in one well of a 24-well plate. This is the first demonstration of the formation of DP spheres in large quantitites.

Repairing goat tibia segmental bone defect using scaffold cultured with mesenchymal stem cells

In this study, we investigated cellular biocompatibility in vitro and segmental bone defect repairing efficacy in vivo of a previously reported fibre-reinforced scaffold, nano-hydroxyapatite/collagen/poly (L-lactic acid) (PLLA)/chitin fibres (nHACP/CF). First, attachment, proliferation, and differentiation of the goat bone mesenchymal stem cells (GBMSCs) cultured on the nHACP/CF scaffolds were evaluated in vitro. The results showed that cells attached to the scaffolds well, and there was no significant difference in cell proliferation between cells on the scaffolds and cells on the polystyrene culture plates that were used as a control. The results also showed that alkaline phosphatase (ALP)/DNA of the cells cultured on the scaffolds was significantly higher than that on the control. The in vivo study compared the bone defect repairing efficacy of nHACP/CF scaffolds with that of autograft bone. Thirty-two adult male goats with 25-mm defects in their tibias at the same anatomic site were divided into four groups. The first group was implanted with the nHACP/CF with GBMSCs. The second group was implanted with autograft bone. The third group was implanted with the nHACP/CF. Nothing was implanted in the fourth group. Bone growth was evaluated by radiography, histology, and biomechanics. The results showed that although the nHACP/CF had new bone formation, it could not repair the defect fully while nHACP/CF with GBMSCs cultured and autograft bone could repair the segmental bone defect by 8 weeks after surgery, suggesting that nHACP/CF is an appropriate scaffold for bone tissue engineering.

Effect of surface morphology of calcium phosphate on osteoblast-like HOS cell responses

Calcium phosphate (CaP) films with different surface roughness are synthesized on a polystyrene surface as a possible candidate for improving the biocompatibility of solid surfaces. These CaP films are used to investigate the influences of the surface roughness and chemical composition on the HOS osteoblast-like cells adhesion, spreading, proliferation, and differentiation. A polystyrene culture plate is used as the control surface. The CaP substrates are designated as a smooth, moderate, and rough surface according to the surface roughness. For smooth and moderate surfaces, a relatively small difference in the surface roughness is observed but the difference in their chemical composition is more significant than the others. Cell adhesion, spreading, proliferation, and differentiation appear to be dependent on surface roughness. These cellular responses are more active on the smoother surfaces than on the rough surface but are more pronounced on the moderate surface. The cell responses are reduced when HOS cells are cultured on the rough surface. The number of cells released from the control surface by trypsinization is greater than the CaP surfaces after a short period of incubation (5 h). However the cells released from smooth and moderate CaP surfaces are equivalent to or greater than the control at the longer period of incubation (17 h). Spreading and proliferation are greater on the CaP surfaces than on the control. The alkaline phosphatase-specific activity is very low during 1 and 2 weeks of culturing but dramatically increases after 3 weeks on all surfaces. The enzyme activity on the control is greater than on the CaP surfaces. The moderate surface shows the greatest enzyme activity in all cases among the CaP surfaces. Although a direct relation between surface chemistry and cell responses has not been established, the surface composition may play a cooperative role in characterizing the cellular responses. According to these results, the moderate surface is the most favorable substrate in terms of surface roughness and chemical composition for cell adhesion, spreading, proliferation and differentiation among all the CaP surfaces.

Evaluation of Cross-Linking Methods for Electrospun Gelatin on Cell Growth and Viability

The creation of a tissue engineering scaffold via electrospinning that has minimal toxicity and uses a solvent system composed of solvents with low toxicity and different cross-linking agents was investigated. First, a solvent system of acetic acid/ethyl acetate/water (50:30:20) with gelatin as a solute was evaluated. The optimum system for electrospinning a scaffold with the desired properties resulted from a gelatin concentration of 10 wt %. Several different methods were used to cross-link the electrospun gelatin fibers, including vapor-phase glutaraldehyde, aqueous phase genipin, and glyceraldehyde, as well as reactive oxygen species from a plasma cleaner. Because glutaraldehyde at high concentrations has been shown to be toxic, we explored other cross-linking methods. Using reactive oxygen species from a plasma cleaner is an easy alternative; however, the degradation reaction dominated the cross-linking reaction and the scaffolds degraded after only a few hours in aqueous medium at 37 °C. Glyceraldehyde and genipin were established as good options for cross-linking agents because of the low toxicity of these cross-linkers and the resistance to dissolution of the cross-linked fibers in cell culture medium at 37 °C. MG63 osteoblastic cells were grown on each of the cross-linked scaffolds. A proliferation assay showed that the cells proliferated as well or better on the cross-linked scaffolds than on traditional two-dimensional polystyrene culture plates.