Tissue Culture Polystyrene Dishes Research Articles

Effect of different biomaterials on the expression pattern of the transcription factor Ets2 in bone-like constructs

Tissue engineering offers an exciting prospect for reconstructive surgery by replacing missing natural scaffolds with artificial ones. For optimal success the artificial scaffold should provide an environment closely resembling the natural tissue. Little is known about the direct influence of the scaffold on the expression of regulators of bone development, such as transcription factors. The aim of this study was to investigate the influence of the scaffold material on the expression of V-ets erythroblastosis virus E26 oncogene homolog 2 (avian) (Ets2), a key transcription factor in bone biology. Human primary bone precursor cells were seeded in three-dimensional constructs consisting of hydroxyapatite (HA) or poly (lactic-co-glycolic acid) (PLGA). Cells grown on tissue culture polystyrene dishes served as controls. After cultivation for up to 21 days the expression of Ets2 and other important bone-specific genes was assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western Blotting. Ets2 mRNA showed significantly higher expression in controls than in bone-like constructs, and more Ets2 mRNA was expressed in cells grown in HA than in PLGA constructs. At protein level however, Ets2 expression was higher in constructs than in controls after prolonged culture. Our study showed for the first time a differential expression of Ets2 in tissue engineered bone constructs in vitro, demonstrating that scaffold chemistry has an influence on the expression of genes regulating osteogenesis.

Characterization of biomaterials polar interactions in physiological conditions using liquid–liquid contact angle measurements: Relation to fibronectin adsorption

Wettability of biomaterials surfaces and protein-coated substrates is generally characterized with the sessile drop technique using polar and apolar liquids. This procedure is often performed in air, which does not reflect the physiological conditions. In this study, liquid/liquid contact angle measurements were carried out to be closer to cell culture conditions. This technique allowed us to evaluate the polar contribution to the work of adhesion between an aqueous medium and four selected biomaterials widely used in tissue culture applications: bacteriological grade polystyrene (PS), tissue culture polystyrene (tPS), poly(2-hydroxyethyl methacrylate) film (PolyHEMA), and hydroxypropylmethylcellulose-carboxymethylcellulose bi-layered Petri dish (CEL). The contributions of polar interactions were also estimated on the same biomaterials after fibronectin (Fn) adsorption. The quantity of Fn adsorbed on PS, tPS, PolyHEMA and CEL surfaces was evaluated by using the fluorescein-labeled protein. PolyHEMA and CEL were found to be hydrophilic, tPS was moderately hydrophilic and PS was highly hydrophobic. After Fn adsorption on PS and tPS, a significant increase of the surface polar interaction was observed. On PolyHEMA and CEL, no significant adsorption of Fn was detected and the polar interactions remained unchanged. Finally, an inverse correlation between the polarity of the surfaces and the quantity of adsorbed Fn was established.

The Potential of N-Rich Plasma-Polymerized Ethylene (PPE:N) Films for Regulating the Phenotype of the Nucleus Pulposus

We recently developed a nitrogen-rich plasma-polymerized biomaterial, designated “PPE:N” (N-doped plasma-polymerized ethylene) that is capable of suppressing cellular hypertrophy while promoting type I collagen and aggrecan expression in mesenchymal stem cells from osteoarthritis patients. We then hypothesized that these surfaces would form an ideal substrate on which the nucleus pulposus (NP) phenotype would be maintained. Recent evidence using microarrays showed that in young rats, the relative mRNA levels of glypican-3 (GPC3) and pleiotrophin binding factor (PTN) were significantly higher in nucleus pulposus (NP) compared to annulus fibrosus (AF) and articular cartilage. Furthermore, vimentin (VIM) mRNA levels were higher in NP versus articular cartilage. In contrast, the levels of expression of cartilage oligomeric matrix protein (COMP) and matrix gla protein precursor (MGP) were lower in NP compared to articular cartilage. The objective of this study was to compare the expression profiles of these genes in NP cells from fetal bovine lumbar discs when cultured on either commercial polystyrene (PS) tissue culture dishes or on PPE:N with time. We found that the expression of these genes varies with the concentration of N ([N]). More specifically, the expression of several genes of NP was sensitive to [N], with a decrease of GPC3, VIM, PTN, and MGP in function of decreasing [N]. The expression of aggrecan, collagen type I, and collagen type II was also studied: no significant differences were observed in the cells on different surfaces with different culture time. The results support the concept that PPE:N may be a suitable scaffold for the culture of NP cells. Further studies are however necessary to better understand their effects on cellular phenotypes.

P.174 Conservative-surgical treatment of parodontitis

Augmentation of the craniofacial region is necessary for many aesthetic and reconstructive procedures. Tissue engineering offers a new option to supplement existing treatment regimens. In this procedure, materials composed of hydroxyapatite (HA), of synthetic or natural origin, are used as scaffolds. The aim of this study was to evaluate the effects of three HA materials on cultured human osteoblasts in vitro.Explant cultures of cells from human alveolar bone were established. Human osteoblasts were cultured on the surface of HA calcified from red algae (C GRAFT™/Algipore®), deproteinized bovine HA (Bio-Oss®) and bovine HA carrying the cell binding peptide P-15 (Pep Gen P-15™). Cultured cells were evaluated with respect to cell attachment, proliferation and differentiation. Cells were cultured for 6 and 21 days under osteogenic differentiation conditions, and tissue-culture polystyrene dishes were used as control. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay, protein synthesis analysis and by scanning electron microscopical examination. Osteogenic differentiation was screened by the expression of alkaline phosphatase. The osteoblastic phenotype of the cells was monitored using mRNA levels of the bone-related proteins including osteocalcin, osteopontin and collagen Type I.We found that cells cultured on C GRAFT™/Algipore® and Pep Gen P-15™ showed a continuous increase in DNA content and protein synthesis. Cells cultured on Bio-Oss®showed a decrease in DNA content from Day 6 (P < 0.05) to Day 21 (P < 0.0001) and protein synthesis on Day 21 (P < 0.005). Alkaline phosphatase activity increased in cells grown on C GRAFT™/Algipore® and Pep Gen P-15™ in contrast to cells grown on Bio-Oss®, in which the lowest levels of activity could be observed on Day 21 (P < 0.05). Reverse transcriptase polymerase chain reaction analysis confirmed the osteoblastic phenotype of the cells grown on all three materials throughout the whole culture period.The results of our in vitro study show that the differences in metabolic activity of cells grown on HA materials are directly related to the substrate on which they are grown. They confirm the excellent properties of HA carrying the cell binding peptide P-15 and HA calcified from red algae as used in maxillofacial surgery procedures.

Temperature-responsive glass coverslips with an ultrathin poly( N-isopropylacrylamide) layer

A temperature-responsive cross-linked polymer gel was covalently grafted onto glass coverslips by electron beam irradiation. The grafted thickness and amount of polymer as well as the surface wettability increased with the initial monomer concentration. When the monomer concentration was 5 wt.%, the grafted polymer density was 0.84 μg cm −2, and cells adhered and spread on the surface at 37 °C, but detached at 20 °C. In contrast, when the monomer concentration was 35 wt.%, the polymer density was 1.28 μg cm −2, and the surfaces were cell repellent even at 37 °C. These results show a remarkable contrast to those obtained from temperature-responsive polymer-grafted tissue culture polystyrene dishes, since various types of cells showed temperature-dependent cell adhesion/detachment when the grafted density was around 2 μg cm −2 on these surfaces. We discuss the possible molecular mechanisms underlying this discrepancy.

Leptin Increases Adult Hippocampal Neurogenesis in Vivo and in Vitro

Leptin, an adipose-derived hormone, has been implicated in several physiological processes involving the hippocampus. However, the role of leptin in adult hippocampal neurogenesis remains unknown. Here we show that leptin regulates neurogenesis in the dentate gyrus of adult mice as well as in cultured adult hippocampal progenitor cells. Chronic administration of leptin to adult mice increased cell proliferation without significant effects on the differentiation and the survival of newly proliferated cells in the dentate gyrus. The expression of the long form leptin receptor, LepRb, was detected in hippocampal progenitor cells by reverse transcription-PCR and immunohistochemistry. Leptin treatment also increased proliferation of cultured adult hippocampal progenitor cells. Analysis of signal transduction pathways revealed that leptin stimulated phosphorylation of Akt and STAT3 but not ERK1/2. Furthermore, pre-treating the cells with specific inhibitors of Akt or STAT3 attenuated leptin-induced cell proliferation in a dose-dependent manner. Taken together, our results support a role for leptin in adult hippocampal neurogenesis and suggest the involvement of the Akt and STAT3 signaling pathways in mediating the actions of leptin on neurogenesis.

Repetitive Firing Triggers Clustering of Kv2.1 Potassium Channels in Aplysia Neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.

PC12 polarity on biopolymer nanogratings

Cell differentiation properties are strongly entangled with the morphology and physical properties of the extracellular environment. A complete understanding of this interaction needs artificial scaffolds with controlled nano-/micro-topography. We induced specific topographies by nanoimprint lithography (NIL) on tissue culture polystyrene (TCPS) dishes substrates and, using light microscopy and high-magnification scanning-electron-microscopy, quantitatively compared the changes in PC12 differentiation phenotype induced by the periodicity of the nanopatterns. This analysis revealed that nanogratings reduce the number of neurites produced by PC12 cells upon treatment with NGF and that neuronal bipolarity correlated with an increased stretching of the cell body and a reduced length of the cell neuronal protrusions.

Bone and vascular endothelial cells in cultures on nanocrystalline diamond films

The adhesion, growth and viability of human osteoblast-like MG 63 cells and bovine pulmonary artery endothelial CPAE cells were investigated in cultures grown on nanocrystalline diamond (NCD) films with either low surface roughness (RMS of 8.2 nm) or hierarchically organized surfaces made of nano-dimensional roughness NCD films deposited on Si surfaces with the original microroughness (RMS of 301.1 nm and 7.6 nm, respectively). The number and viability of both cell types on NCD films was similar to the values obtained on control tissue culture polystyrene dishes, or even better. It can be concluded that nanocrystalline diamond films give good support for adhesion and growth of osteogenic and endothelial cells, and could be used in tissue engineering applications.

The use of biotin–avidin binding to facilitate biomodification of thermoresponsive culture surfaces

Here, we report biomodification of temperature-responsive culture surfaces with biotinylated biomolecules utilizing streptavidin and biotinylation of the surfaces. Poly( N-isopropylacrylamide- co-2-carboxyisopropylacrylamide) was covalently grafted onto tissue culture polystyrene (TCPS) dishes. Biotinylated Arg-Gly-Asp-Ser (RGDS) peptides with different spacer lengths (biotin-conjugated G n RGDS ( n=1, 6, 12, 16)) were examined. Human umbilical vein endothelial cells (HUVECs) adhered and were well spread on G 12RGDS-immobilized surfaces in the absence of serum at 37 °C, while much less cell adhesion was observed with the other peptides. Adhered HUVECs were detached on reducing temperature to 20 °C, or on adding free RGDS peptide. Interestingly, cell detachment was accelerated by applying both these techniques. Consequently, by optimizing the spacer length, biomolecules can be functionally immobilized onto thermoresponsive surfaces via the affinity binding between avidin and biotin.

Novel cell sheet carriers using polyion complex gel modified membranes for tissue engineering technology for cell sheet manipulation and transplantation

A novel hydrogel membrane composed of polyion complex gels was developed to transfer cultured cell sheets from thermoresponsive cell culture dishes. Polyion complexes have been prepared by mixing poly( N, N-dimethylacrylamide- co-2-acrylamido-2-methylpropane sulfonic acid) (P(DMAAm- co-AMPS)) as anionic water-soluble polymers and poly( N, N-dimethylacrylamide- co-2-acryloxyethyltrimethylammonium chloride) (P(DMAAm- co-AETA-Cl)) as cationic water-soluble polymers. Various polyion complexes have been prepared by changing the composition of two polyelectrolytes. Through a set of Teflon ® ring device, polyion complexes have been modified on porous membranes by electron beams irradiated cross-linking and covalent grafting technique. NIH/3T3 mouse cells as demo cells have been cultured to prepare cell sheets. This cell sheets could be recovered by using the resulting polyion complex gels as cell sheet carriers from temperature-responsive cell culture surfaces and be transferred to new collagen-coated tissue culture polystyrene dishes. This new technique has potential applications not only for transplantation but for creation of three dimensional layered cell sheet tissues.

A functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits non-specific protein, bacterial, and mammalian cell adhesion.

This paper describes a new bioassay surface chemistry that effectively inhibits non-specific biomolecular and cell binding interactions, while providing a capacity for specific immobilization of desired biomolecules. Poly(ethylene glycol) (PEG) as the primary component in nonfouling film chemistry is well-established, but the multicomponent formulation described here is unique in that it (1) is applied in a single, reproducible, solution-based coating step; (2) can be applied to diverse substrate materials without the use of special primers; and (3) is readily functionalized to provide specific attachment chemistries. Surface analysis data are presented, detailing surface roughness, polymer film thickness, and film chemistry. Protein non-specific binding assays demonstrate significant inhibition of serum, fibrinogen, and lysozyme adsorption to coated glass, indium tin oxide, and tissue culture polystyrene dishes. Inhibition of S. aureus and K. pneumoniae microbial adhesion in a microfluidic flow cell, and inhibition of fibroblast cell adhesion from serum-based cell culture is shown. Effective functionalization of the coating is demonstrated by directing fibroblast adhesion to polymer surfaces activated with an RGD peptide. Batch-to-batch reproducibility data are included. The in situ cross-linked PEG-based coating chemistry is unique in its formulation, and its surface properties are attractive for a broad range of in vitro bioassay applications.

Rapid Cell Sheet Recovery by Successive Grafting of PHEMA and PIPAAm onto TCPS Dish Surfaces

Rapid recovery of cell sheets is considered important to maintain the biological function and viability of recovered cell sheets. To accelerate required culture substrate hydrophilic/ hydrophobic structural changes in response to culture temperature alteration, poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(N-isopropylacrylamide) (PIPAAm) were grafted successively onto tissue culture polystyrene (TCPS) dishes by electron beam irradiation. Analysis by attenuated total reflection-Fourier transform infrared revealed that PHEMA and PIPAAm were successfully grafted to surfaces of TCPS dish. PIPAAm-PHEMA-grafted TCPS (PIPAAm-PHEMA-TCPS) dishes were compared with PIPAAm-grafted TCPS dishes for cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-TCPS dish, compared to only 13 min to detach cell sheets from PIPAAm-PHEMA-TCPS dish, which is successively grafted with PHEMA and PIPAAm. PHEMA is a well-known as a high hygroscopic polymer. In the case of PIPAAm-PHEMA-TCPS dish, PHEMA layer acted as a water pool to accelerate the hydration of PIPAAm layer due to the effective and simultaneous water supply to PIPAAm layer, resulting in rapid hydration of grafted PIPAAm molecules and detachment of cell sheet compare to PIPAAm-TCPS dish without PHEMA layer.

In vitro Mineralization by Mesenchymal Stem Cells Cultured on Titanium Scaffolds

Titanium has been utilized in the field of orthopaedic and dental reconstructive surgery, but mineralization through osteogenic differentiation of osteogenic cells on titanium surfaces has not been fully investigated. Here we cultured rat mesenchymal stem cells (MSCs) on the surfaces of titanium dishes in osteogenic media containing calcein which is a calcium-binding fluorescence dye. On titanium dishes, MSCs showed high viability to adhere to the surfaces and excellent proliferation. At day 14 of culture, MSCs differentiated into osteoblasts to form mineralized matrices on titanium dishes as well as tissue culture polystyrene (TCPS) dishes which are widely recognized as optimal culture substrates. Calcein was incorporated into the bone minerals fabricated by MSCs cultured on both substrates to show green emission under fluorescence microscopy. The fluorescence intensity was quantified with an image analyser during culture periods. These results indicate that the surfaces of titanium showed a high adhesion/proliferation potential to MSCs and that the titanium effectively supported the osteogenic differentiation of MSCs comparable to TCPS dishes. Therefore, the titanium is an effective scaffold that is applicable in bone reconstruction surgery.

Exogenous Recombinant Human BMP-2 Has Little Initial Effects on Human Osteoblastic Cells Cultured on Collagen Type I Coated/Noncoated Hydroxyapatite Ceramic Granules

Tissue engineering of bone entails the successful interplay between osteoinductive factors, osteogenic cells, their extracellular environment, and an osteoconductive biomaterial scaffold. Naturally produced ceramics, like hydroxyapatite (HA) calcified from red algae, are the most promising materials for use as scaffolds in this field. We hypothesized that extracellular matrix compartments and osteoinductive factors could further ameliorate the bioactivity of the scaffold. Osteosarcoma cells with proven osteogenic phenotype (SaOS-2) were cultured onto type I collagen coated (Coll I/HA) and noncollagen coated HA granules (NC/HA) gained from red algae (C GRAFT/Algipore). Cells grown on tissue culture polystyrene dishes (TCPS) were used as controls. Second, SaOS-2 cells cultured on Coll I/HA, NC/HA, and TCPS were treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) in different concentrations (10, 100, and 500 ng/mL). Non rhBMP-2-treated cultures were used as controls. Cultures of both experiments were grown under osteogenic differentiation conditions and after 24, 48, and 72 hours assays for cell viability, apoptosis, alkaline phosphatase activity (ALP), and osteocalcin (OC) secretion were done. Coating of HA granules with type I collagen showed higher cell viability in rhBMP-2-treated and nontreated cells. Supplementation of cultured cells with exogenous rhBMP-2 showed a dose-dependent effect only in the TCPS group. No alterations of the apoptotic rate within 1 investigation group were found. Addition of rhBMP-2 did not significantly alter the specific OC secretion of cells grown on Coll I/HA and TCPS. These in vitro findings show that in the initial period of cultivation and up to 72 hours, the coating of HA granules with collagen type I had positive effects on cell viability and osteoblastic characteristics of osteoblastic cells. In contrast, the supplementation with exogenous rhBMP-2 shows no dose-dependent effects. The combination of collagen type I and exogenous rhBMP-2 did not ameliorate the bioactivity of hydroxyapatite calcified from red algae in the initial period of cultivation.

Preimplantation factor (PIF) upregulates first trimester toll like receptor-2, supporting the role of PIF as an embryo derived factor influencing maternal innate immunity

RECEPTOR-2, SUPPORTING THE ROLE OF PIF AS AN EMBRYO DERIVED FACTOR INFLUENCING MATERNAL INNATE IMMUNITY MICHAEL PAIDAS, GRACIELA KRIKUN, LYNN BUCHWALDER, FREDERICK SCHATZ, KARL GODLEWSKI, EYTAN R. BARNEA, Yale University School of Medicine, Department Obstetrics, Gynecology and Reproductive Sciences, CoDirector, Yale Blood Center for Women and Children, New Haven, Connecticut, Yale University, Obstetrics and Gynecology & Reproductive Sciences, New Haven, Connecticut, UMDNJ/Robert Wood Johnson Medical School, CAMcare Health Center, Cherry Hill, New Jersey OBJECTIVE: Preimplantation factor (PIF), is a novel, embryo-derived, small peptide which appears very early in pregnancy. The maternal host response in pregnancy must allow for tolerance of the implanting embryo, and preservation of maternal defense mechanisms against viral and bacterial pathogens. We posit that: a) PIF may act as an immune modulating agent, capable of augmenting maternal defense mechanisms via upregulation of Toll-like receptor (TLR)-2, a transmembrane protein involved in pathogen recognition; b) PIF may will influence decidual cell (DC) production of interleukins (IL), metalloproteinases (MMP) involved in extracellular matrix degradation, and angiogenesis. STUDY DESIGN: DCs were obtained from women undergoing first trimester (TRI) elective terminations or third TRI repeat Cesarean sections. DCs were isolated, purified, and seeded on polystyrene tissue culture dishes. The effects of PIF on TLR-2 protein expression was carried out using first TRI DCs. Third TRI DCs were used to study the effects of PIF on well characterized markers of DC function, as measured by ELISA. Western blotting for TLR-2 and beta actin was conducted using established methods. Densitometric analyses were carried out with the Image J NIH analysis software. PIF (10nM) was used as an agonist to DC culture. Values were expressed as TLR2/beta actin ratio, correcting for total DNA content. RESULTS: PIF incubation was associated with a two-fold increase in TLR-2 expression. We were unable to detect any change in secreted levels of the following third TRI DC products: IL-6, IL-8, IL-11, plasminogen activator inhibitor-1, MMP-3, vascular endothelial growth factor. CONCLUSION: In this preliminary study, PIF significantly upregulates first trimester TLR-2 expression, suggesting that PIF may augment the maternal innate immune response. Maintainence of host defense mechanisms must occur as the host prepares to accept the implanting embryo. Further studies are needed to confirm PIF’s effects on TLR-2, evaluate downstream effects, and determine whether PIF regulates first TRI DC function.

Nano-scaled hydroxyapatite/silk fibroin sheets support osteogenic differentiation of rat bone marrow mesenchymal cells

A novel biomaterial that was composed of nano-scaled sintered hydroxyapatite (HAp) and silk fibroin (SF) was fabricated. We cultured rat marrow mesenchymal cells (MMCs) on this biomaterial (nano-HAp/SF sheet), on bare SF sheets, and on tissue culture polystyrene (TCPS) dishes as controls, then evaluated cell adhesion, proliferation, and differentiation of the MMCs. After 1 h of culture, a large number of viable cells were observed on the nano-HAp/SF sheets in comparison to the controls. In addition, after 3 h of culture, the morphology of the cells on the nano-HAp/SF sheets was quite different from that on the SF sheets. MMCs extrude their cytoplasmic processes to nano-HAp particles and are well attached to the sheets. After 14 days of culture, under osteogenic conditions, the alkaline phosphatase (ALP) activity and bone-specific osteocalcin secretion of the cells on nano-HAp/SF sheets were higher than were those on the controls. These results indicated that the surface of the nano-HAp/SF sheets is covered with appropriate HAp crystal for MMC adhesion/proliferation and that the sheets effectively support the osteogenic differentiation of MMCs. Therefore, the nano-HAp/SF sheet is an effective biomaterial that is applicable in bone reconstruction surgery.

Subcellular topological effect of particle monolayers on cell shapes and functions

We studied topological effects of subcellular roughness displayed by a closely packed particle monolayer on adhesion and growth of endothelial cells. Poly(styrene- co-acrylamide) (SA) particles were prepared by soap-free emulsion copolymerization. Particle monolayers were prepared by Langmuir–Blodgett deposition using particles, which were 527 (SA053) and 1270 nm (SA127) in diameter. After 24-h incubation, cells tightly adhered on a tissue culture polystyrene dish and randomly spread. On the other hand, cells attached on particle monolayers were stretched into a narrow stalk-like shape. Lamellipodia spread from the leading edge of cells attached on SA053 monolayer to the top of the particles and gradually gathered to form clusters. This shows that cell–cell adhesion became stronger than cell–substrate interaction. Cells attached to SA127 monolayer extended to the reverse side of a particle monolayer and engulfed particles. They remained immobile without migration 24 h after incubation. This shows that the inhibition of extensions on SA127 monolayer could inhibit cell migration and cell proliferation. Cell growth on the particle monolayers was suppressed compared with a flat TCPS dish. The number of cells on SA053 gradually increased, whereas that on SA127 decreased with time. When the cell seeding density was increased to 200,000 cells cm −2, some adherent cells gradually became into contact with adjacent cells. F-actin condensations were formed at the frame of adherent cells and the thin filaments grew from the edges to connect each other with time. For the cell culture on SA053 monolayer, elongated cells showed a little alignment. Cells showed not arrangement of actin stress fibers but F-actin condensation at the contact regions with neighboring cells. Interestingly, the formed cell monolayer could be readily peeled from the particle monolayer. These results indicate that endothelial cells could recognize the surface roughness displayed by particle monolayers and the response was dependent on the pitch of particle monolayers.

Bio-functionalized thermoresponsive interfaces facilitating cell adhesion and proliferation

Bio-functionalized thermoresponsive culture interfaces co-immobilized with cell adhesive peptide, RGDS, and cell growth factor, insulin (INS), are investigated to promote initial cell adhesion and cell growth for further cell sheet engineering applications. These bio-functionalized interfaces were prepared by electron beam-induced copolymerization of N-isopropylacrylamide (IPAAm) with its carboxyl-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), and grafting onto tissue culture polystyrene dishes, followed by immobilization of RGDS and/or INS to CIPAAm carboxyls. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on the RGDS-INS co-immobilized thermoresponsive interfaces. Immobilized RGDS facilitated initial EC adhesion on the surfaces and INS modification was demonstrated to induce EC proliferation, respectively. More pronounced EC growth was indicated by co-immobilization of appropriate amount of RGDS and INS. This may be due to synergistic effect of direct co-stimulation of adhered ECs by surface-immobilized RGDS and INS molecules. ECs grown on the RGDS-INS co-immobilized thermoresponsive interfaces can also be recovered spontaneously as viable tissue monolayers by solely reducing culture temperature. RGDS-INS co-immobilized thermoresponsive interfaces strongly supported initial EC adhesion and growth than unmodified thermoresponsive surfaces even under serum-free culture. Addition of soluble growth factors to serum-free culture medium effectively induced EC proliferation to confluency. Co-immobilization of cell adhesion peptides and growth factors on thermoresponsive surfaces should be effective for rapid preparation of intact cell sheets and their utilization to regenerative medicine.

Nano-Scaled Hydroxyapatite/Silk Fibroin Composites as Mesenchymal Cell Culture Scaffolds

The objective of this study was to evaluate cell adhesion and proliferation on the hydroxyapatite (HAp)-coated silk fibroin (SF) fabric. Nano-scaled sintered HAp particles were covalently coated on SF chemically modified by graft polymerization. After the fabrication of the HAp/SF composite, mesenchymal cells (MCs) derived from EGFP-expressing transgenic rat bone marrow were seeded on the composite and cultured for 10 days. Fluorescence and scanning electron microscopy (SEM) revealed that the cells adhered and actively proliferated on the composites comparable to those on tissue culture polystyrene (TCPS) dishes. The results suggest that the composites are suitable for mesenchymal cell culture scaffolds and useful materials for regenerative medicine.