Polystyrene Dishes Research Articles

Action potential-triggered somatic exocytosis in mesencephalic trigeminal nucleus neurons in rat brain slices

The neurons in the mesencephalic trigeminal nucleus (MeV) play essential roles in proprioceptive sensation of the face and oral cavity. The somata of MeV neurons are generally assumed to carry out neuronal functions but not to play a direct role in synaptic transmission. Using whole-cell recording and membrane capacitance (C(m)) measurements, we found that the somata of MeV neurons underwent robust exocytosis (C(m) jumps) upon depolarization and with the normal firing of action potentials in brain slices. Both removing [Ca(2+)](o) and buffering [Ca(2+)](i) with BAPTA blocked this exocytosis, indicating that it was completely Ca(2+) dependent. In addition, an electron microscopic study showed synaptic-like vesicles approximated to the plasma membrane in somata. There was a single Ca(2+)-dependent releasable vesicle pool with a peak release rate of 1912 fF s(-1). Importantly, following depolarization-induced somatic exocytosis, GABA-mediated postsynaptic currents were transiently reduced by 31%, suggesting that the somatic vesicular release had a retrograde effect on afferent GABAergic transmission. These results provide strong evidence that the somata of MeV neurons undergo robust somatic secretion and may play a crucial role in bidirectional communication between somata and their synaptic inputs in the central nervous system.

Effect of different surface nanoroughness of titanium dioxide films on the growth of human osteoblast‐like MG63 cells

Cell behavior depends strongly on the physical and chemical properties of the material surface, for example, its chemistry and topography. The authors have therefore assessed the influence of materials of different chemical composition (i.e., glass substrates with and without TiO(2) films in anatase form) and different surface roughness (R(a) = 0, 40, 100, or 170 nm) on the adhesion, proliferation, and osteogenic differentiation of human osteoblast-like MG63 cells. On day 1 after seeding, the largest cell spreading area was found on flat TiO(2) films (R(a) = 0 nm). On TiO(2) films with R(a) = 170 nm, the cell spreading area was larger and the number of initially adhering cells was higher than the values on the corresponding uncoated glass. On day 3 after seeding, the cell number was higher on the TiO(2) films (R(a) = 0 and 40 nm) than on the corresponding glass substrates and the standard polystyrene dishes. On day 7, all TiO(2) films contained higher cell numbers than the corresponding glass substrates, and the cells on the TiO(2) films with R(a) = 40 and 100 nm also contained a higher concentration of β-actin. These results indicate that TiO(2) coating had a positive influence on the adhesion and subsequent proliferation of MG63 cells. In addition, on all investigated materials, the cell population density achieved on day 7 decreased with increasing surface roughness. The concentration of osteocalcin, measured per mg of protein, was significantly lower in the cells on rougher TiO(2) films (R(a) = 100 and 170 nm) than in the cells on the polystyrene dishes. Thus, it can be concluded that the adhesion, growth, and phenotypic maturation of MG63 cells were controlled by the interplay between the material chemistry and surface topography, and were usually better on smoother and TiO(2)-coated surfaces than on rougher and uncoated glass substrates.

Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering

Nanofibrous scaffolds loaded with bioactive nanoparticles are promising materials for bone tissue engineering. In this study, composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA) and diamond nanoparticles were fabricated by an electrospinning technique. PLGA was dissolved in a mixture of methylene chloride and dimethyl formamide (2:3) at a concentration of 2.3 wt%, and nanodiamond (ND) powder was added at a concentration of 0.7 wt% (about 23 wt% in dry PLGA). In the composite scaffolds, the ND particles were either arranged like beads in the central part of the fibers or formed clusters protruding from the fibers. In the PLGA-ND membranes, the fibers were thicker (diameter 270 ± 9 nm) than in pure PLGA meshes (diameter 218 ± 4 nm), but the areas of pores among these fibers were smaller than in pure PLGA samples (0.46 ± 0.02 μm(2) versus 1.28 ± 0.09 μm(2) in pure PLGA samples). The PLGA-ND membranes showed higher mechanical resistance, as demonstrated by rupture tests of load and deflection of rupture probe at failure. Both types of membranes enabled the attachment, spreading, and subsequent proliferation of human osteoblast-like MG-63 cells to a similar extent, although these values were usually lower than on polystyrene dishes. Nevertheless, the cells on both types of membranes were polygonal or spindle-like in shape, and were distributed homogeneously on the samples. From days 1-7 after seeding, their number rose continuously, and at the end of the experiment, these cells were able to create a confluent layer. At the same time, the cell viability, evaluated by a LIVE/DEAD viability/cytotoxicity kit, ranged from 92% to 97% on both types of membranes. In addition, on PLGA-ND membranes, the cells formed well developed talin-containing focal adhesion plaques. As estimated by the determination of tumor necrosis factor-alpha levels in the culture medium and concentration of intercellular adhesion molecule-1, MG-63 cells, and RAW 264.7 macrophages on these membranes did not show considerable inflammatory activity. This study shows that nanofibrous PLGA membranes loaded with diamond nanoparticles have interesting potential for use in bone tissue engineering.

Cardiac differentiation of embryonic stem cells by substrate immobilization of insulin-like growth factor binding protein 4 with elastin-like polypeptides

The establishment of cardiomyocyte differentiation of embryonic stem cells (ESCs) is a useful strategy for cardiovascular regenerative medicine. Here, we report a strategy for cardiomyocyte differentiation of ESCs using substrate immobilization of insulin-like growth factor binding protein 4 (IGFBP4) with elastin-like polypeptides. Recently, IGFBP4 was reported to promote cardiomyocyte differentiation of ESCs through inhibition of the Wnt/β-catenin signaling. However, high amounts of IGFBP4 (approximately 1 μg/mL) were required to inhibit the Wnt/β-catenin signaling and induce differentiation to cardiomyocytes. We report herein induction of cardiomyocyte differentiation using IGFBP4-immobilized substrates. IGFBP4-immobilized substrates were created by fusion with elastin-like polypeptides. IGFBP4 was stably immobilized to polystyrene dishes through fusion of elastin-like polypeptides. Cardiomyocyte differentiation of ESCs was effectively promoted by strong and continuous inhibition of Wnt/β-catenin signaling with IGFBP4-immobilized substrates. These results demonstrated that IGFBP4 could be immobilized using fusion of elastin-like polypeptides. Our results also demonstrate that substrate immobilization of IGFBP4 is a powerful tool for differentiation of ESCs into cardiomyocytes. These findings suggest that substrate immobilization of soluble factors is a useful technique for differentiation of ESCs in regenerative medicine and tissue engineering.

Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes

Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell–substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes ( Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho/Rho-associated kinase (ROCK) signaling pathway may be involved in spheroid formation.

Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells

The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH coating on tissue culture polystyrene dishes, and for the feeder-free culture of hES cells on PMEDSAH coating in defined culture medium. This culture system represents a key step toward the fully defined and xenogeneic-free culture of hES cells.

Maintenance of hemiround colonies and undifferentiated state of mouse induced pluripotent stem cells on carbon nanotube-coated dishes

Induced pluripotent stem (iPS) cells have attracted worldwide interest. However, there have been only a few studies investigating effective culture substrates for feeder-free culturing for the maintenance of iPS cells. In this study, we cultured mouse iPS cells under feeder-free conditions on carbon nanotube (CNT)-coated dishes and then evaluated the colony morphology and differentiation state of the cells on the dishes. After 5 d of cultivation in a medium containing 15% fetal bovine serum (FBS) and leukemia inhibitory factor (LIF), the colonies on thick films of multi-walled CNTs (MWCNTs) were observed to be hemiround; further, the cells expressed early undifferentiation markers. On the other hand, the colonies on a cell culture polystyrene dish and a collagen-coated polystyrene dish showed indistinct outline and spread well, and most spreading cells only weakly expressed early undifferentiation markers. These results indicate that the thick films of MWCNTs could maintain hemiround colonies and undifferentiated state of mouse iPS cells cultured under feeder-free conditions.

Adhesion and Growth of Human Osteoblast-Like Cell in Cultures on Nanocomposite Carbon-Based Materials

This paper summarizes our recent research on carbon nanoparticles (fullerenes, nanotubes, nanodiamond) as substrates for the adhesion, growth and phenotypic maturation of osteogenic cells. Fullerenes C60 were deposited on microscopic glass coverslips in the form of continuous layers or layers micropatterned with bulge-like prominences of 128 ± 8 nm, 238 ± 3 nm, 326 ± 5n m and 1043 ± 57 nm in height. On continuous layers and on layers with prominences up to 326 ± 5 nm, the adhesion and proliferation of human osteoblast-like MG 63 cells was similar as in control cells on polystyrene dishes. On layers with prominences 1043 ± 57 nm in height, the cells grew preferentially in grooves among the prominences. Similar cell responses were found in MG 63 cells cultured on continuous and micropatterned films made of binary C60/Ti composites. In the second set of experiments, single wall carbon nanohorns and multi-wall carbon nanotubes were mixed with a terpolymer of polytetrafluoroethylene, polyvinyldifluoride and polypropylene (in concentrations from 2 to 8 wt%) or with polysulfone (concentrations from 0.5 to 2 wt%). Adding carbon nanotubes to the terpolymer markedly improved the cell adhesion, spreading and subsequent growth of MG 63 cells, while the adhesion and growth of these cells on the pure and modified polysulfone were similar, which was probably due to a more hydrophilic character of polysulfone compared to the terpolymer. Nanocrystalline diamond (NCD) was deposited on silicon substrates and provided an excellent substrate for the adhesion, growth and osteogenic differentiation of MG 63 cells, measured by the concentration of osteocalcin. These beneficial effects of NCD films were further enhanced by boron doping, which can be attributed to increased electroactivity (i.e., electrical potential and conductivity) of these films.

Surface chemical patterning for long‐term single‐cell culture

Surface chemical patterning of polystyrene (PS) dishes for long-term single-cell culture was accomplished by oxygen plasma treatment through the windows of a polydimethylsiloxane membrane mask that produced hydrophilic areas of different shapes and sizes, followed by overnight incubation with either Pluronic F108 solution or a mixture of Pluronic F108 solution and fibronectin. Selective cell attachment on pattern areas of PS dishes was investigated in light of cell seeding experiments and X-ray photoelectron spectroscopy measurements. Activation of the hydrophilic areas of patterned PS surfaces by serum proteins in the culture medium was conducive to cell attachment on the pattern areas of dishes incubated with only Pluronic solution. Preferential adsorption of fibronectin on hydrophilic pattern areas enhanced selective cell attachment on patterned dishes incubated with a mixture of Pluronic solution and fibronectin. Cell-culture experiments demonstrated an effect of surface patterning on both cell and nucleus shape and confirmed the long-term (>2 weeks) stability of the produced single-cell patterns in serum medium.

Efficacy of surface sampling methods for different types of beryllium compounds

The objective of the research work was to evaluate the efficiency of three different sampling methods (Ghost Wipe™, micro-vacuum, and ChemTest®) in the recovery of Be dust by assessing: (1) four Be compounds (beryllium acetate, beryllium chloride, beryllium oxide and beryllium aluminium), (2) three different surfaces (polystyrene, glass and aluminium) and (3) inter-operator variation. The three sampling methods were also tested on site in a laboratory of a dental school for validation purposes. The Ghost Wipe™ method showed recovery ranging from 43.3% to 85.8% for all four Be compounds and for all three quantities of Be spiked on Petri dishes, while recovery with the micro-vacuum method ranged from 0.1% to 12.4%. On polystyrene dishes with 0.4 µg Be, the recovery ranged from 48.3% to 81.7%, with an average recovery of 59.4% for Operator 1 and 68.4% for Operator 2. The ChemTest® wipe method with beryllium acetate, beryllium chloride, and AlBeMet® showed analogous results that are in line with the manufacturer's manual, but collection of beryllium oxide was negative. In the dental laboratory, Ghost Wipe™ samplings showed better recovery than the micro-vacuum method. The ratios between the recovered quantities of Be in each location where the Ghost Wipe™ was tested differed substantially, ranging from 1.45 to 64. In the dental laboratory, a faint blue color indicating the presence of Be was observed on the ChemTest® wipes used in two locations out of six. In summary, the Ghost Wipe™ method was more efficient than micro-vacuuming in collecting the Be dust from smooth, non-porous surfaces such as Petri dishes by a factor of approximately 18. The results obtained on site in a dental laboratory also showed better recovery with Ghost Wipes™. However, the ratio of Be recovered by Ghost Wipes™ versus micro-vacuuming was much lower for surfaces where a large amount of dust was present. Wet wiping is preferred over micro-vacuuming for beryllium forms, but this conclusion probably applies to the ultra-low particulate loading levels (0.4 micrograms or less) which was tested in this study.

Design of culture substrates for large-scale expansion of neural stem cells

Neural stem cells (NSCs) have been frequently used to investigate in vitro the molecular and cellular mechanisms underlying the development of the central nervous system (CNS). In addition, NSCs are regarded as one of the potential sources for the cell replacement therapy of CNS disorders. Most of these studies have utilized NSCs prepared by neurosphere culture. However, this method normally yields a heterogeneous population containing differentiated neural cells as well as NSCs. In addition, the rate of cell expansion is not high enough for obtaining a large quantity of NSCs in a short period. Here we report the design of culture substrates that allow highly selective and rapid expansion of NSCs. We synthesize epidermal growth factor fused with a hexahistidine sequence (EGF-His) and a polystyrene-binding peptide (EGF-PSt), and these engineered growth factors were surface-anchored to a nickel-chelated glass plate and a polystyrene dish, respectively. The EGF-His-chelated glass substrate was further used to assemble a culture module. Neurosphere-forming cells prepared from the fetal rat striatum were used to examine the selective expansion of NSCs using the EGF-His-chelated module and the EGF-PSt-bound polystyrene dish. Our results show that the culture module enables to selectively expand NSCs in a closed system more efficiently than the standard neurosphere culture. The EGF-PSt-bound polystyrene dish also permits efficient expansion of NSCs, providing a straightforward means to acquire a large quantity of pure NSCs in standard laboratories.

A Suspension Induction for Myocardial Differentiation of Rat Mesenchymal Stem Cells on Various Extracellular Matrix Proteins

The microenvironment of bone marrow-derived mesenchymal stem cells (MSCs) strictly regulates their differentiation. In this study, we have developed a new suspension induction method for myocardial differentiation of bone marrow-derived rat MSCs (rMSCs) in vitro on various extracellular matrix (ECM) proteins. Myocardial differentiation of rMSCs was induced with a conventional monolayer method and our suspension method. In our suspension induction, a cell suspension was treated with the medium in the presence of an inducer, incubated for 2 h under a suspension conditions, and moved to a monolayer culture on gelatin-coated, collagen type I-coated, fibronectin-coated, or polystyrene dishes until the total induction time was 24 h. We evaluated the myocardial differentiation by counting the number of colonies of beating cells, performing immunohistochemical staining, and measuring the expression of cardiac-specific gene mRNA using real-time quantitative polymerase chain reaction. We found that rMSCs induced with the conventional monolayer method did not differentiate efficiently, whereas beating cell colonies were found on ECM-coated dishes of suspension-induced cells, after 3 weeks of culture, especially on gelatin-coated dishes. The beating cells were positively stained with anti-troponin T-C antibody and expressed specific cardiac markers. In conclusion, these results demonstrated that the suspension induction followed by subsequent culture on gelatin ECM substrates is a promising method for differentiating rMSCs into cardiomyocytes in vitro.

Biochemical Characterization of the Cell-Biomaterial Interface by Quantitative Proteomics

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered.

A high throughput micro-array system of polymer surfaces for the manipulation of primary pancreatic islet cells

We developed a high throughput micro-arrayed polymer system for the study of polymer surfaces for islet cell culture. A micro-arrayed library with 496 different polymers was synthesized and used to examine attachment and insulin expression of islet cells. While most polymers were not supportive, several related polymers were identified as suitable (“hit’s”). The “hit” arrays composed of “hit” polymers with 36 replicates were fabricated to confirm their capacities to support the attachment of islet cells, and these capacities were further validated in large surfaces. Notably, the attachment of islet cells on these synthetic polymeric films has been found to be as supportive as 804G supernatant coated tissue culture polystyrene dishes, one of the most extensively used substrates for the islet cell attachment. Interestingly, the polymeric surfaces optimal for a different cell type, hES derived cells, were distinct, highlighting the utility of these approaches for identifying cell type specific surfaces.

Patterned Hydrogels for Controlled Platelet Adhesion from Whole Blood and Plasma

AbstractThis work describes the preparation and properties of hydrogel surface chemistries enabling controlled and well‐defined cell adhesion. The hydrogels may be prepared directly on plastic substrates, such as polystyrene slides or dishes, using a quick and experimentally simple photopolymerization process, compatible with photolithographic and microfluidic patterning methods. The intended application for these materials is as substrates for diagnostic cell adhesion assays, particularly for the analysis of human platelet function. The non‐specific adsorption of fibrinogen, a platelet adhesion promoting protein, is shown to be completely inhibited by the hydrogel, provided that the film thickness is sufficient (>5 nm). This allows the hydrogel to be used as a matrix for presenting selected bioactive ligands without risking interference from non‐specifically adsorbed platelet adhesion factors, even in undiluted whole blood and blood plasma. This concept is demonstrated by preparing patterns of proteins on hydrogel surfaces, resulting in highly controlled platelet adhesion. Further insights into the protein immobilization and platelet adhesion processes are provided by studies using imaging surface plasmon resonance. The hydrogel surfaces used in this work appear to provide an ideal platform for cell adhesion studies of platelets, and potentially also for other cell types.

Immobilization of galactose ligands on thermoresponsive culture surface and its influence on cell adhesion/detachment

A novel bio-functionalized thermoresponsive surface was prepared by UV-induced copolymerization of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) and grafting onto tissue culture polystyrene (TCPS) dishes, followed by immobilization of galactose ligands. The results indicated that the roughness of surfaces was increased after copolymerization with NIPAAm and immobilization of galactose ligands. The amount of monomers grafted on TCPS was increased with increasing photografting time. The temperature sensitivity of surface was improved with increasing amount of NIPAAm grafted on TCPS, and surface hydrophilicity was enhanced by the introduction of carboxyl groups and galactose ligands, which accelerated cell detachment. Adhesion, proliferation, detachment and transshipment of human hepatoma cell line (HepG2) on the modified surfaces were examined. The immobilization of galactose ligands facilitated the cell adhesion and proliferation. HepG2 cells growing on the modified surfaces could be recovered spontaneously by only reducing culture temperature. The activity of cells obtained by temperature induction was higher than that obtained by enzymatic digestion.

Self-Organization of the Compositional Gradient Structure in Hyaluronic Acid and Poly(N-isopropylacrylamide) Blend Film

A compositional gradient structure in hyaluronic acid (HA) and poly(N-isopropylacrylamide) (PIPAAm) blend film was self-organized from a homogeneous aqueous solution in a plasma-treated polystyrene dish (PTPSD), and the formation mechanisms of the gradient structure were studied by casting the same solution on PTPSD and a non-treated polystyrene dish (NTPSD) under ambient and vacuum conditions. The formation of a compositional gradient structure in HA/PIPAAm blend film was confirmed by scanning electron microscopy, energy dispersive X-ray (EDX) mapping analysis and step-scan photoacoustic Fourier transformed infrared spectroscopy (PAS–FT-IR) measurements. The EDX mapping measurements for Na element revealed that the HA component gradually decreases from the dish-side to the air-side of the film cast on PTPSD, while for the film cast on NTPSD no such obvious change was observed on the cross-section. Further studies on the films prepared on PTPSD and NPTPSD under ambient and vacuum conditions demonstrated that the hydrophilic interaction and the solvent evaporation rate were the most significant factors leading to the formation of a compositional gradient structure in the HA/PIPAAm blend system.

Measurements of Movement and Diffusion Coefficients of Single Cells on Polymeric Surface from Image Analysis

Time-lapse digital images were taken every 30 s of PC12 cells cultured on polystyrene dishes, collagen-coated dishes and poly(L-lysine)-coated dishes in high-serum medium, low-serum medium and neurite outgrowth factor (NGF)-containing medium to investigate their diffusion coefficients (i.e., self-diffusion coefficients), D and specific movement (i.e., specific frequent movement) using image analysis and Fast Fourier Transform (FFT) analysis. D for these cells was found to fluctuate as a function of time, D varying between 0 and 0.08 μm2/s. The trend observed upon examination of average D values was: D in high-serum medium ≥ D in low-serum medium ≥ D in NGF-containing medium. Image analysis and FFT analysis of single cells cultured on polymeric dishes in these three media did not have any specific frequency of cell movement between 0 and 0.0167 Hz. The high diffusion coefficient and high amplitude of power spectra of PC12 cells in high-serum medium might be attributed to the high energy necessary for their continual suppression of the mitogen-activated protein kinase (MAPK) cascade and for them to maintain their undifferentiated state.

Identification and Characterization of Unique Proline-rich Peptides Binding to the Mitochondrial Fission Protein hFis1

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.

A biomimetic hydrogel based on methacrylated dextran- graft-lysine and gelatin for 3D smooth muscle cell culture

Many synthetic hydrogels for cell encapsulation have hitherto been based on polyethylene glycol which is non-natural, non-biodegradable and only terminal-functionalizable, all of which are drawbacks for tissue engineering or cell delivery. The polysaccharide dextran is also highly hydrophilic but biodegradable and pendant-functionalizable and more closely resembles glycosaminoglycans to mimic the natural extracellular matrix. This study reports synthesis of a methacrylate and lysine functionalized dextran and development of hydrogel composite systems based on this material and methacrylamide modified gelatin. The mechanical stiffness and degree of swelling of the hydrogels were varied by manipulation of the degree of functionalization of dextran and gelatin and concentration/composition of precursor solution. Human umbilical artery smooth muscle cells (SMCs) were encapsulated inside hydrogels during gel hardening with photopolymerization. Rapid cell spreading, extensive cellular network formation and high SMC proliferation occurred within softer hydrogels (with shear storage moduli ranging from 898 to 3124 Pa). The encapsulated SMCs appear to be relatively contractile in the initial culture than on tissue culture polystyrene dish due to physical constraint imposed by the hydrogels but they become more synthetic with time possibly due to the inability of cells to reach confluence inside these cell-mediated degradable hydrogels. From the impressive cell proliferation and network formation, these new hydrogels combining polysaccharide and protein derivatives appear to be excellent candidates for further development as bioactive scaffolds for use in vascular tissue engineering and regeneration.