Chemolabile cellular microarrays for screening small molecules and peptides

Microarrays that mediate the uptake of small molecules into living cells are described. Tissue culture cells were seeded onto glass substrates functionalized locally with fluorescently labelled test substances. In order to enable a localized transfer of substances after contact of cells with the substrate, substances were immobilized on the surface either by non-covalent interactions or chemolabile linker groups. These chemolabile linker groups were incorporated into covalently immobilized compounds. Different ester linkages were evaluated as chemolabile linker groups. As model compounds, esters of the carboxy group of a cysteine with the hydroxy groups of carboxyfluorescein-labelled serine amide and tyrosine amide residues or the thiol group of another fluorescein-labelled cysteine amide were generated. Covalent immobilization occurred on maleimide-functionalized glass cover slips. The surface functionalization and release kinetics were assessed by confocal laser scanning microscopy. The fastest release was obtained for the phenolic tyrosine ester. Alternatively, fluorescently labelled peptides were immobilized by non-covalent interactions on glass and on a hydrogel matrix. In order to increase the efficiency of cellular uptake, peptides were N-terminally extended with a cell-penetrating peptide. Uptake of these peptides into cells was confined to the functionalized spots, and was specific for peptides extended with the cell-penetrating peptide.     

Development of peptide-based patterns by laser transfer

Peptide-based arrays and patterns have provided a powerful tool in the study of protein recognition and function. A variety of applications have been identified, including the interactions between peptides-enzymes, peptides-proteins, peptides-DNA, peptides-small molecules and peptides-cells. One of the main and most critical unresolved issues is the generation of high-density arrays which maintain the biological function of the peptides. In this study, we employ nanosecond laser-induced forward transfer for the generation of high-density peptide arrays and patterns on modified glass surfaces. We show that peptide-based microarrays can be fabricated on solid surfaces and specifically recognized by appropriate fluorescent tags, with the transfer not affecting the ability of the peptides to form fibrils. These initial results are poised to the construction of larger peptide patterns as scaffolds for the incorporation and display of ligands critical for cell attachment and growth, or for the templating of inorganic materials.     

Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays

Kinases represent one of the largest enzyme families and key regulatory proteins in the cell. Only a small subset of these enzymes has been characterised so far. We have prepared different types of phosphopeptide and peptide microarrays displaying peptides deduced from annotated human phosphorylation sites and cytoplasmic domains of all annotated human membrane proteins. This approach was enabled by fully-automated high throughput micro-scale synthesis of peptides by the SPOT technology combined with chemo-selective immobilisation on modified glass slides. The phosphopeptide microarrays displaying 2923 peptides in total have been used for the characterisation of commercially available generic anti-phosphopeptide antibodies. This enabled us to detect Abl kinase activity on a microarray with anti-phosphotyrosine antibodies yielding results comparable to those obtained from a radioactive assay. More than 13 000 peptides deposited on six glass slides were used to profile casein kinase 2 (CK2) using a radioactive assay, since no generic antibody for the reliable detection of serine or threonine phosphorylation could be identified. All previously identified substrates were detected in the microarray experiment. In order to confirm whether substrates on the microarray are substrates in solution phase assays, more than 700 peptides were synthesised and tested with CK2 in a solution phase assay. All substrates identified in the solution phase assay were also detected on the microarray.     

Surface modification with fibronectin or collagen to improve the cell adhesion

Objective

The surface of biomaterials plays a critical role in determining bioactivity. The aim of this study was to evaluate the cell adhesion and proliferation of ADSCs on the surface of biomaterial which is modified with fibronectin or collagen.

Materials and methods

Adipose-derived stromal cells (ADSCs) were obtained from SD rats, expanded in culture, and seeded onto scaffold surface-modified with fibronectin or collagen. To characterize cellular attachment, cells were incubated on scaffold for 1 and 2 h and then counted the cells attached onto the scaffold. The MTT assay was chosen to evaluate the proliferation at days 1, 4, 7 and 14. After 7 d of culture, scanning electron microscope was chosen to observe cell morphology and attachment of ADSCs on the scaffolds.

Results

Attachment at 1 and 2 h of cells on scaffold modified with fibronectin was significantly greater than in control, but not with collagen. The MTT assay revealed that ADSCs proliferation tendency was nearly parallel to that in control. The scanning electron microscope (SEM) showed that ADSCs in experiment expanded thoroughly and excreted much extracellular materials.

Conclusions

Surface modification with fibronectin or collagen can enhance the attachment of cultured ADSCs on the scaffold, but it had not evident effect to proliferation.     

3D polycarprolactone (PCL) scaffold with hierarchical structure fabricated by a piezoelectric transducer (PZT)-assisted bioplotter

The 3D bioplotter, which is one of the rapid-prototyping systems, enables us to produce the design-based scaffolds which could control good mechanical properties and pore structures for mimicking human organs. Although the plotting system has several advantages to fabricate a variety of designed scaffolds, the main disadvantage of scaffolds fabricated by the system is that the strand surfaces are too smooth and tend to discourage initial cell attachment within the scaffolds. To overcome the problem, we suggest a new 3D plotting method supplemented by piezoelectric vibration system for fabricating scaffolds that have hierarchical surface structures, which increase the surface roughness of the scaffold without any additional chemical process. The surface-modified 3D scaffold exhibited various positive qualities including enhanced compressive modulus and improved initial cell attachment and proliferation. Cell culturing results demonstrated that the interactions between chondrocytes and the scaffold were much more favorable than those between the cells and conventionally plotted 3D scaffolds. This process provides a feasible new technique for fabricating high-quality 3D scaffolds for tissue engineering applications.     

High density peptide microarrays. In situ synthesis and applications

The technologies enabling the creation of large scale, miniaturized peptide or protein microarrays are emerging. The focuses of this review are the synthesis and applications of peptide and peptidomimetic microarrays, especially the light directed parallel synthesis of individually addressable high density peptide microarrays using a novel photogenerated reagent chemistry and digital photolithography (Gao et al., 1998, J. Am. Chem. Soc. 120, 12698; Pellois et al. 2002, Nat. Biotechnol. 20, 922). Concepts related to the synthesis are discussed, such as the reactions of photogenerated acids in the deprotection step of peptide synthesis or oligonucleotide synthesis, and the applications of high density peptide chips in antibody binding assays are discussed. Peptide chips provide versatile tools for probing antigen-antibody, protein-protein, peptide-ligand interactions and are basic components for miniaturization, automation, and system integration in research and clinical diagnosis applications.     

Preparation and Characterization of Nanocomposite Scaffolds Based on Polycaprolactone-Polyethylene Glycol/Methylene Diphenyl Diisocyanate/Diethylene Glycol and Nano-Bioactive Glass

Designing and fabricating nanocomposite scaffolds based on biodegradable polymers and bioactive materials are an important topic in the area of bone regeneration. A novel nanocomposite scaffold composed of polyurethane (BPU) and nano-bioactive glass (NBAG) was prepared. The effects of the NBAG content on the properties of the BPU/NBAG composite scaffolds, including the morphologies, porosity and compressive strength, were investigated. The BPU/NBAG composite scaffolds showed an interconnected pore structure with the pore size ranging from 50 to 500?μm for all samples. The porosity percent and swelling ability decreased with increasing NBAG content; however, the compressive strength was enhanced.     

Protein‐Affinitive Polydopamine Nanoparticles as an Efficient Surface Modification Strategy for Versatile Porous Scaffolds Enhancing Tissue Regeneration

Porous scaffolds for tissue regeneration are often functionalized with extracellular matrix proteins to enhance surface/cell interactions and tissue regeneration. However, continuous coatings produced by commonly used surface modification strategies may preclude cells from contacting and sensing the chemical and physical cues of the scaffold. Here, it is shown that polydopamine nanoparticles (PDA‐NPs) tightly adhere on various scaffolds to form nanostructures, and the coverage can be finely tuned. Furthermore, the PDA‐NPs have good affinity to a variety of proteins and peptides. Thus, the PDA‐NPs act as an anchor to immobilize signal biomolecules on scaffolds, and consequently promote cell activity and tissue regeneration. β‐Tricalcium phosphate (TCP) scaffolds decorated with PDA‐NPs demonstrate excellent osteoinductivity and bone‐regeneration performance due to the protein affinity of PDA‐NPs and the intrinsic bioactive characteristics of TCP scaffolds. In summary, PDA‐NPs with excellent affinity for protein adhesion represent a versatile platform to modify porous scaffolds while not compromising the biological functions of the scaffolds, and might have potential applications in tissue regeneration.     

Adsorption of anionic polyelectrolytes to dioctadecyldimethylammonium bromide monolayers

Monolayers of dioctadecyldimethylammonium bromide (DODA) at the air/water interface were used as model for charged surfaces to study the adsorption of anionic polyelectrolytes. After spreading on a pure water surface the monolayers were compressed and subsequently transferred onto a polyelectrolyte solution employing the Fromherz technique. The polyelectrolyte adsorption was monitored by recording the changes in surface pressure at constant area. For poly(styrene sulfonate) and carboxymethylcellulose the plot of the surface pressure as function of time gave curves which indicate a direct correlation between the adsorbed amount and surface pressure as well as a solely diffusion controlled process. In the case of rigid rod-like poly(p-phenylene sulfonate)s the situation is more complicated. Plotting the surface pressure as function of time results in a curve with sigmoidal shape, characterized by an induction period. The induction period can be explained by a domain formation, which can be treated like a crystallization process. Employing the Avrami expression developed for polymer crystallization, the change in the surface pressure upon adsorption of rigid rod-like poly(p-phenylene sulfonate)s can be described. Received 1st July 2000 and Received in final form 7 December 2000     

Variation in thickness of wetting water films in nonuniform electric field

We study the effect of an increase in the thickness of a water film at the inner surface of a glass capillary filled with a NaCl solution and with a disperse octane particle in the form of a column bounded by circular menisci under the action of an external dc electric field. Experimental data are obtained on the film thickness depending on the field characteristics. Analysis of experimental data shows the conformity of experimental data to early model ideas of authors. The projection of the constant dipole moment of a water molecule onto the field direction is estimated on the basis of these ideas.     

Superhydrophobic surfaces prepared from water glass and non-fluorinated alkylsilane on cotton substrates

Superhydrophobic surfaces have been successfully prepared by sol-gel method using water glass as starting material. Such surfaces were obtained first by dip-coating the silica hydrosols prepared via hydrolysis and condensation of water glass onto cotton substrates, then the surface of the silica coating was modified with a non-fluoro compound, hexadecyltrimethoxysilane (HDTMS), to gain a thin film through self-assembly, superhydrophobicity with a water contact angle higher than 151° can be achieved. The morphology and surface roughness were characterized by SEM and AFM.     

Influence of Nano-Bioactive Glass (NBG) Content on Properties of Gelatin-Hyaluronic Acid/NBG Composite Scaffolds

The development of three-dimensional (3-D) scaffolds with highly open porous structure is one of the most important issues in tissue engineering. A novel nanocomposite scaffold of gelatin (Gel), hyaluronic acid (HA), and nano-bioactive glass (NBG) was prepared by blending NBG with a Gel and HA solution followed by lyophilization. The effects of NBG content on the properties of the Gel-HA/NBG composite scaffolds, including the morphologies, porosity, compressive strength, swelling behavior, cell viability and alkaline phosphatase (ALP) activity, were investigated. Porous composite scaffolds with interconnected pores were obtained and the pores became cylindrical with increasing NBG content. The porosity percent and swelling ability decreased with increasing NBG content; however, the compressive strength, cell viability and ALP activity were enhanced. All the results showed the addition of NBG particles can improve the physicochemical and biological properties and the Gel-HA/NBG composite scaffolds exhibited good potential for tissue engineering applications.     

Morphological and surface compositional changes in poly(lactide-co-glycolide) tissue engineering scaffolds upon radio frequency glow discharge plasma treatment

Chemical functionalisation of polymeric scaffolds with functional groups such as amine could provide optimal conditions for loading of signalling biomolecules over the entire volume of the porous scaffolds. Three-dimensional (both surface and bulk) functionlisation of large volume scaffolds is highly desirable, but preferably without any change to the basic morphological, structural and bulk chemical properties of the scaffolds. In this work, we have carried out and compared treatments of poly(lactide-co-glycolide) tissue engineering scaffolds by two methods, that is, a wet chemical method using ethylenediamine and a glow discharge plasma method using heptylamine as a precursor. The samples thus prepared were analysed by scanning electron microscopy and X-ray photoelectron spectroscopy. The plasma treatment generated amide and protonated amine (NH⁺) groups which were present in the bulk and on the surface of the scaffold. Amination also occurred for the wet chemical treatments but the structural and chemical integrity were adversely affected.     

Diffusion of dimethyl sulfoxide in tissue engineered collagen scaffolds visualized by computer tomography

Cryopreservation is a convenient method for long-term preservation of natural and engineered tissues in regenerative medicine. Homogeneous loading of tissues with CPAs, however, forms one of the major hurdles in tissue cryopreservation. In this study, computer tomography (CT) as a non-invasive imaging method was used to determine the effective diffusion of Me2SO in tissue-engineered collagen scaffolds. The dimensions of the scaffolds were 30 x 30 x 10 mm3 with a homogeneous pore size of 100 microm and a porosity of 98%. CT images were acquired after equilibrating the scaffolds in phosphate buffered saline (PBS) and transferring them directly in 10% (v/v)Me2SO. The Me2SO loading process of the scaffold could thus be measured and visualized in real time. The experimental data were fitted using a diffusion equation. The calculated effective diffusion constant for Me2SO in the PBS loaded scaffold was determined from experimental diffusion studies to be 2.4 x 10(-6) cm2/s at 20 degrees C.     

Self-motion of an oil droplet: a simple physicochemical model of active Brownian motion

The self-motion of an oil droplet in an aqueous phase on a glass surface is reported. The aqueous phase contains a cationic surfactant, which tends to be adsorbed onto the glass surface. The oil droplet contains potassium iodide and iodine, which prefers to make an ion pair with the cationic surfactant. Since the ion pair is soluble in the oil droplet, dissolution of the surfactant into the oil droplet is promoted, i.e., the system is far from equilibrium with regard to surfactant concentration. The oil droplet is self-driven in a reactive manner by the spatial gradient of the glass surface tension. We discuss the intrinsic nature of this self-motion by developing a simple mathematical model that incorporates adsorption and desorption of the surfactant on the glass surface. Using this mathematical model we were able to construct an equation of motion that reproduces the observed self-motion of an oil droplet. This equation describes active Brownian motion. Theoretical considerations were used to predict the generation of the regular mode of oil-droplet motion, which was subsequently confirmed by experiments.     

In-Vitro Degradation Behaviors of Composite Scaffolds Based on 1,4-Butadnediamine Modified Poly(lactide-co-glycolide) and Nanobioceramics

In-vitro degradation behaviors of composite scaffold materials composed of 1,4-butanediamine modified poly(lactide-co-glycolide) (BMPLGA), nanobioactive glass (NBG) and β-tricalcium phosphate (β-TCP) were systematically investigated in phosphate-buffered solution (PBS) at 37?°C. The properties of the BMPLGA/NBG-β-TCP and BMPLGA scaffolds, including the changes of pH value, mass, water uptake, compressive strength and molecular mass, were investigated as a function of degradation time. The results showed that the introduction of the NBG and β-TCP particles played important roles in the degradation of BMPLGA matrix. The degradation rate of the BMPLGA/NBG-β-TCP scaffolds was slower than that of the BMPLGA scaffolds.     

Bilayer porous scaffold based on poly-(?-caprolactone) nanofibrous membrane and gelatin sponge for favoring cell proliferation

Electrospun poly-(?-caprolactone) (PCL) nanofibers has been widely used in the medical prosthesis. However, poor hydrophilicity and the lack of natural recognition sites for covalent cell-recognition signal molecules to promote cell attachment have limited its utility as tissue scaffolds. In this study, Bilayer porous scaffolds based on PCL electrospun membranes and gelatin (GE) sponges were fabricated through soft hydrolysis of PCL electrospun followed by grafting gelatin onto the fiber surface, through crosslinking and freeze drying treatment of additional gelatin coat and grafted gelatin surface. GE sponges were stably anchored on PCL membrane surface with the aid of grafted GE molecules. The morphologies of bilayer porous scaffolds were observed through SEM. The contact angle of the scaffolds was 0°, the mechanical properties of scaffolds were measured by tensile test, Young's moduli of PCL scaffolds before and after hydrolysis are 66–77.3 MPa and 62.3–75.4 MPa, respectively. Thus, the bilayer porous scaffolds showed excellent hydrophilic surface and desirable mechanical strength due to the soft hydrolysis and GE coat. The cell culture results showed that the adipose derived mesenchymal stem cells did more favor to adhere and grow on the bilayer porous scaffolds than on PCL electrospun membranes. The better cell affinity of the final bilayer scaffolds not only attributed to the surface chemistry but also the introduction of bilayer porous structure.     

Development of chemically modified glass surfaces for nucleic acid,protein and small molecule microarrays

Microarrays have become a widely used tool to investigate the living cell at different levels. DNA microarrays enable the expression analysis of thousand of genes simultaneously, while protein arrays investigate the properties and interactions of proteins with other proteins and with non-proteinaceous molecules. One crucial step in producing such microarrays is the permanent immobilization of samples on a solid surface. Our goal was to develop diverse linker systems capable of anchoring different biological samples, especially DNA and drug-like small molecules. We developed 6 different chemical surfaces having a 3-D-like linker system for biomolecule immobilization, and compared them to previously described immobilization strategies. The attachment chemistry utilizes the amino reactive properties of acrylic and epoxy functions. The capacity of the support was increased by creating a branching structure holding the reactive functions. The method of anchoring was investigated through a model reaction. From HPLC and mass spectrometry measurements we concluded that the covalent binding of DNA occurs through nucleobases. The tested systems offer the capability to permanently immobilize several biomolecular species in an array format.     

Functionalized alginate/chitosan biocomposites consisted of cylindrical struts and biologically designed for chitosan release

A novel alginate/chitosan composite scaffold was developed. The composite scaffolds were fabricated at low temperature using a three-axis robot system connected to a micro-dispenser and a core/shell nozzle. The structure of the composite scaffolds included hollow struts; deposited chitosan on the inner walls (core region) of the struts reacted electrostatically with the alginate layer (shell region). The fabricated, highly porous composite scaffolds exhibited excellent mechanical properties and controllable chitosan release, which was closely dependent on the weight fraction of the alginate in the shell region. The tensile strength in the dry state was ∼1.8-fold greater than that of pure alginate scaffold due to the ionic interaction between alginate and chitosan. To determine the feasibility of using the developed scaffold in tissue regeneration applications, in vitro cellular responses were evaluated using osteoblast-like-cells (MG63). The cell proliferation on the composite scaffold was ∼3.4-fold greater than that on the pure alginate scaffold. Alkaline phosphate activity and calcium deposition of the composite scaffold after 14 and 21 days of cell culture were significantly enhanced (1.6- and 1.8-fold greater, respectively) compared with those of the pure alginate scaffold. These results suggested that the alginate/chitosan composite scaffolds with a controlled chitosan release have great potential for use in regenerating various tissues.     

Direct formation of a surface-relief grating on glass by ultraviolet visible laser irradiation

Surface-relief gratings were directly fabricated onto a glass surface by UV-visible laser irradiation. The glass surface was pretreated by molten salt, including Ag ions. Periodic intensity modulation of the laser light was conducted with a phase mask or by an interference technique. A pattern generated by intensity modulation was precisely transcribed onto the glass surface and a surface-relief grating was formed. The period and depth of the grating were 0.5 to 10 microm and less than 0.8 microm, respectively. The cross-sectional profile of the grating was sinusoidal or triangular, with very smooth surface morphology.