Fabrication of hydrogel microstructures using polymerization controlled by microcontact printing (PCμCP)

Hydrogels are widely applied as functional biomaterials in the diagnostic and therapeutic fields. For example, intelligent hydrogels containing ionic groups (pH responsive) and poly(ethylene glycol) have promising applications as pH responsive materials in the biomedical and pharmaceutical fields. For potential use of hydrogels in micro- and nano devices, methods are needed to fabricate structures of various geometries at the micro- and nano scale. In this work, polymerization controlled by microcontact printing (PCμCP) is utilized, which is a method that uses microcontact printing to spatially define polymerization zones. Specifically, gold surfaces were modified by a hydrophobic thiol self assembled monolayer via microcontact printing and then a hydrophilic prepolymer solution was applied and only spatially occupied the regions confined by the hydrophobic thiol. Subsequently, polymerization reactions were carried out to create hydrogel microstructures. The patterned hydrogel produced using these methods are highly uniform in size and shape, having potential application in the field of biomedical microdevices.     

Photolithographic patterning of polyethylene glycol hydrogels

A simple, inexpensive photolithographic method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithographic masks were prepared by printing the desired patterns onto transparencies using a laser jet printer. Precursor solutions containing monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The acrylated moieties in the precursor solution were then conjugated in monolayers to specific hydrogel regions by exposure to UV light through the transparency mask. The effects of UV irradiation time and precursor solution concentration on the levels of immobilized peptide were characterized, demonstrating that bound peptide concentration can be controlled by tuning these parameters. Multiple peptides can be immobilized to a single hydrogel surface in distinct patterns by sequential application of this technique, opening up its potential use in co-cultures. In addition, 3D structures can be generated by incorporating PEG-diacrylate into the precursor solution. To evaluate the feasibility of using these patterned surfaces for guiding cell behavior, human dermal fibroblast adhesion on hydrogel surfaces patterned with acryloyl-PEG-RGDS was investigated. This patterning method may find use in tissue engineering, the elucidation of fundamental structure-function relationships, and the formation of immobilized cell and protein arrays for biotechnology.     

The calcium silicate/alginate composite: Preparation and evaluation of its behavior as bioactive injectable hydrogels

In this study, an injectable calcium silicate (CS)/sodium alginate (SA) hybrid hydrogel was prepared using a novel material composition design. CS was incorporated into an alginate solution and internal in situ gelling was induced by the calcium ions directly released from CS with the addition of d-gluconic acid δ-lactone (GDL). The gelling time could be controlled, from about 30 s to 10 min, by varying the amounts of CS and GDL added. The mechanical properties of the hydrogels with different amounts of CS and GDL were systematically analyzed. The compressive strength of 5% CS/SA hydrogels was higher than that of 10% CS/SA for the same amount of GDL. The swelling behaviors of 5% CS/SA hydrogels with different contents of GDL were therefore investigated. The swelling ratios of the hydrogels decreased with increasing GDL, and 5% CS/SA hydrogel with 1% GDL swelled by only less than 5%. Scanning electron microscopy (SEM) observation of the scaffolds showed an optimal interconnected porous structure, with the pore size ranging between 50 and 200 μm. Fourier transform infrared spectroscopy and SEM showed that the CS/SA composite hydrogel induced the formation of hydroxyapatite on the surface of the materials in simulated body fluid. In addition, rat bone mesenchymal stem cells (rtBMSCs) cultured in the presence of hydrogels and their ionic extracts were able to maintain the viability and proliferation. Furthermore, the CS/SA composite hydrogel and its ionic extracts stimulated rtBMSCs to produce alkaline phosphatase, and its ionic extracts could also promote angiogenesis of human umbilical vein endothelial cells. Overall, all these results indicate that the CS/SA composite hydrogel efficiently supported the adhesion, proliferation and differentiation of osteogenic and angiogenic cells. Together with its porous three-dimensional structure and injectable properties, CS/SA composite hydrogel possesses great potential for bone regeneration and tissue engineering applications.     

Corneal epithelial cell growth over tethered-protein/peptide surface-modified hydrogels

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for protein-coated or plain hydrogel surfaces.     

Highly stretchable HA/SA hydrogels for tissue engineering

A highly stretchable hyaluronic acid (HA)/sodium alginate (SA) hydrogel was developed in this study based on an interpenetrating polymer network. HA/SA hydrogels were prepared by mixing two polysaccharides followed by covalent crosslinking via epoxy groups on HA molecules and ionic crosslinking via divalent ions on SA chains sequentially. The effect of HA/SA ratio on the pore size and distribution, swelling ratio, elongation and rheological properties as well as protein loading and release properties of HA/SA hydrogels was explored. Moreover, a surface modification method, layer-by-layer (LBL) assembly technique, was applied to modify the hydrogel to evaluate the hydrogel’s tenability in varying biological performance. It was then shown that the hydrogels had the pore sizes ranging from 100 to 50 μm. With the increase in SA content of the resulting hydrogels, the pore size, swelling ratio, and storage modulus (G′) and loss modulus (G″) of the hydrogel all decreased, whereas the in vitro bulk weight loss was fastened. Moreover, elongation at break (EB) value increased first, reached a peak value and then decreased, that is HA8/SA1 (HA:SA = 8:1) had the highest EB value of 417%. This hydrogel could retain 33.2% of the pre-loaded protein even after 72 h, which could be further attenuated when LBL was used to shell the hydrogel. The growth of fibroblasts on HA8/SA1 hydrogel gave preliminary assessment on its suitability as a cellular carrier, while the LBL modified HA8/SA1 hydrogel also favored the anchoring of keratinocytes, further enhancing its cell carrier role for tissue regeneration, especially skin engineering.     

Covalent Binding of Maleic Anhydride Copolymer Monolayers to Polyacrylamide Hydrogels

Physicochemical surface modifications of soft hydrogel layers are beneficial tools for modulating biomimetic substrate properties in cell adhesion studies. Recently, a layered system of a polyacrylamide hydrogel with a monolayer coating of maleic anhydride copolymers was introduced, however, without a detailed study of the chemical coupling mechanism. Herein it is shown that a stable coupling results from covalent binding of maleic anhydride residues to the primary amides of polyacrylamide via imide bond formation. The many anhydride moieties of the copolymer chains allow for coupling with a low‐yield reaction at mild conditions. Furthermore, hydrogen bonding and polar interactions are excluded to account for a stable binding of maleic anhydride copolymers to polyacrylamide hydrogels.     

Functionalized hydrogel surfaces for the patterning of multiple biomolecules

Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple signals to control cell attachment and growth. Acrylamide-based hydrogels were photo-polymerized in the presence of streptavidin-acrylamide, resulting in planar gel surfaces functionalized with the streptavidin protein. This surface was capable of binding biotin-labeled biomolecules. The proteins fibronectin and laminin, the enzyme alkaline phosphatase, and the photo-protein R-phycoerythrin were patterned using soft lithographic techniques. Polydimethylsiloxane stamps were used to transfer biotinylated proteins onto streptavidin-conjugated hydrogel surfaces. Stamped biomolecules were spatially resolved to feature sizes of 10 mum. Fluorescence measurements were used to assess protein transfer and enzyme functionality on modified surfaces. Our results demonstrate that hydrogel surfaces can be patterned with multiple proteins and enzymes, with retention of biological and catalytic activity. These surfaces are biocompatible and provide cues for cell attachment and growth. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 2007.     

制备表面图案化的聚丙烯酰胺-丙烯酸水凝胶

背景:聚丙烯酰胺水凝胶具备良好的生物相容性,但力学性能较差,影响了其在生物材料领域的应用。目的:通过微模塑图形化压印制备具有特殊尺寸的聚丙烯酰胺-丙烯酸水凝胶。方法:依次将不同体积的聚丙烯酰胺溶液、丙烯酸与过硫酸铵溶液混合,加入含有微模塑图形化印章的孔板中,制备聚丙烯酰胺-丙烯酸水凝胶:A组聚丙烯酰胺溶液1.4 mL,丙烯酸0.1 mL;B组聚丙烯酰胺溶液1.3 mL,丙烯酸0.2 mL;C组聚丙烯酰胺溶液1.2 mL,丙烯酸0.3 mL;D组聚丙烯酰胺溶液1.1 mL,丙烯酸0.4 mL;E组聚丙烯酰胺溶液1.0 mL,丙烯酸0.5 mL;F组聚丙烯酰胺溶液0.9 mL,丙烯酸0.6 mL。6组过硫酸铵溶液均为50 L。光镜下观察水凝胶的图案化结构,电子万能试验机检测水凝胶的力学性能。结果与结论:光镜显示各组水凝胶表面的条纹清晰可见;丙烯酸的加入有效改良了水凝胶的力学性能,随着丙烯酸比例的不断增加,水凝胶的力学性能逐渐增强。结果表明,聚丙烯酸-丙烯酰胺水凝胶具有良好的力学性能,有望在组织工程损伤修复领域具有良好的应用前景。     

Surface functionalization of hyaluronic acid hydrogels by polyelectrolyte multilayer films

Hyaluronic acid (HA), an anionic polysaccharide, is one of the major components of the natural extracellular matrix (ECM). Although HA has been widely used for tissue engineering applications, it does not support cell attachment and spreading and needs chemical modification to support cellular adhesion. Here, we present a simple approach to functionalize photocrosslinked HA hydrogels by deposition of poly(l-lysine) (PLL) and HA multilayer films made by the layer-by-layer (LbL) technique. PLL/HA multilayer film formation was assessed by using fluorescence microscopy, contact angle measurements, cationic dye loading and confocal microscopy. The effect of polyelectrolyte multilayer film (PEM) formation on the physicochemical and mechanical properties of hydrogels revealed polyelectrolyte diffusion inside the hydrogel pores, increased hydrophobicity of the surface, reduced equilibrium swelling, and reduced compressive moduli of the modified hydrogels. Furthermore, NIH-3T3 fibroblasts seeded on the surface showed improved cell attachment and spreading on the multilayer functionalized hydrogels. Thus, modification of HA hydrogel surfaces with multilayer films affected their physicochemical properties and improved cell adhesion and spreading on these surfaces. This new hydrogel/PEM composite system may offer possibilities for various biomedical and tissue engineering applications, including growth factor delivery and co-culture systems.     

MC3T3-E1成骨细胞在微格式化表面的黏附

细胞在材料表面的黏附对细胞的增殖和分化志重要作用。格式化表面提供了对细胞在基底的空间分布和黏附进行控制的方法。本文利用微制作利用微制作形成的格式模板，分别以微接触转印法和微流道法形成格式化表面，使MC3T3－E1成骨细胞以一定的格式黏附于表面上，在微接触转印法形成的含二氯二甲基硅烷（DMS）的疏水区域和不含DMS的亲水区域相间隔的表面，细胞优先在亲水区域黏附，在微流道法形成的胶原和白蛋白格式化表面，细胞优先黏附于含胶原区域，结果还表明微格式化表面可以用于研究表面的物理化学性质对细胞的黏附等功能的影响。     

Corneal epithelial adhesion strength to tethered-protein/peptide modified hydrogel surfaces

In this study, we investigated the suitability of microjet impingement for use on hydrogel materials to determine the cellular adhesion strength of corneal epithelial cells grown on novel hydrogels with extracellular matrix proteins (laminin and/or fibronectin) or a peptide sequence (fibronectin adhesion promoting peptide, FAP) tethered to their surface with poly(ethylene glycol) chains. The deformation of the hydrogel surface in response to the force of the microjet was analyzed both visually and mathematically. After the results of these experiments and calculations determined that no deformation occurred and that the pressure required for indentation (1.25 x 10(6) Pa) was three factors of 10 greater than the maximum pressure of the microjet, the relative mean adhesion strength of primary rabbit corneal epithelial cells grown on the novel poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogels was determined and compared with that of the same type of cells grown on control glass surfaces. Only confluent cell layers were tested. Cells grown on control glass surfaces adhered with a mean relative adhesion strength of 488 +/- 28 dynes/cm2. Under identical conditions, cells grown on laminin- and FAP-tethered hydrogel surfaces were unable to be removed, indicating an adhesion strength greater than 516 dynes/cm2. Cells grown on fibronectin- and fibronectin/laminin (1:1)-tethered surfaces showed significantly lower relative adhesion strengths (201 +/- 50 and 189 +/- 11 dynes/cm2, respectively), compared with laminin- and FAP-tethered surfaces (p = 0.001). Our results demonstrate that the microjet impingement method of cell adhesion analysis is applicable to hydrogel substrates. Additionally, analysis of our test surfaces indicates that fibronectin tethered to this hydrogel in the quantity and by the method used here does not induce stable ligand/receptor bonding to the epithelial cell membrane to the same degree as does laminin or FAP.     

Blood compatibility of photografted hydrogel coatings

In this work, we have evaluated the haemocompatibility of different surface modifications, intended for biomaterials and bioanalytical applications. Polystyrene slides were coated with thin hydrogel films by self-initiated photografting and photopolymerization (SIPGP) of four different monomers. The hydrogel surface modifications were thoroughly characterized and tested for their protein resistance and ability to resist platelet adhesion and activation of the coagulation system. There was very little protein adsorption from human plasma on the hydrogels prepared from poly(ethylene glycol) methacrylate and 2-hydroxyethyl methacrylate. Platelet adhesion tests performed under both static and flow conditions showed that these coatings also demonstrated very high resistance towards platelet adhesion. A small amount of platelets were found to adhere to hydrogels formed from ethylene glycol methyl ether methacrylate and 2-carboxyethyl methacrylate. The polystyrene substrates themselves facilitated large amounts of platelet adhesion under both static and flow conditions. Utilizing a novel setup for imaging of coagulation, it was confirmed that none of the hydrogel surfaces activated the coagulation system to any great extent. We suggest that this simple fabrication method can be used to produce hydrogel coatings with unusually high blood compatibility, suitable for demanding biomaterials applications.     

Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins

To modulate the surface properties of collagen and subsequent cell–surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels.     

Human mesenchymal stem cell culture on heparin-based hydrogels and the modulation of interactions by gel elasticity and heparin amount

Human adipose-derived stem cells (hADSCs) are a promising cell source for tissue engineering and regenerative medicine with no ethnical issue and easy access of large quantities. Conventional surfaces for hADSC culture, such as tissue culture plates (TCPs), do not provide optimal environmental cues, leading to limited expansion, loss of pluripotency and undesirable differentiation of stem cells. The present study demonstrated that heparin-based hydrogels without additional modification provided an excellent surface for adhesion and proliferation of hADSCs, which were further tunable by both the amount of heparin (in a positive way) and the elasticity of hydrogel (in a negative way). The optimized heparin-based hydrogel could selectively modulate the adhesion of hADSCs and human bone marrow stem cells (but not all kinds of cells), and resulted in a significant increase in cell proliferation compared to TCP. Furthermore, in terms of the maintenance of pluripotency and specific differentiation, heparin-based hydrogel was much superior to TCP. The selective binding and proliferation of human mesenchymal stem cells on heparin-based hydrogel over other hydrogels were largely mediated by integrin β1 and selectin, and these superior characteristics were observed regardless of the presence of serum proteins in the culture medium. Consequently, heparin-based hydrogel could be a powerful platform for cultivation of mesenchymal stem cells in various applications.     

Modified carrageenan. 2. Hydrolyzed crosslinked kappa-carrageenan-g-PAAm as a novel smart superabsorbent hydrogel with low salt sensitivity

A novel pH-responsive superabsorbing hydrogel based on K-carrageenan (kappaC) was prepared through polyacrylamide crosslinking grafting followed by alkaline hydrolysis. The hydrogel structure was confirmed using FT-IR spectroscopy. The hydrolysis conditions were systematically optimized to obtain a hydrogel with maximum swelling capacity. Thus, the reaction variables, including the hydrolysis time and temperature, concentration of sodium hydroxide, amount of hydrogel hydrolyzed and post-neutralization pH, were optimized. The swelling measurements of the hydrogels were conducted in 0.15 M aqueous solutions of LiCl, NaCl, KCI, CaCl2 and AlCl3. As observed for the hydrolyzed hydrogel (H-carragPAM), it was found that a 'charge screening' action of small cations and carboxylate anions affected the swelling in univalent salt solutions. In the case of the non-hydrolyzed hydrogel (carragPAM), however, a converse trend was observed. As a result, carragPAM and H-carragPAM superabsorbent hydrogels showed a maximum swelling of 45 and 135 g/g in LiCl and KCl solutions, respectively. Due to the high swelling capacity in salt solutions, the hydrogels may be referred to as anti-salt superabsrbents. The swelling of superabsorbing hydrogels was examined in buffer solutions with pH values ranging between 1 and 13. The H-carragPAM hydrogel exhibited a pH-responsie character so that a swelling-deswelling pulsatile behavior was recorded at pH 4 and 9. The swelling kinetics of H-carragPAM were preliminary investigated.     

The modulation of corneal keratocyte and epithelial cell responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases collagenase production in vitro.

We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.     

Neural Progenitor Cells Survival and Neuronal Differentiation in Peptide-Based Hydrogels

We designed nanofibrous hydrogels as 2-D and 3-D scaffolds for anchorage-dependent cells. The IKVAV-containing peptide amphiphile molecules spontaneously self-assembled into higher-order nanofiber hydrogels under cell-containing media. Neural progenitor cells (NPCs) were incubated in peptide-based hydrogels. Effects of self-assembling hydrogels on survival and neural differentiation of NPCs were observed. Peptide was synthesized using a solid-phase method. TEM study of the hydrogel revealed a network of nanofibers. Phase-contrast light micrographs showed that the described hydrogel had no observable cytotoxicity to NPCs. Additionally this hydrogel could induce cells to differentiate into neuron-like cells and glial-like cells. Moreover, the cells encapsulated within hydrogel had a higher neuronal differentiation rate than in the surface of the hydrogel. This self-assembled hydrogel might serve as nerve tissue-engineering scaffold.     

Thermoresponsive nanocomposite hydrogels with cell-releasing behavior

Poly(N-isopropylacrylamide) (PNIPAAm) hydrogels become more hydrophobic when they reversibly switch from a water-swollen to a deswollen state above the volume phase transition temperature (VPTT, approximately 33 degrees C) which has been used to modulate cell adhesion. In the current work, we prepared novel thermoresponsive nanocomposite hydrogels comprised of a PNIPAAm hydrogel matrix and polysiloxane colloidal nanoparticles ( approximately 220 nm average diameter) via in situ photopolymerization of aqueous solutions of NIPAAm monomer, N,N'-methylenebisacrylamide (BIS, crosslinker), photoinitiator and polysiloxane nanoparticles (0.5-2.0 wt% based on solution weight) at approximately 7 degrees C. The VPTT of the nanocomposite hydrogels is not altered versus the pure PNIPAAm hydrogel. Dynamic mechanical analysis and tensile tests revealed that higher nanoparticle content generally produced improved hydrogel mechanical properties. Surfaces of nanocomposite hydrogels became increasingly more hydrophobic at all temperatures between 10 and 40 degrees C as the amount of hydrophobic polysiloxane nanoparticles was increased. When cooled from 37 to 25 degrees C, mouse smooth muscle precursor cells (10T1/2) were effectively detached from nanocomposite hydrogel surfaces. The utility of photopatterning to create surface micropillars comprised of nanocomposite hydrogels was demonstrated.     

Microcontact printing of quantum dot bioconjugate arrays for localized capture and detection of biomolecules

The nanometer size scale of quantum dots (QDs) along with their unique luminescent properties offers great potential as photostable, color-metrically addressable nanoparticle platforms for high-throughput detection and identification of proteins. Here we apply microcontact printing for assembling quantum dot nanoparticle arrays with retained biomolecular capture functionality onto glass surfaces. This method allows the creation of addressable QD arrays on macroscopic glass surfaces. Using fluorescence and AFM imaging, we find that microcontact-printed QDs self-assemble predominantly as monolayers with highly resolved definition. Microcontact-printed streptavidin-conjugated red QDs exhibit retained adsorption onto silane-treated glass and exhibit functionality as demonstrated by the capture of discrete groups of biotin-conjugated red QDs by printed streptavidin-green QD bioconjugates that is at the detection limit of a few discrete protein binding events. These results indicate that microcontact printing of QD bioconjugate arrays serves as a simple technique that allows localized spatial capture and sensitive detection of proteins. This technique may be useful for development of future fluorescent QD-based systems aimed at the parallel capture and detection of trace concentrations of protein.