Preparation and evaluation of thiol-modified gelatin nanoparticles for intracellular DNA delivery in response to glutathione

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we have developed thiolated gelatin nanoparticles that can release the payload in the highly reducing environment, such as in response to glutathione. Thiolated gelatin was synthesized by covalent modification of the primary amino groups of Type B gelatin using 2-iminothiolane (Traut's reagent). The degree of thiolation of the polymers ranged from 0 to 43.71 mmol of reduced sulfhydryl (SH) groups when the amount of 2-iminothiolane was increased up to 100 mg per gram of the biopolymer. Cytotoxicity evaluations carried out by the formazan (MTS) assay showed that the thiolated gelatin prepared with 20 mg and 40 mg of 2-iminothiolane (SHGel-20 and SHGel-40) per gram of gelatin had comparable cell viability profile to that of the unmodified gelatin. In vitro release studies of fluorescein isothiocyanate (FITC)-labeled dextran (mol wt. 70 000 Da), when encapsulated in gelatin and thiolated gelatin nanoparticles (150-250 nm in diameter), was found to be affected by the presence of glutathione (GSH) in the medium. The presence of GSH was found to enhance the release by about 40% in case of thiolated gelatin and about 20% in gelatin nanoparticles under similar conditions of temperature and GSH concentrations. Qualitative and quantitative analysis of transfection in NIH-3T3 murine fibroblast cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by fluorescence confocal microscopy and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 h. Quantitative results from FACS showed that the thiolated gelatin nanoparticles (SHGel-20) were significantly more effective in transfecting NIH-3T3 cells than other carrier systems examined. The results of this study show that thiolated gelatin nanoparticles would serve as a biocompatible intracellular delivery system that can release the payload in a highly reducing environment.     

Simplified off-gas analyses in animal cell cultures for process monitoring and control purposes

Batch-to-batch reproducibility of animal cell cultures can significantly be enhanced using process control procedures. Most informative signals for advanced process control can be derived from the volume fractions of oxygen and carbon dioxide in the vent line of the reactors. Here we employed simple low-cost sensors, previously not considered for off-gas analysis at a laboratory-scale cell cultures, and compared them with a simultaneously used quadrupole mass spectrometer, i.e., the standard equipment. A decisive advantage is that the sensors did not need any calibration and are easy to use. We show that monitoring and advanced control of cell cultures can significantly be simplified using the devices tested here and that the same batch-to-batch reproducibility can be obtained with much less effort than before.     

Chemically cross-linked chitosan hydrogel loaded with gelatin for chondrocyte encapsulation

Composite hydrogels can be used as a scaffolding material for chondrogenesis, which requires a biomimetic environment to maintain chondrocyte morphology and phenotype. In this study, gelatin molecules were loaded into a hydrogel polymerized from a chitosan derivative (CML) to form a semi-interpenetrating polymer network. While the porous structure of the hydrogels in the dry state was not dependent on the gelatin content, the collapse extent and pore size decreased as the gelatin content increased. The gelatin loading also reduced the swelling ratio of the CML hydrogel and enhanced the hydrogel strength at 20°C due to gelation of the gelatin. The release behavior of the gelatin from the CML hydrogel could be controlled by many factors, such as the amount of gelatin, temperature, and solution pH. The weight loss of the composite hydrogel was expedited after gelatin loading and showed a positive relationship with the gelatin content. The results of in vitro cell culture in the hydrogels revealed that gelatin loading improved cell viability and promoted proliferation and glycosaminoglycans secretion of chondrocytes. This new scaffold production technology for chondrocyte encapsulation provides a further step towards CML applications in tissue engineering and other biomedical areas.     

Fabrication of agar-gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications

Scaffolds of agar and gelatin were developed using a novel entrapment method where agar and gelatin molecules mutually entrapped one another forming stable cell adhesive matrices. Glutaraldehyde was used as a crosslinking agent for gelatin. Three types of hybrid matrices were prepared using agar and gelatin in different proportions in the weight ratio of 1:1, 2:1, and 3:1. Surface characterization of dry scaffolds was carried out by scanning electron microscope. Swelling studies were carried out in phosphate buffer saline (PBS) at physiological pH 7.4. The integral stability of the scaffolds was evaluated by estimating the released disintegrated gelatin from them in PBS at pH 7.4. The attachment kinetics of the cells was evaluated by culturing mouse fibroblast cell line NIH 3T3 on films. The cytocompatibility of these matrices was determined by studying growth kinetics of NIH 3T3 cells on them and morphology of cells was observed through optical photographs taken at various days of culture. It was found that the matrices containing agar and gelatin in 2:1 weight ratio exhibited best growth kinetics. The results obtained from these studies have suggested that the above-described method is a cheap and easy way to fabricate agar-gelatin hybrid scaffolds to grow cells which can be used in various in vitro tissue engineering applications like screening of drugs.     

Devices facilitating the preparation of biological specimens for electron microscopy

The construction of a matrix is described which facilitates the process of placing biological objects into polymer media in order to prepare ultrathin sections without the employment of gelatin, starch and polyethylene capsules that can be used only once. The construction of a reactor for cytochemical assays is presented. The apparatus can be used to locate enzymes within the cell, and to identify microorganisms. A modification of the dialysis technique for microbiological objects is proposed which accelerates and simplifies the process.     

Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization.

The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios. This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion. A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process. The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.     

Enzymatic hydrolysis of gelatin layers on used lith film using thermostable alkaline protease for recovery of silver and PET film

To develop a new efficient and potential industrial enzymatic process for the recovery of silver and poly(ethylene terephthalate) (PET) from used lith film for printing, which has not been recycled at all, enzymatic hydrolysis of gelatin layers on lith film was investigated using the thermostabilized mutant enzyme of the alkaline protease from alkaliphilic Bacillus sp. B21-2. The rate of gelatin hydrolysis of lith film in a stirred-tank reactor increased with the temperature and enzyme concentration. The time required to complete the hydrolysis of gelatin on lith film was longer than that on X-ray film because of the tightly cross-linked structure of the gelatin layers of lith film. The time required to complete the hydrolysis by using the mutant enzyme was less than that using the wild-type enzyme. The gelatin hydrolysis of lith film was well explained by a model that took into consideration a number of physical processes in addition to the chemical process.     

Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications

Chitin is a natural biopolymer have been used for several biomedical applications due to its biodegradability and biocompatibility. By using the calcium solvent system, chitin regenerated hydrogel (RG) was prepared by using -chitin. And also, the swelling hydrogel (SG) was prepared by using β-chitin with water. Then, both RG and SG were mixed with gelatin and N-acetyl-d-(+)-glucosamine (GlcNAc) at 120 °C for 2 h. The chitin/gelatin membranes with GlcNAc were also prepared by using RG and SG with GlcNAc. The prepared chitin/gelatin membranes with or without GlcNAc were characterized by mechanical, swelling, enzymatic degradation, thermal and growth of NIH/3T3 fibroblast cell studies. The stress and elongation of chitin/gelatin membrane with GlcNAc prepared from RG was showed higher than the chitin/gelatin membranes without GlcNAc. But, the chitin/gelatin membranes prepared from SG with GlcNAc was showed higher stress and elongation than the chitin/gelatin membranes without GlcNAc. It is due to the crosslinking effect of GlcNAc. The chitin/gelatin membranes prepared from SG showed higher swelling than the chitin/gelatin membranes prepared from RG. In contrast, the chitin/gelatin membranes prepared from RG showed higher degradation than the chitin/gelatin membranes prepared from SG. And also, these chitin/gelatin membranes are showing good growth of NIH/3T3 fibroblast cell. So these novel chitin/gelatin membranes are useful for tissue engineering applications.     

Preparation and characterization of novel chitosan/gelatin membranes using chitosan hydrogel

Chitin and chitosan are novel biomaterials. The novel chitosan/gelatin membranes were prepared using the suspension of chitosan hydrogel mixed with gelatin. The prepared chitosan/gelatin membranes were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical, swelling, and thermal studies. The morphology of these chitosan/gelatin membranes was found to be very smooth and homogeneous. The XRD studies showed that the chitosan/gelatin membranes have good compatibility and interaction between the chitosan and gelatin. The stress and elongation of chitosan/gelatin membranes on wet condition showed excellent when the mixture ratio of gelatin was 0.50. The prepared chitosan/gelatin membranes showed good swelling, mechanical and thermal properties. Cell adhesion studies were also carried out using human MG-63 osteoblast-like cells. The cells incubated with chitosan/gelatin membranes for 24 h were capable of forming cell adhesion. Thus the prepared chitosan/gelatin membranes are bioactive and are suitable for cell adhesion suggesting that these membranes can be used for tissue-engineering applications. Therefore, these novel chitosan/gelatin membranes are useful for biomedical applications.     

N-Channel field-effect transistors with floating gates for extracellular recordings

A field-effect transistor (FET) for recording extracellular signals from electrogenic cells is presented. The so-called floating gate architecture combines a complementary metal oxide semiconductor (CMOS)-type n-channel transistor with an independent sensing area. This concept allows the transistor and sensing area to be optimised separately. The devices are robust and can be reused several times. The noise level of the devices was smaller than of comparable non-metallised gate FETs. In addition to the usual drift of FET devices, we observed a long-term drift that has to be controlled for future long-term measurements. The device performance for extracellular signal recording was tested using embryonic rat cardiac myocytes cultured on fibronectin-coated chips. The extracellular cell signals were recorded before and after the addition of the cardioactive isoproterenol. The signal shapes of the measured action potentials were comparable to the non-metallised gate FETs previously used in similar experiments. The fabrication of the devices involved the process steps of standard CMOS that were necessary to create n-channel transistors. The implementation of a complete CMOS process would facilitate the integration of the logical circuits necessary for signal pre-processing on a chip, which is a prerequisite for a greater number of sensor spots in future layouts.     

Culturing chick muscle cells on glycosaminoglycan substrates: attachment and differentiation

The effects of different glycosaminoglycans (GAGs) on myogenesis were tested by culturing embryonic chick myoblasts on tissue culture dishes to which either hyaluronic acid (HA) or chondroitin sulfate (ChS) was covalently bound. Both in cell number and in apparent cell type distribution, the population of cells bound to GAGs is similar to that on gelatin and significantly different from that observed with uncoated dishes. When plated on ChS, myoblasts proliferate, align, and fuse at a rate similar to cells plated on gelatin. The final fused cells appear as sheets rather than long, thin myotubes. On HA, the cells proliferate but are inhibited from differentiation. The extent of inhibition is dependent on the amount of HA present. The inhibition of myogenesis is maintained through four subcultures on HA, but can be reversed at any time by culturing cells on gelatin. These experiments indicate that different GAGs have different effects on myogenesis and that HA can actively inhibit the process.     

Fibronectin quantification without antibodies: a bioassay for the detection of the gelatin-captured macromolecule

We have developed a new cell-adhesion-bioassay (CAA) for the quantitative determination of fibronectin in biological fluids. The assay is based on two particular properties of fibronectin: it specifically binds to gelatin with high affinity and simultaneously it can anchor to different surface molecules of a cell. First fibronectin, derived from very different biological fluids, is purified in situ, within the wells of the microtiter plates applied for the assay, using solid surface bound gelatin. After capturing the macromolecule, it is quantified based on its cell adhesive properties. In contrast to ELISA the CAA does not require specific antibodies, and as the Jurkat cells used as indicator cells, seem to recognize fibronectin from different species equally; species specificity of the reagent plays smaller, perhaps negligible, role in the determination of the amount of the macromolecule. The CAA method may not replace fibronectin specific ELISA-s, but using its principle, improved applications, for example a capture EIA for determining fibronectin can easily be envisioned and CAA may serve as a viable alternative for EIA-s when specific antibodies are not available or when relative measurement of not only the soluble but cell surface associated fibronectin is necessary.     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

Enzymatic methods for in situ cell entrapment and cell release

We report an enzyme-based method for the in situ entrapment of cells within a biopolymeric hydrogel matrix. Specifically, we used a calcium-independent microbial transglutaminase that is known to cross-link proteins and observed that it catalyzes the formation of gels from a pre-gel solution containing 10% gelatin and E. coli cells. Hydrogel formation occurs 2-3 h after adding transglutaminase, and no additional external intervention is required to initiate gel formation. The in situ entrapped cells grow rapidly and to high cell densities within the gelatin hydrogel. Additionally, the entrapped cells respond to isopropylthiogalactoside induction. The cross-linked gelatin network can be rapidly hydrolyzed (within 1 h) by the protease, proteinase K. Treatment of the network by this protease releases the entrapped E. coli cells. These cells appear unharmed by proteinase K; they can grow and be induced after protease treatment. The ability to in situ entrap, grow, and release cells under mild conditions provides unique opportunities for a range of applications and should be especially useful for microfluidic biosensor systems.     

Recombinant collagen for animal product-free dextran microcarriers

Manufacturers of vaccines and other biologicals are under increasing pressure from regulatory agencies to develop production methods that are completely animal-component-free. In order to comply with this demand, alternative cell culture substrates to those now on the market, primarily collagen or gelatin, must be found. In this paper, we have tested a number of possible substitutes including recombinant collagen, a 100-kDa recombinant gelatin fragment and a peptide derived from a cell-binding region of type I collagen. The small 15-amino acid peptide did not support attachment of human fibroblasts in monolayer culture. The 100-kDa gelatin fragment supported cell attachment in monolayer culture, but was significantly less active than intact porcine gelatin. Recombinant type I collagen was as successful in promoting cell attachment as native collagen, and both were more effective than porcine gelatin. Based on these data, dextran microspheres were treated with the same attachment proteins—porcine gelatin, native collagen, or recombinant collagen. The same trends were observed as in monolayer culture. Concentrations of the recombinant collagen (as well as native collagen) supported cell attachment on dextran microspheres at concentrations as low as 0.01 μg/cm². Treatment of the dextran with a low level of polyethylenimine, a cationic moiety, further enhanced attachment when used in conjunction with the low concentration of recombinant collagen. Where there was increased cell attachment, increased proliferation followed. We are confident, based on these findings, that a fully recombinant substitute could replace gelatin in current microcarrier preparations without losing the cell growth benefits provided by the native protein.     

Laser fabrication of three-dimensional CAD scaffolds from photosensitive gelatin for applications in tissue engineering

In the present work, 3D CAD scaffolds for tissue engineering applications were developed starting from methacrylamide-modified gelatin (GelMOD) using two-photon polymerization (2PP). The scaffolds were cross-linked employing the biocompatible photoinitiator Irgacure 2959. Because gelatin is derived from collagen (i.e., the main constituent of the ECM), the developed materials mimic the cellular microenvironment from a chemical point of view. In addition, by applying the 2PP technique, structural properties of the cellular microenvironment can also be mimicked. Furthermore, in vitro degradation assays indicated that the enzymatic degradation capability of gelatin is preserved for the methacrylamide-modified derivative. An in depth morphological analysis of the 2PP-fabricated scaffolds demonstrated that the parameters of the CAD model are reproduced with great precision, including the ridge-like surface topography on the order of 1.5 μm. The developed scaffolds showed an excellent stability in culture medium. In a final part of the present work, the suitability of the developed scaffolds for tissue engineering applications was verified. The results indicated that the applied materials are suitable to support porcine mesenchymal stem cell adhesion and subsequent proliferation. Upon applying osteogenic stimulation, the seeded cells differentiated into the anticipated lineage. Energy dispersive X-ray (EDX) analysis showed the induced calcification of the scaffolds. The results clearly indicate that 2PP is capable of manufacturing precisely constructed 3D tissue engineering scaffolds using photosensitive polymers as starting material.     

Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3-4 times more enriched in salt stressed epicotyls. SDS-PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.     

A Layered‐Nanospace‐Confinement Strategy for the Synthesis of Two‐Dimensional Porous Carbon Nanosheets for High‐Rate Performance Supercapacitors

A general approach is developed for the synthesis of 2D porous carbon nanosheets (PCNS) from bio‐sources derived carbon precursors (gelatin) by an integrated procedure of intercalation, pyrolysis, and activation. Montmorillonite with layered nanospace is used as a nanotemplate or nanoreactor to confine and modulate the transformation of gelatin, further leading to the formation of 2D nanosheet‐shaped carbon materials. The as‐made 2D PCNS exhibits a significantly improved rate performance, with a high specific capacitance of 246 F g^?1 and capacitance retention of 82% at 100 A g^?1, being nearly twice that of microsized activated carbon particulates directly from gelatin (131 F g^?1, 44%). The shortened ion transport distance in the nanoscaled dimension and modulated porous structure is responsible for such an enhanced superior rate capability. More importantly, the present strategy can be extended to other bio‐sources to create 2D PCNS as electrode materials with high‐rate performance. This will also provide a potential strategy for configuring 2D nanostructured carbon electrode materials with a short ion transport distance for supercapacitors and other carbon‐related energy storage and conversion devices.     

Biomacromolecules electrostatic self-assembly on 3-dimensional tissue engineering scaffold

A poly(ethylenimine) (PEI) was employed to obtain a stable positively charged surface on a poly(D,L-lactide) (PDL-LA) tissue engineering scaffold. An extracellular matrix (ECM)-like biomacromolecule, gelatin, was selected as polyelectrolyte and deposit alternately with PEI on the activated PDL-LA scaffold via ESA technique. The zeta-potential result showed alternating charge of polyelectrolytes (PEI/gelatin) layering on PDL-LA microspheres. Quartz crystal microbalance (QCM) measurement further verified the gradual deposition of PEI/gelatin on the PDL-LA thin film. The combination of PEI aminolysis and the layer-by-layer technique was then explored to construct gelatin coating onto the 3-D porous PDL-LA scaffold. Scanning electronic microscopy showed that there is no notable difference between modified and unmodified PLA scaffolds, with regard to the porosity, pore diameter, and scaffold integration. The dual-tunnel confocal laser scanning microscopy indicated uniform gelatin distribution on the inner surface of the 3-D porous scaffold. The gradual build-up of protein layer on scaffold was investigated by radioiodination technique. Chondrocyte was chosen to test the cell behavior on modified and unmodified PDL-LA scaffolds. The results of the cell viability, total intracellular protein content, and cell morphology on the PEI/gelatin multilayers modified PDL-LA scaffold showed to promote chondrocyte growth. Comparing conventional coating methods, polyelectrolyte multilayers are easy and stable to prepare. It may be a promising choice for the surface modification of complex biomedical devices. These very flexible systems allow broad medical applications for drug delivery and tissue engineering.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.