Glutaraldehyde Cross-linked Gelatin Research Articles

Reuse of biogas sludge for the slow-release fertilizer production to improve fertilizer use efficiency and soil fertility

Reuse of biogas sludge for the slow-release fertilizer production to improve fertilizer use efficiency and soil fertility

Swelling and DC Conductivity Behaviour of Gelatin-Based Ferrogels

Gelatin-based systems have great potential in bio-medical applications. Here, we report the solvent uptake and DC conductivity of glutaraldehyde-crosslinked gelatin hydrogels and ferrogels. Both the parameters are found to depend on cross-linker (glutaraldehyde) and dopant (carbonyl iron) concentrations. Higher cross-linker proportions cause decrease in solvent uptake, equilibrium swelling ratio and DC conductivity. Solvent uptake is seen to increase while equilibrium swelling ratio and DC conductivity decrease with dopant concentration in the ferrogels. The solvent uptake and DC conductivity behaviour of the studied systems are explained on the basis of variations in pore size and polymer segmental mobility which, themselves, are functions of cross-linker and dopant concentrations. The diffusion process in the hydrogels and ferrogels obeys the second-order kinetic model.

Controlled Delivery of Immunomodulators from a Biomaterial Scaffold Niche to Induce a Tolerogenic Phenotype in Human Dendritic Cells.

Engineering a regulatory phenotype in dendritic cells (DCs) is a potential approach to circumvent an immune response against self-antigens in autoimmunity or alloantigens in allograft rejection. Cell microenvironments influence the differentiation of DC precursors into either proinflammatory/immunostimulatory or tolerogenic/regulatory DCs. Biomaterial-based vehicles can be used to re-engineer cell microenvironments and re-educate the DC phenotype. This study presents the development and validation of a biomaterial-based multicomponent immunomodulatory (MI) scaffold for the purpose of promoting a tolerogenic/regulatory DC phenotype. Glutaraldehyde-crosslinked gelatin microparticles, loaded with specific immunomodulators, were embedded into a porous agarose scaffold. Using the Weibull equation and the Bayesian approach, an empirical mathematical model was derived from the release profile data of "model" molecules. The scaffold design was generated from the model to achieve distinct temporal release profiles of the loaded immunomodulator(s): granulocyte monocyte colony-stimulating factor (GM-CSF), dexamethasone (DEX), and/or peptidoglycan (PGN). The MI scaffold-treated DCs (MI DCs) showed an increase in the expression of tolerogenic markers such as surface immunoglobulin-like transcript 3 (ILT-3) and secreted interleukin-10 (IL-10), with a simultaneous decrease in maturation markers such as CD86 and secreted interferon-γ (IFN-γ). In cell culture studies, these MI DCs were able to suppress T-cell proliferation. This approach is expected to enhance the generation of endogenous regulatory DCs when applied in vivo. This technology serves as a basis for future immunotherapeutic applications in the autoimmunity and allogeneic therapies. It also shows that empirical mathematical modeling can be used to engineer scaffold designs for distinct temporal release of one or more immunomodulators.

Electrospray-Based Microencapsulation of Epigallocatechin 3-Gallate for Local Delivery into the Intervertebral Disc.

Locally delivered anti-inflammatory compounds can restore the homeostasis of the degenerated intervertebral disc (IVD). With beneficial effects on IVD cells, epigallocatechin 3-gallate (EGCG) is a promising therapeutic candidate. However, EGCG is prone to rapid degradation and/or depletion. Therefore, the purpose of this study was to develop a method for controlled EGCG delivery in the degenerated IVD. Primary IVD cells were isolated from human donors undergoing IVD surgeries. EGCG was encapsulated into microparticles by electrospraying of glutaraldehyde-crosslinked gelatin. The resulting particles were characterized in terms of cytocompatibility and anti-inflammatory activity, and combined with a thermoresponsive carrier to produce an injectable EGCG delivery system. Subsequently, electrospraying was scaled up using the industrial NANOSPIDER™ technology. The produced EGCG microparticles reduced the expression of inflammatory (IL-6, IL-8, COX-2) and catabolic (MMP1, MMP3, MMP13) mediators in pro-inflammatory 3D cell cultures. Combining the EGCG microparticles with the carrier showed a trend towards modulating EGCG activity/release. Electrospray upscaling was achieved, leading to particles with homogenous spherical morphologies. In conclusion, electrospray-based encapsulation of EGCG resulted in cytocompatible microparticles that preserved the activity of EGCG and showed the potential to control EGCG release, thus favoring IVD health by downregulating local inflammation. Future studies will focus on further exploring the biological activity of the developed delivery system for potential clinical use.

Water absorption by decellularized dermis

Water absorption by decellularized dermis was investigated and compared with biopolymer and synthetic polymer hydrogels (glutaraldehyde-crosslinked gelatin and crosslinked poly(acrylamide) hydrogel, respectively). Porcine dermis was decellularized in an aqueous sodium dodecyl sulfate (SDS) solution. Histological evaluation revealed that the SDS-treated dermis has much larger gaps between collagen fibrils than non-treated dermis, and that water absorption depends on these gaps. Decellularized dermis has low water absorptivity and the absorption obeys Fick's second law. During absorption, the water diffusion rate decreases with time and occurs in two steps. The first is rapid absorption into the large gaps, followed by slow absorption by the collagen fiber layer. Because of the gaps, decellularized dermis can absorb more water than native dermis and shows different water absorption behavior to glutaraldehyde-crosslinked gelatin and crosslinked poly(acrylamide) hydrogels.

Cell infiltrative hydrogel fibrous scaffolds for accelerated wound healing.

In present study, we generate three-dimensional photocrosslinkable gelatin (GelMA)-based fibrous scaffolds with tunable physical and biological properties by using a combined photocrosslinking/electrospinning approach. The developed GelMA fibrous scaffolds can not only support cell viability and cell adhesion, but also facilitate cell migration and proliferation, accelerating regeneration and formation of cutaneous tissues. In addition, the physical properties of the engineered fibrous GelMA hydrogel including water retention capability, mechanical properties and biodegradability can be tuned to accommodate different patients' needs, making it a promising candidate for skin tissue engineering.

Gelatin Porous Scaffolds as Delivery Systems of Calcium Alendronate

The systemic administration of bisphosphonates (BPs) for the treatment of metabolic diseases characterized by abnormal bone loss suffers from several adverse side effects, which can be reduced by implementation of alternative modes of administration. In this work, glutaraldehyde cross-linked gelatin scaffolds are proposed as delivery systems of calcium alendronate monohydrate (CaAL•H2 O). The 3D highly porous scaffolds display a relevant interconnected porosity (>94%), independently from CaAL•H2 O content (0, 3, and 6 wt%). At variance, pore size varies with composition. The relative increase of the number of smaller pores on increasing BP content is in agreement with the parallel significant increase of the compressive modulus and collapse strength. The scaffolds exhibit a sustained CaAL•H2 O release profile, and a significant amount of the drug is retained in the scaffolds even after 14 d. In vitro tests are carried out using cocultures of osteoblast (OB) and osteoclast (OC). The evaluation of differentiation markers is performed both on the supernatants of cell culture and by means of quantitative polymerase chain reaction. The results indicate that BP containing scaffolds support osteoblast proliferation and differentiation, whereas they inhibit osteoclast viability and activity, displaying a promising beneficial role on bone repair processes.

Temporal and spatial distribution of macrophage phenotype markers in the foreign body response to glutaraldehyde-crosslinked gelatin hydrogels

Currently, it is not well understood how changes in biomaterial properties affect the foreign body response (FBR) or macrophage behavior. Because failed attempts at biomaterial degradation by macrophages have been linked to frustrated phagocytosis, a defining feature of the FBR, we hypothesized that increased hydrogel crosslinking density (and decreased degradability) would exacerbate the FBR. Gelatin hydrogels were crosslinked with glutaraldehyde (0.05, 0.1, and 0.3%) and implanted subcutaneously in C57BL/6 mice over the course of 3 weeks. Interestingly, changes in hydrogel crosslinking did not affect the thickness of the fibrous capsule surrounding the hydrogels, expression of the pan-macrophage marker F480, expression of three macrophage phenotype markers (iNOS, Arg1, CD163), or expression of the myofibroblast marker aSMA, determined using semi-quantitative immunohistochemical analysis. With respect to temporal changes, the level of expression of the M1 marker (iNOS) remained relatively constant throughout the study, while the M2 markers Arg1 and CD163 increased over time. Expression of these M2 markers was highly correlated with fibrous capsule thickness. Differences in spatial distribution of staining also were noted, with the strongest staining for iNOS at the hydrogel surface and increasing expression of the myofibroblast marker aSMA toward the outer edge of the fibrous capsule. These results confirm previous reports that macrophages in the FBR exhibit characteristics of both M1 and M2 phenotypes. Understanding the effects (or lack of effects) of biomaterial properties on the FBR and macrophage phenotype may aid in the rational design of biomaterials to integrate with surrounding tissue.

Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme.

Uric-acid-utilizing soil bacteria were isolated, and 16s rRNA sequence was studied for strain identification. The most prominent uricase-producing bacterium was identified as Comamonas sp BT UA. Crude enzyme was extracted, freeze-dried and its Km and Vmax were determined as 40 meu M and 0.047 meu M min-1ml-1 using Line-weaver Burke plot. An activity of 80 U/mg of total protein was observed when cultured at 37 degree C for 84 h at pH 7. The purified enzyme was used to measure uric acid by spectrophotometric method and electrochemical biosensor. In the biosensing system the enzyme was immobilized on the platinum electrode with a biodegradable glutaraldehyde-crosslinked gelatin film having a swelling percentage of 109+/- 3.08, and response was observed by amperometry applying fixed potential. The electrochemical process as obtained by the anodic peak current and scan rate relationship was further configured by electrochemical impedance spectroscopy (EIS). The polymer matrix on the working electrode gave capacitive response for the electrode-electrolyte interaction. The sensitivity of the biosensor was measured as 6.93 meu A meu M -1 with a sensor affinity [Km(app)] of 50 mu M and 95 percent reproducibility after 50 measurements. The spectrophotometric method could be used in the range of 6-1000 mu M, whereas the biosensor generated linear response in the 1.5- 1000 mu M range with a response time of 24 s and limit of detection of 0.56 meu M. Uric acid was estimated in human blood samples by the biosensor and satisfactory results were obtained.

An Antithrombogenic Citric Acid‐Crosslinked Gelatin with Endothelialization Activity

A novel citric acid-crosslinked gelatin matrix with endothelialization activity and anti-adhesive properties for platelets is prepared. The matrix is characterized using an endothelial cell culture and an antithrombogenic activity test. The number of endothelial cells cultured on the surface of the trisuccinimidyl citrate (TSC)-crosslinked gelatin increases as the concentration of TSC increases to 20 mM, and then decreases with further increases in TSC concentration. Compared with glutaraldehyde-crosslinked gelatin, platelet number and fibrin network formation on the TSC-crosslinked gelatin are minimal at high TSC concentration. The biocompatibility of the matrix is evaluated by bioluminescence imaging. This indicates that the inflammation reaction of the TSC-crosslinked gelatin is lower than that of glutaraldehyde-crosslinked gelatin. Physicochemical analysis of TSC-crosslinked gelatin with different TSC concentrations shows that the high concentration of the cell adhesion sequence, arginine-glycine-aspartic acid, contributes to the promotion of endothelial cell adhesion and subsequent endothelial cell growth. Analysis of the carboxyl groups in the TSC-crosslinked gelatin showed that the antithrombogenic activity is due to the increased negative charge derived from the hydrolyzed active ester groups of TSC. These findings show that TSC-crosslinked gelatin has the potential for use in biomedical devices in contact with blood, such as stents, artificial blood vessels, and artificial heart valves.

Evaluation of cross-linked gelatin membranes as delivery carriers for retinal sheets

Abstract The delivery of intact sheet transplants to the subretinal space can prevent cell loss that is generally associated with the injection of cell suspensions or cell aggregates. The aim of this study was to develop chemically modified gelatin matrices that enhance the delivery efficiency and analyze whether the gelatin membranes cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) can be considered as potential carriers for retinal sheets. The characteristics of EDC cross-linked gelatin membranes were determined by mechanical and in vitro degradation tests, melting point measurements, cell proliferation assays, cytokine expression analyses, and tissue delivery studies. Gelatin membranes without cross-linking and glutaraldehyde cross-linked gelatin samples were used for comparison. Results of this study indicated that introduction of cross-links is capable of rendering the gelatin network more stable against mechanical stresses and deformations as well as rapid hydrolysis during intraocular delivery of delicate tissue sheets. In comparison with the glutaraldehyde treated samples, the EDC cross-linked gelatin membranes showed a better degradation profile and a relatively higher cytocompatibility. In addition, after EDC cross-linking, the gelatin matrices having an acceptable melting point could be used for the fabrication of a sandwich-like carrier with a high transfer and encapsulation efficiency. These findings suggest that the cross-linking agent type gives an influence on delivery functionality of gelatin membranes. In summary, the EDC cross-linked gelatin is an ideal candidate for use as a carrier material in retinal sheet delivery applications.

Gelatin hydrogels: enhanced biocompatibility, drug release and cell viability

Biodegradability and enhanced biocompatibility with pH-sensitivity of hydrogels are becoming very important issues for biomaterials applications so as to minimize the host-body reactions such as, inflammatory, antigenic, and immunogenic problems. This study involves development of hydrogel matrices of gelatin conjugated/modified with highly hydrophilic, pH-sensitive and biocompatible polymer, poly (2-ethyl-2-oxazoline) and glyoxylic acid respectively. Various compositions of gelatin conjugated/modified with poly (2-ethyl-2-oxazoline) (gp) and glyoxylic acid (gg) were synthesized. The swelling kinetics, cell viability and drug release capability from the gels at pH 4.5 and 7.4 were investigated. The results of swelling kinetics showed that, both the degree of swelling (DS) and the maximum degree of swelling (MDS) increased as function of modification (increase in modification) and pH with an increase of time, which is due to increase in ionic groups. The drug-release (1% chlorhexidine) studies at pH 7.4 and 4.5 confirmed a proportional drug release with an increase in degree of swelling. The results of in-vitro cytotoxicity tests using mouse embryonic 3T3 fibroblast cells indicated, an improved cell viability for gelatin gels conjugated/modified with poly (2-ethyl-2-oxazoline) (gp) and glyoxylic acid (gg) gels, when compared with 1% glutaraldehyde cross-linked gelatin gels (gx). Hence, cross-linked gelatin gels can be replaced with gp/gg for potential use in biomedical applications as a matrix for drug delivery.

Creep of glutaraldehyde-crosslinked gelatin films

Abstract Creep behavior of glutaraldehyde-crosslinked gelatin films was evaluated by short-time flexural tests at 30 °C. Samples were characterized by the number-average molecular weight between two crosslinking points, determined by using the Flory–Renher equation. Creep response decreased with increasing degree of crosslinking, which is an indication that crosslinking improves the film stiffness. Experimental creep data were compared with calculated ones by using two different approaches, the creep power-law and the four-parameters model. Good fitting of the experimental results with both models was found, leading to a relationship between the observed creep behavior and the structure of the crosslinked gelatin films. Chemical crosslinkages seemed to induce a decrease of the viscous creep and an enhancement of the elastic contribution.

Tricalcium phosphate and glutaraldehyde crosslinked gelatin incorporating bone morphogenetic protein—A viable scaffold for bone tissue engineering

Bone defects caused by various etiologies must be filled with suitable substances to promote bone repair. Autogenous iliac crest graft is most frequently used, but is often associated with morbidities. Several bone graft substitutes have been developed to provide osteoconductive matrices as well as to enhance osteoinductivity. A tricalcium phosphate and glutaraldehyde crosslinked gelatin (GTG) scaffold, incorporated with bone morphogenetic proteins (BMPs), was developed to provide an alternative mean of bone tissue engineering. This study investigated differences between GTG and BMP-4 immobilized GTG (GTG-BMP) scaffolds on neonatal rat calvaria osteoblast activities. The GTG scaffold possessed an average pore size of 200 microm and a porosity of 75%. HE staining revealed uniform cell distribution throughout the scaffold 24 h post cell seeding. Alkaline phosphatase (ALP) activity of the GTG samples increased initially and then stabilized at 3 weeks postseeding. ALP activity of the GTG-BMP samples was similar to that of the GTG samples in the second and third weeks, but it continued increasing and became significantly greater than that of the GTG samples by the fourth week. Gla-type osteocalcin (Gla-OC) activity of the GTG-BMP samples was initially lower, but also became significantly greater than that of the GTG samples by the fourth week. An HE stain revealed greater numbers of attached cells and a richer matrix deposits in the GTG-BMP samples. A von Kossa stain showed larger mineralizing nodules, in greater numbers, after 4 weeks of in vitro cultivation. These findings suggest that the GTG scaffold provides an excellent porous structure, conductive to greater cell attachment and osteoblast differentiation, and that utility can be significantly enhanced by the inclusion of BMPs. A GTG-BMP scaffold holds promise as a superior bioactive material for bone tissue engineering.

Enzymatic stabilization of gelatin‐based scaffolds

The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.

Osteogenic evaluation of glutaraldehyde crosslinked gelatin composite with fetal rat calvarial culture model.

The cytotoxicity of the synthetic bone substitute composed of tricalcium phosphate and glutaraldehyde crosslinked gelatin (GTG) were evaluated by osteoblast cell culture. In a previous study, the GTG composites were soaked in distilled water for 1, 2, 4, 7, 14, 28, and 42 days, and then the solutions (or extracts) were cocultured with osteoblasts to evaluate the cytotoxicity of GTG composites by alive cell counting. In this study, the extracts were cocultured with the osteoblasts; thereafter, the concentration of transforming growth factor-beta (TGF-beta1) and prostaglandin E2 (PGE2) in the medium was analyzed to strictly reflect the biological effects of GTG composites on the growth of osteoblasts. In order to investigate the osteoconductive potential of the GTG composites on new bone formation in a relative short term, a model of neonatal rat calvarial organ culture was designed prior to animal experiments. Three experimental materials of 4, 8, and 12% GTG composites were evaluated by fetal rat calvarial organ culture for their ability for bone regeneration. Deproteinized bovine and porcine cancellous bone matrixes were used as the controlled materials. All the organ culture units were maintained in cultured medium for 5 weeks. Following the culture period, the morphology of tissue was observed under an optical microscope, and the quantitative evaluation of the new generation bone was determined by using a semiautomatic histomorphometeric method. Except in the initial 4 days, the concentration of TGF-beta1 of 4% and 8% GTG composites was higher than that of the blank group for all the other experimental time periods. The PGE2 concentration for 4% and 8% GTG composites was lower than that of the blank group. It revealed that the 4% and 8% GTG composites would not lead to inflammation and would promote osteoblast growth. The morphology and activity of the osteoblasts were not transformed or changed by the 2 GTG composites. For the 12% GTG composite, the performance of the in vitro condition was inferior to the blank group and the other 2 GTG composites. Although the concentration of TGF-beta1 and PGE2 was gradually back to normal after 14 days, the morphology of the osteoblasts was abnormal with features such as contracted cytoplast structures. The osteoblast was damaged perhaps in the initial stage. We suggested that the 4% and 8% GTG composites should be soaked in distilled water at least for 4 days before medical applications. The 12% GTG composite and the composites with a concentration of glutaraldehyde solution higher than 12% were not recommended as a medical prostheses in any condition. The fetal rat calvaria culture also showed the same results with the analysis of TGF-beta1 and PGE2. From the study, we could predict the results of animal experiments in the future.

Perforated support foils with pre-defined hole size, shape and arrangement

Perforated support foils with holes of pre-defined size, shape and arrangement and with hole sizes down to the sub-micrometer range, named Quantifoil, are presented. The foils are fabricated using semiconductor lithographic techniques. For the first time, a sacrificial layer that consists of a biopolymer, i.e. glutaraldehyde cross-linked gelatin, is used. This layer is removed by proteinase treatment, thus introducing an enzymatic reaction as a tool in microsystems technology. The foils are particularly beneficial in electron microscopy, when a specimen support is required with holes smaller than those attainable with metal grids (∼10 μm). Their advantages in cryo-electron microscopy, for example, are illustrated. Furthermore, foils with a specific hole size and arrangement permit a further automation of electron microscopic (EM) data acquisition procedures.

Studies on the enzymatic and fungal degradation of cross-linked gelatin and its graft copolymers

Glutaraldehyde cross-linked gelatin was graft copolymerized with acrylic acid, acrylamide, vinyl acetate, methyl acrylate, and methyl methacrylate either individually or in combination. The enzymatic and fungal degradation of these graft copolymers with trypsin, pepsin, and mixed cultures ofAspergillus niger, Penicillium ochrochloron, Penicillium funiculosum, andTrichoderma viride was studied for short and extended periods. The weight loss suffered by the samples, the weight of biomass formed, the nitrogen content, and the pH of the culture medium were determined. With the help of these data, the extent of utilization of graft copolymers by fungi as a sole source of carbon was estimated. The samples with less than 100% grafting and with a ratio of polymethyl methacrylate content (L) to polyacrylic acid (H) content (L/H values) lower than 1.0 were readily and extensively degraded.

Xanthine oxidase activity of arthrobacter x-4 cells immobilized in glutaraldehyde-crosslinked gelatin

Cells of Arthrobacter X-4 were immobilized by entrapment in gelatin crosslinked with glutaraldehyde. The xanthine oxidase activity and stability were determined at various temperatures. In comparison with bovine milk xanthine oxidase the bacterial enzyme is more stable and has a different substrate specificity. 1-Methylxanthine was oxidized on a preparative scale.