Bioadhesion and biocompatibility evaluations of gelatin and polyacrylic acid as a crosslinked hydrogel in vitro

This study describes the potentiality of crosslinked hydrogels comprised of gelatin and polyacrylic acid (CHGP) as a biological glue for soft tissues and compares its bonding strength with that of fibrin glue. Water-soluble carbodimide (WSC) was used to crosslink the mixture of gelatin and polyacrylic acid (PAA). An addition of PAA to gelatin increases bonding strength and reduces the gelation time and WSC concentration. Increasing the gelatin, WSC and PAA concentration increases the bonding strength. There is a critical concentration to have a maximum bonding strength. The cured hydrogel exhibited sufficient adhesion to mouse skin with a higher bonding strength than fibrin glue. The in vitro test has been done for evaluating CHGP toxicity.     

In vitro mineralization of gelatin-polyacrylic acid complex matrices

Gelatin-polyacrylic acid (gel-PAA) matrices were obtained by slow diffusion of polyacrylic acid into gelatin gels. The matrices were submitted to uniaxial stretching, which induces a preferential orientation of the collagen molecules, and used as biomimetic substrates for the nucleation of hydroxyapatite from simulated body fluid (SBF). The relative amount of hydroxyapatite deposited from 1.5SBF increases as a function of polyelectrolyte content in the matrices, up to about 30 wt%. In the absence of PAA, the inorganic phase is laid down on the surface of the gelatin matrices as hemispherical aggregates. At variance, hydroxyapatite deposition in the gel-PAA composite matrices at relatively low PAA content occurs preferentially in the spaces between the layers on the surface of the matrices and displays a tablet-like morphology. At high polyelectrolyte concentration, an almost uniform layer of hydroxyapatite covers the whole surface of the matrices. The preferential orientation of the (002) hydroxyapatite reflection indicates a close relationship between the inorganic crystals and the collagen molecules.     

Polymer composition and acidification effects on the swelling and mechanical properties of poly(acrylamide-co-acrylic acid) superporous hydrogels

Poly(acrylamide-co-acrylic acid) (poly(AM-co-AA)) superporous hydrogels (SPHs) were synthesized and the acidification effects on the swelling and mechanical properties were studied. Gelation exotherms were measured to determine the optimum introduction time for adding a blowing agent. The gelation kinetics decreased with increasing the AA concentration. The maximum equilibrium swelling was observed around an AA weight fraction of 0.4, but the compressive strength decreased monotonically with increasing the AA concentration. Poly(AM-co-AA) SPHs were much less swollen in acidic solution than in distilled water of pH 6.7. The swelling ratio decreased with increasing acidity (decreasing pH). Reduction of water absorption content by acidification led to considerable increase in the mechanical strength.     

Poly(lactic acid) scaffold fabricated by gelatin particle leaching has good biocompatibility for chondrogenesis

Three-dimensional poly(L-lactic acid) (PLLA) scaffolds with high porosity and an average pore size of 280–450 μm were fabricated using gelatin particles as porogen. The particles were bonded together by incubation in saturated water vapor at 70°C for 3.5 h. After casting the PLLA/1,4-dioxane solution, freeze-drying and porogen leaching with 70°C water, a porous scaffold with well-interconnected pores and some nano-fibers was obtained. The biological performance of the scaffold was evaluated by in vitro chondrocyte culture and in vivo implantation. In comparison with the control scaffold fabricated with NaCl particles as porogen under the same conditions, the experimental scaffold had better biological performance because the gelatin molecules were stably entrapped onto the pore surfaces. A larger number of cells in the experimental scaffold were observed by confocal laser scanning microscopy after the viable cells had been stained with fluorescein diacetate. The chondrocytes showed more spreading morphology. Higher cytoviability and secretion of glycosaminoglycan (GAG) were also determined in the experimental scaffold. After implantation of the chondrocytes/PLLA scaffold construct to the subcutaneous dorsum of nude mice for 30–120 days, cartilage-like specimens were harvested. Histological examination showed that the regenerated cartilages had a large quantity of collagen and GAG.     

Controlled release of growth factors based on biodegradation of gelatin hydrogel

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-β1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-β1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.     

Cross-linking and characterisation of gelatin matrices for biomedical applications

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/ EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOH_gelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOH_gelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40°C.     

Preparation of a hyaluronic acid hydrogel through polyion complex formation using cationic polylactide-based microspheres as a biodegradable cross-linking agent

A novel hyaluronic acid (HA)-based hydrogel was prepared through polyion complex (PIC) formation between cationic polylactide (PLA)-based microspheres (MS⁺) and hyaluronic acid (HA^?) as an anionic polyelectrolyte. The MS⁺ and HA formed a biodegradable PIC hydrogel (HA^?/MS⁺) when mixed in aqueous media. The swelling behavior and mechanical properties of the PIC hydrogel could be controlled by changing the charge ratio between HA^? and MS⁺. In addition, the HA^?/MS⁺ PIC hydrogel resulted in a lower inflammatory response compared with a collagen hydrogel in vivo.     

Preparation and characterization of pH-sensitive hydrogel of chitosan/poly(acrylic acid) co-polymer

A pH-sensitive co-polymer hydrogel of chitosan/poly(acrylic acid) (CS/PAAc) was prepared by irradiating the aqueous solution mixture of chitosan and acrylic acid with ⁶⁰Co γ-ray irradiation. The effect of the composition of chitosan and AAc on the properties of the hydrogel, such as swelling ratio and pH-sensitivity, were determined. Fourier Transform Infrared (FT-IR) spectrometry was applied in the attenuated total reflectance (ATR) mode for analyzing the structure change of the hydrogels after the treatment in different pH buffer solutions.     

Biocompatibility and biodegradation of cross-linked gelatin/hyaluronic acid sponge in rat subcutaneous tissue

A gelatin/hyaluronic acid (GH) sponge has been fabricated by freeze-drying and cross-linking. The GH sponge was insoluble when cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The morphologies of sponges were investigated using a field emission scanning electron microscope. The porosity of the GH sponge increased with hyaluronic acid content. The GH sponge was biodegradable, as evidenced by implantation in Wistar rat subcutaneous connective tissue. Fibroblasts infiltrated into the sponge matrix, and regenerated collagen in the matrix to a level of 25% by 15 days after surgery. The GH73 sponge induced an acute inflammatory response compared with the GH91 sponge. This inflammatory response could have been stimulated by the presence of hyaluronic acid up to Day 10, as it decreased afterwards. The C-reactive protein of blood samples also indicated the same result. The blood tests and histological results show that GH sponges have good biocompatibility and low antigenicity for tissue engineering scaffolds.     

Biocompatibility and performance in vitro of a hemostatic gelatin sponge

The biocompatibility of a hemostatic gelatin sponge (Cutanplast Standard) was evaluated in vitro. Cytotoxicity was assessed by neutral red uptake and amido black staining tests; genotoxicity was assayed using the Ames test, Sister Chromatides Exchanges (SCE) and chromosomal aberrations. The ability of the hemostatic gelatin sponge to induce platelet adhesion and release reaction was also determined. The undiluted extract of the test material was found to be cytotoxic, but cell viability was not affected by 1:2 and 1:10 diluted extract. The same extract was found to be non-genotoxic using the three assays for genotoxicity. A significant decrease of platelet number, as well as a significant release of platelet factor 4 was found to be caused by the solid material. In conclusion, Cutanplast Standard is neither cytotoxic nor genotoxic, while inducing platelet adhesion and release reaction when challenged with blood.     

Soft tissue adhesive composed of modified gelatin and polysaccharides

Although fibrin glue has been clinically used as a surgical adhesive, hemostatic agent, and sealant, it has the risk of virus infection because its components, fibrinogen and thrombin, are obtained from human blood. To circumvent this problem, we employed bioabsorbable gelatin and polysaccharides to prepare a safer hemostatic glue. Gelatin was modified with ethylenediamine using water-soluble carbodiimide to introduce additional amino groups into the original gelatin, while dextran and hydroxyethyl-starch were oxidized by sodium periodate to convert 1,2-hydroxyl groups into dialdehyde groups. Upon mixing of the two polymer components in aqueous solution, Schiff base was formed between the amino groups in the modified gelatin and the aldehyde groups in the modified polysaccharides, which thus resulted in intermolecular cross-linking and gel formation. The fastest gel formation took place within 2 s, and its bonding strength to porcine skin was about 225 gfcm^-2 when 20 wt% of an amino-gelatin (55% amino) and 10 wt% of aldehyde-HES (>84% dialdehyde) aqueous solutions were mixed. In contrast, the gelation time and bonding strength of fibrin glue was 5 s and 120 gfcm^-2, respectively.     

Evaluation of gelatin hydrogel crosslinked with various crosslinking agents as bioadhesives: in vitro study.

Bioadhesives are used for tissue adhesion and hemostasis in surgery. A gelatin-resorcinol mixture crosslinked with formaldehyde (GRF glue) and/or glutaraldehyde (GRG) is used for this purpose. Although the bonding strength of the GRF glue to tissue is satisfactory, concerns about the cytotoxicity of formaldehyde are reported in the literature. It was suggested that the cytotoxicity problem of the GRF glue may be overcome by changing its crosslinking method. The study was therefore undertaken to assess the feasibility of using an epoxy compound (GRE glue), a water-soluble carbodiimide (GAC glue), or genipin (GG glue) to crosslink with a gelatin hydrogel as new bioadhesives. GRF glue and GRG glue were used as controls. The results of our cytotoxicity study suggested that the cellular compatibility of the GAC and GG glues was superior to the GRF, GRG, and GRE glues. The gelation time for the GG glue was relatively longer than the GRF and GRG glues, while no gelation time could be determined for the GAC glue. Additionally, it took approximately 17 h for the GRE glue to become adhesive. The GRF and GRG glues had the greatest bonding strengths to tissue among all test adhesives, while the bonding strengths of the GAC and GG glues were comparable. In contrast, there was almost no bonding strength to tissue for the GRE glue. However, the GRF and GRG glues were less flexible than the GAC and GG glues. Subsequent to the bonding strength measurement, each test adhesive was found to adhere firmly to the tissue surface and underwent cohesive failure during the bond breaking. In conclusion, the GRF and GRG glues may be used as tissue adhesives when their ability to bind tissue rapidly and tightly is required; the GAC and GG glues are preferable when the adhesive action must be accompanied with minimal cytotoxicity and stiffness; and the GRE glue is not suitable for bioadhesion in clinical applications.     

Polyamide 6 composite membranes: Properties and in vitro biocompatibility evaluation

The aim of the present study was to develop polyamide 6 membrane blended with gelatin and chondroitin sulfate using the phase precipitation method and evaluate its in vitro biocompatibility. Morphology of membranes was studied by laser scanning confocal microscopy which allowed the nondestructive visualization of internal bulk morphology of membranes. Membranes exhibited porous morphology with pores spanning across the membrane width with interconnections at various depths. Membranes showed adequate mechanical properties with tensile strengths of 20.10 ± 0.64 MPa, % strain of 3.01±0.07, and modulus of 1082.50±23.50 MPa. In vitro biocompatibility of membranes by direct contact test did not show degenerative effects on NIH3T3 cells and also its leach-out products (LOP), as determined by tetrazolium (MTT) and neutral red uptake (NRU) assay. Mouse peritoneal macrophage cultured in contact with membranes and PTFE control showed comparable expression of activation markers such as CD11b/CD18, CD45, CD14, and CD86 suggesting the membranes' non-activating nature. Membrane LOP did not induce excessive proliferation of mouse splenocytes suggesting its non-antigenic nature. Preliminary blood compatibility of membranes was observed with no detectable hemolysis in static incubation assay. Taken collectively, the present data demonstrate that polyamide 6 composite membranes are biocompatible and prospective candidates for tissue engineering applications.     

Synthesis and characterization of a novel hyaluronic acid hydrogel

Hyaluronic acid (hyaluronan, HA) has many medical applications as a biomaterial. To enhance its biostability, a novel hydrogel of cross-linked hyaluronic acid was prepared using a double cross-linking process, which involves building cross-linkages between hydroxyl group pairs and carboxyl group pairs. The present study explored a number of cross-linking processes in order to obtain different degrees of cross-linking, which were evaluated by the measurement of water absorption capacity as an index of the gel network density. To gain a better understanding of the stability of the gel, the chemical structure and particularly the rheological behaviour of the cross-linked HA, which included the influences of factors, such as degree of cross-linking, HA concentration and gel particle size, were investigated. The in vitro biostability against hyaluronidase and free radical degradation was tested to show that the cross-linked hydrogel had improved resistance to in vitro hyaluronidase and free radical degradation.     

Preparation of poly(ethylene glycol)-introduced cationized gelatin as a non-viral gene carrier

The objective of this study was to prepare cationized gelatins grafted with poly(ethylene glycol) (PEG) (PEG-cationized gelatin) and evaluate the in vivo efficiency as a non-viral gene carrier. Cationized gelatin was prepared by chemical introduction of ethylenediamine to the carboxyl groups of gelatin. PEG with one terminal of active ester group was coupled to the amino groups of cationized gelatin to prepare PEG-cationized gelatins. Electrophoretic experiments revealed that the PEG-cationized gelatin with low PEGylation degrees was complexed with a plasmid DNA of luciferase, in remarked contrast to that with high PEGylation degrees. When the plasmid DNA complexed with the cationized gelatin or PEG-cationized gelatin was mixed with deoxyribonuclease I (DNase I) in solution to evaluate the resistance to enzymatic degradation, stronger protection effect of the PEG-cationized gelatin was observed than that of the cationized gelatin. The complex of plasmid DNA and PEG-cationized gelatin had an apparent molecular size of about 300 nm and almost zero surface charge. These findings indicate that the PEG-cationized gelatin–plasmid DNA complex has a nano-order structure where the plasmid DNA is covered with PEG molecules. When the PEG-cationized gelatin–plasmid DNA complex was intramuscularly injected, the level of gene expression was significantly increased compared with the injection of plasmid DNA solution. It is concluded that the PEG-cationized gelatin was a promising non-viral gene carrier to enhance gene expression in vivo.     

Influence of gelatin complexation on cell proliferation activity and proteolytic resistance of basic fibroblast growth factor

The objective of this study is to investigate the influence of gelatin complexation on the biological activity of basic fibroblast growth factor (bFGF) and its resistance to trypsin digestion. When bFGF was mixed at 37°C with acidic gelatin with an isoelectric point(IEP)of 5.0, the activity to promote in vitro proliferation of BHK cells became lower compared with that of free bFGF,in contrast to mixing with the basic gelatin with an IEP of 9.0. A maximum reduction in the bFGF activity was observed for the bFGF-gelatin complex prepared at a mixing molar ratio of 1/1. The bFGF activity of cell proliferation reduced at the initial period after mixing with the acidic gelatin at 37°C, followed by no substantial change. Complexation with the acidic gelatin at 4°C had no influence on the bFGF activity, irrespective of the bFGF/gelatin ratio and complexation time. The biological activity of bFGF was reduced by the trypsin treatment, but the reduced extent was suppressed through gelatin complexation at 37°C. In an electrophoresis study, the protective effect of gelatin complexation on the trypsin digestion was also confirmed in terms of the molecular weight loss. It is possible that the complexing gelatin covers bFGF molecules, resulting in suppression of their interaction with the cell surface receptor as well as protection from their enzymatic attack.     

Insoluble ionic complexes of polyacrylic acid with a cationic drug for use as a mucoadhesive,ophthalmic drug delivery system

We have developed a new mucoadhesive drug delivery formulation based on an ionic complex of partially neutralized poly(acrylic acid) (PAA) and a highly potent beta blocker drug, levobetaxolol · hydrochloride (LB · HCl), for use in the treatment of glaucoma. PAA was neutralized with sodium hydroxide to varying degrees of neutralization. Aqueous solutions containing concentrations of LB · HCl equivalent to the degree of PAA neutralization were added to the PAA solutions and formed insoluble complexes, which were isolated. The complex formation was followed by turbidimetric titration, and the complexes were characterized by IR and ¹H NMR spectroscopy. Complexes were prepared with varying degrees of drug loading, such that the same PAA chain would have free COOH groups for mucoadhesion along with ionic complexes of LB · H⁺ with COO^- groups. Thin films of the complexes dissociated to release the drug by ion exchange with synthetic tear fluid. The films shrunk continuously during release of the drug and dissolved completely in 1 h. Solid inserts of these films could be useful as a mucoadhesive ophthalmic drug delivery system.     

Spontaneous Formation of a Hydrogel Composed of Water-Soluble Phospholipid Polymers Grafted with Enantiomeric Oligo(lactic acid) Chains

We designed and synthesized water-soluble biocompatible and biodegradable polymers composed of 2-methacryroyloxyethyl phosphorylcholine and oligo(L- or D-lactic acid) macromonomers to develop an injectable hydrogel matrix. Aqueous solutions containing the polymers with oligo(L-lactic acid) (OLLA) and oligo(D-lactic acid) (ODLA) chains underwent spontaneous gelation when mixed together. This was due to the formation of a stereocomplex between the OLLA and ODLA side-chains, which act as cross-linking components in the hydrogel. Therefore, the hydrogel could be re-dissolved in a buffer solution by hydrolysis of the oligo(lactic acid) chains. We obtained an injectable, biocompatible and degradable hydrogel, and we anticipate that it will be used in applications involving the controlled release of bioactive molecules and cell-based tissue engineering.     

Survival of lactic acid bacteria in simulated gastrointestinal juice protected by a DNA-based complex gel

Salmon milt DNA was utilized in the approach to protect functional materials, proteins, microorganisms, drugs, etc., by forming a DNA-based complex gel with gelatin and κ-carrageenan. The multiplier effect of the combination of DNA, gelatin and κ-carrageenan was examined by measuring the release of methyl orange from the gel after being incubated in simulated gastric juice. The results indicated that the hydrogel made of DNA, gelatin and κ-carrageenan can efficiently maintain the functional materials. Furthermore, a complex gel was developed and optimized by applying an emulsion of lactic acid bacteria and cacao oil onto the already prepared hydrogel. The existence of the lactic acid bacteria in the oil emulsion particles was confirmed by scanning electron microscopy. The protection capabilities in the simulated gastrointestinal juice, which had a viability of 1.50 × 10⁶ CFU/g gel after 2 h incubation in the simulated gastric juice and 2.53 × 10⁷ CFU/g gel after 6 h continued incubation in the simulated intestinal juice, were measured, in contrast to 2.58 × 10⁷ CPU/g in the original gel before treatment.     

In vitro and in vivo biocompatibility evaluation of a polyalkylimide hydrogel for soft tissue augmentation

Injectable fillers are commonly used in Plastic and Reconstructive Surgery to correct serious and slight aesthetic defects due to their low invasiveness and an easy implant technique procedure. Synthetic hydrogels are proposed as filler materials for their similarity with soft tissue and to avoid many disadvantages of naturally derived materials such as short persistence, allergenicity, and immunogenicity. Our studies are focused on the biocompatibility evaluation of a polyacrylic hydrogel containing alkylimide-amide groups and pyrogen free water (96%) (Bio-Alcamid by means of the in vitro cytotoxicity and mutagenicity assays and the in vivo skin irritation, sensitization test, and subcutaneous implant. All tests conducted on Bio-Alcamid showed no toxicity. It is a substance easy to inject and remove; it does not migrate, and its safety allows it to be a suitable filler for the correction of slight and also very serious aesthetic defects.