Crosslinking structures of gelatin hydrogels crosslinked with genipin or a water‐soluble carbodiimide

It was suggested in our previous studies that carbodiimide‐ and genipin‐crosslinked gelatin hydrogels could be used as bioadhesives to overcome the cytotoxicity problem associated with formaldehyde‐crosslinked gelatin hydrogels. In this study, we investigated the crosslinking structures of carbodiimide‐ and genipin‐crosslinked gelatin hydrogels. We found that crosslinking gelatin hydrogels with carbodiimide or genipin could produce distinct crosslinking structures because of the differences in their crosslinking types. Carbodiimide could form intramolecular crosslinks within a gelatin molecule or short‐range intermolecular crosslinks between two adjacent gelatin molecules. On the basis of gel permeation chromatography, we found that the polymerization of genipin molecules could occur under the conditions used in crosslinking gelatin hydrogels via a possible aldol condensation. Therefore, besides intramolecular and short‐range intermolecular crosslinks, additional long‐range intermolecular crosslinks could be introduced into genipin‐crosslinked gelatin hydrogels. Crosslinking a gelatin hydrogel with carbodiimide was more rapid than crosslinking with genipin. Therefore, the gelation time for the carbodiimide‐crosslinked gelatin hydrogels was significantly shorter than that of the genipin‐crosslinked gelatin hydrogels. However, the cohesive (interconnected) structure of the carbodiimide‐crosslinked gelatin hydrogels was readily broken because, unlike the genipin‐crosslinked gelatin hydrogels, there were simply intramolecular and short‐range intermolecular crosslinks present in the carbodiimide‐crosslinked hydrogel. In the cytotoxicity study, the carbodiimide‐crosslinked gelatin hydrogels were dissolved into small fragments in the cultural medium within 10 min. In contrast, the genipin‐crosslinked gelatin hydrogels remained intact in the medium throughout the entire course of the study. Again, this may be attributed to the differences in their crosslinking structures. The genipin‐crosslinked gelatin hydrogels were less cytotoxic than the carbodiimide‐crosslinked gelatin hydrogels. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 4017–4026, 2004     

Effect of gelatin on the swelling behavior of organic hybrid gels based on N‐isopropylacrylamide and gelatin

Organic hybrid gels based on poly(N‐isopropylacrylamide) and a natural polymer, gelatin, were prepared through two‐step crosslinking with genipin or glutaraldehyde. The effects of the gelatin content on the swelling behaviors and physical properties of these hybrid gels were investigated. The results indicated that the swelling ratio decreased with an increase in the content of gelatin in these hybrid gels. The swelling ratio for the gel crosslinked by genipin was significantly smaller than that for the gel crosslinked by glutaraldehyde. The results also showed that the gel crosslinked with genipin had a higher crosslinking density and a higher gel strength. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 1092–1099, 2005     

Multifunctional gentamicin supplementation of poly(γ‐glutamic acid)‐based hydrogels for wound dressing application

The process of wound healing is composed of coagulation, inflammation, fibroplasia, collagenation, epithelization, and wound contraction. The wound dressing should protect the wound from bacterial infection, maintain a moist healing environment, and promote cell migration to reconstruct damaged tissue, and be easy to apply and remove to improve patient comfort. The purpose of our study was to develop multifunctional hydrogels composed of genipin‐crosslinked biodegradable biomaterials of poly(γ‐glutamic acid) and gelatin, encapsulating gentamicin to accelerate wound healing. The results of swelling ratio measurements clearly indicate that hydrogel composition of poly(γ‐glutamic acid)–gelatin had a higher swelling ratio and lower peel adhesion properties than gelatin hydrogel alone. In an in vitro study, the gentamicin incorporated in prepared hydrogels effectively inhibited target microorganisms and produced a higher expression of Type I collagen in fibroblast cells. Confocal laser scanning microscopy revealed that the fibroblast cells cultured in the hydrogel membranes produced fibroblast cell migration and showed a continuous lined cytoskeletal distributing status. In the in vivo study, it was found that the gentamicin incorporated in genipin‐crosslinked γ‐PGA–gelatin wound dressing demonstrates the potential of such biologically functionalized dressing to accelerate wound closure and, hence, its potential clinical usefulness. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Microencapsulation of Zanthoxylum limonella oil (ZLO) in genipin crosslinked chitosan–gelatin complex for mosquito repellent application

Essential oil containing chitosan gelatin complex microcapsules crosslinked with genipin were prepared by complex coacervation process. The effects of various parameters such as oil loading, ratio of chitosan to gelatin, degree of crosslinking on oil content, encapsulation efficiency, and the release rate of the essential oil were studied. Scanning electron microscopy study indicated that the surface of the microcapsules were more irregular as the amount of oil loading increased. Thermal stability of microcapsules improved with the increase in the amount of chitosan in chitosan–gelatin matrix as revealed by thermogravimetric analysis. FT‐IR spectroscopy and differential scanning calorimetry study indicated that there was no significant interaction between chitosan–gelatin complex and oil. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

A novel wound dressing composed of nonwoven fabric coated with chitosan and herbal extract membrane for wound healing

This study presents a novel design of an easily stripped bilayer composite that consists of an upper layer of soybean protein nonwoven fabric coated with a lower layer, genipin‐crosslinked chitosan and Bletilla striata herbal extract (GCB) film as a wound dressing material (GCB/NWF). Preliminary tests investigated the in vitro characteristics of the genipin‐crosslinked chitosan (GC) film. Experimental results showed that the dark bluish color of the GC film may have resulted from the reaction of genipin with the amino acid group in the chitosan solution. Additionally, adding more wt% genipin caused the bluish color of the GC films to be darker. This new wound dressing material also exhibited good mechanical properties. Genipin and chitosan were released from the soaked GC film. Cytotoxic test revealed that 20 ppm of the genipin in the culture medium could be criteria, over which cytotoxicity to L929 fibroblasts could be produced. However, the chitosan and B. striata herbal extract not only were not toxic but also promoted the viability and growth of L929 fibroblasts. Additionally, the GCB film more effectively supported cell attachment and growth. The in vivo histological assessment results revealed that epithelialization and reconstruction of the wound were achieved by covering the wound with the GCB/NWF wound dressing material, and it would be easily stripped from the wound surface without damaging newly regenerated tissue. POLYM. COMPOS., 2010. © 2009 Society of Plastics Engineers     

Effects of Human and Porcine Adipose Extracellular Matrices Decellularized by Enzymatic or Chemical Methods on Macrophage Polarization and Immunocompetence

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage’s functional role.     

京尼平交联明胶多孔支架的制备及降解

采用天然交联剂京尼平,结合相分离技术与冷冻干燥方法,制备不同交联度的明胶多孔支架。结果表明,支架的交联度随着京尼平浓度的增加而升高,最高可以达到65．3％;不同交联度的明胶支架微观呈相互连通的多孔结构,且孔径随交联度的升高而降低;通过调控明胶支架的交联度,可以调控支架的降解时间。     

Fabrication,swelling behavior,and water absorption kinetics of genipin-crosslinked gelatin–chitosan hydrogels

Gelatin/chitosan hydrogels have attracted considerable attention over the last 2 decades in various fields of applications. In this paper, chemically crosslinked composite hydrogels with different gelatin-to-chitosan weight ratios were fabricated and crosslinked with different amounts of genipin via the solvent casting technique combined with freeze-drying. Fourier-transform infrared, scanning electron microscopy (SEM), liquid displacement method, and gravimetric analysis were used to examine the chemical, microstructural, and physical properties of the hydrogels. IR spectra confirmed the formation of covalent bonds between the amino groups of the parent's macromolecules and genipin. SEM micrographs indicated that the hydrogels possessed a highly porous structure with well-defined pore geometries. The swelling capacity and degradation rate of the specimens reduced with increasing the amounts of chitosan and/or genipin. In-depth swelling measurements revealed that the first-order kinetic model was only applicable in the early stage of the swelling study; however, the water absorption behavior of the hydrogels was best described by the pseudo-second-order kinetic model (Schott's model) throughout the swelling experiment. The genipin-crosslinked hydrogels were found to support MC3T3-E1 cell proliferation. The results of this paper thus suggest the 1.5% genipin-crosslinked gelatin/chitosan hydrogels as promising candidates for on-demand drug delivery applications or more precisely osteoarthritis drug delivery systems.     

Thermosensitive elastin-derived polypeptide hydrogels crosslinked by genipin

In this paper, first, elastin-derived peptides (EPs) with low molecular weight were prepared by acid degradation. Second, elastin-derived peptide hydrogels were fabricated by crosslinking EP with genipin. EP exhibited an inverse transition temperature, and the inverse transition temperature (T_t) could be adjusted by changing the concentration and molecular weight of EP, pH, and adding salt. The freeze-dried EP hydrogels were obviously three-dimensional network structure and the property of EP hydrogel can be optimized by dosage of genipin. Differential scanning calorimetry (DSC) tests and the swelling ratio changing with temperature showed the crosslinked EP hydrogels were still thermosensitive and exhibited a negative swelling behavior. The compression modulus of EP hydrogels can even reach 39.4?kPa, surpassing many other elastin-based hydrogels. This genipin crosslinked EP hydrogel is a good biological material and has great potentiality in tissue engineering.     

Gelatin hydrogels with PAMAM nanostructured surface and high density surface-localized amino groups

Amino-terminated PAMAM dendrimers with 16 and respectively 64 amino groups have been chemically immobilized onto the surface of glutaraldehyde (abbreviated GA) crosslinked thin gelatin scaffolds based on Schiff-base linkage formation between the amines and free aldehyde from partially unreacted GA. Crosslinking and dendrimers surface modification of hydrogels have been chemically and physically investigated using FT-IR spectroscopy and microscopy, UV–VIS, LM, AFM, swelling tests and a colorimetric technique. Two types of gelatin-based hydrogels with homogeneous nanorough surfaces and presenting an increased number of amino groups available for further chemical reactions have been obtained. The synthesis strategy presented in this work seems to open a new route for nanorough crosslinked gelatin scaffolds that could be further used for biomedical and especially for hard tissue engineering application.     

Fabrication, characterization and bioactivity evaluation of calcium pyrophosphate/polymeric biocomposites

The design of the biocomposites offers the opportunity to create grafting materials with excellent bioactivity, resorbability and improved mechanical properties. In this study, we are concerned with the preparation of calcium pyrophosphate (CPP) and its composites with polymeric matrix to enhance these properties. The fabricated biocomposites were characterized by X-ray diffraction (XRD), Fourier transformer infrared spectra (FT-IR), thermogravimetric (TGA) analyses and scanning electron microscope with X-ray elemental analysis (SEM-EDAX). The characterization results confirmed homogeneity, interaction and integration between the CPP filler and polymeric matrix. The mechanical properties of biocomposites had enhanced values compared to the original copolymer matrices and were comparable to those of cancellous bone. In vitro test results via calcium and phosphorous ions measurements, showed that the biocomposites had enhanced ability to accelerate the mineralization of calcium phosphate layer on their surfaces. FT-IR and SEM-EDAX post-immersion confirmed that the CPP/polymeric composites containing chitosan or chitosan–gelatin matrix had ability to induce a bone-like apatite layer onto their surfaces. Finally, a novel CPP/polymeric biocomposites have good bioactivity and suitable mechanical properties; therefore, they could be used in bone grafting and tissue engineering applications in future.     

Montmorillonite reinforced type A gelatin nanocomposites

This study investigates the structural, morphological, thermal, and mechanical properties of type A gelatin/montmorillonite (MMT) films as a function of MMT concentration. The variations of the X‐ray diffraction pattern suggest that the structure of the nanocomposites turns from intercalated to exfoliated on increasing clay loading up to 20 wt %. Simultaneously, gelatin interaction with clay negative sheets during gelling provokes a reduction of the triple helix content of the composite films, in agreement with the reduction of the relative intensity of the 1.1 nm diffraction reflection of gelatin and of the values of denaturation enthalpy. On the other hand, the increase of the denaturation and decomposition temperatures, the significant rise of the Young's modulus, as well as the swelling decrease observed as clay content increases, demonstrate a relevant stabilizing effect of MMT on gelatin. The reinforcement action of MMT allows to utilize a relatively low concentration of the crosslinking agent genipin to further stabilize the films. The synergic action of clay and genipin prevents dissolution of the nanocomposites in aqueous solution and enhances their mechanical properties. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40301.     

Effects of coacervation acidity on the genipin crosslinking action and intestine-targeted delivery potency of the O-carboxymethyl chitosan–gum arabic coacervates

O-carboxymethyl chitosan (O-CMC)–gum arabic (GA) coacervates separated in different acidities were crosslinked by genipin and subjected to FTIR analysis, viscoelasticity measurement, DSC characterization, pH response evaluation, and in vitro BSA release profiling to explore the influence of coacervation acidity on genipin crosslinking and intestine-targeted delivery potency of the coacervates. Genipin crosslinking greatly improved the stability of the coacervates against various simulated gastrointestinal fluids and the coacervation acidity exerted different effects on the pH response of coacervates and BSA-loaded microcapsules. It was concluded that genipin-crosslinked O-CMC–GA coacervates could be used to deliver hydrophobic compounds to the intestine.     

Role of Macrophages and RhoA Pathway in Atherosclerosis

The development, progression, or stabilization of the atherosclerotic plaque depends on the pro-inflammatory and anti-inflammatory macrophages. The influx of the macrophages and the regulation of macrophage phenotype, inflammatory or anti-inflammatory, are controlled by the small GTPase RhoA and its downstream effectors. Therefore, macrophages and the components of the RhoA pathway are attractive targets for anti-atherosclerotic therapies, which would inhibit macrophage influx and inflammatory phenotype, maintain an anti-inflammatory environment, and promote tissue remodeling and repair. Here, we discuss the recent findings on the role of macrophages and RhoA pathway in the atherosclerotic plaque formation and resolution and the novel therapeutic approaches.     

Study of hemocompatibility and endothelial cell interaction of tecoflex-based electrospun vascular grafts

Abstract

Ensuring long-term functioning and efficient endothelialization of small diameter vascular grafts (VG) is an urgent task of tissue engineering. A solution may be to use electrospun VGs prepared from blends polyurethane with gelatin and/or bivalirudin. Here, properties of 3D matrices were explored by SEM, contact angle measurements and IR spectroscopy, and their interaction with blood and endothelial cells was studied. Introduction of gelatin into matrices enhanced adhesion and proliferation of endotheliocytes and enabled adhesion of platelets, whereas bivalirudin inhibited platelet adhesion while having no negative effect on the adhesion and proliferation of endothelial cells.     

Immunomodulatory Properties and Osteogenic Activity of Polyetheretherketone Coated with Titanate Nanonetwork Structures

Polyetheretherketone (PEEK) is a potential substitute for conventional metallic biomedical implants owing to its superior mechanical and chemical properties, as well as biocompatibility. However, its inherent bio-inertness and poor osseointegration limit its use in clinical applications. Herein, thin titanium films were deposited on the PEEK substrate by plasma sputtering, and porous nanonetwork structures were incorporated on the PEEK surface by alkali treatment (PEEK-TNS). Changes in the physical and chemical characteristics of the PEEK surface were analyzed to establish the interactions with cell behaviors. The osteoimmunomodulatory properties were evaluated using macrophage cells and osteoblast lineage cells. The functionalized nanostructured surface of PEEK-TNS effectively promoted initial cell adhesion and proliferation, suppressed inflammatory responses, and induced macrophages to anti-inflammatory M2 polarization. Compared with PEEK, PEEK-TNS provided a more beneficial osteoimmune environment, including increased levels of osteogenic, angiogenic, and fibrogenic gene expression, and balanced osteoclast activities. Furthermore, the crosstalk between macrophages and osteoblast cells showed that PEEK-TNS could provide favorable osteoimmunodulatory environment for bone regeneration. PEEK-TNS exhibited high osteogenic activity, as indicated by alkaline phosphatase activity, osteogenic factor production, and the osteogenesis/osteoclastogenesis-related gene expression of osteoblasts. The study establishes that the fabrication of titanate nanonetwork structures on PEEK surfaces could extract an adequate immune response and favorable osteogenesis for functional bone regeneration. Furthermore, it indicates the potential of PEEK-TNS in implant applications.     

Understanding the in situ crosslinked gelatin hydrogel

The present study focuses on an in‐depth investigation at the molecular level of in situ crosslinked gelatin hydrogel by oxidized carboxymethylcellulose (OCMC), intended for biomedical purposes. The crosslinking parameters, namely the crosslinking temperature, crosslinking time and the amount of crosslinker, were varied. The surface and bulk properties were investigated to construct a representative model postulating the interaction of gelatin and OCMC encompassing their physical and covalent linking which determines the properties of the hydrogel. It was inferred that covalent crosslinking facilitates the interfacial interaction between gelatin and OCMC which beyond 30% OCMC composition is impeded due to depletion: microdomain formation leading to phase separation. This study might enable the fabrication of tunable hydrogel with crosslinking parameters in accordance with its purpose. The preliminary biocompatibility studies of the hydrogel conclude that the crosslinked hydrogel generates favourable conditions for cells to adhere and proliferate showing potential for biomedical purposes. © 2015 Society of Chemical Industry     

Dual RNA Sequencing of Mycobacterium tuberculosis-Infected Human Splenic Macrophages Reveals a Strain-Dependent Host–Pathogen Response to Infection

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host–pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host–pathogen interaction.     

Thermal crosslinking of collagen immobilized on poly(acrylic acid) grafted poly(ethylene terephthalate) films

Graft polymerization of acrylic acid onto plasma‐treated poly(ethylene terephthalate) (PET) films was used to prepare surfaces suitable for collagen immobilization by dip‐coating. Such surfaces could be used as matrices for smooth muscle cell cultures in tissue engineering. Contact angle measurements showed that plasma‐treated and grafted PET films undergo considerable surface reorganization during storage under ambient conditions. However, after collagen immobilization the contact angle remained relatively stable. The amount of collagen initially attached to the film surface increased with increasing poly(acrylic acid) graft density, but subsequent washing in water led to significant collagen loss. This loss could nevertheless be substantially reduced by thermal crosslinking of the collagen in the range 110–130 °C. Atomic force microscopy (AFM) observations suggested that the washed crosslinked collagen has a very similar structure to that of the un‐crosslinked collagen. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 1874–1880, 2002     

Biodegradation of chemically modified gelatin films in lake and river waters

Gelatin was chemically modified by crosslinking samples with one of a number of bifunctional reagents as was done earlier in a processing technique used to improve mechanical properties through chain orientation. The effects of this crosslinking on the biodegradability of the resulting films were evaluated in the laboratory by exposing them to lake and river waters for 10 days with or without inoculation with periphyton organisms. Biodegradabilities were assessed by weight losses of the films and by measurements of dehydrogenase activity of biomasses taken from their surfaces. The extent of biodegradation depended on the type of crosslinking agent and the presence or absence of the periphyton. The gelatin films crosslinked with formaldehyde, glyoxal, or glutaraldehyde were the slowest to biodegrade; complete degradation required 8–10 days. In contrast, the most biodegradable was the gelatin crosslinked with hexamethylene diisocyanate, which required only 3–4 days. The uncrosslinked gelatin and the gelatin crosslinked with butadiene diepoxide and diepoxyoctane were intermediate, degrading in 5–7 days. The dehydrogenase activity paralleled the weight losses but rapidly decreased when the amount of gelatin remaining was small. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 29–37, 2000