Preparation of Chitosan Cryostructurates with Controlled Porous Morphology and Their Use as 3D-Scaffolds for the Cultivation of Animal Cells

The influence of the conditions of the formation of genipin cross-linked chitosan cryostructurates on the porous morphology and physicochemical properties of these cryostructurates and on the possibility of their use as biopolymer 3D scaffolds for tissue engineering was studied. The chitosan cryostructurates were obtained by freeze-drying a chitosan acetate solution, treating the resulting sponge with an alcohol solution of ammonia to transform the polyaminosaccharide from a salt into a chitosan-base, and then cross-linking the polymer with genipin (the molar ratios of genipin to the number of chitosan amino groups were 0.05, 0.033, and 0.02, respectively). The pore sizes, water-holding capacity, and in vitro biodegradation rate of the cryostructurates were shown to depend on the aforementioned ratio. The properties of the prepared chitosan cryostructurates, the hydrogels formed by chitosan cross-linking with genipin at positive temperatures, and the films cast from genipin-containing chitosan solutions after solvent evaporation were studied and compared. The biocompatibility of the obtained macroporous sponge materials was demonstrated using L929 mouse fibroblasts. Confocal laser microscopy showed that the cells in all of the 3D scaffolds obtained were evenly distributed; they grew and proliferated when cultured in vitro for seven days.     

Hydrogels and electrospun nanofibrous scaffolds of N-methylene phosphonic chitosan as bioinspired osteoconductive materials for bone grafting

In this work, N-methylene phosphonic chitosan (NMPC) based hydrogels and electrospun nanofibrous scaffolds are reported with objective to obtain osteoconductive and osteoinductive matrixes for bone grafting applications. NMPC, a phosphorylated derivative of chitosan, is known to mimic the function of non collagenous phosphoproteins in providing nucleation sites for biomineralization. NMPC hydrogels were prepared by crosslinking between NMPC and genipin. A detailed investigation of physicochemical properties of NMPC solutions is also carried out in order to obtain beads free nanofibers. Both NMPC gels and nanofibers were further evaluated for their biomineralization potential and biocompatibility with human osteoblast like cells. Results indicated that hydrogels and nanofibrous scaffolds NMPC are biocompatible and significantly osteoinductive compared to tissue culture plate controls. However, cells seeded on nanofibrous scaffolds exhibited greater proliferation measured by MTT assay, and higher expression of early markers for osteogenic differentiation proving the superior applicability of nanofibrous scaffolds for bone grafting applications.     

Synthesis and characterization of pH-sensitive hydrogel composed of carboxymethyl chitosan for colon targeted delivery of ornidazole

In the present study, carboxymethyl chitosan was prepared from chitosan, crosslinked with glutaraldehyde and evaluated in vitro as a potential carrier for colon targeted drug delivery of ornidazole. Ornidazole was incorporated at the time of crosslinking of carboxymethyl chitosan. The chitosan was evaluated for its degree of deacetylation (DD) and average molecular weight; which were found to be 84.6% and 3.5×10(4) Da, respectively. The degree of substitution on prepared carboxymethyl chitosan was found to be 0.68. All hydrogel formulations showed more than 85% and 74% yield and drug loading, respectively. The swelling behaviour of prepared hydrogels checked in different pH values, 1.2, 6.8 and 7.4, indicated pH responsive swelling characteristic with very less swelling at pH 1.2 and quick swelling at pH 6.8 followed by linear swelling at pH 7.4 with slight increase. In vitro release profile was carried out at the same conditions as in swelling and drug release was found to be dependant on swelling of hydrogels and showed biphasic release pattern with non-fickian diffusion kinetics at higher pH. The carboxymethylation of chitosan, entrapment of drug and its interaction in prepared hydrogels were checked by FTIR, (1)H NMR, DSC and p-XRD studies, which confirmed formation of carboxymethyl chitosan from chitosan and absence of any significant chemical change in ornidazole after being entrapped in crosslinked hydrogel formulations. The surface morphology of formulation S6 checked before and after dissolution, revealed open channel like pores formation after dissolution.     

Rheological study of genipin cross-linked chitosan hydrogels

This paper reports the rheological behavior of chitosan solutions that have been cross-linked with different amounts of genipin, at body temperature and physiological pH. The effect of the cross-linker loading on the rheological properties of hydrogels has been evaluated. The oscillatory time sweep method was used to monitor the dynamic viscoelastic parameters during in situ (i.e., in the rheometer) gelation experiments, enabling the determination of the gelation time. The stress and frequency sweeps were employed to measure G' of the cured hydrogels. It was found that the solutions of chitosan cross-linked with genipin, under physiological conditions, could form relatively strong elastic gels when compared to those of pure chitosan. Moreover, the gelation time obtained from the crossover of G' and G' was in excellent agreement with the value obtained from the Winter-Chambon criterion. A significant reduction on this parameter was achieved even at low genipin concentrations. This behavior suggests that these formulations are able to be produced in situ and thus constitute promising matrices for cells and bioactive molecule encapsulations.     

Inter-connecting pores of chitosan scaffold with basic fibroblast growth factor modulate biological activity on human mesenchymal stem cells

In this study, we developed bio-active molecules immobilized chitosan scaffolds with controlled pore architectures for enhanced viability of human mesenchymal stem cells (hMSCs). The decreasing in molecular weight of chitosan by ultrasonication of chitosan solution was effective in the formation of porous chitosan scaffolds, resulting in an increase of inter-connecting micropores (∼10 μm) between macropores. Using a layer-by-layer method, we then prepared heparin-coated chitosan scaffolds as depots for basic fibroblast growth factors (bFGF). Enzyme-linked immunosorbent assays confirmed that heparin-coated chitosan scaffolds could bind higher amount of bFGF (24.21 ng/mg) compared to 2.53 ng/mg of non-coated scaffold. Moreover, we were able to manipulate the release profiles of bFGF from the scaffolds for 7 days. In vitro studies showed that chitosan scaffolds induced the improved viability and proliferation of hMSCs through their synergetic effects of the inter-connecting micropores and the sustained released of bFGF. Our results suggest that bFGF immobilized chitosan scaffolds with controlled inter-connecting pores, in combination with other heparin-binding growth factors, have potential implants for controlling biological functions in regenerative medicine.     

The effects of different crossing-linking conditions of genipin on type I collagen scaffolds: an in vitro evaluation

The purpose of this paper is to analyze the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin under different conditions. The porous genipin cross-linked scaffolds are obtained through a two step freeze-drying process. To find out the optimal cross-link condition, we used different genipin concentrations and various cross-linked temperatures to prepare the scaffolds in this study. The morphologies of the scaffolds were characterized by scanning electron microscope, and the mechanical properties of the scaffolds were evaluated under dynamic compression. Additionally, the cross-linking degree was assessed by ninhydrin assay. To investigate the swelling ratio and the in vitro degradation of the collagen scaffold, the tests were also carried out by immersion of the scaffolds in a PBS solution or digestion in a type I collagenase respectively. The morphologies of the non-cross-linked scaffolds presented a lattice-like structure while the cross-linked ones displayed a sheet-like framework. The morphology of the genipin cross-linked scaffolds could be significantly changed by either increasing genipin concentration or the temperature. The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked).The ninhydrin assay demonstrated that the higher temperature and genipin concentration could obviously increase the cross-linking efficiency. The in vitro degradation studies indicated that genipin cross-linking can effectively elevate the biostability of the scaffolds. The biocompatibility and cytotoxicity of the scaffolds was evaluated by culturing rat chondrocytes on the scaffold in vitro and by MTT. The results of MTT and the fact that the chondrocytes adhered well to the scaffolds demonstrated that genipin cross-linked scaffolds possessed an excellent biocompatibility and low cytotoxicity. Based on these results, 0.3 % genipin concentrations and 37 °C cross-linked temperatures are recommended.     

Crosslinking of Chitosan with Dialdehyde Derivatives of Nucleosides and Nucleotides. Mechanism and Comparison with Glutaraldehyde

In medical and pharmaceutical applications, chitosan is used as a component of hydrogels–macromolecular networks swollen in water. Chemical hydrogels are formed by covalent links between the crosslinking reagents and amino functionalities of chitosan. To date, the most commonly used chitosan crosslinkers are dialdehydes, such as glutaraldehyde (GA). We have developed novel GA like crosslinkers with additional functional groups–dialdehyde derivatives of uridine (oUrd) and nucleotides (oUMP and oAMP)–leading to chitosan-based biomaterials with new properties. The process of chitosan crosslinking was investigated in details and compared to crosslinking with GA. The rates of crosslinking with oUMP, oAMP, and GA were essentially the same, though much higher than in the case of oUrd. The remarkable difference in the crosslinking properties of nucleoside and nucleotide dialdehydes can be clearly attributed to the presence of the phosphate group in nucleotides that participates in the gelation process through ionic interactions with the amino groups of chitosan. Using NMR spectroscopy, we have not observed the formation of aldimine bonds. It can be concluded that the real number of crosslinks needed to cause gelation of chitosan chains may be less than 1%.     

Photopolymerized water-soluble chitosan-based hydrogel as potential use in tissue engineering

Novel biodegradable hydrogels by photocrosslinking macromers based on chitosan derivative are reported. Photocrosslinkable macromers, a water-soluble (methacryloyloxy) ethyl carboxyethyl chitosan were prepared by Michael-addition reaction between chitosan and ethylene glycol acrylate methacrylate. The macromers were characterized by Fourier transform infrared spectroscopy, (1)H NMR and (13)C NMR. Hydrogels were fabricated by exposing aqueous solutions of macromers with 0.1% (w/v) photoinitiator to UV light irradiation, and their swelling kinetics as well as degradation behaviors was evaluated. The results demonstrated that the degradation rates were affected strongly by crosslinking density. The hydrogel was compatible to Vero cells, not exhibiting significant cytotoxicity. Cell culture assay also demonstrated that the hydrogels were good in promoting the cell attachment and proliferation, showing their potential as tissue engineering scaffolds.     

Molecularly imprinted chitosan-genipin hydrogels with recognition capacity toward o-xylene

A molecularly imprinted material was developed from hydrogels of chitosan (CS) cross-linked with genipin (GNP) using o-xylene as the template molecule. Gelling time, mechanical, and diffusion properties of CS-GNP hydrogels were initially investigated to establish optimal conditions to prepare molecularly imprinted hydrogels (MIHs). The elastic modulus was found to be directly proportional to the degree of cross-linking (R = moles of genipin/moles of glucosamine) while the diffusion of water, as monitored by magnetic resonance imaging, decreased with R. CS-GNP hydrogels of varying R were imprinted with o-xylene (MIH(o-xylene)). The adsorption capacity of o-xylene by MIH(o-xylene) was greater than the corresponding control hydrogels, particularly at R = 0.25. Freundlich isotherms yielded a better fitness than Langmuir ones and afforded n and Q(max)values of 2.55 and 103.3 mg/g, respectively. The imprinted hydrogel showed the highest adsorption capacity for o-xylene; however, the material was not highly selective as it also exhibited the capacity to adsorb m- and p-xylene isomers. In turn, the MIH(o-xylene) showed a low adsorption when 2-fluorotoluene was used in rebinding experiments, suggesting that molecular recognition by the binding sites is influenced by the electronic and steric properties of the analyte molecule, thus effectively confirming the imprinting effect within the MIH(o-xylene) network. This work opens the possibility to future development of materials with the capacity to adsorb o-xylene analogue molecules such as contaminants bearing chlorinated aromatic structures.     

壳聚糖球固定化酵母细胞的研究

以戊二醛为交联剂,将壳聚糖球交联引入醛基,然后将交联的壳聚糖球浸泡在酵母细胞悬浮液中,制备了固定化酵母细胞壳聚糖球。以苯乙酮酸为底物,催化合成了D-扁桃酸。最优固定化条件是戊二醛的质量分数w(GA)=1%,酵母细胞与交联壳聚糖球的质量比m(Y):m(CB)0=0.5,交联时间为6h,固定化时间为18h,底物浓度为10mmol/L,在此条件下反应最大转化率和产物光学纯度分别高达67.86%和98.05?。固定化酵母壳聚糖球具有良好的重复使用性和贮存稳定性。     

Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate

Work to date shows that structurally distinct chitosans have reacted inefficiently and unpredictably with fluorescein isothiocyanate (FITC) in an acid–methanol solvent that maintains both chitosan and fluorophore solubility. Since isothiocyanate preferentially reacts with neutral amine groups, and chitosan solubility typically depends upon a minimal degree of protonation, we tested the hypothesis that precise derivatization of chitosan with rhodamine isothiocyanate (RITC) can be achieved by controlling the reaction time and the degree of protonation. Addition of 50% v/v methanol reduced the chitosan degree of protonation in acetic acid but not HCl solutions. At various degrees of protonation, chitosan reacted inefficiently with RITC as previously observed with FITC. Nevertheless, precise derivatization was achieved by allowing the reaction to proceed overnight at a given degree of protonation (p < 0.0001, n = 18) and fixed initial fluorophore concentration. A reproducible 2% to 4% fraction of neutral amines reacted with RITC in proportion to the initial fluorophore concentration (p < 0.005). Using our optimized protocol, chitosans with different degree of deacetylation and molecular weight were derivatized to either 1% or 0.5% mol/mol RITC/chitosan-monomer with a precision of 0.1% mol/mol. The average molecular weight of fluorescent RITC-chitosan was similar to the unlabeled parent chitosan. Precise molar derivatization of structurally distinct chitosans with RITC can be achieved by controlling chitosan degree of protonation, initial fluorophore concentration, and reaction time.     

In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.     

Swelling and mechanical properties of biopolymer hydrogels containing chitosan and bovine serum albumin

Experimental and theoretical investigations of the swelling and mechanical properties of hydrogels formed from chitosan, bovine serum albumin (BSA), and chitosan/BSA mixtures cross-linked with genipin were performed. The properties of cross-linked chitosan hydrogels were explained in terms of its polyelectrolyte behavior, which led to a gradual increase in swelling ratio below the pK value, but whereby its swelling ability was eliminated by the presence of salt that screened the charges. Comparison of theoretical and experimental calculations of the swelling ratio, however, indicated that complications arising from wastage of cross-links, and formation of polymerized genipin cross-links must be considered before quantitative prediction can be achieved. Cross-linked BSA hydrogels swelled even in the presence of salt, and a marked increase in swelling was observed below pH = 3 that was explained as the result of an acid induced denaturation of the protein that led to unfolding of the molecule. Swollen BSA hydrogels were mechanically weak, however. Composite gels made from a cross-linked mixture of chitosan and BSA exhibited the swelling behavior of BSA combined with the mechanical properties of chitosan and were therefore considered most suitable for use in a gastric environment.     

Comparative analysis of gelatin scaffolds crosslinked by genipin and silane coupling agent

Scaffolds based on gelatin (G) are considered promising for tissue engineering, able to mimic the natural extracellular matrix. G drawback is its poor structural consistency in wet conditions. Therefore, crosslinking is necessary to fabricate stable G scaffolds. In this work, a comparative study between the performance of two different crosslinkers, genipin (GP) and γ-glycidoxypropyltrimethoxysilane (GPTMS), is presented. Flat membranes by solvent casting and porous crosslinked scaffolds by freeze-drying were prepared. Infrared spectroscopy and thermal analysis were applied to confirm G chain crosslinking. Moreover, GP and GPTMS increased the stability of G in aqueous media and improved the mechanical properties. Crosslinking reduced the wettability, especially in the case of G_GPTMS samples, due to the introduction of hydrophobic siloxane chains. Both G_GP and G_GPTMS scaffolds supported MG-63 osteoblast-like cell adhesion and proliferation.     

Growth and osteogenic differentiation of adipose-derived and bone marrow-derived stem cells on chitosan and chitooligosaccharide films

Very low molecular weight chitooligosaccharide (COS, 1.4 kDa) and high molecular weight chitosan (1000 kDa) were comparatively studied in terms of physical and biological characteristics. Thin films of COS, chitosan and gelatin were prepared and crosslinked by dehydrothermal treatment at 140 °C for 24 h. COS film presented more hydrophilic property than chitosan film. Behaviors of rat adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (MSCs) were investigated on COS and chitosan films, comparing to those on gelatin film. The results on cell spreading suggested that both ASCs and MSCs preferred to attach on COS film than chitosan film with 6–7 times larger cell areas. Numbers of both stem cells proliferated on COS film were approximately 3-fold higher than those on chitosan film. In addition, COS film enhanced osteogenic differentiating potential of MSCs, as observed from the alkaline phosphatase activity and calcium deposition. Therefore, in this work, COS was shown to be a more favorable material for the growth and osteogenic differentiation of both ASCs and MSCs, compared to high molecular weight chitosan.     

Preparation and characterization of chitosan-carbon nanotube scaffolds for bone tissue engineering

In recent years, significant development has been given to chitosan for orthopedic application. In this study, we have prepared scaffolds with the use of low and high molecular weight chitosan with 0.0025%, 0.005% and 0.01% weight of f-multiwalled carbon nanotube (f-MWCNT) by freezing and lyophilization method and physiochemically characterized as bone graft substitutes. Fourier Transform Infrared Spectroscopy, X-Ray Diffraction Analysis, Thermal Gravimetric Analysis, Scanning Electron Microscopy and Optical Microscopy results indicated that the f-MWCNT was uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and f-MWCNT. The water uptake ability and porosity of scaffolds increased with an increase the amount of f-MWCNT. The cell proliferation, protein content, alkaline phosphatase and mineralization of the composite scaffolds were higher than chitosan scaffold due to the addition of f-MWCNT. Herewith, we are suggesting that chitosan/f-MWCNT scaffolds are promising biomaterials for bone tissue engineering.     

Improvement of biomaterials used in tissue engineering by an ageing treatment

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60 % (scaffold without ageing treatment) to 80 %. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.     

Controlling chitosan’s molecular weight via multistage deacetylation

Chitosan samples with different molecular weights (Mw) and degree of deacetylation (DD) were prepared by controlling operating conditions throughout the multistage alkali treatment. The temperature of the reaction, time duration and number of reaction steps were considered effective parameters. A database was developed for chitosan preparation in order to achieve high degrees of deacetylation and control the molecular weight of chitosan without changing other molecular structures. The number of treatments and the duration of each step of deacetylation significantly affected molecular weight so that two samples were obtained with a DD of 99% and two different molecular weights ranging from 4.66×10⁵ to 2.93×10⁵ Based on these results, the highest molecular weight obtained using the multistage treatment without decreasing DD was 5.32×10⁵, with a DD of 96.67%. Also, the morphological studies indicate that the molecular weight of chitosan has a significant effect on the pore size of the prepared scaffolds. However, this effect is critical. In other words, the pore size will increase by increasing molecular weight of chitosan from low upto medium molecular weight and when it reached to high molecular weight the pore size is decreased.     

Effect of crosslinking in chitosan/aloe vera-based membranes for biomedical applications

The positive interaction between polysaccharides with active phytochemicals found in medicinal plants may represent a strategy to create active wound dressing materials useful for skin repair. In the present work, blended membranes composed of chitosan (Cht) and aloe vera gel were prepared through the solvent casting, and were crosslinked with genipin to improve their properties. Topography, swelling, wettability, mechanical properties and in vitro cellular response of the membranes were investigated. With the incorporation of aloe vera gel into chitosan solution, the developed chitosan/aloe-based membranes displayed increased roughness and wettability; while the genipin crosslinking promoted the formation of stiffer membranes in comparison to those of the non-modified membranes. Moreover, in vitro cell culture studies evidenced that the L929 cells have high cell viability, confirmed by MTS test and calcein-AM staining. The findings suggested that both blend compositions and crosslinking affected the physico-chemical properties and cellular behavior of the developed membranes.     

MMP-Sensitive PEG Diacrylate Hydrogels with Spatial Variations in Matrix Properties Stimulate Directional Vascular Sprout Formation

The spatial presentation of immobilized extracellular matrix (ECM) cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP)-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP). Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10–12 hours in the more crosslinked regions to 4–6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.