Physico-chemical and mechanical properties of porous membranes of carboxymethylchitosan and chitosan-based hydrogel for application in tissue engineering

Este trabalho teve como principal objetivo produzir membranas porosas de carboximetilquitosana e hidrogéis de quitosana com propriedades físico-químicas e mecânicas adequadas para aplicações em Engenharia de Tecidos. Para isso, quitosanas com diferentes graus de acetilação (4,0%<GA<40%) e de elevada massa molar média viscosimétrica (Mv>750.000 g.mol-1) foram produzidas através da aplicação de processos consecutivos de desacetilação assistida por irradiação de ultrassom de alta intensidade (DAIUS) à beta-quitina extraída de gládios de lulas Doryteuthis spp. A carboximetilação de quitosana extensivamente desacetilada (Qs-3; GA=4%) foi realizada pela reação com ácido monocloroacético em meio isopropanol/solução aquosa de NaOH, gerando a amostra CMQs-0 (GS≈0,98; Mv≈190.000 g.mol-1). A irradiação de ultrassom de alta intensidade foi empregada para tratar solução aquosa de CMQs-0 durante 1 h e 3 h, resultando nas amostras CMQs-1 (Mv≈94.000 g.mol-1) e CMQs-3 (Mv≈43.000 g.mol-1), respectivamente. Para a produção de membranas reticuladas, genipina foi adicionada em diferentes concentrações (1,0x10-4 mol.L-1, 3,0x10-4 mol.L-1 ou 5,0x10-4 mol.L-1) às soluções aquosas das CMQs, que foram vertidas em placas de Petri e a reação de reticulação procedeu por 24 h. Em seguida, as membranas reticuladas (M-CMQs) foram liofilizadas, neutralizadas, lavadas e liofilizadas novamente, resultando em nove amostras, que foram caracterizadas quanto ao grau médio de reticulação (GR), grau médio de hidratação (GH), morfologia, propriedades mecânicas e quanto à susceptibilidade à degradação por lisozima. O grau médio de reticulação (GR) foi tanto maior quanto maior a concentração de genipina empregada na reação, variando de GR≈3,3% (M-CMQs-01) a GR≈17,8% (M-CMQs-35). As análises de MEV revelaram que as membranas reticuladas M-CMQs são estruturas porosas que apresentam maior densidade de poros aparentes quanto maiores os valores de Mve GR. Entretanto, as membranas preparadas a partir de CMQs de elevada massa molar (Mv>94.000 g.mol-1) e pouco reticuladas (GR<10%), apresentaram propriedades mecânicas superiores em termos de resistência máxima à tração (>170 kPa) e elongação máxima à ruptura (>40%). Por outro lado, as membranas mais susceptíveis à degradação enzimática foram aquelas preparadas a partir de CMQs de baixa massa molar (Mv≈43.000 g.mol-1) e que exibiram baixos graus de reticulação (GR<11%). Hidrogéis estáveis de quitosana sem o uso de qualquer agente de reticulação externo foram produzidos a partir da gelificação de soluções aquosas de quitosana com solução de NaOH ou vapor de NH3. Os hidrogéis produzidos a partir de soluções de quitosana de elevada massa molar média ponderal (Mw≈640.000 g.mol-1) e extensivamente desacetilada (DA≈2,8%) em concentrações poliméricas acima 2,0%, exibiram melhores propriedades mecânicas com o aumento da concentração polimérica, devido à formação de numerosos emaranhamentos físicos das cadeias poliméricas em solução. Os resultados mostram que as propriedades físico-químicas e mecânicas dos hidrogéis de quitosana podem ser controladas variando a concentração do polímero e o processo de gelificação. A avaliação biológica de tais hidrogéis para a regeneração de miocárdio infartado de ratos revelou que os hidrogéis de quitosana preparados a partir de soluções de polímero a 1,5% foram perfeitamente incorporados sobre a superfície do epicárdio do coração e apresentaram degradação parcial acompanhada por infiltração de células mononucleares.The aim of this study was to produce and characterize porous membranes of carboxymethylchitosan and chitosan-based hydrogel with physicochemical and mechanical properties appropriate for applications in tissue engineering. For this, chitosans with different degrees of acetylation (4,0%<GA<40%) and high viscosity average molecular weight (Mv>750.000 g.mol-1) were produced by application of consecutive processes of ultrasound-assisted deacetylation (USAD) of the beta-chitin extracted from squid pens (Doryteuthis spp.). The carboxymethylation of extensively deacetylated chitosan (Qs-3; DA=4%) was carried out by reaction with monochloroacetic acid in isopropanol/aqueous NaOH, producing CMQs-0 sample (GS≈0,98; Mv≈190.000 g.mol-1). The ultrasonic irradiation was employed to depolymerize the CMQs-0 samples by irradiation for 1 h and 3 h, resulting in CMQs-1 samples (Mv≈94.000 g.mol-1) and CMQs-3 (Mv≈43.000 g.mol-1), respectively. For the production of crosslinked membranes, genipin was added at different concentrations (1,0x10-4 mol.L-1, 3,0x10-4 mol.L-1 ou 5,0x10-4 mol.L-1) in the aqueous solutions of CMQs, which were poured into Petri dishes and the crosslinking reaction proceeded for 24 h. Then, the crosslinked membranes (M-CMQs) were lyophilized, neutralized, washed, and lyophilized again resulting in nine samples which were characterized by crosslinking degree (CrD), swelling ration (SR), morphology, mechanical properties and the susceptibility to enzymatic degradation by lysozyme. The crosslinking degree (CrD) increased with increasing concentration of genipin used in the reaction, varying from CrD≈3.3% (M-CMQs-01) to CrD≈17.8% (M-CMQs-35). The SEM analysis showed that the crosslinked membranes M-CMQs are porous structures that have a higher apparent pores density with increasing values of Mv and CrD. However, the membranes prepared from high molecular weight CMQs (Mv>94.000 g.mol-1) and low crosslinked (GR<10%) showed superior mechanical properties in terms of ultimate tensile strength (>170 kPa) and maximum elongation at break (>40%). However, the more susceptible membrane to enzymatic degradation was prepared from low molecular weight CMQs (Mv≈43.000 g.mol-1) and low cross-linking degrees (GR<11%). Stable chitosan hydrogels without any external crosslinking agent was successfully achieved by inducing the gelation of a viscous chitosan solution with aqueous NaOH or gaseous NH3. The hydrogels produced from high molecular weight (Mw≈640.000 g.mol-1) and extensively deacetylated chitosan (DA≈2,8%) at polymer concentrations above ≈2.0% exhibited improved mechanical properties due to the increase of the chain entanglements and intermolecular junctions. The results also show that the physicochemical and mechanical properties of chitosan hydrogels can be controlled by varying their polymer concentration and by controlling the gelation kinetics, i.e. by using different gelation routes. The biological evaluation of such hydrogels for regeneration of infarcted myocardium revealed that chitosan hydrogels prepared from 1.5% polymer solutions was perfectly incorporated onto the epicardial surface of the heart and presented partial degradation accompanied by mononuclear cell infiltration

Porous membranes of N,O-carboxymethylchitosan/chitosan for applying in the prevention of postsurgical pericardial adhesions

Este trabalho teve como objetivo produzir e caracterizar membranas de quitosana e de N,O-carboximetilquitosana reticuladas, que apresentassem propriedades físicas e químicas adequadas para desempenhar o papel de matriz para proliferação das células mesoteliais. As características estruturais e morfológicas das amostras purificadas de quitosana (amostra Q, adquirida da Yue Planting, China) e carboximetilquitosana na forma sódica (amostra NaCMQH, adquirida da Heppe Medical, Alemanha, e amostra NaCMQD, adquirida da Dayang Chemicals, China) foram investigadas através da espectroscopias de ressonância magnética nuclear e no infravermelho, condutimetria, solubilidade em função do pH e viscosimetria. As membranas de carboximetilquitosanas (amostras M-CMQHs e M-CMQDs) foram confeccionadas via liofilização, e glutaraldeído foi empregado como agente reticulante em diferentes concentrações para avaliar o seu efeito sobre o grau de reticulação e propriedades das membranas. As membranas foram caracterizadas quanto ao grau de reticulação, grau de hidratação, microscopia eletrônica de varredura (MEV), termogravimetria, teste mecânico de tração e quanto a susceptibilidade à degradação enzimática. A amostra Q apresentou grau médio de acetilação (GA) de 23,60%, sendo solúvel em pH ≤ 6,5. A amostra NaCMQH apresentou GA = 16,32% e grau médio de substituição (GS) de 1,68, sendo insolúvel no intervalo 2,5 ≤ pH ≤ 6,5, a amostra de NaCMQD apresentou GA = 3,31% e GS = 1,43, sendo insolúvel no intervalo 3,0 ≤ pH ≤ 7,0. A reticulação das membranas de carboximetilquitosana (amostras M-CMQHs e M-CMQDs) foi realizada com a finalidade de reduzir sua solubilidade e melhorar as propriedades mecânicas. O grau médio de reticulação (GR) foi tanto maior quanto maior a concentração de glutaraldeído empregada na reação, variando de GR = 10,39 ± 0,37% ([glutaraldeído] = 2,5x10-3 mol L-1) a GR = 62,38 ± 1,71% ([glutaraldeído] = 5,0x10-3 mol L-1). As características morfológicas das amostras M-Q, M-CMQHs e M-CMQDs foram observadas pelo emprego de MEV, sendo observada a formação de estruturas porosas, com maior quantidade de poros aparentes quanto maior o GM de 175 poros mm-2 a 291 poros mm-2 com o aumento do grau de reticulação de 12,30% (amostra M-CMQH-2,5) para 35,82%, (amostra M-CMQH-50). A amostra M-Q apresentou baixa taxa de hidratação (321,16 ± 18,68%) e alto percentual de massa recuperada (90,62 ± 2,13%) após imersão por 24 horas em solução PBS, quando comparada às amostras M-CMQHs e M-CMQDs. As amostras M-CMQHs e M-CMQDs apresentaram aumento da resistência máxima à tração com o aumento de GR, aumentando de 0,21 ± 0,16 MPa (amostra M-CMQD-2,5; GR ≈ 10,39%) para 0,82 ± 0,33 MPa (amostra M-CMQH-50; GR ≈ 62,38%). Entretanto, amostras com menor GR apresentaram aumento dos valores de percentual de elongação, sendo que a amostra M-CMQH-2,5 (GR ≈ 12,30%) apresentou elongação máxima de 73,08 ± 2,20%. A amostra M-Q foi pouco susceptível à hidrólise enzimática ([GlcN] = 47x10-4 ± 1x10-4 mol L-1) devido à baixa solubilidade da quitosana em pH > 6,5. Já com relação ao efeito do GR, houve redução da taxa de hidrólise enzimática de [GlcN] = 449x10-4 ± 15x10-4 mol L-1 para [GlcN] = 105x10-4 ± 11x10-4 mol L-1, quando o GR aumentou de 12,30% (amostra M-CMQH-2,5) para 28,64% (amostra M-CMQH-25). As amostras M-CMQH-5, M-CMQH-10, M-CMQD-10 e M-CMQD-25 apresentam as propriedades mais adequadas para o emprego como membranas para a prevenção das adesões pericárdicas, pois apresentam superfícies altamente porosas, com baixas taxa de hidratação e de solubilidade, resistência máxima à tração superior a 0,67 MPa, percentual de elongação superior à 30%, e degradação enzimática inferior a [GlcN] = 400x10-4 mol L-1 após 15 dias de incubação.The aim of this study was to produce and characterize membranes of chitosan and cross-linked N,O-carboxymethylchitosan, displaying appropriate physical and chemical properties to act as matrices for the proliferation of mesothelial cells. The structural and morphological characteristics of the purified samples of chitosan (sample Q, acquired from Yue Planting, China) and sodium carboxymethylchitosan (sample NaCMQH, acquired from Heppe Medical, Germany, and sample NaCMQD, acquired from Dayang Chemicals, China) were determined by nuclear magnetic resonance spectroscopy (NMR1H), infrared spectroscopy, conductometry, viscometry and pH-solubility tests. The carboxymethylchitosan membranes (M-CMQHs and M-CMQDs) were made up by means of lyophilization, with glutaraldehyde being used as a cross-linking agent at different concentrations to evaluate its effect on the cross-linking degree and on the membranes properties. The membranes were characterized in terms of cross-linking degree and hydration rate, by scanning electronic microscopy (SEM), thermogravimetry, ultimate tensile strength and the susceptibility to enzymatic degradation. The sample Q showed average degree of acetylation (DA) of 23.60%, being soluble at pH ≤ 6.5. The sample NaCMQH presented DA=16.32% and average degree of substitution (DS) of 1.68, being insoluble in the region of 2.5 ≤ pH ≤ 6.5. The sample NaCMQD presented DA=3.31% and DS=1.43, being insoluble in the region of 3.0 ≤ pH ≤ 7.0. The cross-linking of carboxymethylchitosan membranes (M-CMQHs and M-CMQDs) was carried out to reduce its solubility and to improve its the physical properties. The higher the glutaraldehyde concentration employed in the reaction, the higher average cross-linking degree (CD), which ranged from 10.39 ± 0.37% ([glutaraldehyde] = 2,5x10-3 mol L-1) to 62.38 ± 1.71% ([glutaraldehyde] = 2,5x10-3 mol L-1). The morphological characteristics of the samples M-Q, M-CMQHs M-CMQDs were observed through SEM, evidencing the formation of porous structures with a larger quantity of apparent pores as DC increased, ranging from 175 pores mm-2 to 291 pores mm-2 when DC increased from 12.30% (sample CMQH-M-2.5) to 35.82% (sample M-CMQH-50). The sample M-Q showed low hydration rate (321.16 ± 18.68%) and high percentage of recovered mass (90.62 ± 2.13%) after immersion for 24 hours, when compared to samples M-CMQHs and M-CMQDs. Increasing the DC of the samples M-CMQHs and M-CMQDs resulted in improved mechanical properties as the ultimate tensile strength increased from 0.21 ± 0.16 MPa (M-CMQD-2.5, DC ≈ 10.39%) to 0.82 ± 0.33 MPa (M-CMQH-50, DC ≈ 62.38%). However, those samples with lower DC values presented an increase in strain at fracture, as the CMQH-M-2.5 sample (DC ≈ 12.30%), which registered a strain at fracture of 73.08 ± 2.20%. The sample M-Q showed a low rate of enzymatic hydrolysis ([GlcN] = 47x10-4 ± 1x10-4 mol L-1) as a consequence of the low solubility of chitosan at pH > 6.5. Concerning the effects of cross-linked degree, there was a reduction in the enzymatic hydrolysis rate from [GlcN] = 449x10-4 ± 15x10-4 mol L-1 to [GlcN] = 105x10-4 ± 11x10-4 mol L-1, when DC increased from 12.30% (M-CMQH-2.5) to 28.64% (M-CMQH-25). The samples M-CMQH-5, M-CMQH-10, M-CMQD-10 and M-CMQD-25 exhibit appropriate properties to act in the prevention of pericardial adhesions, owing to its highly porous surfaces, low hydration rate and insolubility, ultimate tensile strength exceeding 0.67 MPa, strain at fracture higher than 30% and enzymatic degradation rate lower than [GlcN] = 400x10-4 mol L-1 after 15 days of incubation

Tuning the Properties of High Molecular Weight Chitosans to Develop Full Water Solubility Within a Wide pH Range

The preparation of chitosans soluble in physiological conditions has been sought for years, but so far solubility in non-acidic aqueous media has only been achieved at the expense of lowering chitosan molecular weight. In this work, we applied the multistep ultrasound-assisted deacetylation process (USAD process) to β-chitin and obtained extensively deacetylated chitosans with high molecular weights (Mw ≥ 1,000,000 g mol-1). The homogeneous N-acetylation of a chitosan sample resulting from three consecutive USAD procedures allowed us to produce chitosans with a high weight average degree of polymerization (DPw ≈ 6,000) and tunable degrees of acetylation (DA from 5 to 80%). N-acetylation was carried out under mild conditions to minimize depolymerization, while preserving a predominantly random distribution of 2-amino-2-deoxy-D-glucopyanose (GlcN) and 2-acetamido-2-deoxy-D-glucopyanose (GlcNAc) units. This close to random distribution, inferred with deconvolution of nuclear magnetic resonance (1H NMR) spectra, is considered as responsible for the solubility within a wide pH range. Two of the highly N-acetylated chitosans (DA ≈ 60 % and ≈ 70 %) exhibited full water solubility even at neutral pH, which can expand the biomedical applications of chitosans. </p

Extensively deacetylated high molecular weight chitosan from the multistep ultrasound-assisted deacetylation of beta-chitin

International audienceHigh intensity ultrasound irradiation was used to convert beta-chitin (BCHt) into chitosan (CHs). Typically, beta-chitin was suspended in 40% (w/w) aqueous sodium hydroxide at a ratio 1/10 (g mL(-1)) and then submitted to ultrasound-assisted deacetylation (USAD) during 50 min at 60 degrees C and a fixed irradiation surface intensity (52.6 W cm(-2)). Hydrogen nuclear magnetic resonance spectroscopy and capillary viscometry were used to determine the average degree of acetylation ((DA) over bar) and viscosity average degree of polymerization ((DPv) over bar), respectively, of the parent beta-chitin ((DA) over bar = 80.7%; (DPv) over bar = 6865) and USAD chitosans. A first USAD reaction resulted in chitosan CHs1 ((DA) over bar = 36.7%; (DPv) over bar = 5838). Chitosans CHs2 ((DA) over bar = 15.0%; (DPv) over bar = 5128) and CHs3 ((DA) over bar = 4.3%; (DPv) over bar = 4889) resulted after repeating the USAD procedure to CHs1 consecutively once and twice, respectively. Size-exclusion chromatography analyzes allowed the determination of the weight average molecular weight ((M-w) over bar) and dispersity (D) of CHs1 ((M-w) over bar = 1,260,000 g mol(-1); D =1.4), CHs2 ((M-w) over bar = 1,137,000 g mol(-1); D =1.4) and CHs3 ((M-w) over bar = 912,000 g mol(-1); D = 1.3). Such results revealed that, thanks to the action of high intensity ultrasound irradiation, the USAD process allowed the preparation of unusually high molecular weight, randomly deacetylated chitosan, an important breakthrough to the development of new high grade chitosan-based materials displaying superior mechanical properties

Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation

International audienceThe formation of chitosan hydrogels without any external cross-linking agent was successfully achieved by inducing the gelation of a viscous chitosan solution with aqueous NaOH or gaseous NH3. The hydrogels produced from high molecular weight (M-w approximate to 640 000 g mol(-1)) and extensively deacetylated chitosan (DA approximate to 2.8%) at polymer concentrations above similar to 2.0% exhibited improved mechanical properties due to the increase of the chain entanglements and intermolecular junctions. The results also show that the physicochemical and mechanical properties of chitosan hydrogels can be controlled by varying their polymer concentration and by controlling the gelation conditions, that is, by using different gelation routes. The biological evaluation of such hydrogels for regeneration of infarcted myocardium revealed that chitosan hydrogels prepared from 1.5% polymer solutions were perfectly incorporated onto the epicardial surface of the heart and presented partial degradation accompanied by mononuclear cell infiltration

Investigation of the Internal Chemical Composition of Chitosan-Based LbL Films by Depth-Profiling X‑ray Photoelectron Spectroscopy (XPS) Analysis

Chitosan-based thin films were assembled using the layer-by-layer technique, and the axial composition was accessed using X-ray photoelectron spectroscopy with depth profiling. Chitosan (CHI) samples possessing different degrees of acetylation (DA̅) and molecular weight (Mv̅) produced via the ultrasound-assisted deacetylation reaction were used in this study along with two different polyanions, namely, sodium poly­styrene­sulfonate (PSS) and carboxy­methyl­cellulose (CMC). When chitosan, a positively charged polymer in aqueous acid medium, was combined with a strong polyanion (PSS), the total positive charge of chitosan, directly related to its DA̅, was the key factor affecting the film formation. However, for CMC/CHI films, the pH of the medium and Mv̅ of chitosan strongly affected the film structure and composition. Consequently, the structure and the axial composition of chitosan-based films can be finely adjusted by choosing the polyanion and defining the chitosan to be used according to its DA and Mv̅ for the desired application, as demonstrated by the antibacterial tests