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Preparation of acrylic grafted chitin for wound dressing application
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Biomaterials 25 (2004) 1453–1460
Preparation of acrylic grafted chitin for wound dressing application Siriporn Tanodekaewa,*, Malinee Prasitsilpa, Somporn Swasdisonb, Boonlom Thavornyutikarna, Thanawit Pothsreea, Rujiporn Pateepasenc a
National Metal and Materials Technology Center, 114 Thailand Science Park, Paholyothin Rd, Klong 1, Klong Luang, Pathumthani 12120, Thailand b Department of Oral Pathology, Faculty of Dentistry, Henri Dunant Rd., Chulalongkorn University, Bangkok 10330, Thailand c Scientific and Technological Research Equipment Center, Chulalongkorn University, Bangkok 10330, Thailand Received 19 February 2003; accepted 11 August 2003
Abstract Chitin grafted with poly(acrylic acid) (chitin–PAA) was prepared with the aim of obtaining a hydrogel characteristic for wound dressing application. The chitin–PAA films were synthesized at various acrylic acid feed contents to investigate its effect on water sorption ability. Acrylic acid (AA) was first linked to chitin, acting as the active grafting sites on the chain that was further polymerized to form a network structure. The evidences of grafting were found from FTIR and solid state 13C NMR spectra. The TGA results exhibited the high degradation temperature of the grafted product suggesting the formation of a network structure. The degree of swelling (DS) of chitin–PAA films was found in the range of 30–60 times of their original weights depending upon the monomer feed content. The chitin–PAA film with 1:4 weight ratio of chitin:AA, possessed optimal physical properties. The cytocompatibility of the film was investigated with a cell line of L929 mouse fibroblasts. The morphology and behavior of the cells on the chitin–PAA film were determined after different time periods of culture up to 14 days. The L929 cells proliferated and attached well onto the film. These results suggested that the 1:4 chitin–PAA has a potential to be used as a wound dressing. r 2003 Elsevier Ltd. All rights reserved. Keywords: Chitin; Poly(acrylic acid); Hydrogel; Biocompatibility; Scanning electron microscopy (SEM); Wound dressing
1. Introduction Chitin is one of the most abundant organic materials in nature. It can be easily prepared from the shells of crab, shrimp and squid pens. Because of its availability, biodegradability as well as biocompatibility, chitin and its derivatives have been used for a variety of applications such as water treatment, textile and paper, cosmetic, food and health supplements, agriculture and biotechnology [1]. In biomedical area, it was found that chitin possessed high activity as a wound healing accelerator [2]. Many types of chitin-based materials adapted for uses in wound dressing application have been patented [3–7]. However, low water sorption ability of chitin which yields an inefficient exudate removal from the wound surface limits its utility as a wound dressing. The chemical structure of chitin has been modified to overcome this undesirable characteristic. *Corresponding author. Tel.: +66-2-564-6500; fax: +66-2-5646445. E-mail address: [email protected] (S. Tanodekaew). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.08.020
Among many attempts, grafting of various monomers containing hydrophilic groups onto chitin chains seems to be a promising method to enhance its water sorption ability. Grafted products gain a hydrogel characteristic with a great water retention capacity whereas the beneficial properties of chitin such as biodegradability and bioactivity still remain. In fact, there have been very few publications on the grafting of hydrophilic monomers onto chitin, probably due to the insolubility of chitin in water and common organic solvents. On the contrary, a lot of research works have been studied on chitosan, a principal derivative of chitin produced by deacetylation process. Chitosan shows enhanced solubility in dilute acids. Chemical modification to alter its properties can be carried out under milder conditions compared with that of the parent chitin. Consequently, there have been many interesting graft copolymers of chitosan reported [8–14]. Acrylic acid (AA), one of well known hydrogel forming monomers, has been widely applied in graft copolymerization of chitosan to increase its hydrophilicity. The detail of hydrogel forming of chitosan and poly(acrylic acid) (PAA) has
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been previously described [11–14]. Some of chitosan– PAA hydrogels involved the use of glutaraldehyde for crosslinking purpose. Although, it strengthened the gel, glutaraldehyde was considered as a toxic chemical. The toxicity of the end products, therefore, is the concerned issue when toxic chemicals have been employed, especially in the preparation of medical products. In the present paper, a novel procedure to prepare acrylic grafted chitin (chitin–PAA), to impart a hydrogel characteristic for wound dressing, is reported. Since acrylic acid was directly grafted onto chitin, a costly and time consuming process to produce soluble chitin derivatives such as chitosan, employed as a starting material, was unnecessary. Chitin–PAA’s were prepared with various acrylic acid feed contents without the use of any toxic crosslinkers. The grafted products were then analyzed by NMR, FTIR and TGA. Degree of swelling as well as in vitro cytotoxicity of the synthesized gels were studied. The cellular morphology and behavior of L929 mouse fibroblasts on the film were also investigated using cell culture and SEM.
2. Materials and methods 2.1. Materials Squid chitin (Taming Enterprises, Thailand) and acrylic acid (Aldrich) were used as starting materials for the preparation of chitin–PAA hydrogels. The degree of deacetylation of chitin was 0.16 as calculated from solid state 13C NMR spectra. Potassium peroxodisulfate (Fluka) was used as a redox initiator. All these chemicals were used as received. 2.2. Preparation of chitin–PAA hydrogels Chitin powder (1 g) was stirred in acrylic acid (4, 6 and 8 g) with a small amount of sulfuric acid (5 M, 2 ml) at 70 C for 1 h. After cooling to room temperature, a paste-like mixture of chitin–AA was obtained. A small portion of chitin–AA paste was removed for its structural analysis. It was precipitated and washed with deionized water until a decanted solution reached pH 7. The dried chitin–AA sample was then characterized by solid state 13C NMR (Bruker DPX-300 spectrometer), FTIR (Perkin Elmer system 2000) and TGA (Perkin Elmer Thermogravimetric Analyzer TGA7, at a heating rate of 20 C/min under nitrogen atmosphere). To make a chitin–PAA film, the paste mixture of chitin–AA added with potassium peroxodisulfate (1.25 wt% to the monomer) and deionized water (4 ml) was cast in a petri-dish and maintained at 65 C for 4 h. After polymerization, the film was neutralized, washed with deionized water to remove any soluble materials,
and dried. The film was then investigated by solid state 13 C NMR, FTIR and TGA. 2.3. Swelling test The preweighed chitin–PAA films (140–200 mg) were immersed in deionized water at room temperature. At certain time, the swollen films were removed from the water, quickly wiped to remove excess water on the surface, and weighed. The degree of swelling was calculated as follows: DS ¼ ðWw Wd Þ=Wd ; where Ww and Wd are weights of wet and dry film, respectively. 2.4. SEM observation 2.4.1. Surface morphology of the chitin–PAA film The morphology of dry and hydrated chitin–PAA films without cells was examined using SEM. The preparation of the hydrated samples involved fixation, dehydration and gold sputtering as described below, while dry samples were only gold sputtered prior to SEM observation. 2.4.2. Cell–material response A cell line of L929 (ECACC No. 85011425), mouse connective tissue, fibroblast-like cells was cultured in the growth medium which was made up of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), together with penicillin (100 units/ml) and streptomycin (100 mg/ml) at 37 C in a 5% CO2 atmosphere. Once 80% confluence was reached, the cells were subcultured for cytotoxicity study. The hydrogel samples were sterilized by g-ray irradiation. The study of the cell response to the 1:4 chitin–PAA film was performed by plating the cells onto the pre-wet film. Initially, a 2 7 mm2 piece of 1:4 chitin–PAA sample was allowed to wet and expand by absorbing small amount of growth medium without leaving any excess medium before being attached centrally onto a 35-mm tissue culture dish with non-toxic dental wax. L929 cells were then seeded at a density of 2 105 cells/ dish. The cultures were incubated for 14 days. The growth medium was changed every 2 days. The cell response was observed everyday with an inverted phase contrast microscope. The cells on materials were then prepared for SEM at the day of 1, 2, 7 and 14 after exposed to the chitin–PAA samples. At each time point, the samples were washed with 0.1 m phosphate buffer (PB) and fixed with 2% glutaraldehyde in 0.1 m PB for 4 h at 4 C. After thorough washing with PB, the samples were dehydrated by graded ethanol changes and critical point dry. The samples were then gold sputtered in
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vacuum and examined by SEM. The experiments were repeated three times with different batches of the chitin– PAA film made.
3. Results and discussion 3.1. Preparation of chitin–PAA hydrogels The chitin–PAA’s were prepared by a two-step reaction, as shown in Fig. 1. Chitin was first mixed with acrylic acid, yielding a heterogeneous mixture. Under acid-catalyzed reaction, the esterification was occurred to form ester linkages between carboxylic groups of acrylic acid and hydroxyl groups of chitin. Chitin–AA, the paste-like mixture containing active grafting sites was obtained. In fact, the esterification by reacting acrylic acid with chitin is not easily occurred. Regardless of the heterogeneous aqueous reaction which is a result of water insolubility of chitin, the chemical structure of chitin itself hinders the accessibility of reacting acrylic acid monomer. However, chitin used in
Fig. 1. Scheme of the preparation of chitin–PAA hydrogel.
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this experiment is in the b-form which chains arrange in a parallel fashion with relatively weak interchain hydrogen bond [15]. This kind of loosely packed structure, thereby, facilitates the interaction between acrylic acid and chitin to yield ester linkages. In the second step, the casting solution of chitin–AA was further polymerized by the addition of initiator. The subsequent addition of acrylic acid molecules to the active grafting sites propagated the growing of acrylic side chains on chitin. Meanwhile, both interchain crosslinking and PAA homopolymerization were concurrently occurring. The former resulted in a forming of network structure which strengthened the gels when swelling in water while the latter affected the swelling ability of the gels, which will be discussed later. 3.2. Structural analysis The existence of ester linkages between acrylic acid and chitin was evidenced by FTIR. The spectrum of chitin–AA (Fig. 2b) shows a new peak at 1726 cm1 corresponding to the carbonyl absorption of acrylic acid, in addition to the saccharide characteristic peaks of chitin (Fig. 2a). This peak was found markedly broad as acrylic side chains increased, as seen in the spectrum of chitin–PAA (Fig. 2c). This broad band corresponded to
Fig. 2. FTIR spectra of (a) chitin, (b) chitin–AA, (c) chitin–PAA, and (d) PAA.
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CQO stretching vibration of carboxylic acid groups from acrylic chains as observed in the spectrum of PAA (Fig. 2d). The evidence of ester linkages, however, was not observed by the solid state 13C NMR technique. The spectrum of chitin–AA (Fig. 3b) was apparently identical to that of pure chitin (Fig. 3a) with an insignificant change in the degree of deacetylation. It suggested that N-acetyl groups of chitin were well protected and still remained after the esterification process. The number of ester linkages existed in chitin–AA was most probably too small. With the limited sensitivity of the solid state technique, it was unable to detect the resonance signals designated for the links. As expected for the large amount of acrylic side chains, the chitin–PAA spectrum (Fig. 3c) appears
Fig. 3. Solid state (c) chitin–PAA.
13
C NMR spectra of (a) chitin, (b) chitin–AA, and
characteristic peaks of chitin and also a peak at 183.5 ppm representing carbonyl groups and broad peaks at 30–50 ppm representing methylene and methine groups of acrylic chains. 3.3. Thermogravimetric analysis The thermograms of chitin, chitin–AA, chitin–PAA, chitin/PAA blend and PAA are shown in Fig. 4. The thermogram of chitin (Fig. 4a) exhibits two decomposition stages. The one in the range of 50–110 C was due to the loss of water, and the other in the range of 300– 380 C has been described to the degradation of saccharide structure, including the dehydration of saccharide rings and the depolymerization and decomposition of acetylated and deacetylated units of chitin [14]. The thermogram of PAA (Fig. 4e) shows three decomposition stages. The first decomposition stage in the range of 50–180 C was attributed to the loss of bound water. The second one in the interval of 215– 300 C has been described to the dehydration and decarboxylation of the polymer which leads to the formation of inter- and intra-molecular anhydride [14,16]. The third decomposition stage in the range of 365–470 C was a result of the degradation of the residual polymer. As seen from the thermogram of chitin–AA (Fig. 4b), the degradation of its saccharide structure appears in the range of 280–340 C which is approximately 50 C lower than that of chitin. It suggested that chitin–AA was less thermal stability than chitin. In other words, it implied that the links of acrylic acid to chitin, as evidenced from FTIR, possibly reduced the thermal stability of chitin. The thermogram of chitin–PAA (weight ratio of chitin:AA=1:4) (Fig. 4c) shows rather complicated decomposition stages whereas a physical blending mixture of chitin and PAA (weight ratio of chitin:
Fig. 4. Thermograms of (a) chitin, (b) chitin–AA, (c) chitin–PAA, (d) chitin/PAA blend, and (e) PAA.
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PAA=1:4) (Fig. 4d) shows straightforward three decomposition stages overlaying on those of chitin and PAA individually. The complex degradation of chitin– PAA began with the loss of water below 200 C. At the temperature above 200 C, a distinct decomposition stage at the temperature range of 440–510 C was observed, besides the normal degradation stages of chitin and PAA. This high decomposition temperature together with the high residue content, up to 40 wt%, at 600 C observed in the chitin–PAA thermogram suggested the presence of rigid structure which supported the three-dimensional network formation of chitin– PAA. 3.4. Swelling test The influence of the chitin:AA feed ratio on the degree of swelling of chitin–PAA hydrogels is shown in Fig. 5. It was expected to observe the greater swelling of the chitin–PAA film with a higher AA feed content. It was, however, apparent that the swelling degrees increased as a function of monomer feed only at the initial period of immersion. After prolonged swelling in water, chitin–PAA 1:6 was found to imbibe the greater amount of water than chitin–PAA 1:8. In addition, tiny pieces were found to detach from both gels whereas the gel with a lower monomer feed content (chitin–PAA 1:4) contained no debris and still remained intact throughout the swelling test. Accordingly, there was no change in the dried weight of the chitin–PAA 1:4 film observed after being in water for a week whereas the weight loss of 6% and 20% were found for the chitin–PAA 1:6 and 1:8 films, respectively. Consequently, the significant errors in the measurements of the degrees of swelling in these two samples existed. The loss of weight might be associated with the presence of PAA homopolymer as mentioned earlier. During polymerization, only certain amount of acrylic acid monomer underwent graft copolymerization while the rest was homopolymerized.
Fig. 5. Degrees of swelling of chitin–PAA at various monomer feed contents: (a) chitin–PAA 1:4, (b) chitin–PAA 1:6, and (c) chitin–PAA 1:8.
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At a higher monomer feed content, the homopolymerization was more favorable. Although, the PAA homopolymer could be washed out during a film washing process, some residue was strongly entrapped in the film, enhancing swelling of the gel. The longer the gel was in water, the greater the amount of water it absorbed. Bearing such a great amount of water, thereby, weakened the strength of the gels. As observed in a very high monomer feed content (chitin–PAA 1:10), the gel was very soft and difficult to be handled without breaking. It was also worth mentioning that the actual amount of acrylic acid incorporated in the chitin–PAA film was governed by the nature of the heterogeneous reaction. Consequently, some variations in swelling ability were expected for each chitin–PAA batch. For the strength aspect, chitin–PAA 1:4 seemed to be the best candidate for wound dressing. The amount of water absorbed in three different batches of chitin:PAA 1:4 were found in the range of 25–35 times of their dry weights. This water sorption ability should be sufficient for the removal of exudate from wounds. The water sorption ability is an essential property for wound dressing application. Large open wounds usually secrete a lot of exudate and this excess fluid may cause complications particularly infections. The swollen gels showed integrity with a negligible loss of weight for oneweek immersion. Chitin–PAA 1:4 was then selected for the investigation of its compatibility with cells. 3.5. SEM observation Surface morphology of the chitin–PAA film is shown in Fig. 6. Ultrastructurally, the surface of the dry film appeared to be irregularly rough or cobble stone-like and rather dense (Fig. 6a) while the hydrated film swelled in the growth medium resulting in more stretched pattern (Fig. 6b). The L929 cells cultured in contact with the chitin– PAA 1:4 hydrogel for 24 h observed under phasecontrast light microscope was found intact and proliferated without an inhibition zone (figures not shown). The results suggested that this chitin–PAA 1:4 hydrogel was non-cytotoxic and absent in unreacted AA monomer molecules or degradation products that may leach into the culture medium and possibly damage the cells. The L929 cells cultured on the chitin–PAA film were closely investigated at different time points of culture using SEM. The cellular behavior on a biomaterial is an important factor determining its biocompatibility. The cell attachment with different cell shapes was found within 24 h after plating the cells. These included spherical cells with numerous small cytoplasmic processes, rounded cells with some blebs and attaching filopodia and flattening, spreading cell with extended lamellipodia (Fig. 7a–d). These different morphologies
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Fig. 6. Surface textures of the chitin–PAA film: (a) dry film and (b) hydrated film.
Fig. 7. SEM micrographs of L929 cells on the chitin–PAA film. (a) cell population at low magnification, (b–d) different cell shapes at high magnification.
probably exhibited different stages of the cells responding to the substrate. A schematic presentation and SEM micrographs of different stages in adhesion and spreading of cells in vitro as seen with phase contrast light microscope and with SEM was previously presented [17]. The whole process of cell adhesion and spreading consists of cell attachment, growth of filopodia, cytoplasmic webbing, flattening of the cell mass and the ruffling of peripheral cytoplasm progressing in a sequential fashion. These results suggested that the chitin–PAA film was free from residual monomer that may cause any toxicity to cells and it was compatible to the fibroblast cells. In addition, the film offered a hospitable surface for the cells to attach. On day 2 of culture, the cells exhibited more fibroblast-like characteristic, i.e. spindle shape (Fig. 8a). After that, they progressively proliferated and covered most of the film surface on day 7 (Fig. 8b). The culture
was extended up to 14 days, the cells were over grown and piling up on one another which is usually found in over confluent cultures on tissue culture plastic. It is well known that wettability of biomaterials influences cell adhesion and proliferation [18]. It was reported that cell attachment and growth of anchorage-dependent cells were inhibited on non-wettable biomaterials [19–21]. This was attributed to the possible conformational changes of adsorbed proteins [22–23]. Fibroblasts have been shown to respond to the surface roughness of various substrates. The alteration of cell size, shape and orientation was observed when the cells were cultured on grooved surfaces [24–27]. Interestingly, macrophages and fibroblasts responded to the materials with different hydrophilicity in a reversed preference. It was found that macrophages migrated preferentially onto more hydrophobic substrates while fibroblasts accumulated on those that are hydrophilic. In addition, macrophages
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Fig. 8. Cellular morphology and growth of L929 on the chitin–PAA film; (a) after 2 days showing healthy fibroblast-like cells and; (b) after 7 days showing cell confluence.
were found accumulated on rough surface in preference to smooth ones while fibroblasts did the reverse. These opposite preferences exhibited by macrophages and fibroblasts may reflect physical and functional differences in their surface [28]. Although the surface of the chitin–PAA film was not smooth, it allowed the L929 cells to attach and adjust the cell surface together with cell mass pliable to the film texture and become confluent on the film. The modification of chitin by incorporating hydrophilic PAA offers at least two advantages; firstly improved the water sorption of the dressing. Secondly, the increased hydrophilicity may facilitate the attachment, growth and migration of dermal fibroblasts at the wound site, which is important in wound healing process. We previously found that chitin did not well support the adhesion of fibroblasts [29].
4. Conclusion Novel chitin–PAA hydrogels were synthesized. Chitin was first modified with acrylic acid via an esterification process. The polymerization of acrylic acid monomer then proceeded simultaneously with the film forming process with a use of redox initiator. FTIR results were taken as evidence of ester linkages forming between chitin and acrylic acid. The films possessed a network structure and exhibited an enhancement of water sorption ability. The swelling behavior and gel strength were found to depend upon monomer feed content. Chitin–PAA 1:4 yielded optimal swelling and gel strength. The overall results of the cellular behavior on the chitin–PAA 1:4 film in this present study suggested that the material has a potential for biomedical applications particularly as temporary skin substitute.
Acknowledgements The authors are grateful to National Metal and Materials Technology Center, Thailand for financial
support and the Office of Atomic Energy for Peace (OAEP), Thailand for the g-sterilization. The authors would also like to thank Dr. Patricia Watts and staff in the Animal Cell Culture Laboratory of BIOTEC for their general assistance.
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Preparation of acrylic grafted chitin for wound dressing application Siriporn Tanodekaewa,*, Malinee Prasitsilpa, Somporn Swasdisonb, Boonlom Thavornyutikarna, Thanawit Pothsreea, Rujiporn Pateepasenc a
National Metal and Materials Technology Center, 114 Thailand Science Park, Paholyothin Rd, Klong 1, Klong Luang, Pathumthani 12120, Thailand b Department of Oral Pathology, Faculty of Dentistry, Henri Dunant Rd., Chulalongkorn University, Bangkok 10330, Thailand c Scientific and Technological Research Equipment Center, Chulalongkorn University, Bangkok 10330, Thailand Received 19 February 2003; accepted 11 August 2003
Abstract Chitin grafted with poly(acrylic acid) (chitin–PAA) was prepared with the aim of obtaining a hydrogel characteristic for wound dressing application. The chitin–PAA films were synthesized at various acrylic acid feed contents to investigate its effect on water sorption ability. Acrylic acid (AA) was first linked to chitin, acting as the active grafting sites on the chain that was further polymerized to form a network structure. The evidences of grafting were found from FTIR and solid state 13C NMR spectra. The TGA results exhibited the high degradation temperature of the grafted product suggesting the formation of a network structure. The degree of swelling (DS) of chitin–PAA films was found in the range of 30–60 times of their original weights depending upon the monomer feed content. The chitin–PAA film with 1:4 weight ratio of chitin:AA, possessed optimal physical properties. The cytocompatibility of the film was investigated with a cell line of L929 mouse fibroblasts. The morphology and behavior of the cells on the chitin–PAA film were determined after different time periods of culture up to 14 days. The L929 cells proliferated and attached well onto the film. These results suggested that the 1:4 chitin–PAA has a potential to be used as a wound dressing. r 2003 Elsevier Ltd. All rights reserved. Keywords: Chitin; Poly(acrylic acid); Hydrogel; Biocompatibility; Scanning electron microscopy (SEM); Wound dressing
1. Introduction Chitin is one of the most abundant organic materials in nature. It can be easily prepared from the shells of crab, shrimp and squid pens. Because of its availability, biodegradability as well as biocompatibility, chitin and its derivatives have been used for a variety of applications such as water treatment, textile and paper, cosmetic, food and health supplements, agriculture and biotechnology [1]. In biomedical area, it was found that chitin possessed high activity as a wound healing accelerator [2]. Many types of chitin-based materials adapted for uses in wound dressing application have been patented [3–7]. However, low water sorption ability of chitin which yields an inefficient exudate removal from the wound surface limits its utility as a wound dressing. The chemical structure of chitin has been modified to overcome this undesirable characteristic. *Corresponding author. Tel.: +66-2-564-6500; fax: +66-2-5646445. E-mail address: [email protected] (S. Tanodekaew). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.08.020
Among many attempts, grafting of various monomers containing hydrophilic groups onto chitin chains seems to be a promising method to enhance its water sorption ability. Grafted products gain a hydrogel characteristic with a great water retention capacity whereas the beneficial properties of chitin such as biodegradability and bioactivity still remain. In fact, there have been very few publications on the grafting of hydrophilic monomers onto chitin, probably due to the insolubility of chitin in water and common organic solvents. On the contrary, a lot of research works have been studied on chitosan, a principal derivative of chitin produced by deacetylation process. Chitosan shows enhanced solubility in dilute acids. Chemical modification to alter its properties can be carried out under milder conditions compared with that of the parent chitin. Consequently, there have been many interesting graft copolymers of chitosan reported [8–14]. Acrylic acid (AA), one of well known hydrogel forming monomers, has been widely applied in graft copolymerization of chitosan to increase its hydrophilicity. The detail of hydrogel forming of chitosan and poly(acrylic acid) (PAA) has
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been previously described [11–14]. Some of chitosan– PAA hydrogels involved the use of glutaraldehyde for crosslinking purpose. Although, it strengthened the gel, glutaraldehyde was considered as a toxic chemical. The toxicity of the end products, therefore, is the concerned issue when toxic chemicals have been employed, especially in the preparation of medical products. In the present paper, a novel procedure to prepare acrylic grafted chitin (chitin–PAA), to impart a hydrogel characteristic for wound dressing, is reported. Since acrylic acid was directly grafted onto chitin, a costly and time consuming process to produce soluble chitin derivatives such as chitosan, employed as a starting material, was unnecessary. Chitin–PAA’s were prepared with various acrylic acid feed contents without the use of any toxic crosslinkers. The grafted products were then analyzed by NMR, FTIR and TGA. Degree of swelling as well as in vitro cytotoxicity of the synthesized gels were studied. The cellular morphology and behavior of L929 mouse fibroblasts on the film were also investigated using cell culture and SEM.
2. Materials and methods 2.1. Materials Squid chitin (Taming Enterprises, Thailand) and acrylic acid (Aldrich) were used as starting materials for the preparation of chitin–PAA hydrogels. The degree of deacetylation of chitin was 0.16 as calculated from solid state 13C NMR spectra. Potassium peroxodisulfate (Fluka) was used as a redox initiator. All these chemicals were used as received. 2.2. Preparation of chitin–PAA hydrogels Chitin powder (1 g) was stirred in acrylic acid (4, 6 and 8 g) with a small amount of sulfuric acid (5 M, 2 ml) at 70 C for 1 h. After cooling to room temperature, a paste-like mixture of chitin–AA was obtained. A small portion of chitin–AA paste was removed for its structural analysis. It was precipitated and washed with deionized water until a decanted solution reached pH 7. The dried chitin–AA sample was then characterized by solid state 13C NMR (Bruker DPX-300 spectrometer), FTIR (Perkin Elmer system 2000) and TGA (Perkin Elmer Thermogravimetric Analyzer TGA7, at a heating rate of 20 C/min under nitrogen atmosphere). To make a chitin–PAA film, the paste mixture of chitin–AA added with potassium peroxodisulfate (1.25 wt% to the monomer) and deionized water (4 ml) was cast in a petri-dish and maintained at 65 C for 4 h. After polymerization, the film was neutralized, washed with deionized water to remove any soluble materials,
and dried. The film was then investigated by solid state 13 C NMR, FTIR and TGA. 2.3. Swelling test The preweighed chitin–PAA films (140–200 mg) were immersed in deionized water at room temperature. At certain time, the swollen films were removed from the water, quickly wiped to remove excess water on the surface, and weighed. The degree of swelling was calculated as follows: DS ¼ ðWw Wd Þ=Wd ; where Ww and Wd are weights of wet and dry film, respectively. 2.4. SEM observation 2.4.1. Surface morphology of the chitin–PAA film The morphology of dry and hydrated chitin–PAA films without cells was examined using SEM. The preparation of the hydrated samples involved fixation, dehydration and gold sputtering as described below, while dry samples were only gold sputtered prior to SEM observation. 2.4.2. Cell–material response A cell line of L929 (ECACC No. 85011425), mouse connective tissue, fibroblast-like cells was cultured in the growth medium which was made up of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), together with penicillin (100 units/ml) and streptomycin (100 mg/ml) at 37 C in a 5% CO2 atmosphere. Once 80% confluence was reached, the cells were subcultured for cytotoxicity study. The hydrogel samples were sterilized by g-ray irradiation. The study of the cell response to the 1:4 chitin–PAA film was performed by plating the cells onto the pre-wet film. Initially, a 2 7 mm2 piece of 1:4 chitin–PAA sample was allowed to wet and expand by absorbing small amount of growth medium without leaving any excess medium before being attached centrally onto a 35-mm tissue culture dish with non-toxic dental wax. L929 cells were then seeded at a density of 2 105 cells/ dish. The cultures were incubated for 14 days. The growth medium was changed every 2 days. The cell response was observed everyday with an inverted phase contrast microscope. The cells on materials were then prepared for SEM at the day of 1, 2, 7 and 14 after exposed to the chitin–PAA samples. At each time point, the samples were washed with 0.1 m phosphate buffer (PB) and fixed with 2% glutaraldehyde in 0.1 m PB for 4 h at 4 C. After thorough washing with PB, the samples were dehydrated by graded ethanol changes and critical point dry. The samples were then gold sputtered in
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vacuum and examined by SEM. The experiments were repeated three times with different batches of the chitin– PAA film made.
3. Results and discussion 3.1. Preparation of chitin–PAA hydrogels The chitin–PAA’s were prepared by a two-step reaction, as shown in Fig. 1. Chitin was first mixed with acrylic acid, yielding a heterogeneous mixture. Under acid-catalyzed reaction, the esterification was occurred to form ester linkages between carboxylic groups of acrylic acid and hydroxyl groups of chitin. Chitin–AA, the paste-like mixture containing active grafting sites was obtained. In fact, the esterification by reacting acrylic acid with chitin is not easily occurred. Regardless of the heterogeneous aqueous reaction which is a result of water insolubility of chitin, the chemical structure of chitin itself hinders the accessibility of reacting acrylic acid monomer. However, chitin used in
Fig. 1. Scheme of the preparation of chitin–PAA hydrogel.
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this experiment is in the b-form which chains arrange in a parallel fashion with relatively weak interchain hydrogen bond [15]. This kind of loosely packed structure, thereby, facilitates the interaction between acrylic acid and chitin to yield ester linkages. In the second step, the casting solution of chitin–AA was further polymerized by the addition of initiator. The subsequent addition of acrylic acid molecules to the active grafting sites propagated the growing of acrylic side chains on chitin. Meanwhile, both interchain crosslinking and PAA homopolymerization were concurrently occurring. The former resulted in a forming of network structure which strengthened the gels when swelling in water while the latter affected the swelling ability of the gels, which will be discussed later. 3.2. Structural analysis The existence of ester linkages between acrylic acid and chitin was evidenced by FTIR. The spectrum of chitin–AA (Fig. 2b) shows a new peak at 1726 cm1 corresponding to the carbonyl absorption of acrylic acid, in addition to the saccharide characteristic peaks of chitin (Fig. 2a). This peak was found markedly broad as acrylic side chains increased, as seen in the spectrum of chitin–PAA (Fig. 2c). This broad band corresponded to
Fig. 2. FTIR spectra of (a) chitin, (b) chitin–AA, (c) chitin–PAA, and (d) PAA.
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CQO stretching vibration of carboxylic acid groups from acrylic chains as observed in the spectrum of PAA (Fig. 2d). The evidence of ester linkages, however, was not observed by the solid state 13C NMR technique. The spectrum of chitin–AA (Fig. 3b) was apparently identical to that of pure chitin (Fig. 3a) with an insignificant change in the degree of deacetylation. It suggested that N-acetyl groups of chitin were well protected and still remained after the esterification process. The number of ester linkages existed in chitin–AA was most probably too small. With the limited sensitivity of the solid state technique, it was unable to detect the resonance signals designated for the links. As expected for the large amount of acrylic side chains, the chitin–PAA spectrum (Fig. 3c) appears
Fig. 3. Solid state (c) chitin–PAA.
13
C NMR spectra of (a) chitin, (b) chitin–AA, and
characteristic peaks of chitin and also a peak at 183.5 ppm representing carbonyl groups and broad peaks at 30–50 ppm representing methylene and methine groups of acrylic chains. 3.3. Thermogravimetric analysis The thermograms of chitin, chitin–AA, chitin–PAA, chitin/PAA blend and PAA are shown in Fig. 4. The thermogram of chitin (Fig. 4a) exhibits two decomposition stages. The one in the range of 50–110 C was due to the loss of water, and the other in the range of 300– 380 C has been described to the degradation of saccharide structure, including the dehydration of saccharide rings and the depolymerization and decomposition of acetylated and deacetylated units of chitin [14]. The thermogram of PAA (Fig. 4e) shows three decomposition stages. The first decomposition stage in the range of 50–180 C was attributed to the loss of bound water. The second one in the interval of 215– 300 C has been described to the dehydration and decarboxylation of the polymer which leads to the formation of inter- and intra-molecular anhydride [14,16]. The third decomposition stage in the range of 365–470 C was a result of the degradation of the residual polymer. As seen from the thermogram of chitin–AA (Fig. 4b), the degradation of its saccharide structure appears in the range of 280–340 C which is approximately 50 C lower than that of chitin. It suggested that chitin–AA was less thermal stability than chitin. In other words, it implied that the links of acrylic acid to chitin, as evidenced from FTIR, possibly reduced the thermal stability of chitin. The thermogram of chitin–PAA (weight ratio of chitin:AA=1:4) (Fig. 4c) shows rather complicated decomposition stages whereas a physical blending mixture of chitin and PAA (weight ratio of chitin:
Fig. 4. Thermograms of (a) chitin, (b) chitin–AA, (c) chitin–PAA, (d) chitin/PAA blend, and (e) PAA.
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PAA=1:4) (Fig. 4d) shows straightforward three decomposition stages overlaying on those of chitin and PAA individually. The complex degradation of chitin– PAA began with the loss of water below 200 C. At the temperature above 200 C, a distinct decomposition stage at the temperature range of 440–510 C was observed, besides the normal degradation stages of chitin and PAA. This high decomposition temperature together with the high residue content, up to 40 wt%, at 600 C observed in the chitin–PAA thermogram suggested the presence of rigid structure which supported the three-dimensional network formation of chitin– PAA. 3.4. Swelling test The influence of the chitin:AA feed ratio on the degree of swelling of chitin–PAA hydrogels is shown in Fig. 5. It was expected to observe the greater swelling of the chitin–PAA film with a higher AA feed content. It was, however, apparent that the swelling degrees increased as a function of monomer feed only at the initial period of immersion. After prolonged swelling in water, chitin–PAA 1:6 was found to imbibe the greater amount of water than chitin–PAA 1:8. In addition, tiny pieces were found to detach from both gels whereas the gel with a lower monomer feed content (chitin–PAA 1:4) contained no debris and still remained intact throughout the swelling test. Accordingly, there was no change in the dried weight of the chitin–PAA 1:4 film observed after being in water for a week whereas the weight loss of 6% and 20% were found for the chitin–PAA 1:6 and 1:8 films, respectively. Consequently, the significant errors in the measurements of the degrees of swelling in these two samples existed. The loss of weight might be associated with the presence of PAA homopolymer as mentioned earlier. During polymerization, only certain amount of acrylic acid monomer underwent graft copolymerization while the rest was homopolymerized.
Fig. 5. Degrees of swelling of chitin–PAA at various monomer feed contents: (a) chitin–PAA 1:4, (b) chitin–PAA 1:6, and (c) chitin–PAA 1:8.
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At a higher monomer feed content, the homopolymerization was more favorable. Although, the PAA homopolymer could be washed out during a film washing process, some residue was strongly entrapped in the film, enhancing swelling of the gel. The longer the gel was in water, the greater the amount of water it absorbed. Bearing such a great amount of water, thereby, weakened the strength of the gels. As observed in a very high monomer feed content (chitin–PAA 1:10), the gel was very soft and difficult to be handled without breaking. It was also worth mentioning that the actual amount of acrylic acid incorporated in the chitin–PAA film was governed by the nature of the heterogeneous reaction. Consequently, some variations in swelling ability were expected for each chitin–PAA batch. For the strength aspect, chitin–PAA 1:4 seemed to be the best candidate for wound dressing. The amount of water absorbed in three different batches of chitin:PAA 1:4 were found in the range of 25–35 times of their dry weights. This water sorption ability should be sufficient for the removal of exudate from wounds. The water sorption ability is an essential property for wound dressing application. Large open wounds usually secrete a lot of exudate and this excess fluid may cause complications particularly infections. The swollen gels showed integrity with a negligible loss of weight for oneweek immersion. Chitin–PAA 1:4 was then selected for the investigation of its compatibility with cells. 3.5. SEM observation Surface morphology of the chitin–PAA film is shown in Fig. 6. Ultrastructurally, the surface of the dry film appeared to be irregularly rough or cobble stone-like and rather dense (Fig. 6a) while the hydrated film swelled in the growth medium resulting in more stretched pattern (Fig. 6b). The L929 cells cultured in contact with the chitin– PAA 1:4 hydrogel for 24 h observed under phasecontrast light microscope was found intact and proliferated without an inhibition zone (figures not shown). The results suggested that this chitin–PAA 1:4 hydrogel was non-cytotoxic and absent in unreacted AA monomer molecules or degradation products that may leach into the culture medium and possibly damage the cells. The L929 cells cultured on the chitin–PAA film were closely investigated at different time points of culture using SEM. The cellular behavior on a biomaterial is an important factor determining its biocompatibility. The cell attachment with different cell shapes was found within 24 h after plating the cells. These included spherical cells with numerous small cytoplasmic processes, rounded cells with some blebs and attaching filopodia and flattening, spreading cell with extended lamellipodia (Fig. 7a–d). These different morphologies
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Fig. 6. Surface textures of the chitin–PAA film: (a) dry film and (b) hydrated film.
Fig. 7. SEM micrographs of L929 cells on the chitin–PAA film. (a) cell population at low magnification, (b–d) different cell shapes at high magnification.
probably exhibited different stages of the cells responding to the substrate. A schematic presentation and SEM micrographs of different stages in adhesion and spreading of cells in vitro as seen with phase contrast light microscope and with SEM was previously presented [17]. The whole process of cell adhesion and spreading consists of cell attachment, growth of filopodia, cytoplasmic webbing, flattening of the cell mass and the ruffling of peripheral cytoplasm progressing in a sequential fashion. These results suggested that the chitin–PAA film was free from residual monomer that may cause any toxicity to cells and it was compatible to the fibroblast cells. In addition, the film offered a hospitable surface for the cells to attach. On day 2 of culture, the cells exhibited more fibroblast-like characteristic, i.e. spindle shape (Fig. 8a). After that, they progressively proliferated and covered most of the film surface on day 7 (Fig. 8b). The culture
was extended up to 14 days, the cells were over grown and piling up on one another which is usually found in over confluent cultures on tissue culture plastic. It is well known that wettability of biomaterials influences cell adhesion and proliferation [18]. It was reported that cell attachment and growth of anchorage-dependent cells were inhibited on non-wettable biomaterials [19–21]. This was attributed to the possible conformational changes of adsorbed proteins [22–23]. Fibroblasts have been shown to respond to the surface roughness of various substrates. The alteration of cell size, shape and orientation was observed when the cells were cultured on grooved surfaces [24–27]. Interestingly, macrophages and fibroblasts responded to the materials with different hydrophilicity in a reversed preference. It was found that macrophages migrated preferentially onto more hydrophobic substrates while fibroblasts accumulated on those that are hydrophilic. In addition, macrophages
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Fig. 8. Cellular morphology and growth of L929 on the chitin–PAA film; (a) after 2 days showing healthy fibroblast-like cells and; (b) after 7 days showing cell confluence.
were found accumulated on rough surface in preference to smooth ones while fibroblasts did the reverse. These opposite preferences exhibited by macrophages and fibroblasts may reflect physical and functional differences in their surface [28]. Although the surface of the chitin–PAA film was not smooth, it allowed the L929 cells to attach and adjust the cell surface together with cell mass pliable to the film texture and become confluent on the film. The modification of chitin by incorporating hydrophilic PAA offers at least two advantages; firstly improved the water sorption of the dressing. Secondly, the increased hydrophilicity may facilitate the attachment, growth and migration of dermal fibroblasts at the wound site, which is important in wound healing process. We previously found that chitin did not well support the adhesion of fibroblasts [29].
4. Conclusion Novel chitin–PAA hydrogels were synthesized. Chitin was first modified with acrylic acid via an esterification process. The polymerization of acrylic acid monomer then proceeded simultaneously with the film forming process with a use of redox initiator. FTIR results were taken as evidence of ester linkages forming between chitin and acrylic acid. The films possessed a network structure and exhibited an enhancement of water sorption ability. The swelling behavior and gel strength were found to depend upon monomer feed content. Chitin–PAA 1:4 yielded optimal swelling and gel strength. The overall results of the cellular behavior on the chitin–PAA 1:4 film in this present study suggested that the material has a potential for biomedical applications particularly as temporary skin substitute.
Acknowledgements The authors are grateful to National Metal and Materials Technology Center, Thailand for financial
support and the Office of Atomic Energy for Peace (OAEP), Thailand for the g-sterilization. The authors would also like to thank Dr. Patricia Watts and staff in the Animal Cell Culture Laboratory of BIOTEC for their general assistance.
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