Biomacromolecules 2000, 1, 61-67
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Novel Fabrication of Open-Pore Chitin Matrixes Kok Sum Chow and Eugene Khor* Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore Received January 12, 2000
A novel method has been developed to produce open-pore chitin matrixes. Chitin solutions were loaded with calcium carbonate (CaCO3) crystals and the mixture cast to form gels. The CaCO3-chitin gels were submerged in 1 N HCl solution to produce highly porous matrixes with good water vapor permeability, water uptake profile, and enhanced mechanical properties. The open-pore system is obtainable because CaCO3 loaded into the chitin gel reacts with 1 N HCl solution to produce gaseous carbon dioxide. Evolution of carbon dioxide during the reaction results in continuous pore structures from the matrix’ bulk to surface. When the concentration of CaCO3 loaded into the chitin gel is controlled, defined homogeneous pores measuring 100-500 and 500-1000 µm, with porosities of ≈76% and 81%, respectively, can be produced. Introduction Porous biodegradable materials are in great demand in various applications such as controlled drug release systems,1-5 enzyme immobilization support,6-8 molecular fractionation,9,10 and transition-metal adsorption.11-13 These materials are also being explored widely in tissue engineering, a field that offers an attractive approach to tissue repair and fabrication.14-16 In particular, regeneration of damaged tissues is a significant objective because of insufficient organ and tissue donors for the high demands.17 The potential of cell transplantation has been investigated by many groups for the regeneration of several tissues such as nerve,18,19 liver,20,21 cartilage,22-24 and bone.25,26 The scaffolding material should be designed to have sufficient porosity, adequate mechanical stability, biocompatibility, and biodegradability or bioadsorbability. Numerous techniques have been used to prepare scaffolds including casting/salt leaching.27,28 A polymer-salt composite is first prepared by solvent casting. After gelation, the salt particle was leached out leaving an open-pore structure. In the phase separation method,29 microcellular materials are formed by cooling polymer solutions until phase separation and solvent freezing occur. The solvent is then removed by sublimation in vacuo to form pores. A solvent evaporation method30 has been demonstrated to produce matrixes with various pore sizes. In this method, thermalsensitive gels were utilized to aggregate in water as a result of both the intramolecular hydrophobic interactions within the system. After the aggregated gel was dried, porous structures formed. Natural biodegradable polymers are an important class of materials that can act as a temporary scaffold for the transplanted cell before being substituted by an extracellular * To whom correspondence should be addressed. Tel.: 65-874-2836. Fax: 65-779-1691. E-mail:
[email protected].
matrix. Various groups have investigated the production of chitosan-based porous materials.31,32 Chitosan is a partially deacetylated derivative of chitin. Chitosan has an amino group that permits its dissolution in an acidic solvent (pH < 6). The high density of amino groups on the polymer backbone imparts on chitosan a hemostatic characteristic.33,34 This property may cause complications by coagulating blood when used as scaffolds in tissue transplantation. Chitin, the acetylated form, has an acetyl group attached to the C2 position instead of the amino moiety. This greatly reduces the polycationic character of the polymer backbone and diminishes the hemostatic property. Ironically, the amide group that gives chitin the advantage over chitosan also lessens greatly its solubility in most organic solvents. The presence of amide functionality imparts a rigid threedimensional hydrogen-bonded molecular structure and renders chitin as only soluble in an aprotic solvent system such as a dimethyl acetamide/lithium chloride mixture.35,36 Because of chitin’s inability to dissolve in common solvent systems, most of the other techniques mentioned previously are not suitable. Freeze-drying and cryogenic-induced phase separation processes that were used by chitosan-based materials31,32 can only produce pore sizes up to 250 µm. This paper introduces a novel method of fabricating porous chitin matrixes with variable open-pore structures from 100 to 1000 µm with good water vapor permeability, water uptake, and mechanical properties. The availability of a larger range of pore sizes provides more flexibility to submit to a wider spectrum of applications, especially for those that require pore sizes above 500 µm. Experimental Section Materials and Methods. Chitin was obtained from Polyscience, USA, and purified by stirring in 1 M NaOH at room temperature for 7 days and in 1 N HCl for 1 h. The degree of acetylation was determined by FT-IR37 and
10.1021/bm005503b CCC: $19.00 © 2000 American Chemical Society Published on Web 02/04/2000
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microanalysis to be ≈80%. Anhydrous silica gel was activated at 200 °C prior to the water vapor transmission test. CaCO3 was vacuum-dried for 1 week and sieved thru a 20-µm sieve. All reagents used for the preparation of the chitin matrixes were of analytical grade unless otherwise stated. Preparation of 0.5% Chitin Solution in 5% Lithium Chloride (LiCl)/Dimethyl Acetamide (DMAc). Anhydrous LiCl (5.0 g) was dried at 130 °C for about 0.5 h, cooled inside a desiccator, and dissolved in 100 mL of DMAc by magnetic stirring. Chitin flakes (0.05 g) were suspended in this solution and shaken overnight at 150 rpm, 4 °C in a refrigerated shaking incubator to give 100 mL of 0.5% (w/ w) chitin solution in a 5% DMAc/LiCl solvent system. The viscous clear solution was filtered through glass wool and stored in glass containers at room temperature. Loading of Calcium Carbonate (CaCO3). CaCO3 (0.3, 1.0, and 3.0 g) was added to various chitin solutions (100 mL each). The mixture was shaken overnight at 150 rpm, 4 °C in a refrigerated shaking incubator to afford 0.3%, 1.0%, and 3.0% (w/w) CaCO3-loaded chitin solutions, respectively. Preparation of CaCO3-Chitin Gels. Gels were obtained by dispensing the 100 mL of 0.3%, 1.0%, and 3.0% CaCO3chitin solutions into molds measuring 227 × 156 × 91 cm3. The solvent was allowed to evaporate slowly in the fume hood, for 2 days to give CaCO3-chitin gels. Treatment of CaCO3-Chitin Gels with 1 N HCl. The CaCO3-chitin gels were soaked in 1 N HCl solution (300 mL) inside a shaking water bath at room temperature, at 50 rpm for 2 h. The resulting gels were soaked with deionized water (500 mL) to ensure complete removal of DMAc, HCl, and other calcium residues. After the soaking step, resultant chitin gels were subjected to an air-drying procedure. Air Drying. The chitin gel was blotted dry by filter paper and placed in a fume hood. To ensure flatness after drying, the gels were placed between two pieces of glass plates. The drying process was carried out for about 5 days to give airdried chitin matrixes. Estimation of Pore Size. The pore size of all the airdried chitin matrixes were estimated from the SEM micrographs obtained using a JEOL JSM-T220A scanning microscope at an accelerating voltage of 15 kV. A Fine Coat Ion Sputter JFC-1100 coater performed gold coating on the matrixes. Mechanical Properties. The air-dried chitin matrixes were cut into dimensions measuring 6.0 × 1.0 cm2. A tensile test was performed on an Instron 4502 tensile tester (Instron Co., Canton, MA), employing a gauge length of 20 mm and crosshead speed of 2 mm/min, giving a strain rate of 0.1 mm/min. To prevent slippage of the specimen at the grips due to the reduction in thickness upon clamping, a pair of rectangular cardboard pieces of thickness 1.5 mm was glued to each end of the specimen. 13C Cross Polarization Magic Angle Spinning (CPMAS) Spectroscopy. 13C CPMAS experiments were conducted on a VARIAN 400 MHz spectrometer by BRUKER using a contact time of 2 ms. Rotor frequency was at 10 kHz with no apparent sidebands observed on the spectrum. The proton cross polarization and decoupling frequency was adjusted
Chow and Khor
to the pure water resonance at 4.65 ppm. The 13C transmitter frequency was adjusted to 100 ppm using adamantine as the reference at 38.5 ppm for the CH2 peak, downfield; 1500 scans were acquired for each run and measurements were taken at room temperature. Water Uptake Procedure. Each dried chitin matrix was soaked inside separate compartments containing deionized water (30 mL). The water uptake procedure was carried out by blotting dry the chitin sample with filter paper and measuring the weight of the chitin matrix at various intervals. water uptake ratio ) W1/W0 where W1 is the wet weight and W0 is the initial dry weight. Water Vapor Transmission Test. The water vapor transmission test was based on the ASTM E96-95 standard test method for the water vapor transmission test for materials38 with slight modifications to suit the test materials and conditions of this study. Air-dried chitin matrixes were cut into circular disks measuring ≈32 cm in diameter. The test specimens used were representative of the sample tested. The overall thickness of each specimen was measured at the center of each quadrant and the results were averaged. Measurements of 0.005 mm or more shall be made to the nearest 0.01 mm. High-density polyethylene (HDPE) containers measuring ≈4.5 × 30 cm2 were used as the specimen dish for the test. Silica gels were used to fill up to half of the HDPE containers. Porous chitin matrixes were attached to the opening of these containers by waterproof adhesive and placed inside an oven maintained at 35 °C and 65% relative humidity. The specimen was weighed at 1-h intervals for 8 h. Before each weighing, each container was shaken to mix the desiccant. The time of the weighing was made to a precision of ≈1% of the time span between each successive weighing. For every hour of weighing, a record of the weight was taken to the nearest 30 s. A HDPE container half-filled with silica gel and without any sample on the opening was placed inside the oven as a control. Results and Discussion Selection of CaCO3 as the Source of Carbon Dioxide. Of all the carbonate salts surveyed, only CaCO3 can form a stable suspension with chitin solution. Carbonates and bicarbonates of potassium and sodium will phase separate and settle to the bottom. Therefore, CaCO3 was chosen to be loaded into chitin solution. CaCO3 was reported to be often associated with chitin in insects and anthropods.39 Studies were also conducted on the chelating effect of chitin with calcium salts.40 So it was not surprising that only CaCO3 was able to form a stable suspension with chitin solution. CaCO3 is not soluble in water, but after it reacts with acid (1 N HCl), calcium chloride is produced. Calcium chloride is more soluble in an aqueous medium and can be removed after several rounds of soaking and washing. Elemental analysis showed no detectable amount of calcium in the final products.
Novel Fabrication of Open-Pore Chitin Matrixes
Figure 1. 13
13C
Biomacromolecules, Vol. 1, No. 1, 2000 63
CPMAS spectrum of CaCO3-chitin.
C Cross Polarization Magic Angle Spinning (CPMAS) Spectroscopy. 13C CPMAS spectroscopy revealed that CaCO3 was coordinated to the carbonyl moiety of the amide functionality in chitin (Figure 1). The 13C spectrum showed the existence of another peak at 168 ppm, 4 ppm upfield from the usual 172 ppm of carbonyl group of amide in chitin. The coordination of calcium to the carbonyl group shielded the group and resulted in an upfield shift. After the materials were treated with 1 N HCl and rinsed in deionized water, the extra peak was no longer found, indicating the removal of CaCO3 or CaCl2. This is in agreement with the results from elemental analysis that showed no traces of calcium. Effect of Carbon Dioxide Evolution on the Morphology of Air-Dried Chitin Matrixes. When the CaCO3-loaded gels were subjected to a 1 N HCl solution, gaseous carbon dioxide was produced together with calcium chloride and water. The evolution of gaseous carbon dioxide gas produces the desired porous morphology on the chitin gel. Uniform large pore structures were obtained with the pore size ranging from 100 to 1000 µm. Because the pores were not generated by crystallization of ice crystallites,41,42 no freeze-drying procedure was required in the process. The chitin matrixes can be obtained by a simple and cost-effective air-drying process. This method is also in marked contrast to the gas-foaming process. Although gas foaming will lead to the formation of highly porous matrixes, the external surfaces are nonporous. Various investigators have reported the collapses of external pores.43,44 A possible reason for the collapse of the pores is the rapid diffusion of dissolved gases from the surface. This will limit the nucleation of voids at the surface of the material. In contrast, loading of CaCO3 and initiation of gas evolution from within the material reduces highly porous matrixes with pores from the bulk to the surface of the matrixes. The pores formed are able to maintain the structure, even after they dried and reswelled to the original gel dimension after soaking in deionized water.
Effect of CaCO3 Concentration on the Pore Morphology. Chitin gels were cast from 0.3%, 1.0%, and 3.0% (w/ w) CaCO3 in 5% chitin solution. The gels cast from the higher concentration exhibited a higher gelation rate but a lower carbon dioxide evolution rate. It was observed that the gels cast from 0.3% and 1.0% CaCO3 concentrations showed faster disappearance of the loaded calcium salt compared to the 3.0% gels. The higher density of CaCO3 inside the gels presented a more compact structure that hindered the diffusion of HCl into the material, limiting the reaction between the calcium salt and HCl. The compact structure also hindered the evolution of gaseous carbon dioxide. At 3.0% CaCO3, pore sizes ranging from 100 to 500 µm (Figure 2.1) were produced in the internal crosssection as well as the external surface of the matrixes. The lower carbon dioxide gaseous evolution rate limited the pore size to below 500 µm. At the lower concentration of 1.0% CaCO3, a faster diffusion of HCl into the materials was observed. The gaseous carbon dioxide evolution rate was also facilitated. This resulted in larger pore sizes ranging from 500 to 1000 µm (Figure 2.2). The pores were well-defined and homogeneous throughout the chitin matrixes. At an even lower CaCO3 concentration of 0.3%, a different phenomenon was observed. The less compact gel produced a nonporous structure after the disappearance of CaCO3 from the material (Figure 2.3). It is believed that 0.3% CaCO3 is too insignificant to produce enough gaseous carbon dioxide to form porous structures. Most of the calcium salt dissolved in the aqueous HCl solution and reacted outside the gel. Therefore, gels loaded with lower CaCO3 concentrations (in this case 0.3%) will produce nonporous materials because of insufficient gaseous carbon dioxide production to form the porous structures. On the basis of this result, when the concentration of CaCO3 loaded into the gels is controlled, the pore size of the resulting materials can be predicted.
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Figure 3. Load-displacement curve of matrixes produced by loading 3.0%, 1.0%, and 0.3% CaCO3 into chitin solution.
Figure 2. SEM micrographs of matrixes produced by loading 3.0%, 1.0%, and 0.3% CaCO3 into chitin solution. (2.1) 3.0% CaCO3. (2.2) 1.0% CaCO3. (2.3) 0.3% CaCO3.
Effect of CaCO3 Concentration on the Mechanical Strength. Porous matrixes from CaCO3-chitin gels produced significantly better mechanical strength than freezedried chitosan products.45 The maximum stress at peaks for 0.3%, 1.0%, and 3.0% CaCO3 concentrations were 46.77, 2.783, and 6.670 MPa, respectively (Figure 3). Porous chitin matrixes generated from 1.0% CaCO3 showed the lowest maximum stress value because of the large pore structures formed. The lower maximum stress value reflected the weaker integrity of this matrix compared to that of the 3.0% CaCO3-generated matrix. Owing to the smaller pores formed,
a stronger matrix with ≈2.4-fold maximum stress was obtained. Both of these matrixes were lower than the nonporous material produced by 0.3% CaCO3-loaded gel. The smooth surface gave a considerably tougher material compared to the porous ones. The nonporous material also exhibited a larger value of maximum displacement at 1.49 mm compared to 1.0% CaCO3- and 3.0% CaCO3-loaded gels at 0.42 and 0.89 mm, respectively. The larger displacement of the nonporous material may be due to its more amorphous polymer structure, which permits a greater degree of elongation. On the contrary, the porous matrixes with large pores will result in crack initiation and eventually fracture. Therefore, adequate control of mechanical strength can be achieved by varying the concentration of CaCO3 loaded into the chitin gels. A more compact structure of 3.0% CaCO3-chitin gels with smaller pore size produced matrixes with better mechanical strength compared to the less compact 1.0% CaCO3-chitin gels. Nonporous matrixes produced the highest value of stress at peak and maximum displacement because of the absence of pores that instigate weakness. Effect of Temperature on the Pore Morphology. Because the evolution of gaseous carbon dioxide is an important aspect in predicting the pore size of the matrix, temperature during the evolution process is also a crucial parameter. A series of 1.0% CaCO3-chitin gels was subjected to 1 N HCl at 5, 28 (room temperature), 60, and 80 °C. At higher than ambient temperatures (60 and 80 °C), large pores of 500-1000 µm were observed. This is expected, as the larger rate of gaseous carbon dioxide evolution at higher temperatures will produce larger pore structures. The downside of producing porous matrixes at high temperatures is the low intractability of the materials. All the matrixes disintegrated during the process and were unable to undergo further testing. At lower than ambient temperature (5 °C), a very slow evolution of gaseous carbon dioxide was observed. This resulted in the production of a nonporous material with a visually transparent appearance. At ambient temperature (≈28 °C), the evolution of gaseous carbon dioxide was maintained at an optimum rate to produce large pore sizes ranging from 500 to 1000 µm. At this evolution rate, the matrix also showed good intractability and flexibility.
Biomacromolecules, Vol. 1, No. 1, 2000 65
Novel Fabrication of Open-Pore Chitin Matrixes
Table 1. Results of Permeability Test of Matrixes Produced by Loading 3.0%, 1.0%, and 0.3% CaCO3 into Chitin Solution test matrixes 3% 1% 0.30% ctrl
thickness (m)
G/t (g/h)
10-4
10-3
4.900 × 8.000 × 10-4 1.600 × 10-4 NA
7.000 × 8.000 × 10-3 7.000 × 10-3 1.300 × 10-2
A (m2) 10-4
9.500 × 9.425 × 10-4 9.616 × 10-4 9.517 × 10-4
S (mmHg)
R1 (%)
R2 (%)
38.64 38.64 38.64 38.64
65.00 65.00 65.00 65.00
38.64 38.64 38.64 38.64
test matrixes
WVT (g/h‚m2)
P (g/Pa‚s‚m2)
ave. permeability (g/Pa‚s‚m)
3% 1% 0.30%
6.650 × 10-6 7.540 × 10-6 6.731 × 10-6
6.529 × 10-9 7.403 × 10-9 6.609 × 10-9
3.199 × 10-12 5.922E × 10-12 1.057 × 10-12
Figure 4. Measurement of weight against time for the permeability test of matrixes produced by loading 3.0%, 1.0%, and 0.3% CaCO3 into chitin solution.
Water Vapor Transmission (WVT). The rate of water vapor transmission was determined graphically by plotting the weight against the elapsed time. A straight-line plot (Figure 4) was obtained for all the specimens; nominally steady-state transmission was observed. WVT ) G/tA, g/h‚m2 where G is the weight change (g), t is the time (h), G/t is the gradient of the plot (g/h), and A is the test area (m2). permeance (P) ) (WVT)/δp ) (WVT)/S(R1 - R2) where δp is the vapor pressure difference (Hg), S is the saturation vapor pressure at the test temperature (mmHg), R1 is the relative humidity at the source, and R2 is the relative humidity at the vapor sink, 0% for desiccant. average permeability ) permeance × thickness The average water vapor permeability is defined as the rate of water vapor transmission through the unit of flat material of unit thickness induced by the unit vapor pressure difference between two specific surfaces, under specific temperature and humidity conditions. The highest average water vapor permeability of 5.922 × 10-12 g/Pa‚s‚m was obtained from porous matrixes of 1.0% CaCO3-chitin gels (Table 1). This is 1.9- and 4.9-fold more than the average permeability from the 3.0% CaCO3- and 0.3% CaCO3-loaded chitin gels of 3.199 × 10-12 and 1.057 × 10-12, respectively. The larger pore size of 500-1000 µm in a 1.0% CaCO3-generated matrix allowed greater water vapor transmission across the material. This is followed by the 3.0% CaCO3 gel with 100500-µm pores and nonporous material from 0.3% CaCO3
Figure 5. Water uptake profile of matrixes produced by loading 3.0%, 1.0%, and 0.3% CaCO3 into chitin solution.
gel. From this study, we are able to control the water vapor transmission across a material by controlling the pore size. This is critical in the design and fabrication of scaffolds for tissue engineering, especially artificial skin generation. Water Uptake Ability. All three types of chitin matrixes absorbed more than twice their weight of water (Figure 5). All matrixes were saturated in the first 50 min of water uptake. On the basis of the water uptake experiments, the amount of water absorbed depends on not only the pore size of the sample but also the ability to retain the water gained. The porous matrixes will be able to absorb more water during the uptake procedure with the largest ratio of about 3.5- and 3.0-fold original weight from the matrixes of 1.0% CaCO3and 3.0% CaCO3-chitin gels, respectively. This is in stark contrast to the 1.7-fold ratio of the nonporous material from 0.3% CaCO3-chitin gel. A matrix generated from 3.0% CaCO3-chitin gel contained a smaller pore size compared to the matrix from 1.0% CaCO3-chitin gel; its compact structure prevented a larger uptake of water during the experiment. This resulted in a lower uptake by 0.5-fold when compared to that of the 1.0% CaCO3-chitin gel. All the chitin materials showed good intractability during the water uptake experiments. The maximum water uptake was maintained for more than a week without any decrease in the wet weight of the chitin matrixes or without disintegration. Porosity Measurement. The porosities of the materials were not determined by a mercury intrusion porosimeter because of the compressible texture of the materials. The densities of the materials were measured to provide an estimation of the porosities. By measurement of the weights and dimensions of the materials before and after soaking into
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Table 2. Dimension Measurements and Porosity of Matrixes Produced by Loading 3.0%, 1.0%, and 0.3% CaCO3 into Chitin Solution sample 3.0% initial swelled
length (cm)
width (cm)
thickness (cm)
vol (cm3)
weight (g)
density (g/cm3)
porosity (%)
2.000 2.000
2.000 2.000
0.070 0.100
0.280 0.400
0.053 0.318
0.190 0.795
76
sample 1.0% initial swelled
length (cm)
width (cm)
thickness (cm)
vol (cm3)
weight (g)
density (g/cm3)
porosity (%)
1.700 1.700
1.800 1.800
0.048 0.082
0.147 0.251
0.045 0.407
0.415 1.623
81
sample 0.3% initial swelled
length(cm)
width(cm)
thickness(cm)
vol (cm3)
weight (g)
density (g/cm3)
porosity(%)
1.800 1.800
1.800 1.800
0.048 0.052
0.156 0.168
0.085 0.153
0.547 0.909
40
deionized water, we can estimate the density of the initial and swelled materials. The porosity () of the sample was estimated by ) Vp/Vt ) (1 - F2/F1) × 100 where Vp is the pore volume, Vt is the total volume, F2 is the density of the initial material, and F1 is the density of the swelled material. The porosities of the materials were estimated to be at 81%, 76%, and 40% for the 1.0%-, 3.0%-, and 0.3%-loaded CaCO3-chitin gels (Table 2). Matrixes produced from 1.0% CaCO3- and 3.0% CaCO3-chitin gels showed a high degree of porosity, comparable to those found in porous chitinous materials formed by cryogenic-induced phase separation. 32 Conclusion This work has presented a new approach to the fabrication of porous matrixes, different from all the other conventional methods. When CaCO3 is loaded into chitin solution, a simple, cost-effective, and rapid way of matrix production can be achieved. This novel method of porous matrix fabrication is particularly suitable for chitin because it has very limited processing options resulting from its insolubility in most solvents and the absence of melt temperature. We are also able to control the pore dimensions and porosity of the chitin matrixes by varying the amount of CaCO3 loaded into the chitin solution. These matrixes showed good water vapor permeability and water uptake ability with good mechanical strength. The formation of open-pore structures on the surface of the matrixes provides an ideal system for cell implantation in tissue engineering and other applications that require a three-dimensional matrix with large surface areas. Acknowledgment. The authors would like to thank The National University of Singapore and National Science and Technology Board, Singapore, for financial support (RP 960666/A). Kok Sum Chow would also like to thank the University for his research scholarship.
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