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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.53 n.1 Concepción mar. 2008 


J. Chil. Chem. Soc, 53, N° 1 (2008)



1Faculty of Veterinary and Animal Sciences, University of Chile and Center for Advanced Interdisciplinary Research in Materials (CIMAT). Santa Rosa 11735, P.O. Box 2-15. La Pintana, Santiago,

2Faculty of Physics and Mathematics Science. Av. Tupper 2069, Box 2777, University of Chile. Santiago, Chile.

*Corresponding author: Dr. Andrónico Neira-Carrillo.


The present work deals with the effect of the constrained volume given by crosslinked chitosan (CHI) as a sphere on the in vitro CaC03 crystallization. Crosslinked CHI was obtained with formaldehyde (FA), glutaraldehyde (GA), epichlorhydrine (EPCH) and poly (propylene glycol) diglycidyl ether (PPDGE) as crosslinking agents. Determination of swelling percentage (%) of the crosslinked CHI spheres was carried out in TRIS buffer at pH 9. Spheres of high molecular weight were prepared using drops of CHI solution on NaOH. In vitro CaC03 crystallization using gas-diffusion method was done. In addition, a synthetically sulphonated containing polymethylsiloxane (S-PMS) was used as modifying additive on the CaC03 crystals growth in a confined space of CHI. The degree of the crosslinking altered the diffusion of C02 gas through the CHI spheres during the CaC03 crystallization resulting in different and specific crystals morphologies.

Keywords: chitosan, crosslinking agents, gas-diffusion method, crystallization and calcite crystals.



Biomineralization is a widespread phenomenon in nature by which living organisms influence the crystallization of inorganic minerals and leads to the formation of precisely controlled inorganic-organic composites (mollusk and egg shells, crustacean carapaces, echinoderm exoskeleton and spines, corals, bones and teeth), in which the minute organic part exerts substantial control on the mineralization process1,2. Thus, the resulted inorganic material shows uniform size particles, novel crystal morphology, specific crystallographic orientation and remarkable properties3,4. The control of mineralization by biological molecules and how crystal polymorphism and structure can be controlled by organic molecules additives has been extensively studied5,6. Small amounts of acid-rich proteins and proteoglycans play a major role in forming the biomineralized composites by influencing mineral crystal nucleation and growth7,8,9,10. Calcium carbonate (CaC03) represents one of the major inorganic mineral and has been extensively investigated. CaC03 crystals have three polymorphs: calcite (hexagonal), aragonite (orthorhombic) and vaterite (hexagonal). In nature it growth is typically heterogeneous crystallization and occurs in association with surfaces in a constrained volume space. It is known that organisms produce a geometrically well define microenvironment, controlling not only the addition of the organic molecules but also the localization and velocity of ions flux, pH and supersaturation.

Furthermore, the mineralization mechanism is altered by different chemical groups (e.g., amine, sulfate, and carboxylate) and functionalized networks11,12. The resulting morphology of the in vitro crystals is an expression of different growth rates in the various crystallographic directions, modulated by the adsorbed additives present in solution. Different approaches have been used to synthesize specific polymorphs of CaC03 in various forms such as: films, spheres, sponge-like structures, ligand-receptor complexes, block copolymers and synthetic polypeptides13,14,15,16. In order to understand the biogenic crystallization of inorganic materials using synthetic systems we investigate the effect of the constrained volume using sphere of crosslinked chitosan (CHI) on the in vitro CaC03 crystallization. CHI spheres were prepared using drops of CHI solution on NaOH. CHI (poly-/?-(1^4)-2-amino-2-deoxy-D-glucose) is obtained through partial deacetylation of chitin (poly-/?(l—>4)-2-acetamido-2-deoxy-D-glucose) which is the second most abundant polysaccharide in nature17"20.

The work includes in this paper is therefore focused on the preparation of the confined space of CHI spheres from commercial CHI of high and low molecular weight and we study their effect on CaC03 in vitro crystallization. The following crosslinking agents: formaldehyde (FA), glutaraldehyde (GA), 2-(chloromethyl) oxirane or epichlorhydrine (EPCH) and poly (propylene glycol) diglycidyl ether (PPDGE) were tested. In addition, poly (methyl ethyl benzene sulfonic acid) siloxane polymer (S-PMS) was used as modifying sulphonated additive on CaC03 crystallization in the confined CHI spheres. The synthesis and characterization of S-PMS was prepared through hydrosilylation and sulphonic reactions and its influence as a template on the CaC03 crystals will be soon reported21.


1. Materials

Chitosan samples of high molecular weight (Mw = 350 kDa, >83% deacetylation) from Aldrich and low molecular weight (Mw = 70 kDa, >75% deacetylation) from Fluka were washed with acetone and methanol and dried to constant weight. Calcium chloride dihydrate, ethanol and Tris(hydroxymethyl) aminomethane were obtained from ACS-Merck and ammonium hydrogen carbonate was from XT. Baker. These reagents were of the high available grade. The distilled water was obtained from capsule filter 0.2 |itn flow (U.S. Filter). The degree of swelling (%) for each CHI spheres was estimated between dry and swollen spheres using an analytical balance (Precisión-Hispana, model AE 200).

2.    Formation of crosslinked CHI sphere and swelling (%) determination

The concentration of the crosslinking agents was 5xlO"2M in NaOH 0.067 M solution at 40°C for 3 h. The spheres of crosslinked CHI were obtained using drops of CHI solution in the range of 0.25-5.0 % in acetic acid at 5% on NaOH. Spheres of CHI are formed in situ when drops of CHI solution is fall down on concentrated NaOH solution (Fig. 1). Moreover, crosslinked spheres of CHI were prepared in the presence of S-PMS at concentration of 1 % (wt/vol). The introduction of S-PMS inside the sphere of CHI was done using microinjection technique with a syringe. The swelling percentage (%) for each crosslinked CHI sphere were determined as follow: a dried sphere of CHI (vacuum for lh at 60 °C) was put in a graduated beaker and then a known volume of buffer TRIS solution at pH 9.0 was added until covering completely the spheres and transferred in a thermostatized bath at 40°C for 3 h. After this time, the spheres were carefully dried with a filter paper and weight again. The determination of the swelling percentage (%) was carried out with all CHI spheres, that is, with and without crosslinked CHI. The swelling percentage (%) was calculated using the following equation:

2. In vitro CaC03 Crystallization

The gas-diffusion crystallizations (Fig.2) were done using a chamber consisting of 85 mm plastic Petri dish having a central hole in its bottom glued to a plastic cylindrical vessel. Inside the chamber, polystyrene microbridges with a crosslinked CHI sphere were filled with 35 uL of 200 mM CaCl2 solution in 200 mM TRIS buffer pH 9. The cylindrical vessel contained 3 ml of 25 mM NH4HC03 solution. All experiments were carried out inside the Petri dish using different pH at 20 °C for 24 h. CaC03 crystals results from the diffusion of C02 gas into the buffered CaCl2 solution. CaC03 grown inside CHI spheres were collected and once rinsed with distilled water and on 50 to 100 % gradient ethanol solution, dried at room temperature and then coated with gold using an EMS-550; automated sputter coater. All CaC03 crystals were observed in a Tesla BS 343 A microscope.


In order to find the optimal experimental condition for the formation of CHI spheres, different concentrations of CHI in acetic acid solutions at 5% in the range of 0.25-5.0 % in NaOH were tested (Table 1).

We suspect that the crosshnking degree in the CHI spheres could alter the diffusion of C02 gas through the sphere during the CaC03 crystallization and will lead to the control of CaC03 nucleation and changing the crystals morphology with some specific crystallographic orientation. The determination of the swelling percentage (%) of the CHI and crosslinked CHI spheres for both molecular weight of CHI in TRIS buffer at pH 9 is shown in the Fig. 3(a,b). The asterisk (*) symbol in case of FA and GA indicates that they were used in acidic media, as well. Fig. 3(a) shows that GA and EPCH have a lower swelling percentage (%) value than PPDGE and FA. Whereas PPDGE and FA showed higher swelling (%) value that the control CHI sample. For both FA* and GA*, the acidic media was a better experimental condition for the crosshnking effectiveness in which the swelling (%) decreased and more crosslinked spheres were obtained. Moreover, GA and EPCH produced good and defined unchanged CHI spheres after the swelling process. In case of (Fig.3b) all the crosshnking agents presented lower swelling (%) value than the control sphere indicating that the effectiveness of these agents was better with LMW of CHI. Like in Fig.3a, EPCH and GA showed higher effectiveness of crosshnking degree.

Fig. 4 (a,b) shows a comparison of the swelling (%) of crosslinked CHI sphere obtained in the presence of S-PMS for both molecular weights of CHI. As before, the asterisk (*) for FA and GA indicates that they were used in acidic media and the symbol +P after the previous crosshnking agents in the graph indicates that the swelling process was done in the presence of S-PMS polymer. In the Fig. 4a, the swelling value for FA, PPDGE and EPCH in the presence of S-PMS was lower than without this polymer. However, for GA in both media, the swelling (%) was higher. This observation suggests that the presence of sulphonated moieties along the backbone of the chain of S-PMS polymer interacts with the amine group of CHI modifying the crosshnking process. However, when the S-PMS was introduced in CHI sphere of LMW (Fig. 4b) the swelling (%) value for FA (in both media) and EPCH do not change. For the crosslinked CHI sphere obtained with GA (in both media) and PPDGE the swelling (%) value was higher. Thus, when the S-PMS polymer was microinjected into the CHI spheres modifyed the crosshnking process showing different swelling (%). In general crosslinked CHI spheres for both high and low molecular weight prepared with GA and EPCH agents showed the lowest swelling (%) value in the presence of S-PMS.

In order to evaluated the effect of the crosshnked CHI as a constrained volume on the formation of CaC03 crystals a set of in vitro crystallization experiments were performed with spheres of CHI using HMW of CHI prepared by soaking the spheres in the buffered CaCl2 solution (see Figure 2). The crystallization of CaC03 was based on the gas-diffusion method in TRIS buffer pH 9 at 20 °C for 24 h. The S-PMS was incorporated in situ during the formation of CHI spheres and used as an additive on the CaC03 crystallization to observe its effect on the crystal morphology compared with crystals obtained without S-PMS. If the presence of S-PMS affect the microenvironment obtained by the crosshnking agents in the crosshnked CHI spheres we will expect that the CaC03 crystals show morphological modifications. In fact, SEM analysis showed that crystals growth in the crosshnked CHI sphere are dramatically influenced by the chemical microenvironment and was possible to reproduce experimentally similar crystals modifications obtained with biological molecules in nature.

By using the S-PMS at 1.6 mg/ml, it was possible to obtained well defined CaC03 crystals which were deposited on the surface of CHI sphere and growth inside ofthe sphere withdifferentmorphologies. Figure 5 Ashows control CaC03 crystals obtained without crosshnking agent, which resulted in rhombohedra] calcite crystals in both outside (5 As) and inside part (5 Ai) of CHI spheres. These crystals are in a size range of 7 to 10 urn. When crystallization was carried out with crosshnked CHI spheres obtained with EPCH in the presence of S-PMS some corners ofthe crystals were rounded and the crystals faces were smooth and exhibited no etch pits (Fig. 5Bs). The size of these crystals was from 10 to 30 urn. However, all crystals grown inside the crosshnked sphere resulted in a mixture of small single crystals, which aggregates forming rosette-like crystals (Fig. 5Bi). Moreover, these aggregated crystals are uniformly distributed in CHI sphere with size from 30 to 50 urn. The resulting aggregation of crystals grown inside indicated that it was possible to obtain in vitro calcite crystals with similar morphology to those occurring in some natural systems e.g.: in the eggshell membranes modulated by proteoglycans and proteins (see Fig. 7yo,22,23 Also, when the same crosshnking agent was used in the presence of S-PMS during the CHI sphere formation, we found similar aggregated CaC03 crystals with major distribution (5Ci).

Figure 6 (A-C) shows the CaC03 crystals grown in the presence of crosshnked CHI sphere with GA at 20°C for 24 h. Figure 6As shows CaC03 crystals deposited on the CHI sphere surface which resulted in rhombohedra calcite crystals but with notorious corner modification. In contrast, fragmented CaC03 crystals deposition was observed when crystals were grown inside the crosshnked CHI sphere (Fig. 6i). However, when the GA* agents with high effectiveness of crosshnking degree was used as template substrate, a very flat plate of CaC03 crystals growth in the crosshnked CHI spheres demonstrating the strong influence ofthe chemical micro-environment. In addition, modified calcite crystals at the surface of crosshnked CHI spheres were observed (Figure 6 Bi). In the case of crosshnked sphere obtained with GA in the presence of S-PMS in solution, similar aggregated crystals composed with single small calcite crystals were observed (Fig. 6 Ci). The resulting crystals grown on the surface ofthe sphere show atypical rhombohedra calcite crystal characteristic of the control samples. The results obtained here are in accordance with the assumption that the presence of sulphonated S-PMS polymers leads to local accumulation of Ca2+ what relates with to the polymers nature, concentration, volume space and pH in the system.

The role of proteins in biomineralization and the mechanism of eggshell formation are not well understood. Different isolated and purified proteins from chicken (ovocleidin 17, and C-type lectins) and gose eggshell matrix (ansocalcin) with homologous amino acid sequence have been obtained23. The poly crystalline aggregates growing on mammillae of the eggshell membrane22 with these biological proteins appear to be similar to calcite crystals randomly deposited inside the crosslinked CHI sphere. Figure 7 illustrate the capability to reproduce in vitro calcite crystals deposition on a synthetic substrate as compared with the natural post- oviposition eggshell membrane incubated in calcium chloride, at pH 7.4 without any additive22. Understandings the role of various functional groups at the surface of synthetic polymers and how proteins control the morphological changes of the crystal in the natural bioceramics will increase our capability to understand the nucleation, growth and orientation processes and to develop functional and advanced biomaterials9,24.

The Figure 8 shows a summary of the calcite crystals grown inside the CHI spheres obtained after the in vitro CaC03 crystallization at 20°C for 24 h. Figure 8 (a-d) represents the crystals grown inside of crosslinked without S-PMS. Figure 8 (e-1) shows the crystals morphology obtained in the presence of S-PMS using the microinjection technique (e-h) and in the presence of S-PMS in solution (i-1) crosslinked with EPCH, FA, GA and PPDGE, respectively.


In summary, the crosslinking degree of the CHI sphere altered dramatically the flux of C02 gas velocity during the in vitro CaC03 crystallization showing inside of the CHI spheres different and specific crystals morphologies. The swelling test carried out with all crosslinking agents was close related with the crystals modification. The effect of GA and EPCH in contrast of PPDGE resulted more effective and weak, respectively. It was found that the presence of S-PMS can effectively control the morphogenesis of CaC03 crystals which is strongly dependent of the chemical environment of the crosslinked CHI sphere. In addition, S-PMS can undergo changes in the charge of sulfonate groups and adopt different orientations in a confined space than in solution, and thereby elicit changes in CaC03 morphology. We surmise that the crystallization of calcite, which is triggered by the sulphonated moieties of S-PMS, results from a local accumulation of Ca2+ ions which correlates closely with the polymer's nature, concentration, incubation time and pH26. Finally the use of functionalized polysiloxanes chemistry as an flexible additive in a confined space templates27,28 provides a viable approach for studying various aspects of biomineralization including production of controlled particles, polymorphism and defined morphologies.


This research was supported by FONDAP 11980002 granted by the Chilean Council for Science and Technology (CONICYT) and Fund. Andes C-14060/31.


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(Received: 3 December 2007 - Accepted: 23 January 2008)