Alginate hydrogels are an import class of ionic biopolymers for medical and biotechnological
applications, produced by the combination of alginate with a proper cross-linking agent.
During the gel formation, cross-links can be formed between the alginate chains and cationic
species, changing the elastic behaviour of the material that controls the volume change
phenomena of the gels.
This paper proposes a new rapid prototyping system to produce three-dimensional alginate
structures by extruding, layer-by-layer, a previously prepared solution of sodium alginate in
water mixed with a solution of calcium chloride, both of known concentration. The building
process to obtain these gel structures is described, from a chemical point of view, and some
obtained structures are shown. The alginate concentration over both the kinetics and accuracy
effects of the process is investigated. The preliminary findings of this research work promise
to open an exciting new area for medical applications.Mechanical Engineerin

Bártolo, Paulo

Lagoa, Ricardo

Mendes, Ausenda

Texas ScholarWorks

ALGINATE-BASED RAPID PROTOTYPING SYSTEMP.J. BARTOLO, R. LAGOA, A. MENDESDepartment of Mechanical Engineering, School of Technology and ManagementPolytechnic Institute of LeiriaMorro do Lena, Alto do Vieiro, Leiria, PortugalReviewed, accepted August 13, 2003AbstractAlginate hydrogels are an import class of ionic biopolymers for medical and biotechnologicalapplications, produced by the combination of alginate with a proper cross-linking agent.During the gel formation, cross-links can be formed between the alginate chains and cationicspecies, changing the elastic behaviour of the material that controls the volume changephenomena of the gels.This paper proposes a new rapid prototyping system to produce three-dimensional alginatestructures by extruding, layer-by-layer, a previously prepared solution of sodium alginate inwater mixed with a solution of calcium chloride, both of known concentration. The buildingprocess to obtain these gel structures is described, from a chemical point of view, and someobtained structures are shown. The alginate concentration over both the kinetics and accuracyeffects of the process is investigated. The preliminary findings of this research work promiseto open an exciting new area for medical applications.Keywords: Alginate, Hydrogels, Biomimetics, Medical Applications, Rapid prototyping.IntroductionThe engineering activity, a design by human process, is usually portrayed as a unique humanprocess, relying on the intuition, experience and judgment of engineers and designers, tocreate a representation of objects meeting a set of requirements. However, there is also adesign by nature (Bartolo and Bartolo, 2003) process, the process through which all theorganisms dynamically adapt to its environment, performing its different functions, such asrespiration, reproduction, sensing, movement, etc.  This design by nature is an evolutionarytransformation process with a unique goal, the survival of each population of organismsthrough the correct adjustment to the environment. This kind of design process has beenoptimised throughout years and years of natural evolution.Just as an ecosystem incorporates change and adapts towards self-sustaining processes,lessons from nature can be used to support and optimise the design by human activity. Thephilosophy under this research project is the belief that engineers can learn with nature. Thisprocess of borrowing the best from nature is called biomimetics (Jeronimidis and Atkins,1995) and this research study focuses on this new concept, investigating a new route toproduce three-dimensional patterns in alginate hydrogels for medical applications, through abiomimetic free form manufacturing system. This system intends to replicate naturalprocedures used by some marine brown algae, namely Laminaria Hyperborea, to producealginate, a structural component of the algae.Hydrogels have been receiving much attention due to their potential use in a wide variety ofbiomedical applications, including tissue engineering scaffolds, drug delivery, contact lenses,corneal implants and wound dressing (Griffith and Naughton, 2002; Garcia and Ghaly, 1996;60Kim et al., 1998). Most hydrogels are usually synthesized using reactive monomers and cross-linkers. However, they do consume non-renewable resources, employ hazardousmanufacturing steps and are not biodegradable, apart from not always being biocompatible sotheir use in medical applications is limited. Consequently, research attention has been centredon generating hydrogels from natural and biocompatible polymeric materials, such ascollagen, gelatin, chitosan and alginate. Biomaterials present properties, such asbiocompatibility, biodegradability, sensitivity to pH, electrical or temperature stimuli, thatenables its use in biotechnology and medical applications.Biologic manufacturing systemThe alginate-based rapid prototyping system, which is in a preliminary research stage, intendsto produce alginate solid structures by extruding a previously prepared solution of sodiumalginate in water mixed with a solution of calcium chloride, both of known concentration.Understanding the reaction kinetics of the gelation process of alginate-based systems is thefirst step for the development of an intelligent system leading to the design, optimisation andcontrol of an alginate-based rapid prototyping system.Alginate is a natural linear polysaccharide that contains 1,4-linked b-D-mannuronic (M) anda-L-guluronic (G) acid residues (Figure 1), arranged in a non-regular and block-wise fashionalong the chain (Kang et al, 2002). The biological properties of this biopolymer have beenexploited in numerous medical and surgical applications, such as drug delivery, cellencapsulation, etc (Gombotz and Wee, 1998; Bucke, 1987).OHOHHOHOHHHCOO b D Mannuronic acida L Guluronic acidOHOHHOHOOFigure 1: Structure of an alginate showing a linkage between the mannuronic and guluronicacid.Gelling occurs when divalent ions (Ca2+, Ba2+, Fe2+, Sr2+, etc.) or trivalent ions (Al, etc.) takepart in the interchain ionic binding between G-blocks in the polymer chain giving rise to athree dimensional network. Such binding zones between the G-blocks are often referred to as“egg boxes” (Figure 2). This ions act as cross-linkers that stabilise alginate chains forming agel structure, which contains cross-linked chains interspersed with more freely movablechains that binds and entraps large quantities of water. The gelling process is characterised bya re-organisation of the gel network accompanied by the expulsion of water (Serp et al.,2002).Its reactivity with calcium and the subsequent gel formation capacity is a direct function ofthe average chain length of the G blocks, showing a great affinity for Ca2+ ions (Gombotz andWee, 1998). Gels made of M-rich alginate are softer and more fragile, and may also havelower porosity. This is due to the lower binding strength between the polymer chains and to61the higher flexibilities of the molecules. The gelling process is highly dependent upondiffusion of gelling ions into the polymer network.Figure 2: Alginate gelling results from reticulation of the chains by calcium ions.MaterialSodium alginate was purchased at Panreac (Barcelona, Spain). Calcium chloride was suppliedby Carlo Erba (Milano, Italy). All solutions were prepared with pure water, with conductivityof 0.054 mS/cm. Alginate solutions were prepared by addition of weighted portions of sodiumalginate to measured volumes of water. Due to their high viscosity, these solutions wereagitated by orbital shaking for three hours at 50 ºC to ensure good homogeneity. Calciumchloride solution 5% (w/v) was obtained dissolving the salt in water. This solution wasdiluted to obtain solutions containing different concentrations of calcium chloride.ResultsTo evaluate the kinetics of the gelling process solutions, containing different concentrationsof alginate and different concentrations of calcium chloride (CaCl2), they were prepared andmixed at room temperature. The effect of the alginate concentration is shown in Figure 3,which describes the weight loss as a function of time for solutions containing differentamounts of alginate (1% and 2%) mixed with a solution of 5% CaCl2. Figure 4 illustrates thevariation of weight loss as a function of time, for a solution containing 2% of alginate mixedwith solutions containing different amounts of CaCl2. The experimental data (discrete values),was fitted using a sigmoidal equation. Fitting the experimental data enables us to have acontinuous range of values to better evaluate the gelation process. To correlate theexperimental data and the values obtained from the sigmoidal equation, a numerical routineusing the Marquardt-Levenberg (Press et al., 1986) multivariable non-linear regressionmethod was employed. A good correlation was achieved by controlling the number of steps,the increment and a small tolerance parameter, corresponding to the difference of values at thestep n+1 and values at the step n, which is used for convergence purposes.Ca2+Sol structure Gel structure62Figure 3: Weight loss vs gelation time for two solutions containing different concentrationsof alginate mixed with a solution of of 5% CaCl2Figure 4: Weight loss vs gelation time for a solution containing 2% of alginate mixed withsolution containing different concentrations CaCl2.The weight loss increases with time and is more significant for samples produced by solutionscontaining low contents of alginate and high contents of calcium chloride is shown in Figures3 and 4. This is due to two main reasons:ß the amount of water present in the initial alginate solution, which is higher in moredilute solutionsß the kinetics of the gelation process, which is higher whenever solutions containinghigher concentrations of CaCl2 are used.The calcium divalent ions act as cross-linkers that stabilise alginate chains forming a gelstructure. Therefore, increasing the concentration of calcium chloride present in the solutionincreases the cross-linking of polymeric chains and the expulsion of water. During the gelformation, calcium ions diffuse from the liquid solution to the forming gel and itscomplexation by the carboxylic groups of G blocks increases the gel stiffness. Thisphenomenon is accompanied by a significant loss in water at counter-current of the flux ofcalcium ions (Figure 5). Ahmad and Huglin (1994) and Hu and Lin (1994) claim that thewater contained in alginate gels can been found in two states: free-water and non-free-water.Alg 1% Time (min)0 20 40 60 80 100 120 1400,00,10,20,30,4Alg 2% Time (min)0 20 40 60 80 100 120 1400,000,050,100,150,200,250,30CaCl21%Time (min)0 20 40 60 80 100 120 1400,000,050,100,150,200,25CaCl2  5% Time (min)0 20 40 60 80 100 120 1400,000,050,100,150,200,250,3063Therefore, it is expected that the water leaving the gel during the gelation process is the onewhich interacts less with the polymer (free-water).Figure 5: The diffusion of chemical elements during the gelation process of alginatestructures.Figure 6 compares the variation of the rate of weight loss with time as a function of bothalginate and CaCl2 concentration. This Figure shows that maximum value of the rate ofweight loss is almost independent of the concentration of alginate or calcium chloride andoccurs very fast, in less than one minute.  However, the rate of weight loss is higher forsamples produced with solutions containing higher concentrations of alginate or solutionscontaining higher concentrations of CaCl2.Figure 6: Variation of the rate of weight loss vs time as a function of a) alginateconcentration for samples produced through a solution containing 5% of CaCl2; and b)calcium chloride concentration for samples produced through a solution containing 2% ofalginate.The variation of the maximum value of the rate of weight loss with the concentration ofcalcium chloride for samples produced using a solution of 2% of alginate was fitted using asigmoidal equation (r2=1) as shown in Figure 7. This variation reveals two main regions. Thefirst region corresponds to the gelation processes performed with solutions containingconcentrations of CaCl2 lower than 3%. In this region, the concentration of CaCl2 has greatimpact on the rate of weight loss and consequently on the gelation kinetics. The second regionappears at high values of CaCl2.  This can be attributed to diffusion limitations of the divalentions of Ca2+ as the gel structure is formed. Analogous behaviour is observed in Figure 8 forAlginatewaterCa2+ExternalenvironmentTime (min.)0 1 2 3 4)0,000,050,100,150,200,252% Alg3% Alg1% AlgTime (min.)0 1 2 3 4fi)0,000,020,040,060,080,100,120,140,160,180,205% CaCl22% CaCl21% CaCl2a) b)6465Figure 9: Variation of the shrinkage as a function of a) time and b) weight loss of a discproduced through a solution with 2% of alginate mixed with a solution containing 5% ofCaCl2.The accuracy in the definition of the geometric shape of alginate structures is also stronglydependent on both the concentrations of alginate and calcium chloride. Figure 10 displays theshapes of different circular structures produced by mixing a solution with 2% of alginate withsolutions containing different concentrations of CaCl2. A better definition is obtained usinghigh contents of alginate and CaCl2. The gelation process of alginate structures will enable toretain the shape of structures during a large period of time (see Figure 11), owing to the fastprocess of he gelation rate during the first 5 minutes, that slows down afterwards slowingdown the rate of weight loss.Figure 10: Circular structures produced by mixing solutions containing differentconcentrations of alginate with solutions containing different concentrations of CaCl2.Time (min.)0 20 40 60 80 100 120 1400,220,240,260,280,300,320,340,360,380,400,42Weight loss (%)0,10 0,15 0,20 0,250,200,240,280,320,360,400,44a)b)66Figure 11: An alginate structure three weeks after its generation.A phenomenological model previously developed for stereo-thermo-lithographic processes(Bartolo and Mitchell, 2003; Bartolo and Mitchell, 2001) was adopted to evaluate the kineticsof the gelation process of alginate systems. According to this model, the rate of weigh loss,dW/dt, is given by the following equation:( )nm W1WdtdW-⋅G=where G is the gelation rate constant, W is the fractional value of weight loss, and m and n arethe exponential coefficients of the kinetic model. All the kinetic parameters have been foundto be dependent on both the alginate and calcium chloride concentration.The rate constant is given by the following equation:[ ] [ ]˜˜¯ˆÁÁËÊ⋅˜˜¯ˆÁÁËÊ⋅=GlgexpCaClexp2 Ajgxwhere x, g and j are constants, [CaCl2] is the concentration of calcium chloride and [Alg] isthe concentration of alginate. The rate constant was found to be dependent on the alginate andcalcium chloride concentrations in an Arrhenius way as shown in Figure 12.1/(concentration of alginate (%))0,2 0,4 0,6 0,8 1,0 1,2(G(i))-2,4-2,2-2,0-1,8-1,6-1,4-1,2-1,0-0,81/(concentration of CaCl2 (%))0,0 0,2 0,4 0,6 0,8 1,0 1,2G ()-1,6-1,4-1,2-1,0-0,8-0,6-0,4-0,2a) b)Figure 12: a) Variation of ln (k) vs. (1/concentration of CaCl2) for samples produced using asolution containing 2% of alginate. b) Variation of ln (k) vs. (1/concentration of alginate) forsamples produced using a solution containing 5% of CaCl2.67The exponential coefficients’ dependencies with the CaCl2 concentration are determinedthrough an exponential regression (r2=1 for the exponential coefficient n and r2=0.98 for thecoefficient m), and are shown in Figure 13. This Figure reveals that the exponentialcoefficient m increases with the concentration of calcium chloride, while the coefficient ndecreases. Similar behaviour is observed for exponential coefficients’ dependencies with thealginate concentration.Figure 13: Variation of the exponential coefficients vs. concentration of CaCl2 for samplesproduced using a solution containing 2% of alginate.The model intends to provide:ß a better understanding of the gelation process;ß a more accurate estimation of build times and weight loss profiles;ß a platform for testing new alginate structures or other bio-polymeric systems andprocess improvements;ß the development of a standard set of parameters and gels for some pre-determinedthree-dimensional model characteristics.ConclusionsA new rapid prototyping system to produce three-dimensional alginate structures has beendescribed from a chemical point of view. The gelation process is strongly dependent on boththe alginate and calcium chloride concentrations. During the gelation process a significantwater loss is observed influencing the shrinkage of the produced structures. Stable alginatestructures are obtained after 20 minutes and the shape of these structures is retained for a longperiod of time.A kinetic model, describing the gelation process, is also proposed. This computational modelwill support the development and optimisation of this alginate-based rapid prototypingsystem, avoiding a great amount of experimental work.Concentration of CaCl2 (%)0 1 2 3 4 5 6Coefficient n1,821,841,861,881,901,921,941,96Concentration of CaCl2 (%)0 1 2 3 4 5 6Coefficient m0,040,060,080,100,120,140,160,1868Future work will focus on the relationship between morphology/mechanical properties andgelation kinetics in order to better understand the generation process of gel structures. Thiswill help to produce appropriated porous structures for tissue engineering.ReferencesAhmad, M.B. and Huglin, M.B., DSC studies on states of water in cross-linked poly(methylmethacrylate-co-N-vinyl-2-pyrrolidine) hydrogels, Polymer Int., 1994, 33, 273-277.Bartolo, H.M and Bartolo, P.J., Learning from biological systems, in InnovativeDevelopments in Architecture, Engineering and Construction, Edited by C.J. Anumba,Millpress, Rotterdam, 2003.Bartolo, P.J. and Mitchel, G., Modelling the curing kinetics of thermosetting resins instereolithographic applications, Proceedings of the 7thEuropean Conference on AdvancedMaterials and Processes, Published by Associazione Italiana di Metallurgia, 2001.Bartolo, P.J. and Mitchell, G., Stereo-thermal-lithography: a new principle for rapidprototyping, Rapid Prototyping Journal, 2003, 9, 150-157.Bucke, C., Cell immobilization in Ca-alginate, Methods Enzymol., 1987, 135, 175-188.Garcia, A.M. and Ghaly, E.S., Preliminary spherical agglomerates of water soluble drug usingnatural polymer and cross-linked technique, J. Control Rel., 1996, 40, 179-186.Gombotz, W.R. and Wee, S.W., Protein release from alginate matrices, Adv. Drug Deliv.Rev., 1998, 31, 267-285.Griffith, L.G. and Naughton, G., Tissue engineering – current challenges and expandingopportunities, Science, 2002, 295, 1009-1014.Hu, D.S.G. and Lin, M.T.S., Water-polymer interactions and critical phenomena of swellingin inhomogeneous poly(acrylonitrile-acrylamide-acrylic acids) gels, Polymer, 1994, 33, 4416-4422.Kang, H., Jeon, G., Lee, M., Yang, J., Effectiveness test of alginate-derived polymericsurfactants, J Chem Technol Biotechnol, 2002, 77, 205-210.Kim, S.S., Utsunomiya, H., Koski, J.A., Wu, B.M., Cima, M.J., Sohn, J., Mukai, K., Griffith,L.G., Vacanti, J.P., Survival and function of hepatocytes on a novel three-dimensionalsynthetic biodegradable polymer scaffold with an intrinsic network of channels, Ann. Surg.,1998, 228, 8-13Press, H., Flannery, B.P., and Teukolsky, S.A., Numerical recipes, Cambridge UniversityPress, Cambridge, 1986.Serp, D., Mueller, M., Stockar, U. and Marison, I.W., Low-temperature electron microscopyfor the study of polysaccharide ultrastructures in hydrogels. II. Effect of temperature on thestructure of Ca2+-alginate beads, Biotechnology and Bioengineering, 2002, 79, 253-259.69

Alginate-Based Rapid Prototyping System

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Alginate-Based Rapid Prototyping System

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