STUDY AND EVALUATION OF SODIUM ALGINATE FIBERS SCAFFOLDS AND SCAFFOLDS WITH α-TCP PLUS SODIUM ALGINATE FIBERS
Letícia Araújo Vasconcellos1, Luís Alberto dos Santos1
1Universidade Federal do Rio Grande do Sul, LABIOMAT, Porto Alegre, RS, Brasil
UFRGS - Campus do Vale - Porto Alegre/RS, Av. Bento Gonçalves, 9500, setor 4, predio 74, sala 125 CEP: 91501-970
E-mail:
The useofbiomaterial calcium phosphatehasrevolutionizedorthopedicsanddentistrytodayto repairdamaged parts oftheskeletal system. Amongthese, thecementsof calcium phosphate(CFCs), which has attractedgreatinterestdue toits biocompatibilityand hardeningin situ, whichallowsgreatereaseofhandling. TheseCFCshavelow mechanical propertieswhencomparedwiththe bonesof thehuman body. A study was conductedtoobtainCFCsand the influence of additionofsodium alginatefibers on the mechanical properties of composite. Thesodium alginatefiberswere obtainedbya new freeze-dryingtechnique. It waspossible toobtain resorbable fiber composites, thusachievingahigh tenacity structuresfor potential use on construction ofscaffoldsfortissue growth.
Keywords: Scaffolds, sodium alginate fibers, calcium phosphate cement, biomaterial
1.INTRODUCTION
The research of new materials has always occupied a prominent position in the scientific community. In this new millennium, materials with special properties for medical applications have been very prominent as they unify efforts of researchers in biotechnology.Biomaterials can be defined as substances of natural or synthetic origin which are tolerated on a temporary or permanent basis by the various tissues that make up the organs of living beings.
Although it is important to highlight that many implants have stability problems on the surface due to interactions with human tissues and physiological fluids that come into contact. An important aspect about possibilities of biomaterials application is that all components and products that may be generated in biodegradation processes must be biocompatible (Miller & San Román, 2004).
Among developed and studied materials in recent year which have these and other important characteristics for the use as biomaterials, there are the calcium phosphate bioceramics. Calcium phosphates get particular interest in medicine, mainly due to its occurrence in normal (dentin, enamel, bone) and pathological (calculations) calcifications.
A fiber embedded in any matrix contributes to increase the capacity of supporting body efforts. Load is transferred from the matrix to the fiber through shear strain in the fiber-matrix interface. The charge transfer is usually a result of differences in physical properties of the fiber and matrix, for example, difference in elasticity modulus between fiber and matrix.
Alginate is an abundant natural polysaccharide with potential to produce biofilms and fibers due to its colloidal properties, and also to produce scaffolds for tissue engineering (soft tissues). It is a linear copolymer consisted of β-D-mannuronic acid blocks (M units) and α-L guluronic acid blocks (G units) extracted from various species of brown algae. Alginate may react with divalent ions of calcium, for example, forming a gel, or may react with polyvalent ions forming crossed links. Due to their linear structure and high molecular weight, alginate is capable to form resistant films and good fibers in a solid state (Rinaudo, 2008).
Sodium alginate has been studied for the use as a matrix for specific cell culture support, immobilization of microorganisms, pharmaceutical excipient, material for dental impression and wound healing (Lee et al., 2004; Augst et al., 2006; Avella et al., 2007; Qin, 2008, Ko et al., 2010).
The structure of the alginate gel is governed not only by the concentration and chemical structure of the gel material, but also by its formation kinetics, which depends on the cation concentration, ionic strength and pH. The formation of calcium alginate gel is complex and depends on the type of alginate used, the association degree between alginate and calcium, the calcium ion source (chloride, phosphate, lactate or calcium acetate) and preparation methods.
Scaffold is a support in which these cells are set to undergo a cell multiplication and also an "orientation" of their tissues. It requires a scaffold with a defined pore size and high porosity in its structure. It also requires a high surface area, biodegradability and proper degradation rate, biocompatibility, mechanical integrity to maintain the structure and interaction with the cells allowing adhesion, growth, migration and, finally, cell differentiation.
Bone marrow contains beyond hematopoietic stem cells and endothelial stem cells, a rare population of multipontencial stem cells able to support hematopoiesis and to differentiate themselves into various cell lines. These cells were originally identified from mononuclear cells of mice’s bone marrow by Alexander Friedenstein and colleagues in 1966, who denominated them as fibroblast colony-forming cells. But recently these cells have been denominated mesenchymal stem cells (MSCs) (ZAGO, 2006).
Tissue engineering studies involve seeding and in vitro cells culture within biodegradable scaffolds before implantation. Scaffolds gradually degrade after implantation and may end up being eliminated. The goal is to provide a basis for cell-seeding, with cells from different sources in biodegradable porous scaffolds (Beyer, et al).
Calcium phosphate cements scaffolds (α-TCP) have great potential for use as materials to manufacture scaffolds, due to their biocompatibility, osteoconductivity and bioactivity. However, there is a great problem which hinders their use: low mechanical properties (for fracture toughness, for example).
In this work we managed to get three different types of scaffolds. The first sodium alginate was obtained by TIPS, the second one by a method which was patented by the authors, by dripping, and the third one that increased mechanical properties of α-tricalcium phosphate (α-TCP) bone cements by the addition of sodium alginate fibers obtained by the patented method. We built scaffolds by the simple method of adding paraffin spheres. With these three different types of scaffolds we obtained positive results in the fields of genetic and biomaterials engineering.
2.MATERIALS AND METHODS
The first step consisted in obtaining the calcium phosphate cement powder (α-TCP) and the sodium alginate fibers with dripping in various reagents under stirring and, after that, freezing and lyophilization, and finally the construction of scaffolds (α-TCP) containing additions of sodium alginate fibers.
2.1.Attainment of calcium phosphate cement powder (α-tricalcium phosphate)
The α-tricalcium phosphate cement (α-TCP) was synthesized in laboratory using calcium carbonate (CaCO3) (Nuclear) and calcium pyrophosphate (γ-Ca2P2O7). Initial homogenization between precursors was performed dry in alumina ball mill for 1 h. The calcination procedure allowed the reaction of two precursors, gamma phase of calcium pyrophosphate and calcium carbonate, which were kept in muffle furnace at a temperature of 1300°C for 5 hours followed by quenching. The values for achieving the synthesis of α-TCP were calculated by the following chemical reactions:
CaCO3 + Ca2P2O7 → α Ca3 (PO4)2 + CO2
After cooling, α-TCP was manually disaggregated with mortar and porcelain pestle and passed through sieve 20 mesh ABNT (850µm). Wet grinding was done using absolute ethyl alcohol (Vetec®) (CH3CH2OH) for 2 hours. The resulting composition was placed in a stainless steel tray and kept 72 hours in an oven at 70°C to promote total evaporation of alcohol.
After milling in alcohol, α-TCP is now called cement, as it reacts with water and allows hardening. The liquid fraction of the cement was formulated in order to obtain an aqueous solution that accelerates cement setting reaction, similar to the composition used in the literature for mixture of α-TCP cements. The most widespread formulation for the liquid fraction corresponds to a solution of 2.5% disodium hydrogen phosphate (Na2HPO4) in distilled and deionized water, this being the one we used. The obtained cement was characterized by the following techniques:
• Crystalline phases: Qualitative X-ray Diffraction (XRD). Phillips X'Pert MPD diffractometer with copper pipe (radiation Kα = 1.5418 Ǻ). Voltage and current used in the tube was 40 kV and 40 mA respectively, to obtain the diffractograms of this work;
• Chemical groups. Infrared spectroscopy (IR). The apparatus used in this work was a 1000 Spectrometer Spectrum in a range from 500 to 4000 cm-1.
• Particle size: laser particle size analysis were made in equipment CILAS 1180 (apparatus’ detection range from 0,04 to 2500 µm using isopropyl alcohol.
The obtained powders were mixed into the liquid phase (2,5% Na2HPO4 - Sodium phosphate dibasic dodecahydrate PA [Vetec®]) for the cement setting reaction, obtaining pure test bodies and with the addition of sodium alginate fibers by volume of 1%, 2%, 3% and 4%. Its physicochemical and mechanical properties were analyzed using the following tests:
• Mechanical resistance: compression test, bending and KIC and JIC calculations: we followed the guidelines of ASTM E 1820-96 "Standard Test Method for Measurement of Fracture Toughness" (ASTM, 1996). For the tests, we used Shimadzu (AG-X/50 kN) universal testing machine with speed of 1mm/min and 0,5 mm/min in compression test and bending in four points.
2.2.Attainment of sodium alginate fibers by dripping
To obtain the fibers, it was prepared sodium alginate (Aldrich®, purity> 99.5%) at 1 and 2% concentrations, dissolved in deionized water in a magnetic stirrer for two hours (600 rpm). The solutions were dripped up to five times in three different reagents (ethanol P.A. (Vetec®), isopropyl alcohol P.A. (Vetec®) and methanol (Synth®) under stirring (~ 600 rpm) for 30 minutes. After removal of the samples using a tweezer, sieve to drain liquid excess and placed in a freezer (-20°C) for 24 hours and then lyophilized. It was determined the characteristics of the fibers by: scanning electron microscopy (SEM) and infrared spectroscopy.
2.3.Attainment of calcium phosphate cement with sodium alginate fibers
To the composition of calcium phosphate cement obtained in item 2.1. It was added fibers obtained in item 2.2., in content of 0, 1, 2, 3, 4% by volume. Test bodies were made and their physicochemical and mechanical properties were determined using the following tests: mechanical resistance, compression tests and KIC and JIC (ASTM E 1820-96), scanning electron microscopy (SEM).
2.4.Preparation of scaffolds with alginate fibers
After α-TCP was obtained and characterized and after sodium alginate lyophilized fibers, we made the preparation of scaffolds. First, sodium alginate lyophilized fibers were selected, separated (to maintain mix’s homogeneity) and mixed to α-TCP with paraffin spheres and added to the liquid phase at 1 ml per 1g of cement. Paraffin balls were added in the following percentages by volume: 57, 63 and 66% (paraffin density = 0,89 g/cm3, calcium phosphate cement density (α-TCP) = 2,35 g/cm3).
Obtained paste was poured and placed in various types of molds following the guidelines of ASTM E 1820-96 (ASTM, 1996) and put in a container at 100% humidity and placed in an oven with controlled temperature of 37°C for 48 hours for its complete cure. After 48 hours it was removed from the molds and subjected to heat and chemical treatment for extraction of paraffin. The extraction of paraffin from the scaffolds with sodium alginate fibers (1, 2, 3, 4%) consisted in placing the samples on absorbent paper in an oven at 50°C for 24 hours and after that scaffolds remained submerged in Hydrogen Peroxide P.A. at room temperature for 48 hours.
2.5.Tests of mechanical strength and fracture toughness
The fracture toughness tests were performed following the guidelines of ASTM E 1820-96 "Standard Test Method for Measurement of Fracture Toughness" (ASTM, 1996). We calculated the values of fracture toughness, KIC, for the crack fragile propagation, and JIC, full surface which includes crack front from a fractured surface to the other, used to characterize the stress-strain field around the crack front, being a criterion of fracture in plastic-elastic regime. This criterion was adapted to characterize fiber reinforced concrete, first by Mindess et al (Mindess et al., 1977) and later by Li et al (Li et al., 1987).
2.6.Water absorption, density and apparent porosity
The procedure for calculating the water absorption, density and apparent porosity was based on the international standard ASTM C 20 (“Standard Test Methods for Apparent porosity, water absorption, apparent specific gravity, and bulk density of burned refractory brick and shapes by boiling water”) by determining the mass of dry sample and the wet and immersed mass. This method is based on the Archimedes’ principle.
To perform the weighing of immersed and wet samples it was necessary that the test bodies remained immersed in water for 24 hours. To perform the submerged weight, it was used Archimedes' principle of fluid displacement determining it. We measured the dry weight (DW), wet weight (WW) and the weight submerged in water (SW) and applying Equations 1-3, we calculated the Water Absorption (WA), Apparent Porosity (AP) and Density Apparent (DA), respectively.
WA = Pu - Ps / Ps [Equation 1]
AP = - Ps / Pu - Pi [Equation 2]
DA = (Ps / Pu - Pi) x ρL [Equation 3]
ρL is the density of the liquid medium. For the calculation of density and apparent porosity it was used 7 test bodies of each composition. The dry mass was determined after drying of test bodies for a period of time not less than 24 hours in an oven at 50°C.
2.7.In vitro cement study
To perform the in vitro study, samples of calcium phosphate cements (neat, 1, 2, 3 and 4% sodium alginate fibers) were placed in a solution of SBF (Simulated Body Fluid), liquid prepared in laboratory simulating the bodily fluids (Kokubo et al., 1990; Ohtuski et al., 1991). The SBF solution was prepared to present a composition close to ions that exist in human blood plasma. The SBF solution was prepared in Tris/HCl (pH 7,4) with addition of NaCl, KCl, CaCl2, MgCl2, NaHCO3, K2HPO4 and Na2SO4.
Samples were placed in polyethylene bottles at a rate of 50 mL/g. The bottles were sealed and remained in water bath at 37 ± 1°C in the following periods of time: 01 day, 07 day, 14 days, and the solution of SBF was changed every 48 hours. Each period of time, samples were removed, rinsed in distilled water and dried at room temperature for at least 24 hours.
2.8.Evaluation of In Vitro Cytotoxicity Potential - Agar diffusion method
The scaffolds approved in the previous steps were evaluated for cytotoxicity in accordance with: ISO 10993.5: 2009 - Biological Evaluation for Medical Devices Tests for Cytotoxicity: In Vitro Methods, ASTM F895-84: 1984-Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity and U.S. Pharmacopeia 32, 2009 - Biological Reactivity Tests In Vitro. For the cytotoxicity test, it was used cell from fibroblasts line. The composites approved in the previous steps were evaluated at Instituto Adolfo Lutz - Section of Cell Cultures, by using Agar diffusion method.
The cell line (NCTC clone 929), mouse connective tissue cells (ATCC CCL-1) are seeded in Petri dishes and incubated for 48 hours to form the cell monolayer. The liquid culture medium is replaced by solid overlay medium, which consists of equal parts of the medium twice concentrated and agar with neutral red. The samples are placed on this overlay medium and the plates are incubated again for 24 hours. It is observed macroscopically and microscopically the index zone (IZ), which is the area not stained by vital dye. The samples are tested in quadruplicate on separate plates. Table 2 presents the description of the index zone (IZ) observed under the microscope.
Solid samples are placed directly on the overlay medium. Positive control was fragmented of 0,5 cm x 0,5 cm of toxic latex and for negative controls it is used nontoxic filter paper disks with 0,5 cm of diameter.
3.RESULTS AND DISCUSSION
3.1.Characterization of calcium phosphate cement (α-TCP)
3.1.1.X-Ray Diffraction (XRD) of α-TCP
The presence of β-TCP as an undesired phase in α-TCP powder obtained by several methods have been reported in literature (Bermudez et al., 1994; Tampieri et al., 1997; Famery et al., 1994; Fernandez et al., 1996). By the time that are no registers about the attainment of pure α-TCP. The purest registered material was obtained by reaction between CaCO3 and DCPA at 1300°C, for 6 hours, followed by quenching (Bermudez et al., 1994) and contained about 5% of β-TCP, because of a low amount to high cooling rate. Usually, α-TCP achieved by this method contains approximately 15% of β-TCP (Geneva et al., 1997).As it can be seen in Figure 1, also the materials obtained in this work by reaction between DCPA and CaCO3 contain β-TCP as an unwanted phase.
Figure 1 shows that for a composition 3CaO.P2O5, the stable phase atlow temperature is the β-TCP, which at 1125°C is transformed into α-TCP. This last phase is stable up to approximately 1400°C, when it becomes α-TCP. According to Carrodeguas et al (2008), the transformation from β to α phase starts between 1150 and 1200°C, and between 1300 and 1350°C its starts a stability region of pure α-TCP that extends up to 1500°C.
3.1.2.Particle size distribution
To determine the particle size it was used the laser diffraction method. This technique uses light scattering to determine particles sizes by their volume. For example, if 11% of distribution is in the size between 6,9 to 7,75 μm it means that the total volume of all particles with diameters in this range represents 11% of the total volume of all particles in the distribution. This method is used for particles with diameters from 0,5 μm to 350μm. Three measurements were made for the same sample. For particles to remain dispersed in solution it was used ultrasound for 1 minute before to analysis.
The results of laser particle size analysis show that the size of calcium phosphate grain (α-TCP) remained in the average of 8μm, as it is shown in Figure 2, observing an ideal size of the cement particle that has to be smaller and/or equal to 10μm. According to Geneva et al., the particle size is a key factor that significantly modifies the final properties of the cement, and that especially affects the kinetics of chemical reaction and mechanical strengthening of the cement. However, it is a parameter that can be useful to adjust the behavior of the cement according to the required clinical application.
Surface properties such as roughness, specific area and interface porosity between the material and the biological environment (“in vivo” or “in vitro”) can be controlled by modification of the particle size in the starter powder used in the cement (Geneva et al., 2004).
3.1.3.Chemical groups
The main bands identified in the spectrum of α-TCP are the functional groups of orthophosphate (PO4), hydroxyl (OH), hydrogen (HPO4) and likely pyrophosphate (P2O7), the latter two ones in trace amount, as their peaks are not considerable neither observed in other spectrum positions, what is a characteristic of these species, as it is shown in Figure 3.
Observed the presence of peaks at 660 and 563 cm-1, what is a characteristic of mode (P-O) and PO4. 967 cm-1 referring to PO-H link HPO4 groups stretching mode; 1030 cm-1 referring to PO4 groups; 1467 and 1423 cm-1 referring to C-O link of CO3 groups, 1640 cm-1 referring to axial deformation of (C=O); 2918 and 2847 cm-1 axial deformations of aliphatic CH; 3456 cm-1 referring to OH link of groups (OH); 3665 and 3075 cm-1 referring to absorbed water (H2O).