Evaluation of in vitro bioactivity of Chitosan/Mimosatenuiora composites

S.A. Martel-Estrada a,n, I. Olivas-Armendriz b, E. Santos-Rodrguez c, C.A. Martnez-Prez b,P.E. Garca-Casillas b, J. Hernndez-Paz b, C.A. Rodrguez-Gonzlez b, C. Chapa-Gonzlez b

a Instituto de Arquitectura, Diseo y Arte, Universidad Autnoma de Cd. Jurez, Av. Del Charro 450 Norte, Col. Universidad, C.P. 32310 Cd. Jurez, Chihuahua,Mexicob Instituto de Ingeniera y Tecnologa, Universidad Autnoma de Cd. Jurez, Av. Del Charro 450 Norte, Col. Universidad, C.P. 32310, Cd. Jurez, Chihuahua,Mexicoc ICTP Meso-American Centre for Theoretical Physics (ICTP-MCTP)/Universidad Autnoma de Chiapas, Ciudad Universitaria, Carretera Zapata Km. 4, Real delBosque (Tern), Tuxtla Gutirrez, Chiapas, C.P. 29040, Mexico

a r t i c l e i n f o

Article history:Received 7 August 2013Accepted 4 January 2014Available online 10 January 2014

Keywords:Bioactive materialsChitosanMimosa tenuioraSBF

a b s t r a c t

[Porous biocomposite scaffolds of Chitosan/Mimosa tenuiora (Ch/Mim) were fabricated for tissueengineering applications by thermally induced phase separation and lyophilization techniques. Thein vitro bioactivity evaluation of the scaffolds was carried out by analyzing the apatite layers produced onthem using SBF as the incubation medium. The apatite formation was analyzed using FTIR spectroscopyand Field Emission Scanning Electron microscopy coupled to Energy-Dispersive Electron X-ray Spectro-scopy. The cumulative results obtained from IR spectra and SEM-EDS suggest that the developedcomposites might have potential applications in tissue engineering.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Mimosa tenuiora (Willd.) Poiret is a species of the Mimosoi-deae, a subfamily of the Fabaceae botanical family that isdistributed from Mexico to Venezuela and Brazil [1]. It is a plantwith a high content in tannins and large amount of starch [2].M. tenuiora is traditionally used as an aqueous extract [3] for thetreatment in wounds and venous leg ulcers due to its healing andantiseptic properties [4]. It has been used in the treatment ofburns and infections in both humans and animals because of itsantimicrobial and cicatrizing effects [4,5].

The chemical composition of the M. tenuiora cortex has beenidentied as a group of alkaloids, mainly N-N-dimethyltryptamine,serotonin, triperpenoid glycosides, and steroidal saponins: 3-O--D-glucopiranosil campesterol, 3-O--D-glucopiranosil estigmas-terol, and 3-O--D-glucopiranosil -sitosterol [4].

On the other hand, Chitosan is a semi-crystalline polysaccharidederived from chitin and possesses antimicrobial activity [6]. Due toits properties, Chitosan is a good candidate for preparation ofbiomaterials that could substitute missing or damaged tissue [7].

One of the most important characteristics of a bioactive materialis the possibility that a biologically active carbonate hydroxyapatite

layer can form on their surface [8]. Calcium phosphate is the mainconstituent of bone tissue [9]. This substance could be formed on thematerial surface by an inorganic chemical reaction similar to thatoccurring in bones. This ability has been related to osteoconductionand bone bonding [10], because the properties of the apatite layercan affect cell viability and proliferation [8]. In order to predictbioactivity, a simulated body uid (SBF) has been used [11].

With these characteristics, it could be reasonable to believe inthe bioactivity of a Chitosan/M. tenuiora composite. So, in thisstudy, a biocomposite of Chitosan/M. tenuiora composite wasmanufactured through a thermally induced phase separationmethod. The composite was used to investigate the in vitrobioactivity of scaffolds by analyzing the apatite layers producedin them using SBF as an incubation medium.

2. Materials and methods

Materials: Chitosan was purchased from Carbomer, Inc (UnitedStates). The bark of M. tenuiora was obtained directly in theregion of Jiquipilas Chiapas. Ethanol was provided by CTR Scientic(Mexico). Glacial acetic acid (Mallinckrodt, United States) was usedas a solvent. SBF was prepared in our laboratory according to apreviously published method [7].

Preparation of composites: Chitosan/M. tenuiora was preparedusing the thermally induced phase separation technique. Briey,

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Materials Letters

0167-577X/$ - see front matter & 2014 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.matlet.2014.01.004

n Corresponding author: +52 656 688 48 20.E-mail address: adriana.martel@uacj.mx (S.A. Martel-Estrada).

Materials Letters 119 (2014) 146149

Chitosan and M. tenuiora were dissolved in 1% (%v/v) aqueousacetic acid solution. The solutions were mixed together at 80/20ratio. Finally, the composite was frozen and freeze dried for 2 days.

FTIR spectroscopy: To evaluate the Chitosan/M. tenuiora com-posites and mineralized scaffold, FTIR spectra were recorded usinga transmission mode in an IR spectrometer (Nicolet 6700, ThermoScientic, USA). For each spectrum, 100 scans at16 cm1 resolu-tion were averaged.

Characterization of morphology and in vitro bioactivity: A pre-viously studied method was used for the in vitro bioactivity study[7]. Briey, porous samples of approximately 250 mm3 weresoaked in 5 mL of tetraethoxyl orthosilicate for 2 h in a vacuumoven at 60 1C. The samples were rinsed with ethanol and weredried in air at room temperature for 1 day. Then, the samples weresoaked in 5 mL of 1.5 SBF, pH 7.4, in an incubator at 37 1C. Afterthe incubation period, the specimens were washed carefully withdeionized water and nally dried.

Field Emission Scanning Electron Microscopy (FE-SEM, JEOLJSM-7000 F) was applied to investigate the morphology of thecomposites and Field Emission Gun Scanning Electron Microscopy(Jeol JSM-7000 F) coupled with Energy Dispersive X-ray Spectro-scopy (15 kV) to analyze the dispersion and distribution of apatiteparticles and to detect the concentration of calcium and phos-phorous on the surface after the treatment for the differentexperimental conditions. The Ca/P ratio was estimated for thevarious conditions. Also, the pore size was measured using theScandium Universal SEM Imaging Platform software.

3. Results and discussion

FTIR of the samples: Fig. 1 shows that the NH band of Chitosanin the composite was affected and shifted from 1543 cm1 to1553 cm1. On the other hand, the peak that corresponds to theprimary alcohol groups of Chitosan (CO stretching) was shiftedfrom 1418 cm1 to 1459 cm1 for the composite. Furthermore, itis possible to observe a reduced intensity on the band at1021 cm1 that could imply an interaction of the ether bond ofChitosan with the M. tenuiora. All these events suggest that thereis an interaction between the amino group of the Chitosan andcarboxyl groups in M. tenuiora or hydroxyl groups in Chitosanand carboxyl groups of the M. tenuiora in the blend.

Characterization of morphology and in vitro bioactivity: Poreswith sizes around 50100 m are essential for bone ingrowth [12].As shown in Table 1, the composite Chitosan/M. tenuiora hasmore homogenous porosity than that of Chitosan, with an averagepore size of 211.6113.74 m. The composite also showed an open-pore structure with high porosity (Fig. 2). The pore size andporosity obtained is shown in Table 1.

According to SEM images (Fig. 2e and f), the particles formedin vitro have a spherical shape. Some large particles with adiameter of about 10 mm and smaller crystals around 3 mm wereobserved. With an increase in immersion period a more compactmineral layer was formed on the surface. The images revealed thatapatite crystals grow in a layer-by-layer manner. The Ca/P peakintensity ratio for Chitosan at 28 days was 1.13, which might

Fig. 1. FTIR spectra of Mimosa tenuiora, Chitosan and 80/20 Ch/Mimosa tenuiora composite before incubation.

Table 1Pore size distribution. It shows the average pore size (mm) and the distribution of porosity in Chitosan and 80/20 Chitosan/Mimosa tenuiora.

Composite Average pore size (mm) o50 mm (%) 50100 mm (%) 100200 mm (%) 200300 mm (%) 300400 mm (%) 400500 mm (%) 4500 mm (%)

Chitosan 150.56714.65 15 30 35 10 4 1 580/20 Ch/Mim 211.61713.74 12 16 24 22 12 12 2

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Fig. 2. SEM micrographs exhibit (a) Chitosan/Mimosa tenuiora composite and (b) Chitosan scaffold without any treatment. Then, the gure shows the Chitosan/Mimosatenuiora scaffolds at 21 days of SBF treatment at (c) 600 , (d) Chitosan at 28 days at 1000 and Chitosan/Mimosa tenuiora at 28 days of treatment at (e) 1000 and(f) 1700 .

Fig. 3. FTIR of the (a) Chitosan/Mimosa tenuiora composite before the treatment and (b) Chitosan and (c) Chitosan/Mimosa tenuiora composite immersed for 28 days in1.5 SBF. The image shows evidence of carbonate (CO23 ). Also the FTIR spectra revealed the presence of the v3 stretching mode of PO bonds.

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indicate the presence of a Ca-decient apatite. After immersion,nucleation and growth have occurred on the surface, resulting in acluster of mineral precipitates. At higher magnication, thespheres appear as needle-like deposits as shown in Fig. 2f. TheEDS analysis revealed that the Ca/P relation on the samplesChitosan/M. tenuiora was 1.36 at 21 days and 1.86 at 28 days.

FTIR of composite before and after soaking in SBF showed theformation of an apatite-like layer (Fig. 3). New bands can beassigned to the phosphate group at 1044 cm1 (PO stretching(3) vibration), 956 cm1 and 624 cm1 (PO bending vibration,4) and to the carbonate group at 1454 cm1 (CO23 stretchinggroup) and 848 cm1 (CO23 ) [9]. It is obvious from the resultsdescribed previously that the composites Chitosan/M. tenuiloracould develop a carbonate containing hydroxyapatite layer on thesurface.

4. Conclusions

Biodegradable Chitosan/M. tenuiora composites have beensuccessfully prepared by using a thermally induced phase separa-tion method. On the Chitosan/M. tenuiora composite scaffoldthere are many amine groups which could act as nucleation sites.As a result, apatite could be formed more efciently on thecomposite scaffolds than on the Chitosan scaffolds.

Also, a porous and interconnected morphology with enhancedbioactivity that could be appropriate for tissue engineering hasbeen found. The ability of the composites to form on their surfacea mineralized layer was evident through the cumulative resultsobtained from IR spectra, SEM and EDS. Future research must befocused on testing the biocompatibility of this composite.

Acknowledgments

The authors acknowledge the nancial support of the MexicanPublic Education Secretary and the Mexican National Council for

Science and Technology (CONACyT) through Projects SEP-CONACyTCB 2012-01-180909 and PROMEP/103.5/13/2347.

References

[1] Anton R, Jiang Y, Weniger B, Beck J, Rivier L. Pharmacognosy of Mimosatenuiora (Willd.) Poiret. J Ethnopharmacol 1993;38:1537.

[2] Sousa T, Leal N, Cavalcanti E, Alburquerque U. A new approach to studymedicinal plants with tannins and avonoids contents from the local knowl-edge. J Ethnopharmacol 2008;12:7280.

[3] Zippel J, Deters A, Hensel A. Arabinogalactans from Mimosa tenuiora (Willd).Poiret bark as active principles for wound healing properties: specicenhancement of dermal broblast activity and minor inuence on HaCaTkeratinocytes. J Ethnopharmacol 2009;124:3916.

[4] Lammogllia-Ordiales L, Vega.Memije M, Herrera-Arellano A, Rivera-Arce E,Agero J, Vargas-Martnez F, et al. A randomised comparative trial on the useof a hydrogel with tepescohuite extract (Mimosa tenuiora cortex extract-2G)in the treatment of venous leg ulcers. Int Wound J 2011;9:4128.

[5] Pimentel E, Cruces M, Zimmering S. Evaluation of the mutagenic potential oftepezcohuite in the Drosphila wing spot test. Mutat Res 1991;264:1156.

[6] Martnez-Camacho A, Cortez-Rocha M, Ezquerra-Brauer J, Graciano-VerdugoA, Rodrguez-Flix F, Castillo-Ortega M, et al. Chitosan composite lms:thermal, structural, mechanical and antifungal properties. Carbohydr Polym2010;82:30515.

[7] Martel-Estrada S, Martnez-Prez C, Chacn-Nava J, Garca-Casillas P, Olivas-Armendriz I. In vitro bioactivity of Chitosan/poly(D,L-lactide-co-glycolide)composites. Mater Lett 2011;65:13741.

[8] Kong L, Gao Y, Lu G, Gong Y, Zhao N, Zhang X. A study on the bioactivity ofChitosan/nano-hydroxyapatite composite scaffolds for bone tissue engineer-ing. Eur Polym J 2006;42:31719.

[9] Martel-Estrada S, Olivas-Armendriz I, Martnez-Prez C, Hernndez T, Acosta-Gmez E, Chacn-Nava J, et al. Chitosan/poly(D,L-lactide-co-glycolide) scaf-folds for tissue engineering. J Mater Sci 2012;23:2893901.

[10] Peltola T, Jokinen M, Veittola S, Simola J, Yli-Urpo A. In vitro bioactivity andstructural features of mildly heat-treated solgel-derived silica bers.J Biomed Mater Res 2000;54:57990.

[11] Lee K, Rhee S. The mechanical properties and bioactivity of poly(methylmethacrylate)/SiO2CaO nanocomposite. Biomaterials 2009;30:34449.

[12] Jones A, Arns C, Hutmacher D, Milthorpe B, Sheppard A, Knackstedt M. Thecorrelation of pore morphology, interconnectivity and physical properties of3D ceramic scaffolds with bone ingrowth. Biomaterials 2009;30:144051.

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Evaluation of in vitro bioactivity of Chitosan/Mimosa tenuiflora compositesIntroductionMaterials and methodsResults and discussionConclusionsAcknowledgmentsReferences