Stability of the Magnesium Carbonate Apatite/Anionic ...

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this study was to prepare self-organized magnesium and carbonate ... scaffolds were prepared by precipitation: calcium acetate (Ca(C2H3O2)2) solution ( 0.02.
Key Engineering Materials Vols. 493-494 (2012) pp 844-848 © (2012) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/KEM.493-494.844

Stability of the Magnesium Carbonate Apatite/Anionic Collagen Scaffolds: Effect of the Cross-link Concentration Marcia S. Sader1,a, Gutemberg Gomes Alves 2,b, Racquel Z. LeGeros3,c and Gloria D. A.Soares1,d 1

Dept of Metallurgical and Materials Eng., COPPE/UFRJ, RJ, Brazil Dept of Cellular Biology, Fluminense Federal Un., Niteroi, RJ, Brazil 3 Dept of Biomaterials & Biomimetics, NYU College of Dentistry, New York a b c d [email protected], [email protected], [email protected], [email protected] c

Keywords: Cross-linked scaffold, Substituted apatite, 3D scaffolds

Abstract. Natural bone constitutes of an inorganic phase (a biological nanoapatite) and an organic phase (mostly type I collagen). The challenge is to develop a material that can regenerate lost bone tissue with degradation and resorption kinetics compatible with the new bone formation. The aim of this study was to prepare self-organized magnesium and carbonate substituted apatite/collagen scaffolds, cross-linked with glutaraldehyde (GA). Bovine tendon was submitted to alkaline treatment resulting in a negatively charged collagen surface. The scaffolds were prepared by precipitation: simultaneous dropwise addition of solution containing calcium (Ca) and magnesium (Mg) ions and collagen into a buffered solution containing carbonate and phosphate ions in reaction vessel maintained at 37 °C, pH=8. The reaction products were cross-linked with 0.125 and 0.25% (v/v) glutaraldehyde (GA) solution and freeze-dried. The samples were characterized by Fouriertransformed infrared spectroscopy (FTIR). In vitro cytotoxicity (based on three parameters assays) and scaffolds degradation in culture medium and osteoblastic cells culture were performed in the cross-linked materials. No cytotoxic effects were observed. The cross-linked samples with the lower GA concentration showed a lower stability when placed in contact with culture medium. Human osteoblasts attached on the scaffolds surface cross-linked with 0.25% GA, forming a continuous layer after 14 days of incubation. These results showed potential application of the designed scaffolds for bone tissue engineering. Introduction Tissue engineering aims to develop promising materials that will replace, restore or regenerate lost tissues. These biomaterials play a significant role as 3D scaffolds providing an essential microenvironment to support cellular attachment, proliferation and differentiation, serving as a temporary support as extracellular matrix. These materials should be biocompatible, bioresorbable, with a degradation rate simultaneous with the new tissue formation, and high interconnectivity to allow cells ingrowth. Moreover, the scaffolds must have physico-chemical properties similar to the host site stimulate its regeneration [1]. Natural bone is a composite constituted of an inorganic phase (a biological nanoapatite) and an organic phase (mostly type I collagen). Apatite has several different crystallographic sites where ionic substitutions can occur. Magnesium (Mg) is one of the trace-element present in biological hard tissue and replaces calcium in the apatite, causing disturbance in the apatite lattice, decrease of crystal size and an increase in degradation rate [2]. The bone mineral is better described as a carbonate apatite. The carbonate (CO3) substitution occurs mainly in the phosphate sites within the crystal structure (B-type substitution), causing changes in the lattice parameters, reduction in crystal size and increase in solubility without changing the surface characteristics that influence the initial cell attachment affecting the collagen matrix deposition [2, 3]. The simultaneous incorporation of Mg and CO3 ions in the biological apatite lattice has additive effect on the lattice parameters and crystal morphology, leading to higher solubility [2]. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP, www.ttp.net. (ID: 200.20.0.142-23/09/11,20:40:14)

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Type I collagen is easily degraded and reabsorbed by the body and allows good cells attachment. However, its low mechanical strength limits its use, requiring the cross-linking for applications in bone tissue engineering [4] in order to maintain the scaffolds integrity when implanted in the body. The development of a material which could mimic the bone structure and properties is the challenge of this new field. Bone defects can be filled with 3D composites that mimic the nanostructure and bone properties pre-seeded with autologous cells for a fast healing [5]. The aim of this study was to develop a self-organized 3D scaffolds of Mg-CO3-apatite/anionic collagen (MCA/COL) composite that may have a potential application for filling bone defects and restoring the traumatized or degenerated connective tissues. Materials and Methods Bovine tendon was immersed in an alkaline solution (3 mL g-1 of tissue) for 24 h at 20 °C containing 6% (v/v) dimethyl sulfoxide, salts of alkaline and alkaline earth metals. The resulting material was balanced in a solution containing sodium sulphate (Na2SO4), sodium chloride (NaCl), potassium chloride (KCl), and calcium sulphate (CaSO4), (6 mL/g of tissue) for 6 h. Excess salts were removed by rinses in 3% (wt./wt.%) boric acid solution, deionized water, followed by 0.3% (wt./wt.%) ethylene diamine tetracetic acid (EDTA) solution, pH 11, in deionized water, and finally, extracted by acetic acid (pH 3.5). This treatment resulted in a collagen with a negatively charged surface. The scaffolds were prepared by precipitation: calcium acetate (Ca(C2H3O2)2) solution ( 0.02 mol/L), magnesium acetate (MgC4H6O4) solution (0.002 mol/L) as well as the collagen solution (3 mg/ml) in acetic acid (0.5 mol/L) were gradually added through peristaltic pumps into a reaction vessel containing sodium bicarbonate (NaHCO3) solution (0.1 mol/L) and sodium phosphate (Na2HPO4/ NaH2PO4) solution (0.1 mol/L) maintained at 37 °C, pH=8. The reaction products were washed with buffer phosphate and deionized water and cross-linked with 0.125 and 0.25% (v/v) glutaraldehyde (GA) solution and washed many times with deionized water to eliminate any crosslinking residue. Cylindrical molds with 6 mm diameter and 4 mm height were filled by the composite and freeze-dried (Liotop L101). The samples were characterized using Fourier transformed infrared spectroscopy, (FTIR, Nicolet Magna IR - 550 Spectrometer II). Scaffolds extracts, immersed in culture medium (100 mg/ml, material/DMEM free of bovine fetal serum) at 37 oC for 24 h, were collected for cytotoxicity assay according to ISO 10993-12 [6] and 10993-5 [7]. Phenol (1%) was used as positive control and polystyrene beads (PE) as negative control. All the tests were performed in quintuplicate. Mean values and standard deviations were submitted to one-way ANOVA and Tukey’s post-test considering significance at 0.05 or 0.01. The shape of the scaffolds with and without cross-linking after 14 days of incubation in DMEM at 37 °C was observed by scanning electron microscopy, (SEM, JEOL - JSM6460LV). The morphology of human bone marrow mesenchymal cells seeded on the cross-linked samples for 14 days was observed using SEM. Results and Discussion FTIR spectra of the anionic collagen (A) and the mineralized scaffolds before (B) and after (C) cross-linking are shown in Fig. 1. Characteristic bands of collagen such as N-H stretching at 3310 cm-1 (amide A) and C-H stretching at 3077 cm-1 (amide B) and the stretching vibration of the carbonyl groups, C=O bond (amide I, 1658 cm-1), the angular deformation of N-H bond (amide II, 1552 cm-1) and the absorption band related to C-N bond vibration (amide III, 1235 cm-1) were identified. The characteristic bands of type B carbonate substitution in the apatite lattice were identified on the mineralized scaffolds before and after cross-linking. The spectra exhibited the CO32- bands at 1560 cm-1 (ν3 C-O) and 875 cm-1 (ν2 C-O) and phosphates bands (PO43-) appears at 1023 cm-1 and 557 cm-1. The intensity of the collagen bands decreased after the mineralization process.

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Figure 1. FTIR spectra of anionic collagen (A) and the mineralized composite before (B) and after cross-linked with 0.25% GA (C). Fig. 2 shows the results of the in vitro cytotoxicity tests based on three parameters assays [XTT, Neutral Red (NR) and Crystal Violet Dye Elution (CVDE)] on the cross-linked scaffolds with 0.25% GA. In order to obtain a better performance, the three parameters lysosomal integrity and membrane permeability; dehydrogenase activity; mainly mitochondrial; and DNA content of cell viability were evaluated in parallel [8]. No cytotoxic effect was verified on the cross-linked composites. Several methods are available to evaluate the cytotoxic effects of biomaterials on cultured cells which affect cell function.

Figure 2. Cytotoxic assay of cross-linked scaffolds with 0.25% GA with mouse fibroblasts (A) XTT reduction (B) Neutral Red uptake (C) Crystal Violet dye elution ** Statistically significant differences between groups (p < 0.01). * Statistically significant differences between groups (p < 0.05).

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The composites stability before and after cross-linking were performed in culture medium (DMEM) during 14 days. The scaffolds shape was observed by SEM (Fig. 3). The scaffolds crosslinked with 0.25% GA kept its stability under wet conditions for 14 days (Fig. 3c) showing an integrated structure with no morphological changes. This indicates the necessity of chemical crosslinking process for the stabilization of the open pores structure.

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Figure 3. SEM images of the scaffolds immersed in DMEM for 14 days without cross-linking (a), cross-linked with 0.125% GA (b) and with 0.25% GA (c). SEM images of bone marrow mesenchymal cells seeded onto the scaffolds cross-linked with 0.25% GA for 14 days showed the cells morphology and adhesion (attachment and spreading phenomena). After the incubation period the cells presented numerous philopodia in all directions on the scaffolds surface and were widely spread over and adhered to the substrate, with flattened morphology forming a continuous layer (Fig. 4b).

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Figure 4. SEM images of the scaffolds cross-linked with 0.25% GA (a) and seeded with bone marrow mesenchymal cells after 14 days of culture ( b). Conclusion MCA/COL composite consisting of Mg and CO3 substituted apatite with a Type B carbonate apatite lattice and cross-linked collagen was prepared. No cytotoxic effect was observed. The composites showed an integrated structure with no morphological changes after being kept under wet conditions for 14 days. The osteoblasts cells showed a flattened shape on the scaffolds surface. These results showed potential application of the designed scaffolds for bone tissue engineering. Acknowledgments The authors acknowledge the financial support given by CNPq, CAPES and FAPERJ (Soares) and NIH/NIAMS AR 056208 (LeGeros).

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References [1] P.X. Ma, Biomimetic materials for tissue engineering, Adv Drug Delivery Rev. 60 (2008) 184198. [2] R.Z. LeGeros, Calcium phosphates in oral biology and medicine, Monographs in oral science, AG Publishers (Ed), Basel 15 (1991). [3] R.Z. LeGeros, G. Dalcusi, R. Kijkowska, B. Kerebel, The effect of magnesium on the formation of apatites and whitlockitas, Magnesium in Health and Disease, J. Libbey & Co. (1989) 11-19. [4] D.A. Wahl, J.T. Czernuszka, Collagen-hydroxyapatite composites for hard tissue repair, European Cells and Materials 11 (2006) 43-56. [5] M. Gelinsky, P.B. Welzel, P. Simon, et al., Preparation and properties of a biomimetic nanocomposite material for tissue engineering of bone, Chem. Eng. Journal 137 (2008) 84-96. [6] ISO 10993-5. Biological evaluation of medical devices. Part 5: Tests for cytotoxicity: In vitro methods. International Organization for Standardization. Geneva; 2009. [7] ISO 10993-12. Biological evaluation of medical devices. Part 12: Sample preparation and reference materials. International Organization for Standardization. Geneva; 2008. [8] E.R. Takamori, E.A. Figueira, R. Taga, et al., Evaluation of the cytocompatibility of mixed bovine bone, Braz Dent J. 18 (2007) 179-184.