Extraction and characterization of highly purified collagen from bovine pericardium for potential bioengineering applications

Materials Science and Engineering C 33 (2013) 790–800

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Extraction and characterization of highly purified collagen from bovine pericardium for potential bioengineering applications Maria Helena Santos a, b,⁎, Rafael M. Silva a, b, Vitor C. Dumont a, b, Juliana S. Neves b, Herman S. Mansur c, Luiz Guilherme D. Heneine d a

Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri—UFVJM, Diamantina/MG 39100–000, Brazil Center for Assessment and Development of Biomaterials—BioMat, Federal University of Vales do Jequitinhonha e Mucuri—UFVJM, Diamantina/MG 39100–000, Brazil Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais—UFMG, Belo Horizonte/MG 31270–901, Brazil d Department of Health Science, Ezequiel Dias Foundation—FUNED, Belo Horizonte/MG 30510–010, Brazil b c

a r t i c l e

i n f o

Article history: Received 1 October 2011 Received in revised form 30 August 2012 Accepted 1 November 2012 Available online 10 November 2012 Keywords: Biomaterials Collagen Protein Electrophoresis Characterization of Biomaterials Tissue replacements Bovine collagen xenografts

a b s t r a c t Bovine pericardium is widely used as a raw material in bioengineering as a source of collagen, a fundamental structural molecule. The physical, chemical, and biocompatibility characteristics of these natural fibers enable their broad use in several areas of the health sciences. For these applications, it is important to obtain collagen of the highest possible purity. The lack of a method to produce these pure biocompatible materials using simple and economically feasible techniques presents a major challenge to their production on an industrial scale. This study aimed to extract, purify, and characterize the type I collagen protein originating from bovine pericardium, considered to be an abundant tissue resource. The pericardium tissue was collected from male animals at slaughter age. Pieces of bovine pericardium were enzymatically digested, followed by a novel protocol developed for protein purification using ion-exchange chromatography. The material was extensively characterized by electrophoresis, scanning electron microscopy, energy dispersive X-ray spectroscopy, and infrared spectroscopy. The results showed a purified material with morphological properties and chemical functionalities compatible with type I collagen and similar to a highly purified commercial collagen. Thus, an innovative and relatively simple processing method was developed to extract and purify type I collagen from bovine tissue with potential applications as a biomaterial for regenerative tissue engineering. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Natural polymers are readily biodegradable macromolecules [1]. Among several natural biopolymers, protein and protein-rich tissues have drawn the attention from the research community as a promising alternative in regenerative medicine. Collagen is an extracellular matrix component that is frequently used as a biomaterial [2–4]. Type I collagen, the most common type of collagen, is the major component of connective tissues such as tendons, ligaments, skin, bone, dentin, cornea, and the fibrous capsules of internal organs [5,6]. The insoluble fibers of this collagen have a high tensile strength that provides mechanical support for multiple physiological functions. Another important role of collagen is to provide guidance for developing tissues [1,2,6,7]. The extensive use of collagen in medicine, dentistry, and pharmacology is also related to its natural properties, including hemostatic activity, biodegradability, low allergenicity with high antigenicity and biocompatibility [8]. Furthermore, it is an efficient porous matrix (scaffold) with ⁎ Corresponding author at: Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri—UFVJM, Diamantina/MG 39100–000, Brazil. Tel.: +55 38 3532 6066. E-mail address: [email protected] (M.H. Santos). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.003

specific physical, chemical, and biological features that are of interest for multiple bioengineering and cell culture applications [9–15]. Type I collagen is currently the gold standard in the field of tissue-engineering [14]. Collagen-based biomaterials can originate from two fundamental techniques to form a functional scaffold. One is a decellularized collagen matrix preserving the original tissue shape and extracellular matrix structure. None of the methods used for tissue decellularization can produce an extracellular matrix completely free of cellular debris and a combination of techniques is often required to obtain a material free of any cell remnant [16]. To realize the potential beneficial effects of biologic scaffolds in the field of tissue engineering and regenerative medicine, optimal methods of decellularization are needed [17]. The other type of collagen-based biomaterial is made from a collagen extraction from biological tissues. Acid solubilization, neutral salt solutions and proteolytic solutions are often used to extract collagen from biological tissues. However, proteolytic extraction alters the collagen molecular structure by cleaving the terminal telopeptide regions, resulting in the decrease in the tropocollagen self-assembled fibrils. To maximize yield, an acid extraction using a pepsin solubilization is the most effective technique, despite the possible occurrence of cleavage or a partial denaturing of the telopeptides [14].

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Proteins derived from animal sources must be purified to ensure their safety for biomedical applications. Multiple techniques are reported in the literature [14,18–21] and new methods of purification have been attempted [22–24]. In general, complex and costly methods are used for designing and developing medical-grade protein-based xenografts. Novel simpler processing methods that comply with the current established validated purification protocols are needed to lower production costs, matching the demand for biomaterials originating from renewable and abundant sources [5,14,25]. In addition, medicalgrade purified collagen is not readily commercially available in many countries. The importation of this biomaterial can be costly and is often restricted by governmental health agencies. The number of industries in these countries that are involved in graded materials for biomedical applications is still deficient. As a type I collagen-rich tissue, Bovine Pericardium (BP) has been widely used as a raw material for biomaterials in various areas of the health sciences [26,27]. It is a heterologous biological material with excellent biocompatibility, suitable biophysical properties, and elastic properties due to the high content of collagen fibers [28–30]. In addition, BP is an easy-to-handle material with worldwide availability and a relative low cost, as it is a by-product of the animal slaughtering process. From an economical perspective, the use of animal tissues that are usually treated as waste by the meat industry is rather attractive. This is a very important issue worldwide. It is estimated that approximately 50% of the animal weight is meat production waste (50–54% for each cow, 52% for each sheep or goat) [31,32]. Specifically in Brazil, the cost of BP is less than USD 0.50 kg. On the other hand, the cost of biomaterials is usually expensive for the end-user, typically sold in the range of USD 500.00–5,000.00 g. From an industrial perspective, there is a very broad commercial margin of over 1000 times the actual cost of the natural raw materials, such as BP. New techniques to isolate and purify these biomaterials for tissue reconstruction are needed to meet the growing demand for their various applications in diverse areas of health sciences. This study aimed at extracting and purifying type I collagen protein from BP tissue using an innovative and relatively simple method for the potential use as a biomaterial in tissue engineering. This method may be transferrable to an industrial-scale process. The collagen-based materials were extensively characterized by gel electrophoresis and Western blotting through mice immunization experiments. Scanning electron microscopy associated with energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy were used to characterize morphology and molecular chemical structures. 2. Materials and methods 2.1. Extraction of collagen Collagen was extracted from intact BP tissue pieces collected from male animals at slaughter age (18–24 months). Tissues adjacent to the dense connective tissue were removed. BP was washed with Ringer's sodium lactate solution (Sanobiol Laboratory, SP, Brazil) twice and cut into flaps of 5×15 cm2 in the longitudinal direction to preserve the natural architecture of its fibers. BP flaps were submerged in double-distilled glycerin (Labsynth, SP, Brazil) at room temperature. Standard personal protective equipment was used during the procedures for handling BP. After 60 days, the BP flaps were shredded and kept fully immersed in 300 mL of a 0.1 M sodium hydroxide (NaOH) solution (Vetec Química Fina Ltda., RJ, Brazil), at a temperature of (4 ± 1)°C. After 48 h, the residue was filtered 3 times. The BP was processed using an Ultra-Turrax macerator (Janke and Kunkel, IKA, Germany) in a cooled vessel. Pepsin (21.45 g) (Vetec) was solubilized in 300 mL of 10 mM hydrochloric acid (HCl) (Vetec) and the solution was poured over 429 g of BP paste (pH 12.0) at a pepsin-to-BP ratio of 1:20. The pH of the mixture was adjusted to 2.0 with concentrated HCl and maintained for 12 h at 4 °C. The mixture was then centrifuged at 10,000 xg for 20 min at 4 °C. The supernatant was collected and stored in a dark container at 4 °C. The BP

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residue was processed two more times. Urea (H2NCONH2) (F. Maia Indústria e Comércio Ltda., SP, Brazil) and sodium chloride (NaCl) (Vetec) were added to the total volume (2.08 L) of the collected supernatant, to reach an approximate concentration of 2 M (249.85 g) and 50 mM (6.08 g), respectively. The pH was adjusted to 7.0 with 1.0 M Tris (Sigma-Aldrich Co., MO, USA) to deactivate pepsin. This mixture, called crude COL, was maintained at 4 °C. Aliquots of the mixture (15 mL) were frozen at − 20 °C. 2.2. Preparation of the crude collagen (crude COL) The best chromatography condition was determined using the test tube method as described in the literature [33]. Four pH values of 6.8, 7.4, 8.2, and 10.0 and different eluting buffer concentrations (0.10, 0.15, 0.25, 0.50, 0.75, and 1.0 M NaCl) were tested. The buffer solution (BS) was prepared using concentrated HCI in 2 L of a 40 mM Tris solution. To this solution, 50 mM NaCl (58.50 g) and 2 M urea (249.85 g) were added to give an ionic strength of approximately 4.75 mS/cm at 26 °C. After centrifugation of the test tubes at 3000 xg, the supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 2.3. Purification of crude COL Purification of the crude COL was performed on a low-pressure liquid chromatography system (Pharmacia Biotech, NJ, USA). The eluent solution (ES) for the elution of the protein adsorbed on the resin was prepared with 300 mL of BS (pH = 10.0) with 0.10 M NaCl (1.76 g), giving an ionic strength of approximately 24.8 mS/cm at 26 °C. Fifty milliliters of DEAE-Sepharose resin (Sepharose-Diethylaminoethyl) CL-6B (DEAE) (Pharmacia Biotech) was prepared and totally immersed in BS (pH = 10.0) for 12 h at 4 °C. The column was slowly filled with DEAE using 400 mL of BS (pH = 10.0) and kept at 4 °C. At the time of use, the column was kept at room temperature and was balanced with 500 mL of BS (pH=10.0) at a flow rate of 2 mL.min−1 in the chromatography system. The material in the column containing DEAE and 3 mL of the crude COL (pH=10.0) were mixed in a rotary shaker for 20 min and returned to the chromatograph system. The column was washed with 400 mL of BS using the same flow rate. The collagen adsorbed on the DEAE was eluted with 200 mL of ES at a flow rate of 3 mL.min−1. After the sample elution, the resin was washed with 100 mL of a 2 M NaCl cleaning solution. This was followed by a wash with 400 mL of a regeneration solution containing 30% isopropyl alcohol (Vetec, Brazil) and with 200 mL of distilled water, both at a flow rate of 5 mL min−1. During the elution, aliquots of 3 mL were collected separately and identified. The aliquots were measured by spectrophotometry at 226 nm and characterized by SDS-PAGE. The pooled aliquots containing collagen were dialyzed against 1.0 mM phosphate-buffered saline at 4 °C for 16 h. The solution was renewed three times during this period. The dialyzed samples were frozen in liquid nitrogen and kept at − 80 °C in a biological freezer (Thermo Fisher Scientific, GA, USA) until they are concentrated in a lyophilizer (Edwards, MA, USA) for 36 h. A simplified flow chart of the procedures for the type I collagen extraction from bovine pericardium to obtain the pure type I collagen is presented in Fig. 1. 2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Crude COL was characterized by PAGE in the presence of SDS. The collagen was diluted in a sample buffer without reduction, concentrated twice with 2% SDS, homogenized and heated in a water bath for 3 min. The high molecular weight (Mw) protein standards (Sigma, USA) contained a mixture of α-macroglobulin (193 kDa), β-galactosidase (112 kDa), fructose-6-phosphate kinase (86 kDa), pyruvate kinase (70 kDa), fumarase (57 kDa), lactate dehydrogenase (39.5 kDa) and

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triosephosphate isomerase (36 kDa). Three microliters of each of the Mw markers and 10 to 30 μL of the crude COL samples were loaded onto the gel and run with a constant electric current at 200 V for approximately 40 min in the electrophoresis equipment (Bio-Rad Laboratories, CA, USA). A silver staining method was used for the visualization and identification of the protein bands in the gel using the Silver stain kit for proteins (Sigma, USA). Samples of the pure COL and the lyophilized pure COL were also characterized by SDS-PAGE. Gel concentrations were 12% (v/v) for the separation process and 4% (p/v) for the stacking process. Commercial type I collagen from calf skin (Sigma, USA) was prepared (1.0 g mL−1) with 1% CH3COOH (v/v) and was used as a purity reference standard.

determine the protein concentration [36]. Samples of the commercial collagen and the lyophilized pure COL were diluted in a solution of 0.01 M CH3COOH at a ratio of 1 mg of protein/1 mL of acid. The quantification of the proteins in the samples at a concentration of 40 μg mL − 1 was determined by the Lowry method [37]. Bovine albumin with a purity of 98%, pH 7.0 (ICN Biomedicals, CA, USA) was used as standard. Each sample was analyzed in duplicate. The extinction coefficient method, a measurement of the collagen samples absorbance readings at 276 nm, was also used. The ratio between the measured values and the extinction coefficient of bovine type I collagen found in the literature [38] was used to quantify the amount of protein in the samples.

2.5. Determination of molecular weight and protein concentration

2.6. Immunization of mice

Using the software Gel-Pro Analyzer (Media Cybernetics, MD, USA), the molecular weights of the different components present in the protein sample were calculated from their characterization in the polyacrylamide gel using the high Mw standards and the collagen samples. Initially, an estimate of the amount of crude COL was obtained using an absorbance reading in a spectrophotometer. Estimates of the protein concentration (mg mL − 1) were determined using the equation (1.55 × A280) − (0.76 × A260) [34,35], where A280 is the absorbance of the sample at 280 nm and A260 is its absorbance at 260 nm. In the first and second stages of the crude COL purification, the collected samples were measured by an absorbance at 226 nm to

A sample of 1 mg pure COL was diluted in 1 mL 1 M CH3COOH (Merck, Brazil) and held at room temperature for 3 h. The commercial collagen sample (pH 8.0) was prepared with a solution of 1.0 M Tris–HCl. An emulsion was prepared by a vigorous mixing of 1.1 mL of a complete Freund's adjuvant (Sigma) and 1.0 mL of the commercial collagen solution (pH 8.0). On the back of 7 female BALB/c mice (weighing 18–22 g), approximately 100 μL of the emulsion was subcutaneously inoculated once a week for 4 weeks. In the first inoculation dose, a complete Freund's adjuvant was used. For the following 3 doses, an incomplete Freund's adjuvant (Sigma, USA) was used. The animals were bled 1 week after the last inoculation. The blood samples

Fig. 1. Flow chart for the extraction and purification of the type I collagen from bovine pericardium.

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were kept at room temperature for 60 min and then stored for 12 h at 4 °C. Sera media were collected and centrifuged in eppendorfs at 100 xg for 15 min at 4 °C. The samples were collected, separated into 100 μL aliquots, and stored at −20 °C. 2.7. Western blotting Two identical gels were prepared by SDS-PAGE with samples of the pure COL. One of the gels was stained with silver to illuminate the protein bands. The other gel was used for Western blotting (WB) to determine the immune reactivity of the serum from the immunized mice. WB was performed using 3.8 μL mouse IgG conjugate and a rabbit anti-IgG (Sigma, USA). The primary antibody was detected with a horseradish peroxidase-labeled secondary antibody and a WB detection kit (mini system Bio-Rad, USA).

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form a 3-dimensional structure with a molecular formula of (α1)2 α2, arranged mostly in the form of a triple helix except for the telopeptides at the ends of the protein [1,2,5]. Pepsin breaks up the collagen molecules in the region of the telopeptides, which contains intermolecular cross links that increase biocompatibility. The form of the BP paste facilitates the pepsin action on the tissue. Enzymatic digestion solubilizes the BP residue, resulting in a cloudy solution with high viscosity. Collagen is released from its natural source and collected after centrifugation to form the crude material extract. Various additives are often included in the crude extract to prevent a chemical or enzymatic degradation of the protein of interest [39]. An addition of a urea solution [19,20,23] increases the linearity of the fiber chains, while NaCl [20] balances the solution without causing a denaturation of the protein. 3.2. Purification of crude COL and characterization by SDS-PAGE

2.8. Energy dispersive X-ray spectroscopy Samples of the pure COL were placed on a glass plate covered with a glass coverslip and examined by transmitted light microscopy and scanning electron microscopy (SEM). BP was immersed in deionized water for 30 min under moderate stirring and then dehydrated with increasing concentrations of acetone/ water solutions (10%, 25%, 50%, 100%). Representative samples of the BP, the commercially supplied collagen, the crude COL, and the lyophilized pure COL were made conductive by coating with a thin layer of sputtered gold. A semiquantitative elemental analysis was performed by energy dispersive X-ray spectroscopy (EDS) (Quest, Thermo Noran, USA) coupled to a scanning electron microscope (at 15 kV). 2.9. Scanning electron microscopy As also performed for the EDS analysis, the scanning electron microscopy samples were coated with Au to analyze their surface morphological structures by capturing images using a scanning electron microscope (JSM-6360 LV; JEOL, Tokyo, Japan, secondary electrons at 10 kV). 2.10. Fourier transform infrared spectroscopy The functional groups of collagen and BP samples were analyzed by Fourier Transform Infrared Spectroscopy (FTIR) using diffuse reflectance (DRIFTS, Perkin-Elmer, Paragon 1000). Samples of BP cut into 0.5 cm2 square fragments were dried in an oven at 60 °C for 60 min. BP, commercial collagen (reference standard), and lyophilized pure COL (0.02 g) were dispersed with pre-dried KBr (110 °C, 2 h, spectroscopy grade, Sigma) using the proportion of 10 wt.% for the measurements (duplicates, n = 2). Background noise was corrected with pure KBr data. The FTIR spectra of the samples were obtained with 32 scans, ranging from 400 to 4000 cm−1 with a resolution of 4 cm−1. 3. Results and discussion 3.1. Extraction of collagen Special precautions were taken in handling BP, a potential source of infectious diseases that may be transmissible to humans. Storing BP in double distilled glycerin and performing the procedures at 4 °C were essential to prevent bacterial growth and to maintain the native form of collagen in the tissue. Alkaline medium (NaOH) solubilizes noncollagenous proteins and negates the effects of endogenous enzymes on collagen [20,26]. To remove proteins and other unnecessary molecules such as lipids and to negate the action of enzymes, the tissue was crushed and immersed in an alkaline solution. Type I collagen consists of polypeptide chains of tropocollagen with a repeated sequence of amino acid residues. These chains

Biomaterials based on collagen and its derivatives require a very high degree of purity. Their final application is intended to interact with biological tissues, with direct involvement in the cell activity promoting tissue repair. The crude extract requires further purification of the protein before being used in the biological applications. In addition to protein stability, other considerations must be taken into account, including the application of the final product, the process efficiency, the economic feasibility and the ease of production [40]. An ion-exchange chromatography process was successfully used in this study. Taking into account the approximate values of the column and accessories used in the technique, this process is considered to be relatively low-cost, second only in cost to gel filtration chromatography [39]. In ion-exchange chromatography, the adsorption column matrices contain ionized chemical groups that interact with the protein. Considering the physicochemical characteristics of collagen, ion-exchange chromatography using a positively charged DEAE resin was chosen as the purification method for the crude COL. Even with a complete characterization of the protein properties, the optimization of a purification protocol involves extensive trial and error experimentation, as the protein behavior can be unpredictable during the purification process. Changes in the protein structure with changes in their physico-chemical characteristics with a modification or loss of biological activity can occur [39]. The pH is directly related to the isoelectric point (pI) and pH control is necessary for the efficient operation of the ion exchange chromatography [41]. A range of pH values from 6.8 to 10.0 was used to determine the optimal pH to be used in the purification of the crude COL. The pH value of 6.8 was chosen as it is above the pI (6.36) of bovine type I procollagen [38]. The physiological pH value of 7.4 and the pH 8.2 used in the protocols of Haralson and Hassel in 1995 [36] and Sato and collaborators in 2003 [20], were also considered as good starting points to improve upon previous studies [34]. We included a pH value of 10.0 in this study because it is above the pI of the chains forming the triple helix of type I bovine collagen (pI of α1(I) = 9.25 and pI of α2(I) = 9.23), according to the ExPASy Proteomics Server [38]. Among the pH values tested, a pH of 10.0 showed better performance, making the sample negatively charged. In addition to the different pH media, various elution solution concentrations were also studied. After the application of a 0.25 M salt solution, there was a significant elution of the protein of interest. This elution did not occur using concentrations of 1.0, 0.75 and 0.50 M NaCl. Fig. 2A graphically shows the complete process of the collagen elution using 0.25 M NaCl. In the graphical representation, the number of fractions represents the number of 3 mL aliquots of the solution collected from the chromatography column during the elution process over a 1 min interval. The absorbance in the ultraviolet range at a wavelength of 226 nm was also measured. The BS solution removes cell debris and proteins which are not adsorbed on the resin [39]. After applying the BS, the pool of eluted

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Fig. 2. Chromatogram of the pure COL elution using 0.25 M NaCl (A). Number of fractions (●) represents the number of 3 mL aliquots of the solution collected from the chromatography column during the elution process over intervals of 1 min (absorbance at λ = 226 nm). SDS-PAGE using a 12% gel concentration for the crude COL (C) shows strong bands visualized near the molecular weight (Mw) of 112 kDa from β-galactosidase of the standard of proteins (B). After the crude COL loading onto the DEAE cellulose column chromatography, BS application (arrow) eluted aliquots (A, pool a) visualized on the 12% gel concentration (D, line a). The entire content of the crude COL remained adsorbed on the resin. The ionic strength of the ES of 0.25 M NaCl (arrow) severed the connections between the DEAE and the crude COL. In the first aliquots (A, pool b) of the formed peak, the identified bands were related to the chains typically found in type I collagen (D, line b). In the SDS-PAGE (D, lines c) a measurement of the aliquots collected just after the peak (A, pools c) did not show the presence of collagen. The second peak was formed with an application of 2 M NaCl (arrow) and the aliquots (A, pool d) showed weaker bands (D, line d), possibly low Mw contaminants such as pepsin and elements used in the processing methods. The cleaning of the column with a 30% isopropyl alcohol solution (arrow) removed all possible hydrophobic proteins, lipoproteins and lipids that were strongly adhered to the resin. The collected aliquots (A, pool e) may be observed on the gel (D, line e). Absorbance at λ = 226 nm for the pure COL elution using the ES of 0.25 M NaCl was 0.129 (A). This measurement was a direct indication of the COL presence in the sample.

aliquots (Fig. 2A, pool a) was collected and analyzed using SDS-PAGE. A gel of the pooled samples showed that the entire content of the crude COL remained adsorbed on the resin (Fig. 2D, line a). The molecular weights of chains α1 (I) and α2 (I) are between 112 and 95.5 kDa [19,20,41,42]. The strongest bands of crude COL (Fig. 2C) were visualized in the gel near the Mw of 112 kDa from β-galactosidase from the standard proteins (Fig. 2B). The Mw of these bands was close to the values described in the literature for the α1 and α2 chains typically found in type I collagen. The ionic strength of the ES with 0.25 M NaCl severed the bonds between the DEAE and the crude COL, causing the collagen to elute as the first significant peak in the chromatogram (Fig. 2A). An SDS-PAGE of the initial pool of aliquots of this peak

(Fig. 2A, pool b) suggested that the bands are related to type I collagen (Fig. 2D, line b). Further aliquots collected just after elution (Fig. 2A, pools c) did not contain collagen as determined using an SDS-PAGE gel (Fig. 2D, lines c). The second relevant peak observed in the chromatogram was formed after the application of a 2 M NaCl solution (Fig. 2A). Weaker bands were also observed for components with molecular weights lower than 112 kDa. These bands are often thought to contain low Mw contaminants from pepsin and elements usually found in the salts used in the processing methods. The higher concentration of salt eliminated the contaminants that remain adsorbed on the resin. Aliquots of the pool may be observed on the gel (Fig. D, line d). The cleaning of the column with an isopropyl alcohol solution (Fig. 2A) removed any

Fig. 3. Chromatogram of the pure COL elution using 0.10 M NaCl (A). Building on the results from the ES of 0.25 M NaCl, a simplified procedure for the elution of pure COL was used. After loading the crude COL onto the DEAE resin, an elution using the ES with 0.10 M NaCl (A, arrow) formed a characteristic peak. Immediately after the peak, the fractions were measured to be close to zero (A). The pooled aliquots of the peak corresponding to fractions 7–11 (A) were characterized by SDS-PAGE (B). Each of the fractions showed the same characteristic pattern of bands (D, lines 7–11). Bands near the Mw of 112 kDa (B, lane S) showed in the gel (B) are close to the values described in the literature for the α1 and α2 chains typically found in type I collagen. Another band in the gel (B) was observed near the Mw of 193 kDa from α-macroglobulin of the standard proteins (B, lane S), which corresponds to β-type chains. Absorbance at λ = 226 nm for the pure COL elution using the ES of 0.10 M NaCl was 0.378 (A).

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Fig. 4. SDS-PAGE at 12% gel concentration of the pooled aliquots corresponding to fractions 7–11 (A) and the lyophilized pure COL (B) using the ES of 0.10 M NaCl, showing similar type I collagen characteristics for the commercial collagen (C). After purification of the crude COL, bands α1 (I) α2 (I) and β-type were visible in the final product (B), with Mw values close to the Mw of the type I collagen bands. The lyophilized pure COL collagen (B) is essentially free of other proteins and purified relative to bovine pericardium and is comparable to type I commercial collagen (C), the standard for purity used in this study.

possible hydrophobic proteins, lipoproteins and lipids that were strongly adhered to the resin. Aliquots of the pool may be observed on the gel (Fig. D, line e). Washing with deionized water prevented the contamination and clogging of the resin for the following purification procedures. To build upon the elution results with 0.25 M NaCl, salt concentrations of 0.15 and 0.10 M were also used for the elution of the collagen. Both salt concentrations demonstrated the same elution pattern in the chromatogram with absorbance readings at 226 nm. ES with the lower salt concentrations still had a high enough ionic strength to disrupt only the bonds formed between the DEAE groups and the crude COL, resulting in an elution of pure COL. These results suggested that the salt concentration of 0.10 M NaCl should be used for the protein elution as this minimizes the contamination of the sample with salt. However, a small amount of NaCl in the purified sample may be acceptable as salt often contributes to protein preservation. The elution of the crude COL on DEAE with a salt concentration of 0.10 M NaCl can be observed in the chromatogram showed in Fig. 3A. This profile is consistent with other chromatograms for the α chains of type I collagen found in the literature [23,36]. Aliquots of the peak (Fig. 3A, fractions 7–11) were analyzed using a polyacrylamide gel (Fig. 3B, lanes 7–11). Lower salt concentrations in the ES resulted in a more defined peak, with each fraction showing the same characteristic pattern of the bands for type I collagen [19,20,23,36]. After using an ES with 0.10 M NaCl (Fig. 3A), the absorbance at 226 nm for the final fractions remained very close to zero. The peptide bonds common to all proteins (amide chromophore) show optical activity with the n–π* and π–π* transitions at wavelengths of approximately 250 nm and 190 nm, respectively [34,35]. The measurement of the absorbance at a wavelength of 226 nm is used to identify the presence of type I collagen. This method was used in the protocols for obtaining and purifying this protein [36]. Absorbance in the ultraviolet range at a wavelength of 226 nm was a direct indication of the COL presence in the samples. The absorbance for the collagen elution from DEAE using an ES with 0.25 M NaCl and 0.10 M NaCL was 0.129 (Fig. 2A) and 0.378 (Fig. 3A), respectively. The aliquots containing pure COL, corresponding to fractions 7 to 11, were mixed and characterized by SDS-PAGE (Fig. 4A). The pooled aliquots showed the presence of pure COL using an elution solution of 0.10 M NaCl. Another band was observed near the Mw of 193 kDa as calibrated

from the standard protein α-macroglobulin (Fig. 4A, lane S). According to the results reported by Lacerda et al. [41], this band most likely corresponds with 2 α chains bound together, termed β-type chains. The final stage of the collagen purification involved dialysis and lyophilization to remove the remaining contaminants still present in the sample. The pooled aliquots collected during the purification process underwent dialysis in membranes with a porosity value less than the size of the protein. The goal of the dialysis was to remove salts or other nonvolatile molecules from the protein solution. After dialysis,

Fig. 5. Graphical representation (A) of the curves for standard Mw (a) and the curves for Mw of the pure COL bands (b), calculated using the software Gel-Pro Analyzer with the values of the Mw standard (lane a) and the sample of the pure COL (lane b) in the SDS-PAGE at 12% gel concentration (B), respectively. The curves in the graphic (A, b) and the bands in the polyacrylamide gel (B, lane b) with Mw values of 125.38 and 117.33 kDa correspond with the typical α1 and α2 chains of the pure type I COL. Some bands located in the area of 193 kDa from the standard curve were measured to have Mw values of 191.72, 173.56, and 161.34 kDa (A, b). One band with significant intensity and an estimated Mw of 173.56 kDa might represent type β chains. Two more bands with lower intensity were detected with Mw of 108.85 kDa and 55.27 kDa (A, b).

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proportion of 1 mg/1 mL of 0.1 N CH3COOH, as specified by the manufacturer for its solubilization. 3.4. Molecular weight evaluation

Fig. 6. WB analysis for type I collagen. The proteins were immobilized on a nitrocellulose membrane. The chromogen reacted with the peroxidase of the conjugate produced with the rabbit anti-mouse IgG that was connected to the anti-type I collagen produced by the mice (lane B), characterizing the lyophilized pure COL as a type I collagen. These results are similar to the commercial bovine type I collagen transferred in lane A. Transfer of the Mw markers (lane S).

the pure COL was lyophilized to remove volatile contaminants [39]. The lyophilized pure COL can be observed in Fig. 4B. SDS-PAGE was used to analyze the purity of the collagen preparations. The analysis indicated that the isolated collagen was essentially free of other proteins and purified from other components of the bovine pericardium. After the purification of the crude COL, bands α1 (I) α2 (I) and β-type remained visible in the pure COL and the lyophilized pure COL (Fig. 4B). The bands corresponding to the contaminants had been practically eliminated. Bands of the lyophilized pure COL were located between the standard protein bands (Fig. 4B, lane S) with molecular weights close to the molecular weights of collagen bands reported in the literature [41,42]. The characteristic bands of the lyophilized pure COL (Fig. 4B) were compared with the bands of commercial collagen (Fig. 4C), the standard for purity used in this study.

3.3. Concentration of pure COL Lyophilized pure COL (Fig. 4B) was measured to have a concentration of 0.23 g mL − 1 by the Lowry method [37]. Using the extinction coefficients of the α2(I) chain, these values ranged from 0.90 to 0.33 mg mL − 1. With the values for the α1(I) chain of type I bovine collagen found in the literature, these values ranged from 0.58 to 0.24 mg mL−1. The lowest concentration found using the extinction coefficient was 0.24 mg mL−1, which was very close to the concentration found using the Lowry method. By the same method, the concentration found for commercial collagen (Fig. 4C) was 0.50 mg mL−1, using the

Fig. 5A represents the Mw standard curve (a) and the Mw curve of the lyophilized pure COL bands (b) calculated using the software Gel-Pro Analyzer from the characterization of the high Mw standards (Fig. 5B, lane a) and the sample of the lyophilized pure COL (Fig. 5B, lane b) in the polyacrylamide gel, respectively. The Mw of the pure COL bands was observed to be located in the area of the highest Mw in the standard curve. Using the standard curve, some of the bands were determined to be located in the area of 193 kDa giving molecular weights of 191.72, 173.56, and 161.34 kDa (Fig. 5A, b). One band with significant intensity had an estimated Mw of 173.56 kDa, most likely type β chains [41,44]. Two bands of higher intensity, most likely corresponding to α1 and α2 chains of pure type I COL, had molecular weights of 125.38 and 117.33 kDa and are observed in the graphical representation of the standard curve (Fig. 5A, b) and the respective polyacrylamide gel (Fig. 5B, lane b). Two more bands with lower intensities were detected, one with a Mw of 108.85 kDa and another with a value of 55.279 kDa (Fig. 5A, b). The molecular weights reported in the literature for α1 and α2 chains of type I collagen obtained by other solubilization methods [41,43] are smaller than the molecular weights found in this study. Using an organic solvent in alkaline medium for the collagen extraction, a selective hydrolysis of the carboxyamide groups of asparagine and glutamine residues occurs. There is a decrease in the intensity of the hydrophobic electrostatic interactions and the hydrogen bridges that facilitates collagen solubilization, resulting in lower molecular weights [44]. The enzyme solubilization method involves the removal of the telopeptides, increasing the fraction of soluble collagen. This may explain the higher Mw found for the pure COL. For the conditions used, an extensive degradation or solubilization of the matrix may not have occurred, as suggested by Lacerda et al. [41]. Despite being classified as a type I collagen, these proteins may also have small differences in their structure across different tissues in the same animal, or in the same tissue between different animals. These potential variances may lead to different molecular weights in the isolated collagen. Protein characterization using polyacrylamide gels at different concentrations can also contribute to the variances in molecular weights, due to the permeability of the particular gel. 3.5. Western blotting Western blotting (WB) is widely used for protein analysis. The proteins immobilized on a nitrocellulose membrane can be detected using enzymes linked to antibodies, which are viewed with a chromogenic substrate [34,45]. Caputo et al. [46] reported the use of WB to characterize collagen, identifying the specific bands of collagen type I. In our study the transfer of the collagen bands to the nitrocellulose membrane was observed by staining sites corresponding to the bands present in the gel. The chromogen reacted with the peroxidase of the conjugated rabbit anti-mouse IgG connected to the anti-type I collagen produced by the mice (Fig. 6B). This method characterized the pure COL as a type I collagen, similar to the commercial bovine type I collagen (Fig. 6A). The high Mw bands of the standard protein control may be identified in Fig. 6S. 3.6. Scanning electron microscopy/energy dispersive X-ray spectroscopy Commercial collagen appeared as a dense membrane. The presence of some fibers was observed in the transmitted light microscopy. The pure COL had a less dense fibrillar structure with translucent fibers. Characterization by SEM/EDS is essential for the determination of the final purity of the collagen. SEM images of the pure COL showed

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Fig. 7. SEM images: the pure COL showing contamination with NaCl crystals (A) and peaks of high intensity for these elements in their respective spectrum (a); particles observed on the surface of the pure COL (B, circles) and an analysis these specific areas by EDS, showing high concentrations of NaCl (b); dialyzed pure COL (C) showing lower concentrations of NaCl in their EDS spectrum (c).

contamination by NaCl crystals before dialysis (Fig. 7A), as indicated by the presence of the high intensity peaks for these elements in their respective EDS spectrum (Fig. 7A). Some particles were observed on the surface of the pure COL before dialysis, as observed in the SEM photomicrograph of Fig. 7B. The analysis focused only on these specific areas that showed the presence of NaCl at high concentrations, as observed in the EDS spectrum of Fig. 7B. The SEM image of the pure COL taken immediately after dialysis (Fig. 7C) showed lower concentrations of NaCl (EDS spectrum, Fig. 7C). In the SEM micrographs, a dense structure of a dehydrated BP was observed, exhibiting fibers with a certain parallelism and ripples in the longitudinal direction. Although not very well organized, the dominant orientation of these fibers follows the circumferential direction of the pericardial sac, demonstrating an anisotropic structure with a fibrous meshwork appearance (Fig. 8A). The images suggest a similarity between the structure of the commercial collagen (Fig. 8B) and the structure of the lyophilized pure COL (Fig. 8C). BP was characterized by the parallel orientation of its intersecting fibrils. Compared with BP, a complete rearrangement of the fibers was observed for the lyophilized pure COL structure. The fibrillar appearance observed in BP changed into an amorphous structure, characterized by a structure composed of strips (Fig. 8C). Certain areas contained disorganized nanofibers (Fig. 8D). A spongy-like structure (Fig. 8E) with irregularly distributed and interconnected pores (Fig. 8F) was observed in other locations.

The EDS spectrum of BP (Fig. 9A) showed the presence of carbon, oxygen and low-intensity peaks of gold from the metallic coating. The spectrum pattern of the lyophilized pure COL (Fig. 9B) showed the presence of the relevant peaks, suggesting a relatively high content of carbon corresponding to the main element of organic components, small amounts of sodium and chlorine corresponding to the elements usually found in the salts used in the processing methods, and sulfur corresponding to the amino acids present in the collagen protein structure. The presence of some peaks of low intensity corresponding to the trace elements in the sample was reported by Andrade et al., in 2004 [47]. The presence of NaCl in collagen provides structural stability, helping to avoid protein denaturation. However, the presence of larger amounts of NaCl, as well as other elements in the collagen product, contributes to the contamination of the material [47]. Other trace elements such as Mg, Al, Si, P, K and Ca were observed in the EDS spectrum of the commercial collagen (Fig. 9C). These contaminants were most likely derived from the chemical process, possibly compromising the purity of the collagen. 3.7. Fourier transform infrared spectroscopy characterization FTIR spectroscopy was used as a supporting tool for characterizing the collagen extracted and purified from the BP tissue. As discussed in the literature [12,13,48–50], collagen is a generic term that covers a

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Fig. 8. SEM images show structures that suggest: intersecting fibers with a parallel orientation and a meshwork appearance in BP (A); rearrangement of the fibers into an amorphous structure in the commercial type I collagen (B) and in the pure COL that showed areas of disorganized strips (C), nanofibers (D) and spongy like structures (E) with irregularly distributed and interconnected pores (F).

broad class of proteins, each with specific structures, functions, and tissue distributions in the extracellular matrix. The functional groups of collagen may be detected by FTIR spectroscopy because the amide groups of the polypeptides and proteins possess a number of characteristic vibrational modes or group frequencies. Essentially, the peptide group has nine amide characteristic bands (A, B, I, II … VII). These important functional groups (Fig. 10A) in living organisms connect the sequence of amino acids to form the α chain of type I collagen molecule (Fig. 10B) [1,2,50]. Amide I and amide II bands are the two major bands of the protein infrared spectrum. The amide I band (ranging from 1700 to 1600 cm−1) is mainly associated with the C\O stretching vibration and is directly related to the backbone conformation. The Amide II band is derived from the N\H bending vibration and from the C\N stretching vibration. The amide III band is usually weak in FTIR spectroscopy but can be usually found in the region at 1350– 1250 cm−1 [51,52]. We found that the FTIR spectra of the commercial collagen (Fig. 10C) and the lyophilized pure COL (Fig. 10B) were comparable to the spectrum of the BP sample (Fig. 10A). These similarities were expected because BP is a collagen-rich biological tissue containing mostly type I collagen [26–30]. More specifically, all major bands related to the relevant functional groups of collagen were detected (Fig. 10C). We observed peaks associated with the amide

regions, amide I (1680–1620 cm−1), amide II (1580–1480 cm−1), and amide III (1300–1200 cm−1). The C\H broad alkyl stretching band (2850–3000 cm−1) and the strong hydroxyl band (3200–3600 cm−1) overlapping with the N\H stretching band (3360–3320 cm − 1, amide-A) were also observed. The samples also had a signal at the 1360–1340 cm−1 band (Fig. 10C, dashed rectangle) Representing more than the carbonyl groups, this band is also characteristic of the wagging vibration of the proline side chains present in the type I collagen found in biological tissues [53]. An absorption band corresponding to the carboxylic functional group (\COOH) at 1740–1720 cm−1 was also found in the commercial collagen spectrum (Fig. 10C) but not in the lyophilized pure COL sample (COL, Fig. 10B). It may be noted that some differences in the bands of the FTIR absorption peaks for collagen and collagen-containing tissues have been reported in the literature [48,49,54,55]. These differences may be due to variability in the material investigated as well as the variety of instruments and methods used for the measurements. Some regions of the spectra may present a broad band, as a result of two or more bands overlapped, or as a set of resolved bands. In that sense, a full characterization of the purified collagen using FTIR spectroscopy is beyond the scope of this investigation. The collagen produced in this study was characterized as a purified type I collagen extracted from bovine pericardium tissue based

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Fig. 10. Representation of the relevant chemical functional groups and molecules: (A) amides; (B) amino acids of type I collagen: glycine, proline or hydroxyproline; (C) FTIR spectra of: (a) BP, (b) lyophilized pure COL and (c) commercial collagen.

production. After obtaining a pure COL, improvements were necessary in some protein processing stages as well as the characterization tools. Further development on the protein quantification methods are needed to obtain the best final yield and a precise quantification of the lyophilized pure COL product. This material might be suitable for use in the field of tissue engineering.

4. Conclusions

Fig. 9. EDS spectra of: BP with peaks corresponding to the presence of carbon, oxygen and peaks of low intensity of gold regarding the metallic coating (A); pure COL with peaks corresponding to a high content of carbon, small amounts of sodium and chlorine and sulfur (B); and commercial type I collagen with the presence of carbon peaks and several trace elements (C).

Bovine Pericardium tissues were utilized as a natural source for the extraction of collagen protein. The BP tissue was subjected to an enzymatic digestion followed by a new process methodology for the purification of type I collagen using ion-exchange chromatography. The purified COL was characterized as a type I collagen that has morphological and spectroscopy characteristics comparable to a commercially available high purity collagen. The pure COL produced using a relatively simple processing route can be considered as a promising source of biomaterial for a myriad of biological applications. The methodology developed in this study is potentially suitable for transferring to an industrial scale.

Acknowledgements

on a combination of analytical methods. The BP tissue is an affordable animal source of protein that can be extracted and purified by a relatively simple methodology that may be applied to the industrial scale

The authors express their gratitude to the researchers and technicians of the Immunology Laboratory of Foundation Ezequiel Dias and SEM Microscopy Laboratory (DEMET-EEUFMG). Additionally, the authors thank CAPES, CNPq and FAPEMIG for financial support.

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