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

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Materials Science and Engineering C

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Extraction and characterization of highly purified collagen from bovine pericardiumfor 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, Brazilb Center for Assessment and Development of Biomaterials—BioMat, Federal University of Vales do Jequitinhonha e Mucuri—UFVJM, Diamantina/MG 39100–000, Brazilc Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais—UFMG, Belo Horizonte/MG 31270–901, Brazild Department of Health Science, Ezequiel Dias Foundation—FUNED, Belo Horizonte/MG 30510–010, Brazil

⁎ Corresponding author at: Department of Dentistry,Jequitinhonha e Mucuri—UFVJM, Diamantina/MG 3913532 6066.

E-mail address: mariahelena.santos@gmail.com (M.H

0928-4931/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.msec.2012.11.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 October 2011Received in revised form 30 August 2012Accepted 1 November 2012Available online 10 November 2012

Keywords:BiomaterialsCollagenProtein ElectrophoresisCharacterization of BiomaterialsTissue replacementsBovine collagen xenografts

Bovine pericardium is widely used as a raw material in bioengineering as a source of collagen, a fundamentalstructural molecule. The physical, chemical, and biocompatibility characteristics of these natural fibers enabletheir broad use in several areas of the health sciences. For these applications, it is important to obtain collagenof the highest possible purity. The lack of a method to produce these pure biocompatible materials using sim-ple and economically feasible techniques presents a major challenge to their production on an industrialscale. This study aimed to extract, purify, and characterize the type I collagen protein originating from bovinepericardium, considered to be an abundant tissue resource. The pericardium tissue was collected from maleanimals at slaughter age. Pieces of bovine pericardium were enzymatically digested, followed by a novel pro-tocol developed for protein purification using ion-exchange chromatography. The material was extensivelycharacterized by electrophoresis, scanning electron microscopy, energy dispersive X-ray spectroscopy, andinfrared spectroscopy. The results showed a purified material with morphological properties and chemicalfunctionalities 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 collagenfrom 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 tissueshave drawn the attention from the research community as a promisingalternative in regenerative medicine. Collagen is an extracellularmatrixcomponent that is frequently used as a biomaterial [2–4]. Type I colla-gen, themost common type of collagen, is themajor component of con-nective tissues such as tendons, ligaments, skin, bone, dentin, cornea,and the fibrous capsules of internal organs [5,6]. The insoluble fibersof this collagen have a high tensile strength that provides mechanicalsupport for multiple physiological functions. Another important roleof collagen is to provide guidance for developing tissues [1,2,6,7]. Theextensive use of collagen in medicine, dentistry, and pharmacology isalso related to its natural properties, including hemostatic activity, bio-degradability, low allergenicity with high antigenicity and biocompati-bility [8]. Furthermore, it is an efficient porous matrix (scaffold) with

Federal University of Vales do00–000, Brazil. Tel.: +55 38

. Santos).

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specific physical, chemical, and biological features that are of interestfor multiple bioengineering and cell culture applications [9–15]. Type Icollagen is currently the gold standard in the field of tissue-engineering[14].

Collagen-based biomaterials can originate from two fundamentaltechniques to form a functional scaffold. One is a decellularized collagenmatrix preserving the original tissue shape and extracellular matrixstructure. None of the methods used for tissue decellularization canproduce an extracellular matrix completely free of cellular debris anda combination of techniques is often required to obtain a material freeof any cell remnant [16]. To realize the potential beneficial effects of bi-ologic scaffolds in the field of tissue engineering and regenerative med-icine, optimal methods of decellularization are needed [17]. The othertype of collagen-based biomaterial is made from a collagen extractionfrom biological tissues. Acid solubilization, neutral salt solutions andproteolytic solutions are often used to extract collagen from biologicaltissues. However, proteolytic extraction alters the collagen molecularstructure by cleaving the terminal telopeptide regions, resulting in thedecrease in the tropocollagen self-assembled fibrils. To maximize yield,an acid extraction using a pepsin solubilization is themost effective tech-nique, despite the possible occurrence of cleavage or a partial denaturingof the telopeptides [14].

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Proteins derived fromanimal sourcesmust be purified to ensure theirsafety for biomedical applications. Multiple techniques are reported inthe literature [14,18–21] and new methods of purification have beenattempted [22–24]. In general, complex and costly methods are usedfor designing and developing medical-grade protein-based xeno-grafts. Novel simpler processing methods that comply with the cur-rent established validated purification protocols are needed to lowerproduction costs, matching the demand for biomaterials originatingfrom renewable and abundant sources [5,14,25]. In addition, medical-grade purified collagen is not readily commercially available in manycountries. The importation of this biomaterial can be costly and is oftenrestricted by governmental health agencies. The number of industriesin these countries that are involved in graded materials for biomedicalapplications is still deficient.

As a type I collagen-rich tissue, Bovine Pericardium (BP) has beenwidely used as a raw material for biomaterials in various areas of thehealth sciences [26,27]. It is a heterologous biological material withexcellent biocompatibility, suitable biophysical properties, and elasticproperties due to the high content of collagen fibers [28–30]. In addi-tion, BP is an easy-to-handle material with worldwide availability anda relative low cost, as it is a by-product of the animal slaughteringprocess. From an economical perspective, the use of animal tissues thatare usually treated as waste by the meat industry is rather attractive.This is a very important issue worldwide. It is estimated that approxi-mately 50% of the animal weight is meat production waste (50–54% foreach cow, 52% for each sheep or goat) [31,32]. Specifically in Brazil, thecost of BP is less than USD 0.50 kg. On the other hand, the cost of bioma-terials is usually expensive for the end-user, typically sold in the range ofUSD 500.00–5,000.00 g. From an industrial perspective, there is a verybroad commercial margin of over 1000 times the actual cost of the natu-ral rawmaterials, such as BP. New techniques to isolate and purify thesebiomaterials for tissue reconstruction are needed to meet the growingdemand for their various applications in diverse areas of health sciences.

This study aimed at extracting and purifying type I collagen proteinfrom BP tissue using an innovative and relatively simplemethod for thepotential use as a biomaterial in tissue engineering. Thismethodmay betransferrable to an industrial-scale process. The collagen-based mate-rials were extensively characterized by gel electrophoresis andWesternblotting through mice immunization experiments. Scanning electronmicroscopy associated with energy dispersive X-ray spectroscopy andFourier transform infrared spectroscopywere used to characterizemor-phology and molecular chemical structures.

2. Materials and methods

2.1. Extraction of collagen

Collagen was extracted from intact BP tissue pieces collected frommale animals at slaughter age (18–24 months). Tissues adjacent to thedense connective tissuewere removed. BPwaswashedwith Ringer's so-dium lactate solution (Sanobiol Laboratory, SP, Brazil) twice and cut intoflaps of 5×15 cm2 in the longitudinal direction to preserve the naturalarchitecture of its fibers. BP flaps were submerged in double-distilledglycerin (Labsynth, SP, Brazil) at room temperature. Standard personalprotective equipment was used during the procedures for handling BP.

After 60 days, the BP flapswere shredded and kept fully immersed in300 mL of a 0.1 M sodium hydroxide (NaOH) solution (Vetec QuímicaFina Ltda., RJ, Brazil), at a temperature of (4 ± 1)°C. After 48 h, the resi-due was filtered 3 times. The BP was processed using an Ultra-Turraxmacerator (Janke and Kunkel, IKA, Germany) in a cooled vessel. Pepsin(21.45 g) (Vetec) was solubilized in 300 mL of 10 mM hydrochloricacid (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 ad-justed to 2.0 with concentrated HCl andmaintained for 12 h at 4 °C. Themixturewas then centrifuged at 10,000 xg for 20 min at 4 °C. The super-natant was collected and stored in a dark container at 4 °C. The BP

residue was processed two more times. Urea (H2NCONH2) (F. MaiaIndústria e Comércio Ltda., SP, Brazil) and sodium chloride (NaCl)(Vetec) were added to the total volume (2.08 L) of the collected su-pernatant, 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 with1.0 M Tris (Sigma-Aldrich Co., MO, USA) to deactivate pepsin. Thismixture, called crude COL, was maintained at 4 °C. Aliquots of themixture (15 mL) were frozen at −20 °C.

2.2. Preparation of the crude collagen (crude COL)

The best chromatography condition was determined using the testtube 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 solu-tion. 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 at26 °C. After centrifugation of the test tubes at 3000 xg, the supernatantswere analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE).

2.3. Purification of crude COL

Purification of the crude COL was performed on a low-pressure liq-uid chromatography system (Pharmacia Biotech, NJ, USA). The eluentsolution (ES) for the elution of the protein adsorbed on the resin waspreparedwith 300 mLof BS (pH=10.0)with 0.10 MNaCl (1.76 g), giv-ing an ionic strength of approximately 24.8 mS/cm at 26 °C. Fifty milli-liters 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 DEAEusing 400 mL of BS (pH=10.0) and kept at 4 °C.

At the timeof use, the columnwas kept at room temperature andwasbalanced with 500 mL of BS (pH=10.0) at a flow rate of 2 mL.min−1 inthe chromatography system. The material in the column containingDEAE and 3 mL of the crude COL (pH=10.0) were mixed in a rotaryshaker for 20 min and returned to the chromatograph system. The col-umn was washed with 400 mL of BS using the same flow rate. The col-lagen adsorbed on the DEAEwas elutedwith 200 mL of ES at a flow rateof 3 mL.min−1. After the sample elution, the resin was washed with100 mL of a 2 M NaCl cleaning solution. This was followed by a washwith 400 mL of a regeneration solution containing 30% isopropyl alco-hol (Vetec, Brazil) and with 200 mL of distilled water, both at a flowrate of 5 mL min−1. During the elution, aliquots of 3 mLwere collectedseparately and identified. The aliquots were measured by spectropho-tometry at 226 nm and characterized by SDS-PAGE.

The pooled aliquots containing collagen were dialyzed against1.0 mM phosphate-buffered saline at 4 °C for 16 h. The solution wasrenewed three times during this period. The dialyzed samples werefrozen in liquid nitrogen and kept at −80 °C in a biological freezer(Thermo Fisher Scientific, GA, USA) until they are concentrated in alyophilizer (Edwards, MA, USA) for 36 h.

A simplified flow chart of the procedures for the type I collagenextraction from bovine pericardium to obtain the pure type I collagenis presented in Fig. 1.

2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Crude COLwas characterized by PAGE in the presence of SDS. The col-lagen was diluted in a sample buffer without reduction, concentratedtwice 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 theMw markers and 10 to 30 μL of the crude COL samples were loadedonto the gel and runwith a constant electric current at 200 V for approx-imately 40 min in the electrophoresis equipment (Bio-Rad Laboratories,CA, USA). A silver staining method was used for the visualization andidentification of the protein bands in the gel using the Silver stain kitfor proteins (Sigma, USA). Samples of the pure COL and the lyophilizedpure COL were also characterized by SDS-PAGE. Gel concentrationswere 12% (v/v) for the separation process and 4% (p/v) for the stackingprocess. Commercial type I collagen from calf skin (Sigma, USA)was pre-pared (1.0 g mL−1) with 1% CH3COOH (v/v) and was used as a purityreference standard.

2.5. Determination of molecular weight and protein concentration

Using the software Gel-Pro Analyzer (Media Cybernetics, MD, USA),themolecularweights of the different components present in the proteinsample were calculated from their characterization in the polyacryl-amide gel using the high Mw standards and the collagen samples.Initially, an estimate of the amount of crude COL was obtainedusing an absorbance reading in a spectrophotometer. Estimates ofthe protein concentration (mg mL−1) were determined using theequation (1.55×A280)−(0.76×A260) [34,35], where A280 is the ab-sorbance of the sample at 280 nm and A260 is its absorbance at260 nm. In the first and second stages of the crude COL purification,the collected samples were measured by an absorbance at 226 nm to

Fig. 1. Flow chart for the extraction and purification

determine the protein concentration [36]. Samples of the commer-cial collagen and the lyophilized pure COL were diluted in a solutionof 0.01 M CH3COOH at a ratio of 1 mg of protein/1 mL of acid. Thequantification of the proteins in the samples at a concentration of40 μg mL−1 was determined by the Lowrymethod [37]. Bovine albu-min with a purity of 98%, pH 7.0 (ICN Biomedicals, CA, USA) was usedas standard. Each sample was analyzed in duplicate. The extinctioncoefficient method, a measurement of the collagen samples absor-bance readings at 276 nm, was also used. The ratio between themea-sured values and the extinction coefficient of bovine type I collagenfound in the literature [38] was used to quantify the amount of pro-tein in the samples.

2.6. Immunization of mice

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 commer-cial collagen sample (pH 8.0) was prepared with a solution of 1.0 MTris–HCl. An emulsion was prepared by a vigorous mixing of 1.1 mLof a complete Freund's adjuvant (Sigma) and 1.0 mL of the commer-cial 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 sub-cutaneously inoculated once a week for 4 weeks. In the first inocula-tion dose, a complete Freund's adjuvant was used. For the following 3doses, an incomplete Freund's adjuvant (Sigma, USA)was used. The an-imals were bled 1 week after the last inoculation. The blood samples

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 at4 °C. Sera media were collected and centrifuged in eppendorfs at 100xg for 15 min at 4 °C. The samples were collected, separated into100 μL aliquots, and stored at−20 °C.

2.7. Western blotting

Two identical gels were prepared by SDS-PAGE with samples of thepure COL. One of the gelswas stainedwith silver to illuminate the proteinbands. The other gel was used for Western blotting (WB) to determinethe immune reactivity of the serum from the immunized mice. WB wasperformed using 3.8 μL mouse IgG conjugate and a rabbit anti-IgG(Sigma, USA). The primary antibody was detected with a horseradishperoxidase-labeled secondary antibody and aWBdetection kit (mini sys-tem Bio-Rad, USA).

2.8. Energy dispersive X-ray spectroscopy

Samples of the pure COL were placed on a glass plate covered witha glass coverslip and examined by transmitted light microscopy andscanning electron microscopy (SEM).

BPwas immersed in deionizedwater for 30 minundermoderate stir-ring and then dehydrated with increasing concentrations of acetone/water solutions (10%, 25%, 50%, 100%). Representative samples of theBP, the commercially supplied collagen, the crude COL, and the lyophi-lized pure COL were made conductive by coating with a thin layer ofsputtered gold. A semiquantitative elemental analysis was performedby 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 mi-croscopy samples were coated with Au to analyze their surface mor-phological structures by capturing images using a scanning electronmicroscope (JSM-6360 LV; JEOL, Tokyo, Japan, secondary electronsat 10 kV).

2.10. Fourier transform infrared spectroscopy

The functional groups of collagen and BP samples were analyzed byFourier Transform Infrared Spectroscopy (FTIR) using diffuse reflec-tance (DRIFTS, Perkin-Elmer, Paragon 1000). Samples of BP cut into0.5 cm2 square fragments were dried in an oven at 60 °C for 60 min.BP, commercial collagen (reference standard), and lyophilized pureCOL (0.02 g) were dispersed with pre-dried KBr (110 °C, 2 h, spectros-copy grade, Sigma) using the proportion of 10 wt.% for the measure-ments (duplicates, n=2). Background noise was corrected with pureKBr data. The FTIR spectra of the samples were obtained with 32scans, 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 sourceof infectious diseases that may be transmissible to humans. StoringBP in double distilled glycerin and performing the procedures at4 °C were essential to prevent bacterial growth and to maintain thenative form of collagen in the tissue. Alkaline medium (NaOH) solubi-lizes noncollagenous proteins and negates the effects of endogenousenzymes on collagen [20,26]. To remove proteins and other unneces-sary molecules such as lipids and to negate the action of enzymes, thetissue was crushed and immersed in an alkaline solution.

Type I collagen consists of polypeptide chains of tropocollagenwith a repeated sequence of amino acid residues. These chains

form a 3-dimensional structure with a molecular formula of (α1)2α2, arranged mostly in the form of a triple helix except for thetelopeptides at the ends of the protein [1,2,5]. Pepsin breaks up thecollagen molecules in the region of the telopeptides, which containsintermolecular cross links that increase biocompatibility. The form ofthe BP paste facilitates the pepsin action on the tissue. Enzymatic di-gestion solubilizes the BP residue, resulting in a cloudy solution withhigh viscosity. Collagen is released from its natural source and col-lected after centrifugation to form the crude material extract. Vari-ous additives are often included in the crude extract to prevent achemical or enzymatic degradation of the protein of interest [39].An addition of a urea solution [19,20,23] increases the linearity ofthe fiber chains, while NaCl [20] balances the solution without caus-ing a denaturation of the protein.

3.2. Purification of crude COL and characterization by SDS-PAGE

Biomaterials based on collagen and its derivatives require a veryhigh degree of purity. Their final application is intended to interactwith biological tissues, with direct involvement in the cell activitypromoting tissue repair. The crude extract requires further purifica-tion of the protein before being used in the biological applications. Inaddition to protein stability, other considerations must be taken intoaccount, including the application of the final product, the processefficiency, the economic feasibility and the ease of production [40].An ion-exchange chromatography process was successfully used inthis study. Taking into account the approximate values of the columnand accessories used in the technique, this process is considered tobe relatively low-cost, second only in cost to gel filtration chroma-tography [39]. In ion-exchange chromatography, the adsorption col-umnmatrices contain ionized chemical groups that interact with theprotein. Considering the physicochemical characteristics of collagen,ion-exchange chromatography using a positively charged DEAE resinwas chosen as the purification method for the crude COL.

Even with a complete characterization of the protein properties, theoptimization of a purification protocol involves extensive trial and errorexperimentation, as the protein behavior can be unpredictable duringthe purification process. Changes in the protein structure with changesin their physico-chemical characteristics with a modification or loss ofbiological activity can occur [39].

The pH is directly related to the isoelectric point (pI) and pH con-trol is necessary for the efficient operation of the ion exchange chro-matography [41]. A range of pH values from 6.8 to 10.0 was used todetermine the optimal pH to be used in the purification of the crudeCOL. The pH value of 6.8 was chosen as it is above the pI (6.36) of bo-vine type I procollagen [38]. The physiological pH value of 7.4 and thepH 8.2 used in the protocols of Haralson and Hassel in 1995 [36] andSato and collaborators in 2003 [20], were also considered as goodstarting points to improve upon previous studies [34]. We includeda pH value of 10.0 in this study because it is above the pI of the chainsforming the triple helix of type I bovine collagen (pI of α1(I)=9.25and 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 perfor-mance, making the sample negatively charged.

In addition to the different pHmedia, various elution solution con-centrations were also studied. After the application of a 0.25 M saltsolution, there was a significant elution of the protein of interest.This elution did not occur using concentrations of 1.0, 0.75 and0.50 M NaCl. Fig. 2A graphically shows the complete process of thecollagen elution using 0.25 M NaCl. In the graphical representation,the number of fractions represents the number of 3 mL aliquots ofthe solution collected from the chromatography column during theelution process over a 1 min interval. The absorbance in the ultravio-let range at a wavelength of 226 nm was also measured.

The BS solution removes cell debris and proteins which are notadsorbed on the resin [39]. After applying the BS, the pool of eluted

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 chroma-tography 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 bandsvisualized 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 chroma-tography, 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 identifiedbands 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) didnot 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 lowMw 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 possiblehydrophobic 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 atλ=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.

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aliquots (Fig. 2A, pool a) was collected and analyzed using SDS-PAGE. Agel of the pooled samples showed that the entire content of the crudeCOL remained adsorbed on the resin (Fig. 2D, line a). The molecularweights 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 visual-ized in the gel near the Mw of 112 kDa from β-galactosidase from thestandard proteins (Fig. 2B). The Mw of these bands was close to thevalues described in the literature for the α1 and α2 chains typicallyfound in type I collagen. The ionic strength of the ES with 0.25 M NaClsevered the bonds between the DEAE and the crude COL, causing thecollagen 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. 3. Chromatogram of the pure COL elution using 0.10 M NaCl (A). Building on the resultsAfter loading the crude COL onto the DEAE resin, an elution using the ES with 0.10 M NaClmeasured to be close to zero (A). The pooled aliquots of the peak corresponding to fractionscharacteristic pattern of bands (D, lines 7–11). Bands near the Mw of 112 kDa (B, lane S) shchains typically found in type I collagen. Another band in the gel (B) was observed near theresponds to β-type chains. Absorbance at λ=226 nm for the pure COL elution using the ES

(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-PAGEgel (Fig. 2D, lines c). The second relevant peak observed in the chro-matogram was formed after the application of a 2 M NaCl solution(Fig. 2A).Weaker bandswere also observed for components withmolec-ular weights lower than 112 kDa. These bands are often thought to con-tain low Mw contaminants from pepsin and elements usually found inthe salts used in the processing methods. The higher concentration ofsalt eliminated the contaminants that remain adsorbed on the resin. Ali-quots of the poolmay be observed on the gel (Fig. D, line d). The cleaningof the column with an isopropyl alcohol solution (Fig. 2A) removed any

from the ES of 0.25 M NaCl, a simplified procedure for the elution of pure COL was used.(A, arrow) formed a characteristic peak. Immediately after the peak, the fractions were7–11 (A) were characterized by SDS-PAGE (B). Each of the fractions showed the same

owed in the gel (B) are close to the values described in the literature for the α1 and α2Mw of 193 kDa from α-macroglobulin of the standard proteins (B, lane S), which cor-of 0.10 M NaCl was 0.378 (A).

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 similartype 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 Mwvalues 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 iscomparable to type I commercial collagen (C), the standard for purity used in this study.

Fig. 5. Graphical representation (A) of the curves for standard Mw (a) and thecurves for Mw of the pure COL bands (b), calculated using the software Gel-Pro An-alyzer 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 inthe graphic (A, b) and the bands in the polyacrylamide gel (B, lane b) with Mwvalues of 125.38 and 117.33 kDa correspond with the typical α1 and α2 chains ofthe pure type I COL. Some bands located in the area of 193 kDa from the standardcurve 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 rep-resent type β chains. Two more bands with lower intensity were detected with Mwof 108.85 kDa and 55.27 kDa (A, b).

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possible hydrophobic proteins, lipoproteins and lipids that were strong-ly adhered to the resin. Aliquots of the pool may be observed on the gel(Fig. D, line e).Washingwith deionizedwater prevented the contamina-tion and clogging of the resin for the following purification procedures.

To build upon the elution results with 0.25 M NaCl, salt concentra-tions of 0.15 and 0.10 Mwere also used for the elution of the collagen.Both salt concentrations demonstrated the same elution pattern inthe chromatogram with absorbance readings at 226 nm. ES with thelower salt concentrations still had a high enough ionic strength to dis-rupt only the bonds formed between the DEAE groups and the crudeCOL, resulting in an elution of pure COL. These results suggested thatthe salt concentration of 0.10 M NaCl should be used for the proteinelution as this minimizes the contamination of the sample with salt.However, a small amount of NaCl in the purified sample may be ac-ceptable as salt often contributes to protein preservation. The elutionof the crude COL on DEAE with a salt concentration of 0.10 M NaClcan be observed in the chromatogram showed in Fig. 3A. This profileis consistent with other chromatograms for the α chains of type I col-lagen 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 moredefined peak, with each fraction showing the same characteristic pat-tern of the bands for type I collagen [19,20,23,36]. After using an ESwith 0.10 M NaCl (Fig. 3A), the absorbance at 226 nm for the finalfractions 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 wave-lengths of approximately 250 nm and 190 nm, respectively [34,35].The measurement of the absorbance at a wavelength of 226 nm isused to identify the presence of type I collagen. This method wasused in the protocols for obtaining and purifying this protein [36]. Ab-sorbance in the ultraviolet range at a wavelength of 226 nm was a di-rect indication of the COL presence in the samples. The absorbance forthe collagen elution from DEAE using an ES with 0.25 M NaCl and0.10 M NaCL was 0.129 (Fig. 2A) and 0.378 (Fig. 3A), respectively.

The aliquots containing pure COL, corresponding to fractions 7 to 11,weremixed and characterized by SDS-PAGE (Fig. 4A). The pooled aliquotsshowed the presence of pure COL using an elution solution of 0.10 MNaCl. Another band was observed near the Mw of 193 kDa as calibrated

from the standard protein α-macroglobulin (Fig. 4A, lane S). Accordingto the results reported by Lacerda et al. [41], this bandmost likely corre-sponds with 2 α chains bound together, termed β-type chains.

The final stage of the collagen purification involved dialysis and ly-ophilization to remove the remaining contaminants still present inthe sample. The pooled aliquots collected during the purification pro-cess underwent dialysis in membranes with a porosity value less thanthe size of the protein. The goal of the dialysis was to remove salts orother nonvolatile molecules from the protein solution. After dialysis,

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

796 M.H. Santos et al. / Materials Science and Engineering C 33 (2013) 790–800

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 prepara-tions. The analysis indicated that the isolated collagen was essentiallyfree of other proteins and purified from other components of the bovinepericardium. After the purification of the crude COL, bandsα1 (I)α2 (I)and β-type remained visible in the pure COL and the lyophilized pureCOL (Fig. 4B). The bands corresponding to the contaminants had beenpractically eliminated. Bands of the lyophilized pure COL were locatedbetween the standard protein bands (Fig. 4B, lane S) with molecularweights close to the molecular weights of collagen bands reported inthe literature [41,42]. The characteristic bands of the lyophilized pureCOL (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 concen-tration of 0.23 g mL−1 by the Lowry method [37]. Using the extinc-tion coefficients of the α2(I) chain, these values ranged from 0.90to 0.33 mg mL−1.With the values for theα1(I) chain of type I bovinecollagen found in the literature, these values ranged from 0.58 to0.24 mg mL−1. The lowest concentration foundusing the extinction co-efficient was 0.24 mg mL−1, which was very close to the concentrationfound using the Lowrymethod. By the samemethod, the concentrationfound for commercial collagen (Fig. 4C) was 0.50 mg mL−1, using the

proportion of 1 mg/1 mL of 0.1 N CH3COOH, as specified by the manu-facturer for its solubilization.

3.4. Molecular weight evaluation

Fig. 5A represents the Mw standard curve (a) and the Mw curve ofthe lyophilized pure COL bands (b) calculated using the softwareGel-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 pureCOL bands was observed to be located in the area of the highest Mwin the standard curve. Using the standard curve, some of the bandswere determined to be located in the area of 193 kDa giving molecu-lar weights of 191.72, 173.56, and 161.34 kDa (Fig. 5A, b). One bandwith significant intensity had an estimated Mw of 173.56 kDa, mostlikely type β chains [41,44]. Two bands of higher intensity, most likelycorresponding to α1 and α2 chains of pure type I COL, had molecularweights of 125.38 and 117.33 kDa and are observed in the graphicalrepresentation of the standard curve (Fig. 5A, b) and the respectivepolyacrylamide gel (Fig. 5B, lane b). Two more bands with lower in-tensities were detected, one with a Mw of 108.85 kDa and anotherwith a value of 55.279 kDa (Fig. 5A, b).

The molecular weights reported in the literature for α1 and α2chains 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 extrac-tion, a selective hydrolysis of the carboxyamide groups of asparagineand glutamine residues occurs. There is a decrease in the intensity ofthe hydrophobic electrostatic interactions and the hydrogen bridgesthat facilitates collagen solubilization, resulting in lower molecularweights [44]. The enzyme solubilization method involves the removalof the telopeptides, increasing the fraction of soluble collagen. Thismay explain the higher Mw found for the pure COL. For the conditionsused, an extensive degradation or solubilization of the matrix maynot have occurred, as suggested by Lacerda et al. [41]. Despite beingclassified as a type I collagen, these proteins may also have small dif-ferences in their structure across different tissues in the same animal,or in the same tissue between different animals. These potential var-iances may lead to different molecular weights in the isolated colla-gen. Protein characterization using polyacrylamide gels at differentconcentrations can also contribute to the variances in molecularweights, due to the permeability of the particular gel.

3.5. Western blotting

Western blotting (WB) is widely used for protein analysis. Theproteins immobilized on a nitrocellulose membrane can be detectedusing enzymes linked to antibodies, which are viewed with a chro-mogenic substrate [34,45]. Caputo et al. [46] reported the use of WBto characterize collagen, identifying the specific bands of collagentype I. In our study the transfer of the collagen bands to the nitrocel-lulose membrane was observed by staining sites corresponding to thebands present in the gel. The chromogen reacted with the peroxidaseof the conjugated rabbit anti-mouse IgG connected to the anti-type Icollagen produced by the mice (Fig. 6B). This method characterizedthe pure COL as a type I collagen, similar to the commercial bovinetype I collagen (Fig. 6A). The high Mw bands of the standard proteincontrol may be identified in Fig. 6S.

3.6. Scanning electron microscopy/energy dispersive X-ray spectroscopy

Commercial collagen appeared as a dense membrane. The presenceof some fibers was observed in the transmitted light microscopy. Thepure COL had a less dense fibrillar structure with translucent fibers.

Characterization by SEM/EDS is essential for the determination ofthe final purity of the collagen. SEM images of the pure COL showed

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 observedon 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 concentrationsof NaCl in their EDS spectrum (c).

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contamination by NaCl crystals before dialysis (Fig. 7A), as indicated bythe presence of the high intensity peaks for these elements in their re-spective EDS spectrum (Fig. 7A). Some particles were observed on thesurface of the pure COL before dialysis, as observed in the SEMphotomi-crograph of Fig. 7B. The analysis focused only on these specific areas thatshowed the presence of NaCl at high concentrations, as observed in theEDS spectrum of Fig. 7B. The SEM image of the pure COL taken immedi-ately after dialysis (Fig. 7C) showed lower concentrations of NaCl (EDSspectrum, Fig. 7C).

In the SEMmicrographs, a dense structure of a dehydrated BPwasobserved, exhibiting fibers with a certain parallelism and ripples inthe longitudinal direction. Although not very well organized, thedominant orientation of these fibers follows the circumferential di-rection of the pericardial sac, demonstrating an anisotropic structurewith a fibrous meshwork appearance (Fig. 8A). The images suggest asimilarity between the structure of the commercial collagen (Fig. 8B)and the structure of the lyophilized pure COL (Fig. 8C). BP wascharacterized by the parallel orientation of its intersecting fibrils.Compared with BP, a complete rearrangement of the fibers wasobserved for the lyophilized pure COL structure. The fibrillar ap-pearance observed in BP changed into an amorphous structure,characterized by a structure composed of strips (Fig. 8C). Certainareas contained disorganized nanofibers (Fig. 8D). A spongy-likestructure (Fig. 8E) with irregularly distributed and interconnectedpores (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. Thespectrum pattern of the lyophilized pure COL (Fig. 9B) showed thepresence of the relevant peaks, suggesting a relatively high contentof carbon corresponding to the main element of organic components,small amounts of sodium and chlorine corresponding to the elementsusually found in the salts used in the processing methods, and sulfurcorresponding to the amino acids present in the collagen proteinstructure. The presence of some peaks of low intensity correspondingto the trace elements in the sample was reported by Andrade et al., in2004 [47].

The presence of NaCl in collagen provides structural stability, help-ing to avoid protein denaturation. However, the presence of largeramounts of NaCl, aswell as other elements in the collagen product, con-tributes to the contamination of thematerial [47]. Other trace elementssuch asMg, Al, Si, P, K and Cawere observed in the EDS spectrum of thecommercial collagen (Fig. 9C). These contaminantsweremost likely de-rived from the chemical process, possibly compromising the purity ofthe collagen.

3.7. Fourier transform infrared spectroscopy characterization

FTIR spectroscopy was used as a supporting tool for characterizingthe collagen extracted and purified from the BP tissue. As discussed inthe literature [12,13,48–50], collagen is a generic term that covers a

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 amor-phous 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 irreg-ularly distributed and interconnected pores (F).

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broad class of proteins, each with specific structures, functions, and tis-sue distributions in the extracellular matrix. The functional groups ofcollagenmay be detected by FTIR spectroscopy because the amide groupsof the polypeptides and proteins possess a number of characteristic vibra-tional modes or group frequencies. Essentially, the peptide group hasnine amide characteristic bands (A, B, I, II … VII). These important func-tional groups (Fig. 10A) in living organisms connect the sequence ofamino 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 majorbands of the protein infrared spectrum. The amide I band (rangingfrom 1700 to 1600 cm−1) is mainly associated with the C\O stretchingvibration and is directly related to the backbone conformation. TheAmide II band is derived from the N\H bending vibration and fromthe C\N stretching vibration. The amide III band is usually weak inFTIR spectroscopy but can be usually found in the region at 1350–1250 cm−1 [51,52]. We found that the FTIR spectra of the commercialcollagen (Fig. 10C) and the lyophilized pure COL (Fig. 10B) werecomparable to the spectrum of the BP sample (Fig. 10A). These sim-ilarities were expected because BP is a collagen-rich biological tissuecontaining mostly type I collagen [26–30]. More specifically, allmajor bands related to the relevant functional groups of collagen weredetected (Fig. 10C). We observed peaks associated with the amide

regions, amide I (1680–1620 cm−1), amide II (1580–1480 cm−1), andamide 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 atthe 1360–1340 cm−1 band (Fig. 10C, dashed rectangle) Representingmore than the carbonyl groups, this band is also characteristic of thewagging vibration of the proline side chains present in the type I colla-gen found in biological tissues [53]. An absorption band correspondingto the carboxylic functional group (\COOH) at 1740–1720 cm−1 wasalso found in the commercial collagen spectrum (Fig. 10C) but not inthe lyophilized pure COL sample (COL, Fig. 10B). It may be noted thatsome differences in the bands of the FTIR absorption peaks for collagenand collagen-containing tissues have been reported in the literature[48,49,54,55]. These differencesmay be due to variability in thematerialinvestigated as well as the variety of instruments andmethods used forthe measurements. Some regions of the spectra may present a broadband, as a result of two ormore bands overlapped, or as a set of resolvedbands. In that sense, a full characterization of the purified collagen usingFTIR spectroscopy is beyond the scope of this investigation.

The collagen produced in this study was characterized as a puri-fied type I collagen extracted from bovine pericardium tissue based

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

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

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on a combination of analytical methods. The BP tissue is an affordableanimal source of protein that can be extracted andpurified by a relative-ly simple methodology that may be applied to the industrial scale

production. After obtaining a pure COL, improvements were necessaryin some protein processing stages as well as the characterization tools.Further development on the protein quantificationmethods are neededto obtain the best final yield and a precise quantification of the lyophi-lized pure COL product. This material might be suitable for use in thefield of tissue engineering.

4. Conclusions

Bovine Pericardium tissues were utilized as a natural source forthe extraction of collagen protein. The BP tissue was subjected to anenzymatic digestion followed by a new process methodology for thepurification of type I collagen using ion-exchange chromatography.The purified COL was characterized as a type I collagen that has mor-phological and spectroscopy characteristics comparable to a commer-cially available high purity collagen. The pure COL produced using arelatively simple processing route can be considered as a promisingsource of biomaterial for a myriad of biological applications. The meth-odology developed in this study is potentially suitable for transferringto an industrial scale.

Acknowledgements

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

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