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Bovine osteoblasts cultured on polyanionic collagen scaffolds: an ultrastructural and immunocytochemical study

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Collagen is the most abundant protein in the body and is also the most important component of the extracellular matrix. Collagen has several advantages as a biomaterial such as lack of toxicity, biocompatibility, biodegradability, and easy
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  Bovine osteoblasts cultured on polyanionic collagen scaffolds: anultrastructural and immunocytochemical study Patrı´ cia da Luz Moreira, 1 Selma Candel  aria Genari, 1,2 Gilberto Goissis, 3 Fernando Galembeck, 4 Yuehuei H. An, 5 Arnaldo Rodrigues Santos, Jr. 6 1 Departamento de Biologia Celular, Instituto de Biologia, UNICAMP, Campinas, Sa˜o Paulo, Brazil 2 Centro Estadual de Educac¸a˜o Tecnol  ogica Paula Souza, Faculdade de Tecnologia de Bauru, Bauru, Sa˜o Paulo, Brazil 3 Biotech Biom  edica Produtos M  edicos e Odontol  ogico Ltda ME (Produc¸a˜o), Sa˜o Carlos, Sa˜o Paulo, Brazil 4 Departamento de Fı´ sico-Quı´ mica, Instituto de Quı´ mica, UNICAMP, Campinas, Sa˜o Paulo, Brazil 5 Orthopaedic Research Laboratoy, Medical University of South Carolina, Charleston, South Carolina 6 Centro de Cie ˆ ncias Naturais e Humanas, Universidade Federal do ABC, Santo Andr  e, Sa˜o Paulo, BrazilReceived 25 January 2011; revised 16 May 2012; accepted 28 June 2012Published online 15 September 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32804 Abstract:  Collagen is the most abundant protein in the bodyand is also the most important component of the extracellularmatrix. Collagen has several advantages as a biomaterial suchas lack of toxicity, biocompatibility, biodegradability, and easyreabsorption. In this study, we examined bovine osteoblastscultured on native or anionic collagen scaffolds prepared frombovine pericardium after selective hydrolysis of glutamine andasparagine side chain amides for periods from 24 (BP24) and48 h (BP48). The cells were cultured in control and mineraliza-tion medium at 37  C in the presence of 5% CO 2 . Transmissionand scanning electron microscopy, energy dispersive spectros-copy, and an immunocytochemical marker were used for anal-ysis. Cells with an irregular morphology forming a confluentmultilayer were observed on matrices kept in control medium.Most of these cells presented a polygonal or elongated flat-tened morphology. Several spherical deposits of calcium crys-tal associated with phosphorus were observed on the nativeand BP48 matrices. Similar results were observed in sampleskept in control medium except with lower calcium/phosphorusratio. Vesicles actively expelled from the cell membrane werealso seen (do this vesicles corresponds to calcium/phosphorusdeposits). Osteocalcin was clearly visible on matrices kept inmineralization medium and was more expression on the sur-face of BP48 matrices. The results showed that anionic colla-gen is able to support osteoblastic differentiation, regardlessof the medium used. Finally, the BP48 matrix promoted betterosteoblast differentiation than the native matrix.  V C 2012 WileyPeriodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 18–27, 2013. Key Words:  anionic collagen, cell culture, osteoblasts, bioma-terials, tissue engineering, cell differentiation How to cite this article:  Moreira PL, Genari SC,Goissis G, Galembeck F, An YH, Santos AR, Jr. 2013. Bovine osteoblasts culturedon polyanionic collagen scaffolds: An ultrastructural and immunocytochemical study. J Biomed Mater Res Part B 2013:101B:18–27. INTRODUCTION Bone fractures result in the loss of the mechanical stabilityof bone, bone tissue discontinuation, and partial destructionof blood supply. Bone repair is a complex process that con-sists of the stabilization of bone fragments, bone consolida-tion, reconstruction of avascular and seminecrotic frag-ments, and finally, internal and external remodeling of thenewly formed tissue. 1,2 External factors can markedly affect the regeneration process, although tissues act according tobiological rules that control cell proliferation and differen-tiation, as well as extracellular matrix production, which canindependently occur despite of external interference,although being influenced by them. 3 In contrast, fracturesaccompanied by bone mass loss require grafts or implants.Natural polymers such as collagen 4–6 and elastin 7,8 are bio-materials that offer advantages for tissue engineering, forexample, the presence of the cellular recognition sequenceArg-Gly-Asp (RGD) that influences cell adhesion. 9 However,in their pure form these materials are unable to re-establishthe initial tissue characteristics.Collagen is the most abundant protein in the body andis also the most important component of the extracellularmatrix. Collagen molecules determine the size, tensilestrength, and cellular arrangement of all structures andorgans. 9–11 Collagen has several advantages as a biomaterialsuch as lack of toxicity, biocompatibility, biodegradability,and easy reabsorption by the body, 12–15 as well as low anti-genicity, high tensile strength, and high affinity for water.Some modifications are necessary to overcome thesedrawbacks, including an increase in the number of crosslinksto improve tensile strength 16–18 and the addition of othermaterials, 19–20 growth factors, 6,21,22 glycosaminoglycans, 23 Correspondence to:  A. R. Santos, Jr. (arnaldo.santos@ufabc.edu.br) 18  V C  2012 WILEY PERIODICALS, INC.  and other molecules 8,24 to the collagen scaffold. These modifi-cations also increase the mechanical stability of collagenwhile, at the same time, decreasing its biodegradability andprotecting the collagen structure from  in vivo  reabsorption. Inaddition to increasing the negative charge and carboxylicgroups in the collagen molecule, 16,25 these modificationsimprove the piezoelectrical properties of collagen, which pro-mote osteogenesis. 26 In vivo  tests performed with these anionic matrices havedemonstrated bone formation and a low inflammatoryresponse even in bones with established osteoporosis, withpromising characteristics for bone defect repair. 27,28 In con-trast, the native collagen matrix caused a mild to intenseinflammatory reaction associated with reabsorption cen-ters. 28,29 Cell interactions with biomaterials, as well as thequality of these interactions, are known to influence theability of cells to proliferate and differentiate when in con-tact with the implant. 21,22 These interactions are influencedby the topography of the material, superficial energy, androughness, among others. 3,6 Among many reports relatingnegative charges with osteogenesis, there is a few  in vitro data that could help explain the effect of negative chargeson differentiation of bone cells. Some reports have shownvariations on osteoblast metabolism when in contact withnegative charges on biomaterial surface. The cellular inter-actions that occur on these anionic collagen matrices arenot completely understood.In the present study, we examined bovine osteoblastscultured on anionic collagen scaffolds prepared from bovinepericardium submitted to alkaline hydrolysis for 24 (BP24)or 48 h (BP48). Analysis of the mineralization process bytransmission electron microscopy (TEM), scanning electronmicroscopy (SEM), and energy dispersive spectroscopy(EDS), together with the evaluation of the morphologicaland functional differentiation of osteoblastic cells on theseanionic collagen matrices as compared to native collagenusing an immunocytochemical marker, may provide usefulinformation for the understanding of the biological proc-esses observed in  in vivo  experiments. 25,27–29 MATERIALS AND METHODS Matrix preparation Bovine pericardium (BP; Braile Biom  edica S/A, Sa˜o Jos  e doRio Preto, Brazil) was used for preparation of the 3D matrix.The samples were prepared at the Sa˜o Carlos Institute of Chemistry, Sa˜o Paulo University, as described by Lacerdaet al. 30 and Goissis et al. 31 Briefly, alkaline hydrolysis wasperformed in aqueous dimethylsulfoxide solution containingchloride and sulfate salts of alkaline and alkaline earth met-als. The matrices were hydrolyzed for 24 (BP24) and 48 h(BP48) and the conditions were such that only amide fromasparagine and glutamine residues was hydrolyzed. Excesssalts were removed by extensive washing with 3% boricacid solution, 0.3% EDTA, pH 11.0, and deionized water, andfinally, the matrices were equilibrated in 0.13 mol/L phos-phate buffer, pH 7.4. The anionic collagen matrices producedhad been characterized by thermal analysis, infrared spec-troscopy, titration, and determination of dielectric propertiesas described elsewhere. 30,31 The following matrices wereanalyzed: (1) 3D native anionic collagen/elastin matrices(not submitted to alkaline hydrolysis treatment), and (2) 3Danionic collagen matrices submitted to alkaline hydrolysis,BP24 and BP48. After analysis, the matrices were cut intosmall circles (6 mm diameter    3 mm thick) and used inthe experiments described below. Cell culture A noncontinuous fetal bovine osteoblast line kindly providedby Dr. William Whitson 32 and maintained at the OrthopedicResearch Laboratory of the Medical University of South Caro-lina was used in this study. The cells were used in all experi-ments until passage 4. The osteoblasts were cultured in Whit-son’s DMEM medium containing 15% fetal calf serum (bothfrom Sigma Chemical, St. Louis, MO) at 37  C in a 5% CO 2 atmosphere. 32 For experimental analysis, Whitson’s mineraliza-tion medium 32 containing 15% fetal calf serum (both fromSigma) was also used for culture of the noncontinuous cell line.This medium differs from Whitson’s DMEM by the addition of  b -glycerophosphate and calcium chloride. The cells were kept in Whitson’s DMEM medium until the time of the experiment,when they were cultured in the two media as described earlier.The medium was changed daily until the cells reached conflu-ence at about 5–7 days, when they were subcultured. The cellswere cultured on the native, BP24 and BP48 scaffolds in Whit-son’s DMEM medium (called control medium in this study) andWhitson’s mineralization medium (called mineralization me-dium), both supplemented with 15% FCS, for 21 days at 37  Cin the presence of 5% CO 2 . Suspensions containing an initialconcentration of 1.0    10 5 cells/mL were added to a 96-wellplate (200  l L/well) (Corning/Costar Corporation, Cambridge,MA) with the matrices. After 4 h of incubation for the purposeof adhesion, the plate to be analyzed with the mineralizationmedium had the DMEM medium replaced by the mineralizationone at certain time points, the matrices were processed foranalysis by the techniques described below. Scanning electron microscopy and energy dispersivespectroscopy (EDS) After fixation in 4% paraformaldehyde/2.5% glutaraldehyde(Sigma) in 0.1 M   phosphate buffer (Merck), pH 7.4, for 30min, the matrices ( n  ¼  5 for each culture conditions used)were washed with phosphate buffer, postfixed in 1% osmiumtetroxide (Sigma), and dehydrated in an ethanol series. Thematerial was critical point dried (Balzers CPD030, Balzers,Elgin, IL) and sputtered with gold (Balzers SCD 050). Thespecimens were examined under a JEOL JSM-5800 LV (Jeol,Tokyo, Japan) SEM equipped with an EDS analysis system. Transmission electron microscopy  The specimens ( n  ¼  5 for each culture conditions used)were fixed in 4% paraformaldehyde/2.5% glutaraldehyde(Sigma) in 0.1 M   phosphate buffer (Merck), pH 7.4, for 30min, postfixed in 1% osmium tetroxide in the same bufferat 4  C, dehydrated in acetone, and embedded in epoxy resin(Embed-812, Electron Microscopy Sciences, EMS, WA). Ultra-thin sections (20–60 nm) were stained with 2% uranyl ORIGINAL RESEARCH REPORT JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS  | JAN 2013 VOL 101B, ISSUE 1 19  acetate (EMS) for 20 min and 2% lead citrate (EMS) for 7min prior to observation under a Leo 906 TEM (Carl Zeiss,Oberkochen, Baden-Wu¨rttemberg, Germany). Immunocytochemical detection of osteocalcin After fixation in 4% paraformaldehyde (Sigma) in 0.1 M   phos-phate buffer (Merck), pH 7.4, for 30 min, the matrices ( n  ¼  4for each culture conditions used) were washed with phos-phate buffer and embedded in paraplast (Fisher Scientific, Vet-erans Memorial Drive, Houston, TX). The blocks were cut into5  l m were made 3 or 4 sections by slide and four slides havebeen produced by sample studied. Thick sections and thespecimens were cleared using routine procedures. The sec-tions were then washed in cold PBS (Nutricell NutrientesCelulares, Campinas, Brazil) and nonspecific binding siteswere inactivated by incubation in 1% BSA (Sigma)/PBS(Nutricell) for 60 min in a humid chamber. After washing incold PBS, the primary rabbit antihuman osteocalcin antibody(BT-593, Biomedical Technologies, Stoughton, MA) diluted1:30 in PBS and 1% BSA was added and the sections wereincubated overnight in the dark in a humid chamber. AfterBSA gentle removal with cold PBS, the secondary antibody(FITC-conjugated goat antirabbit IgG, BT-557, BiomedicalTechnologies, Stoughtou, MA) diluted 1:50 in PBS was addedand the sections were incubated for 1 h in the dark. Negativecontrols consisted of omission of the primary antibody wereanalyzed all fields in each slide. The same observer analyzedall samples. Analysis and photographic documentation wereperformed with an Olympus IX50 microscope (Olympus,Tokyo, Japan) equipped with a FITC filter. RESULTS Scanning electron microscopy and energy dispersivespectroscopy  Cells with an irregular morphology forming a confluent mul-tilayer were observed on matrices kept in control medium FIGURE 1.  (A) Scanning electron photomicrographs of osteoblasts cultured on the native, (B) BP24, and (C) BP48 matrices in control (A1–C1)and mineralization medium (A2–C2). In detail, spots of spherical mineral deposits on BP48 matrices. Scale bar  ¼  2.5  l m. 20 MOREIRA ET AL. BOVINE OSTEOBLASTS CULTURED ON NATIVE OR ANIONIC COLLAGEN SCAFFOLDS  [Figure 1 (A1–C1)]. Most of these cells presented a polygo-nal or elongated flattened morphology maintained through-out the culture period. A small number of vesicles and/ormicrovilli were noted on the cell surface. The cells also pre-sented evident filopodia and lamellipodia. Multilayered po-lygonal/elongated confluent cells rich in vesicles and/or mi-crovilli were observed on matrices kept in mineralizationmedium [Figure (1A2–C2)]. The morphological resultsobserved were representative and reproductive through thedifferent groups studied. Several spots of spherical calciumcrystal deposits were noted on the native and BP48 matri-ces [Figure (1A2–C2)]. Identification and mapping of theelements by EDS analysis suggested the concomitant pres-ence of P and Ca in the same regions in all matrices(Figures 2 and 3). Samples kept in control medium pre-sented different amounts of Ca and P [Table I and Figure(2A1–C1)]. Mapping revealed superposition of these ele-ments [Figure (2A2–C2)]. A Ca/P ratio of 0.27, 0.10, and0.05 was obtained for native collagen, BP24, and BP48,respectively (Figure 2). On the other hand, differences in Caand P deposition were observed for matrices kept in miner-alization medium (Table I and Figure (3A1–C1)], but super-position of the elements was the same [Figure (3A2–C2)].The Ca/P ratio was 1.46, 0.10, and 2.26 for BP48, BP24,and native collagen, respectively (Figure 3). The atomic con-centration (%) of some elements in the different experimen-tal conditions can be seen in Table I. Using the medium of mineralization, the P concentration in samples BP24 andBP48 were always higher than in native collagen. In thesame culture condition, the Ca concentration was alwaysgreater in BP48 than other samples. Transmission electron microscopy  All matrices presented a similar ultrastructure when cul-tured in control [Figure 4(A)] and mineralization [Figure4(B)] medium. The collagen matrices consisted of fibersarranged in layers that tended to run perpendicular to thesection [Figure 4(A)]. At higher magnifications, when a par-allel plane of the section was obtained, collagen showed aregular banding pattern. When mineralization nutritional FIGURE 2.  EDS analysis of matrices cultured in control medium: (A) native, (B) BP24, and (C) BP48. A1 to C1 show the EDS spectra and calcium/ phosphorous ratio. Note the absence of Ca 2 þ peaks. A2 to C2 show the element mapping of calcium (Ca), phosphorous (P), and oxygen (O).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] ORIGINAL RESEARCH REPORT JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS  | JAN 2013 VOL 101B, ISSUE 1 21  conditions were supplied, accumulation of electron densematerial was observed on the matrices [Figure 4(B), aster-isk]. Cells were mainly seen on the surface of the matrices[Figure 4(C,D)] and presented several filopodia penetratingthe matrices [Figure 4(F)] perpendicular to the plane of thesection. No chromatin condensation was observed [Figure4(C)], a finding suggesting the presence of an active nucleuswith vesicles actively expelled from the plasma membrane FIGURE 3.  EDS analysis of matrices cultured in mineralization medium: (A) native, (B) BP24, and (C) BP48. A1 to C1 show the EDS spectra andcalcium/phosphorous ratio. Note the Ca 2 þ peaks on BP48. A2 to C2 show the element mapping of calcium (Ca), phosphorous (P), and oxygen(O). Observe the same position of Ca and P on BP48. [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.] TABLE I. Atomic Concentration (%) of Some Elements in the Different Samples ElementsSamplesNative BP24 BP48C M C M C MCarbon (C) 54.94 20.39 41.80 46.98 74.91 34.15Oxygen (O) 15.20 4.90 5.00 8.70 12.07 15.86Phosphorus (P) 0.40 2.16 1.17 2.94 2.10 12.47Calcium (Ca) 0.11 4.90 0.12 0.32 0.12 18.26Gold (Au) 7.19 50.76 44.80 33.65 8.08 17.89 C: control medium; M: mineralization medium. 22 MOREIRA ET AL. BOVINE OSTEOBLASTS CULTURED ON NATIVE OR ANIONIC COLLAGEN SCAFFOLDS
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