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© 2012, Copyright the Authors Artificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Cytocompatibility of Porous Biphasic Calcium Phosphate Granules With Human Mesenchymal Cells by a Multiparametric Assay *†Fabio Mitri, ‡§Gutemberg Alves, §Gustavo Fernandes, ¶Bruno König, **Alexandre Jr Rossi, and ‡§Jose Granjeiro *Area of Morphology, Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Minas Gerais; †Postgraduation Program in Medical Sciences, Fluminense Federal University; ‡Cell and Molecular Biology Department, Fluminense Federal University; §Clinical Research Unit, Antônio Pedro Hospital, Fluminense Federal University, Niterói; **Laboratory of Bioceramic Biomaterials, Brazilian Center for Physics Research, Rio de Janeiro; and ¶Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
Abstract: This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/betatricalcium phosphate (b-TCP) (60:40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100°C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules
revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/b-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications. Key Words: Stem cells—Biocompatibility —Biphasic calcium phosphate—Tissue engineering.
Biomaterials are being increasingly employed as substitutes to autogenic grafts in regenerative medicine. One such material, namely hydroxyapatite (HA), is widely utilized in bone restoration due to physical properties which are similar to those of the mineral constituent from bone tissue, and because of
its remarkable osteoconductive capacity (1). Recently, HA has been also studied as a promising scaffold for the delivery of stem cells directly onto the grafted area, strongly favoring a rapid bone regeneration (2). However, HA presents a major drawback by having very low biodegradation rates, which may compromise its rapid substitution by newly formed bone tissue (3). In order to minimize this drawback, a second more soluble material, beta-tricalcium phosphate (b-TCP), may be added to HA, generating a biphasic calcium phosphate (BCP) composite with improved feature due to the relatively high resorption ratios, as well as a well-described bioactivity of TCP (4,5). Therefore, biphasic HA/b-TCP composites may play an important role during assisted bone regeneration, serving as better scaffolds to promote stem cell-induced osteogenesis (6).
doi:10.1111/j.1525-1594.2011.01409.x Received March 2011; revised August 2011. Address correspondence and reprint requests to Dr. Gutemberg Alves, Cell and Molecular Biology Department, Fluminense Federal University, Instituto de Biologia Outeiro S. J. Batista s/n Campus Valonguinho, Centro, Niteroi, Rio de Janeiro 24210-130, Brazil. E-mail:
[email protected] Presented in part at the 6th Latin American Congress of Artificial Organs and Biomaterials (COLAOB 2010), held August 17–20, 2010 in Gramado, Rio Grande do Sul, Brazil.
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In this context, some physical properties of the biphasic scaffold become rather important, as they may directly impact the biological performance of the grafted material. Among those included is the material porosity. The incorporation of spaces inside the matrix of the biphasic composite enables the formation of a scaffold to vascular infiltration, optimal bone oxygenation, and cell attachment and proliferation, both fundamental to fast material integration, resorption, and subsequent substitution by bone tissue (7). Variations on the chemical composition (proportion of HA/TCP) may reduce the compression strength of the scaffold (8), as well as modify both degradation and resorption ratios of a given material, with possible biological effects (9). Processes such as thermal treatment at high temperatures or pressing may lead to the closing of pores and reduction in the overall dissolution levels (10), reducing the expected good biological responses of the material (11–13). Therefore, structural modifications may impact on the material mechanical properties, biodegradation, and biocompatibility, all of which constitute basic criteria to the development of scaffolds in bone engineering. In spite of all the improvements on solubility and bioactivity obtained with BCPs, it is important to consider the possible impact of their composition on processes linked to cell proliferation and survival. It is essential to ensure that the proportional amounts of each component, as well as the addition of porogeneic agents, do not impoverish the cytocompatibility of the final composite, due to the release of toxic or irritating components. Therefore, in vitro cytotoxicity tests represent critical requirements previous to the clinical application of such materials (14). Although cell and tissue responses have been reported for several modified biphasic (HA/b-TCP) ceramics (15–19), there are still conflicting results on the in vitro cytocompatibility and cell metabolism alterations induced by both dense and porous BCPs. Direct-contact assays showed no cytotoxic effects of modified BCP ceramics (16,19,20) on L929 fibroblasts and human osteosarcoma cells. In contrast, porous BCPs were described elsewhere as strongly cytotoxic (21), after exposition of fibroblasts to extracts of similar ceramics. It is important to note that most works employed the same cell viability parameter (the reduction of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT]), and tested either murine fibroblasts or tumoral-derived cells. Therefore, it becomes evident that (i) there is still controversy on the in vitro cytocompatibility of porous BCPs as measured by MTT, and (ii) there is a lack of information on the Artif Organs, Vol. 36, No. 6, 2012
biological response to these composites by primary cell cultures or stem cells, such as human primary mesenchymal cells, which are strongly related to bone tissue engineering (2). In this work, an interesting multiparametric assay was applied employing three different sequential tests (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5[(phenylamino) carbonyl]-2H-tetrazolium hydroxide [XTT], Neutral Red, and Crystal Violet Dye Exclusion) to evaluate viability and integrity of human primary mesenchymal cells after exposition to extracts of moldable, injectable biomaterials based on granules of both dense and porous biphasic HA/ b-TCP. In this manner, it could be observed that changes on a rather important physical property of BCPs, namely the induction of increased porosity, do not alter significantly the viability of these important human primary cells. MATERIALS AND METHODS Material synthesis Four types of ceramics were developed at the Brazilian Center for Physics Research (CBPF), using stoichiometric HA and beta-tricalcium phosphate. For the synthesis of the BCP granules, betatricalcium phosphate [Ca3(PO4)2 + 2% H2O2] was obtained commercially (Merck, Brazil), while HA [Ca10(PO4)6OH2] was synthesized at the CBPF by the precipitation method (22). Briefly, diluted solutions of calcium nitrate and ammonium phosphate were slowly mixed dropwise, and maintained under constant agitation and temperature. The precipitate was filtered, washed, and dried at 80°C. b-TCP and HA were then mixed at a mass ratio of 60 HA:40 b-TCP, and added to a porogeneic agent. With a uniaxial press and a metallic matrix with 3.5-cm diameter, tablets were prepared with or without porogeneic agent (wax spheres, Licowax PE 520; 355 micrometers, Clariant, Charlotte, NC, USA) at a 75% volume ratio, and different applied strengths (20 or 100 MPa) in order to influence material porosity (Table 1). Subsequently, tablets were submitted to thermal treatment at 800°C for 10 h, and then calcined at 1150°C for 8 h. Afterwards, the material was crushed and sieved to obtain granules with the desired granulometric range (425 > f < 710 mm). The granules thus obtained were sterilized in an autoclave before use. Physicochemical evaluation The relative density and apparent (open) porosity were determined by the Archimedes method in test samples of 5 g of each material, in triplicate, with
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TABLE 1. Manufacturing conditions of sample tablets Material
Strength
Density g/cm3
Apparent porosity %
Dense HA: Stoichiometric HA Dense BCP: Stoichiometric HA + b-TCP: mass ratio 60 HA:40 b-TCP Porous HA: Stoichiometric HA + wax (75% volume ratio) Porous BCP: Stoichiometric HA + b-TCP: mass ratio 60 HA:40 b-TCP + wax (75% volume ratio)
100 MPa 100 MPa 20 MPa 20 MPa
3.07 2.75 1.20 2.07
1.2 10 62 26
distilled water as liquid medium. Elemental analyses for the presence of Ca and P were made using X-ray fluorescence spectroscopy (PW2400 X-ray fluorescence spectrometer, Philips, Amsterdam, The Netherlands). Scanning electron microscopy (SEM) of granules Dense and porous granules of biphasic ceramic were gold coated on an ion sputter (E101-Hitachi, Hitachi Co., Tokyo, Japan) and examined for surface and microstructure on a Zeiss DSM 940 scanning electron microscope (Carl Zeiss, Inc., Munich, Bayern, Germany) at low accelerating voltages (1–2 kV). Five random fields were analyzed for the estimation of the range of size of microporosity. Preparation of sample extracts For the cytocompatibility tests, extracts of each sample were prepared according to international standards for medical devices evaluation (23): After sterilization, 1 g of each material (in the form of grains) was incubated on 10 mL alpha minimum essential medium (Alpha-MEM, Cultilab, Brazil) for 24 h at 37°C. The extracts thus obtained were collected and assayed immediately for cytocompatibility. Phenol at 2% and extracts from high-density polystyrene beads were prepared as positive and negative controls for cytotoxicity, respectively. Cell culture Human highly purified mononuclear cells were provided and characterized by the Biological Samples Bank from the Antônio Pedro Hospital at the Fluminense Federal University—HUAP/UFF (Niterói). They were obtained from bone marrow aspirates from a single patient, sorted, and concentrated using a mononuclear cell program by density gradient on Ficoll-Hypaque according to a standardized method (24). Adherent mesenchymal cells from human bone marrow were cultured in 24-well culture plates until subconfluency. Cells were tripsinized, suspended in complete culture medium (Alpha-MEM), and subcultured at a cell density of 8.5 ¥ 103 cells/cm2 on 96-well culture plates (Corning, Union City, CA, USA) with 200 mL
of culture medium. Subcultures at the second passage were incubated at 37°C in a humidified atmosphere of 5% CO2. After 24 h, the medium was removed from each well and replaced by 180 mL of each sample extract plus 20 mL of fetal bovine serum (final concentration of 10%), in four replicates. After 24 h, the extracts were removed and cells were tested for viability. The content of calcium and phosphate ions in each extract was also quantified colorimetrically, through the methods of cresolphtalein (25) and Fiske and Subbarow (26), respectively. Cell viability assay The viability of human primary mesenchymal cells exposed to the different extracts was evaluated by a multiparametric methodology, as described previously (27) (In Cytotox Kit, Xenometrix, Allschwil, Switzerland). This assay evaluates three different viability parameters, sequentially and on the same cells, allowing for improved comparisons of the results thus obtained. Mitochondrial activity Mitochondrial dehydrogenase activity was measured by the ability of such enzymes to reduce the reagent XTT to soluble formazan salts, with differing color. The reduction was detected colorimetrically by the absorbance of samples at 480 nm, on a Synergy II microplate reader (Biotek Instruments, Winooski, VT, USA). As formazan is in a soluble form, there is no need for extraction, allowing for the survival of cells and, consequently, proceeding to the next evaluation (membrane integrity). Membrane integrity After removing the XTT reagent and washing, the membrane integrity of exposed cells was measured by the uptake of Neutral Red dye on lysosomes of membrane intact viable cells. After 3 h of exposure to Neutral Red dye, cells were fixed and the dye extracted. The absorbance of the supernatant was measured at 540 nm. Cell density The density of cells still attached to the culture plate was estimated immediately after the Neutral Artif Organs, Vol. 36, No. 6, 2012
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Red test. Plates were washed and exposed to Crystal Violet dye, which stains DNA, regardless of the living state of cells (which allows cell estimation even after fixation in the previous step). After exhaustive washing, the dye was extracted in a methanol/acetic acid mixture, and the amount of Crystal Violet dye bound to nuclear proteins and DNA of adhered cells was measured by absorbance at 540 nm. Statistical analysis Mean values and standard deviation obtained for each test (n = 3 experiments) were calculated and submitted to a D’Agostino and Pearson omnibus normality test. After detection of a normal distribution, one-way analysis of variance was performed with Tukey posttest (alpha error type set to 0.05), using GraphPad Prysm 5 (GraphPad Software, Inc., La Jolla, CA, USA). RESULTS AND DISCUSSION The elemental analysis of the HA and BCP granules proves that while HA presented a calcium and phosphorous ratio very similar to that expected for stoichiometric HA, the combination of HA and TCP produced granules deficient in calcium, regardless of the porogeneic treatment. However, the different methods employed in the present work have generated materials with very diverse physical properties. As shown in Table 1, the dense HA group presented a density very similar to the theoretical HA density of 3.16 g/cm3 (28) indicating the success of the sintering steps, with a very low apparent porosity (1.2%) resulting from the densification. The density of the dense BCP samples was approximately 87% of that of the isolated HA and b-TCP, probably due to the differing thermal behavior of these materials when submitted to higher temperatures, creating empty spaces among pockets of HA and TCP, which both decrease the final density and proportionate the presence of a inherent porosity (increased to 10% in the BCP group). The porous groups (submitted to lower pressing strengths in the presence of wax spheres) both presented a decrease in density and a strong increase in apparent porosity, when compared to the groups treated at 100 MPa. This porosity could be observed on both dense and porous BCP granules by SEM. Figure 1 shows that dense BCP granules present a rather smooth, tough irregular surface (Fig. 1A), as compared to the explicit porous surface of granules synthesized in the presence of wax and lower pressure, where craters also evidence the previous presence of bigger pores of 350 mm caused by the presence of the wax spheres, which were Artif Organs, Vol. 36, No. 6, 2012
FIG. 1. SEM of dense and porous granules of biphasic calcium phosphates. (A) Dense BCP granules. (B) Porous BCP granules. (C) Porous BCP granules at higher magnification, showing depressions resulting from the wax spheres, and micropores which ranged from 20 to 130mm in diameter.
disrupted after grinding (Fig. 1B). The micropores are more evident when observed at a higher magnification (Fig. 1C), presenting variation in their size from 20 to 130 mm. This surface porosity may represent a well-desired feature of BCP materials intended for bioengineering uses such as cell delivery in bone therapy (6). In order to detect if such differences on ceramic surfaces have an impact on the biocompatibility of
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FIG. 2. Effects of dense and porous BCP or HA extracts on cell viability. (A) Mitochondrial dehydrogenase activity (XTT assay); (B) membrane integrity (Neutral Red Uptake assay); (C) estimated cell density (CVDE, Crystal Violet Dye Elution assay). Results presented as a percentage of control group (culture medium). (a) Statistically different from control group (P < 0.05); (b) statistically different from all other groups (P < 0.05); (c) statistical difference between groups (P < 0.05). Two percent phenol and high-density polystyrene beads were used as positive and negative controls for cytotoxicity, respectively.
the biphasic ceramics, a multiparametric analysis of extracts from each material was performed, using stoichiometric HA for comparison (Fig. 2). As shown in Fig. 2A, no significant differences were found among dense and porous ceramics on mitochondrial dehydrogenase activity, as measured by XTT assay. Curi-
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ously, all extracts from ceramic groups seemed to exert some effects on mitochondrial activity, as they significantly differed from culture plastic control (P < 0.05). Figure 2B shows some significant differences (P < 0.05) on Neutral Red dye uptake by membrane intact viable cells exposed to extracts from dense and porous groups. Porous biphasic ceramic presented levels of Neutral Red Assay (NR) uptake significantly higher than those from both dense HA and dense BCP. While analyzing Crystal Violet dye exclusion test (Fig. 2C), however, no statistical difference was found among the extracts from ceramic and control (culture plastic) groups, indicating similar relative densities of cells, regardless of the material porosity or composition. As expected, the only exception was the positive control for cytotoxicity (2% phenol), which presented less than 50% of survival as compared to control. The use of BCP ceramics as bone graft substitutes brings the advantage of associating the high biocompatibility from HA and the desirable resorption properties from b-TCP. Combining such good attributes with the already described improvements brought by surface microporosity (29) may contribute to achieve better materials for bone grafting. However, it is implied that such modifications on the material properties should not interfere in its final biocompatibility. In this regard, the present results show in a preliminary but consistent manner that induction of porosity in BCP does not affect the cytocompatibility of the biphasic compound material through the liberation of soluble toxic components, as evaluated by three different cell viability parameters. In this work, tests were performed on human mesenchymal cells. The relevance of these cells on bone remodeling and regeneration comes from their ability of differentiating bone, cartilage, tendon, and other connective tissues (30,31). They also proved previously to be very responsive to the methodology employed in this work (27). These cells were exposed to extracts prepared according to international standards (23), focusing on effects caused by the eventual release of substances in biologic media, which is especially relevant if we consider the increased solubility of BCP in comparison to HA (4), or the possibility of remaining residues of the porogeneic agent. A good cytocompatibility with these important stem cells would, therefore, be one of the prerequisites prior to the application of porous BCP granules in the strategic delivery of osteogenic cells for faster tissue regeneration. Artif Organs, Vol. 36, No. 6, 2012
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XTT is a soluble variation of the widely employed MTT test, which accounts for mitochondrial activity in the tested material (32). Curiously, all tested ceramic extracts induced a significant increase on mitochondrial dehydrogenase activity, in comparison to culture plastic (control). These results contrast with other reports, where extracts of porous BCP induced a cytotoxicity which could be reverted by previous centrifugation (21). The authors attributed this phenomenon to the possible liberation of BCP microparticles during sintering. However, as no pressing treatment is described in that work, it is possible that the strength applied to the material prior to heating treatment prevented such a release in the present model. It is interesting to note that other authors have found a similar increase in mitochondrial activity, even tough on models employing dense BCPs modified with the addition of agarose (15,16,21). Evidence was also presented, showing that the increase in mitochondrial activity is not accompanied by production of reactive oxygen species (ROS)—a risk to be considered in cells with altered respiratory metabolism (33). Further studies would be necessary to evaluate if ROS production also remains unaffected in the present study model. Neutral Red uptake assay refers directly to cell survival, as it is described that only membrane intact viable cells are able to incorporate and bind the supravital dye in their lysosomes (34). The present results indicate that cell viability after exposition to soluble extracts of porous BCP remains the same as that promoted by tissue plastic or even highly biocompatible polystyrene beads (negative control). On the other hand, both dense ceramics (HA and BCP) extracts induced a slight cytotoxicity, with cell survival under 75% of control. Curiously, dense samples also promoted a significant (P < 0.05) depletion of the available calcium ions in their respective culture media (Table 3). These results are very similar to those described elsewhere with silicon-doped calcium phosphates, where a time-dependent depletion of calcium was demonstrated in the culture
TABLE 2. Elemental analysis of the HA and BCP granules Material
Ca %
MOL
P%
MOL
Ca/P
Dense HA Dense BCP Porous HA Porous BCP
56.041 54.363 55.666 54.463
1.001 0.971 0.994 0.973
42.134 43.495 41.956 43.286
0.593 0.613 0.591 0.610
1.68 1.58 1.68 1.59
medium, resulting from calcium phosphate precipitation over the material, and partially impeding cell proliferation (35). It is important to note that in such cases, the confined conditions of the in vitro test would present no parallel to the physiological medium during grafting, where a rapid reposition of calcium and other ions would occur, even after adsorption or precipitation over the biomaterial. Crystal Violet Dye Elution assay was unable to correlate directly with NR results, as no cytotoxicity was detected by the estimation of cell density in any group, with the exception of the positive control (2% phenol). Nevertheless, around 40% viability was detected after 24-h treatment with 2% phenol, while the same cells were clearly unable to accumulate Neutral Red dye (0% survival as compared to control). It is possible that phenol, as well as dense ceramic extracts, may be affecting cell survival without cell detachment during 24-h exposure, leading to the incorporation of Crystal Violet in nuclear material from dead or dying attached cells. This possibility exposes some interesting questions on the use of a single parameter to evaluate in vitro biocompatibility. CONCLUSIONS The present in vitro results show that the treatment with a porogenic agent and light pressing on biphasic calcium phosphate granules does not interfere negatively on the cytocompatibility of such ceramics with human primary mesenchymal cells, as confirmed by three different parameters. Moreover, porous BCP
TABLE 3. Content of calcium and phosphate ions on 24-h extracts before cell exposition Medium Alpha-MEM Dense HA extract Dense BCP extract Porous HA extract Porous BCP extract
Phosphate (mg/L)
Calcium (mg/L)
110 ! 10 95 ! 12 92 ! 3 111 ! 14 121 ! 20
194 ! 8 94 ! 11* 105 ! 12* 163 ! 25 177 ! 8
Results presented as mean ! SD. * Significantly different from Alpha-MEM (P < 0.05). Artif Organs, Vol. 36, No. 6, 2012
CYTOCOMPATIBILITY OF POROUS BIPHASIC CALCIUM PHOSPHATE GRANULES has shown better results than both dense HA and BCP. In this manner, these ceramics are well suited for further in vitro and in vivo studies in order to determine their specific biocompatibility and future clinical applications. Acknowledgments: The authors acknowledge the financial support of CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro), FINEP (Financiadora de Estuods e Projetos), MCT (Ministério da Ciência e Tecnologia), and DECIT-MS (Departamento de Ciência e Tecnologia do Ministério da Saúde), and the technical assistance of Dr. Vinícius Shot, Dr. Adriana Linhares, and Leticia de Oliveira Castro at HUAP-UFF, Brazil, and Dr. Maria Aparecida Pinheiro dos Santos from IPQM, Brazil.
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