2010 cytocompatibility of bio-inspired

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Cytocompatibility of bio-inspired silicon carbide ceramics M. Lo ´ pez-A ´ lvarez, 1 A. de Carlos, 2 P. Gonza ´ lez, 1 J. Serra, 1 B. Leo ´n 1 1 Applied Physics Department, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain 2 Biochemistry, Genetics and Immunology Department, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain Received 7 January 2010; accepted 16 May 2010 Published online 24 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31700 Abstract: Due to its good mechanical and biochemical prop- erties and, also, because of its unique interconnected poros- ity, bio-inspired silicon carbide (bioSiC) can be considered as a promising material for biomedical applications, including controlled drug delivery devices and tissue engineering scaf- folds. This innovative material is produced by molten-Si infil- tration of carbon templates, obtained by controlled pyrolysis of vegetable precursors. The final SiC ceramic presents a po- rous-interconnected microstructure that mimics the natural hierarchical structure of bone tissue and allows the internal growth of tissue, as well as favors angiogenesis. In the pres- ent work, the in vitro cytocompatibility of the bio-inspired SiC ceramics obtained, in this case, from the tree sapelli (Entandrophragma cylindricum) was evaluated. The attach- ment, spreading, cytoskeleton organization, proliferation, and mineralization of the preosteoblastic cell line MC3T3-E1 were analyzed for up to 28 days of incubation by scanning electron microscopy, interferometric profilometry, confocal laser scan- ning microscopy, MTT assay, as well as red alizarin staining and quantification. Cells seeded onto these ceramics were able to attach, spread, and proliferate properly with the main- tenance of the typical preosteoblastic morphology through- out the time of culture. A certain level of mineralization on the surface of the sapelli-based SiC ceramics is observed. These results demonstrated the cytocompatibility of this porous and hierarchical material. V C 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 95B: 177–183, 2010. Key Words: bio-inspired ceramics, silicon carbide, MC3T3-E1, cytocompatibility, in vitro mineralization INTRODUCTION Tissue engineering and regenerative medicine have emerged as an interdisciplinary field, which require the development of new biomaterials and fabrication methods to engineer appropriate scaffolds for regeneration of specific organs and tissue functions. In particular, the design of an ideal scaffold, focused on regener- ation of bone tissue, should replicate the architecture and hier- archical 3-D structure of the bone, with predetermined density, pore shape and size, and interconnected pathways. 1–4 Following these considerations, bio-inspired porous materials represent an exciting approach to fulfill all the demands since they mimic the smart structures present in Nature, taking inspiration from the most complex organized biological systems. In this context, bio-inspired silicon carbide (bioSiC) obtained from vegetable structures emerges as an innovative porous scaffold for bone tissue engineering. This promising biomaterial is produced by molten silicon infiltration of car- bon templates obtained by controlled pyrolysis of vegetable precursors, such as woods, algae, and plants. These porous scaffolds retain the unique microstructural properties of the bio-structures with hierarchical distribution of macro and microporosity, organized in a set of structural units and dis- tributed in levels with an increasing size, and interconnected porosity kept from the vegetable vascular system. Thus, the bio-derived material presents the high porosity and pore interconnection necessaries to support migration and prolif- eration of osteoblasts and mesenchymal cells, bone tissue ingrowth, vascular invasion, nutrient delivery, and matrix deposition in the empty spaces. As well, it maintains the suit- able structure–morphology to encourage osseointegration and to provide an optimum transfer of biological fluids. 5–10 Moreover, the biodiversity of the natural grown vegetable structures offers a large variety of templates with different density, morphology, pore shape, pore size, and interconnec- tion, leading to bioinspired materials with optimized micro- structure and tailorable properties, similar to those of the tis- sue to be regenerated. 11–15 In fact, its fabrication has been reported from soft and hard woods, such as beech (Fagus syl- vatica), eucalyptus (Eucalyptus globulus), sapelli (Entandro- phragma cylindricum), oak (Quercus rober), and so on. 16–19 The aim of this work is to assess the in vitro suitability of the bio-inspired sapelli-based SiC ceramics. The preosteo- blastic cell line MC3T3-E1 established from newborn mouse calvaria was the selected cell line because of its capacity to differentiate into osteoblasts and form mineralized extracel- lular matrix (ECM) in vitro. Thus, cell proliferation and min- eralization in vitro tests onto the bio-inspired SiC ceramics surface were carried out. MATERIALS AND METHODS SiC preparation The biomorphic silicon carbide tested in this experiment was obtained from the wood of sapelli (Entandrophragma cylindri- cum). The wood pieces were previously dried and then were pyrolyzed in a furnace in argon flowing atmosphere at 1000 C with controlled heating and cooling ramps. The carbonaceous Correspondence to: M. Lo ´ pez-A ´ lvarez; e-mail: [email protected] V C 2010 WILEY PERIODICALS, INC. 177

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Cytocompatibility of bio-inspired silicon carbide ceramics

M. Lopez-Alvarez,1 A. de Carlos,2 P. Gonzalez,1 J. Serra,1 B. Leon1

1Applied Physics Department, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain2Biochemistry, Genetics and Immunology Department, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain

Received 7 January 2010; accepted 16 May 2010

Published online 24 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31700

Abstract: Due to its good mechanical and biochemical prop-

erties and, also, because of its unique interconnected poros-

ity, bio-inspired silicon carbide (bioSiC) can be considered as

a promising material for biomedical applications, including

controlled drug delivery devices and tissue engineering scaf-

folds. This innovative material is produced by molten-Si infil-

tration of carbon templates, obtained by controlled pyrolysis

of vegetable precursors. The final SiC ceramic presents a po-

rous-interconnected microstructure that mimics the natural

hierarchical structure of bone tissue and allows the internal

growth of tissue, as well as favors angiogenesis. In the pres-

ent work, the in vitro cytocompatibility of the bio-inspired

SiC ceramics obtained, in this case, from the tree sapelli

(Entandrophragma cylindricum) was evaluated. The attach-

ment, spreading, cytoskeleton organization, proliferation, and

mineralization of the preosteoblastic cell line MC3T3-E1 were

analyzed for up to 28 days of incubation by scanning electron

microscopy, interferometric profilometry, confocal laser scan-

ning microscopy, MTT assay, as well as red alizarin staining

and quantification. Cells seeded onto these ceramics were

able to attach, spread, and proliferate properly with the main-

tenance of the typical preosteoblastic morphology through-

out the time of culture. A certain level of mineralization on

the surface of the sapelli-based SiC ceramics is observed.

These results demonstrated the cytocompatibility of this

porous and hierarchical material. VC 2010 Wiley Periodicals, Inc.

J Biomed Mater Res Part B: Appl Biomater 95B: 177–183, 2010.

Key Words: bio-inspired ceramics, silicon carbide, MC3T3-E1,

cytocompatibility, in vitro mineralization

INTRODUCTION

Tissue engineering and regenerative medicine have emerged asan interdisciplinary field, which require the development of newbiomaterials and fabrication methods to engineer appropriatescaffolds for regeneration of specific organs and tissue functions.In particular, the design of an ideal scaffold, focused on regener-ation of bone tissue, should replicate the architecture and hier-archical 3-D structure of the bone, with predetermined density,pore shape and size, and interconnected pathways.1–4 Followingthese considerations, bio-inspired porous materials representan exciting approach to fulfill all the demands since they mimicthe smart structures present in Nature, taking inspiration fromthe most complex organized biological systems.

In this context, bio-inspired silicon carbide (bioSiC)obtained from vegetable structures emerges as an innovativeporous scaffold for bone tissue engineering. This promisingbiomaterial is produced by molten silicon infiltration of car-bon templates obtained by controlled pyrolysis of vegetableprecursors, such as woods, algae, and plants. These porousscaffolds retain the unique microstructural properties of thebio-structures with hierarchical distribution of macro andmicroporosity, organized in a set of structural units and dis-tributed in levels with an increasing size, and interconnectedporosity kept from the vegetable vascular system. Thus, thebio-derived material presents the high porosity and poreinterconnection necessaries to support migration and prolif-eration of osteoblasts and mesenchymal cells, bone tissueingrowth, vascular invasion, nutrient delivery, and matrix

deposition in the empty spaces. As well, it maintains the suit-able structure–morphology to encourage osseointegrationand to provide an optimum transfer of biological fluids.5–10

Moreover, the biodiversity of the natural grown vegetablestructures offers a large variety of templates with differentdensity, morphology, pore shape, pore size, and interconnec-tion, leading to bioinspired materials with optimized micro-structure and tailorable properties, similar to those of the tis-sue to be regenerated.11–15 In fact, its fabrication has beenreported from soft and hard woods, such as beech (Fagus syl-vatica), eucalyptus (Eucalyptus globulus), sapelli (Entandro-phragma cylindricum), oak (Quercus rober), and so on.16–19

The aim of this work is to assess the in vitro suitabilityof the bio-inspired sapelli-based SiC ceramics. The preosteo-blastic cell line MC3T3-E1 established from newborn mousecalvaria was the selected cell line because of its capacity todifferentiate into osteoblasts and form mineralized extracel-lular matrix (ECM) in vitro. Thus, cell proliferation and min-eralization in vitro tests onto the bio-inspired SiC ceramicssurface were carried out.

MATERIALS AND METHODS

SiC preparationThe biomorphic silicon carbide tested in this experiment wasobtained from the wood of sapelli (Entandrophragma cylindri-cum). The wood pieces were previously dried and then werepyrolyzed in a furnace in argon flowing atmosphere at 1000�Cwith controlled heating and cooling ramps. The carbonaceous

Correspondence to: M. Lopez-Alvarez; e-mail: [email protected]

VC 2010 WILEY PERIODICALS, INC. 177

porous preforms were then infiltrated under vacuum condi-tions with pure silicon at 1550�C, exceeding the silicon melt-ing point. That temperature was achieved following a heatingramp with 30 minutes of permanence on it. Then, the furnacetemperature was decreased by a well-controlled ramp and thesilicon carbide was obtained.14,20 The final material was cut toobtain pieces of 3.8 � 3.8 � 2.0 mm3.

The samples were sterilized applying several cycles of15 minutes in an ultrasound waterbath with milli-Q water,ethanol 100%, and acetone. Then, samples were autoclavedat 121�C for 20 minutes.

Cell cultureThe preosteoblastic cell line MC3T3-E1 was obtained from theEuropean Collection of Cell Cultures (ECACC, UK). A cell suspen-sion of 1.7� 105 cells/mL in 100 lL of MEM-alpha (Sigma) sup-plemented with 10% fetal bovine serum (Invitrogen) was addeddirectly over the surface of the sterilized samples. Previously,the samples were placed in 96-well tissue cultured plates withthe open porosity at the lateral sides and longitudinal channelsat the top of the scaffold. Cells were seeded for 1, 3, 7, 14, 21,and 28 days in a humidified atmosphere with 5% CO2 and at37�C.

To induce the differentiation 1 day after seeding, ascor-bic acid 2-phosphate (2 mM) and glycerol 2-phosphate (10mM) were added to the MEM-alpha, and 7 days after thecell seeding a melatonin solution (50 nM) was also added.The culture medium was changed every 2–3 days. Standardtissue culture polystyrene (TCP) of the bottom of the wellswas also seeded with the same cellular suspension to vali-date the different tests. TCP is considered the experimental‘‘gold standard’’ for in vitro studies of osteoblasts. Theexperiment was repeated twice under same conditions.

Cell proliferationCell proliferation was assessed and quantified with the CellProliferation Kit I (MTT) from Roche Applied Science along thetime of culture. This colorimetric assay is based on the reduc-tion of the yellow tetrazolium salt MTT (3-(4,5-dimethyltya-zolyl-2)-2,5-diphenyl tetrazolium bromide) into insoluble pur-ple formazan crystals by the mitochondrial enzyme succinatedehydrogenase, only present in living cells.

After the incubation period (1, 3, 7, 14, and 21 days), 10 lLof MTT labeling reagent in phosphate buffered saline (PBS)was added to each well. The microplate was incubated for 4 h(37�C and 5% CO2). After that time, the formazan crystalsformed were solubilized by adding 100 lL of 10% sodium do-decyl sulphate (SDS) in 0.01M HCl. The plate was incubatedovernight. Then, the plate was shaken to favor the release ofsolubilized formazan crystals in the inside of the porous ce-ramic and, after removing the sample, the resulting colored so-lution was quantified at 570 nm using a Bio-Rad Model 550microplate spectrophotometer.

Cell proliferation analysisCell adhesion and morphology were analyzed by scanning elec-tron microscopy (SEM). After each incubation time, the threereplicates of each experiment were fixed with 2% glutaralde-hyde in 0.1M pH 7.4 cacodylate buffer for 2 h at 4�C. Samples

were then washed three times for 30 minutes each with caco-dylate buffer 0.1M and dehydrated in graded ethanol solutions(30%, 50%, 70%, 80%, 95%) for 30 minutes in each solutionand in absolute ethanol for 1 h. After dehydration, the sampleswere submitted to an increasing amylacetate: ethanol mixture(25:75, 50:50, 75:25, 15 minutes each) and to pure amylacetatetwice for 15 minutes. The critical point of CO2, at 75 atm and31.3�C was the final step. The samples were finally mountedon metal stubs and sputter-coated with gold prior to their anal-ysis using a Philips XL 30 scanning electron microscope.

In addition to SEM analysis, the samples were submitted tointerferometric profilometry technique (WYKO NT-1100) tostudy the evolution of the cellular coverage of the sample sur-face through the changes in roughness that occurred duringthe incubation time (1, 3, and7 days). A bioSiCVR piece withoutcells was also analyzed. Three-dimensional images of the sam-ples were taken and six measurements per sample were takento obtain representative mean values of the surface roughness(Ra) at each incubation time.

Confocal laser scanning microscopy (CLSM) was carriedout by fixing the cells with 4% paraformaldehyde solution for10 minutes at room temperature. After that, the cells were per-meabilized for 5 minutes with 0.1% (v/v) Triton X-100 in PBSbuffer 1X. Alexa Fluor 488 Phalloidin solution was added for20 minutes in the dark to visualize the cell cytoskeleton fila-mentous actin (F-actin). After that incubation, propidiumiodide solution was added for 5 minutes in the dark for cellnuclei labeling. Finally, cells were observed with a confocalmicroscope Bio-Rad MRC 1024.

Alizarin red staining and quantificationMineralized tissue deposition was determined by stainingwith alizarin red solution (Chemicon International). Thismineralization was also quantified by the extraction of thestain and measurement of alizarin red uptake.

For staining after each incubation time, the three repli-cates (per experiment) with cells were washed with PBSand then fixed by covering with 70% ethanol and incubatingat room temperature for 15 minutes. Cells were conse-quently rinsed three times (5 minutes each) with an excessof distilled water. Alizarin red stain solution was added andincubated at room temperature for 20 minutes. After thattime, cells were washed four times with milli-Q water. Theimage acquisition was carried out with a digital cameraattached to an optical microscope Nikon SMZ 10 A.

For quantitative analysis, after the image acquisition,10% acetic acid was added to each well and cells were incu-bated for 30 minutes with shaking. Cells were scraped andtransferred to a 1.5 mL microcentrifuge tube. After shakingfor 30 seconds, they were heated to 85�C for 10 minutesand transferred to ice for 5 minutes. Cells were then centri-fuged for 15 minutes at 12,000g. The quantity of alizarinwas measured in a spectrophotometer at 405 nm. Valueswere compared with standards, previously prepared.

Statistical analysisData are presented as mean 6 standard deviation (withthree replicates per experiment, except the roughness

178 LOPEZ-ALVAREZ ET AL. CYTOCOMPATIBILITY OF BIO-INSPIRED SiC CERAMICS

analysis with six replicates per sample). Error bars in fig-ures represent standard deviations. Differences betweengroups were analyzed according to a Student t-test, with p< 0.05 considered statistically significant. The experimentwas repeated twice under same conditions.

RESULTS

Cell proliferationResults regarding cell proliferation (MTT assay) on biomor-phic silicon carbide ceramics are shown in Figure 1. SEMmicrographs show that MC3T3 cells proliferated properlythroughout the culture time on the tested material. A slightdecrease in the proliferation of the cells, compared with the‘‘gold standard’’, was observed from 1 to 3 days of culture.From 3 to 14 days of incubation, the cells presented an ex-ponential cell growth. On the 21st day an expected reductionin proliferation on TCP was also quantified on SiC ceramics.The behavior of the cells followed the same pattern as thosegrown on the TCP. When statistical differences during theincubation time were analyzed the proliferation increasedsignificantly (p < 0.01) from 3 to 7 days and decreased

FIGURE 1. Cell proliferation results (MTT assay) after incubation of

MC3T3 cells over 1, 3, 7, 14, and 21 days on silicon carbide ceramics

and TCP as control. (Statistical significant differences: * p < 0.05 and

** p < 0.01).

FIGURE 2. Scanning electron microscopy (SEM) images showing the attachment and spreading of MC3T3-E1 cell line on sapelli-based biomor-

phic silicon carbide ceramics. A, D, G for 1 day; B, E, H for 3 days; and C, F, I for 7 days. Three magnifications are shown: 100� (A, B, C), 500�(D, E, F) and 1000� (G, H, I).

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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | OCT 2010 VOL 95B, ISSUE 1 179

significantly from 14 to 21 days on the bio-inspired SiCceramics.

Figure 2 presents, in three magnifications, scanning elec-tron microscope (SEM) micrographs of the preosteoblasticcell line MC3T3-E1 adhesion and morphology throughoutthe time of culture (A, D, G for 1 day, B, E, H for 3 days andC, F, I for 7 days) on sapelli-based SiC ceramics. In the lowmagnification micrographies (A, B and C), the microstruc-tural properties of this bio-inspired material are shown. Thelong tubular cells and sap channels (tracheae), used by theangiosperm species to transport and store food, can be per-fectly observed. On the first day of incubation (A, D, G), cellsappeared closely attached to the SiC ceramics with the flat-tened morphology typical of healthy osteoblasts. After 3

days of incubation (B, E, H), the cells connected to eachother by filopodia, and lamellipodia almost covered thewhole surface, including some of the channels. Whenobserved on the 7th day of incubation (C, F, I), several layersof cells covered the surface and the channels were almostcompletely filled. Cells attached and spread properly, with-out any signal of cytotoxicity.

Figure 3 shows the interferometric profilometry imagesfor bio-inspired SiC without cells (A), with 1 day of cellincubation (B), 3 days (C), and 7 days (D). The table repre-sented below the images corresponds to the surface rough-ness of the different samples. When observing the images, itis shown that as the incubation time increased, the lowerprofiles represented by blue colors were less obvious as a

FIGURE 3. Interferometric profilometry 3D images of bio-inspired SiC ceramics without cells (A), and with cells after 1 day (B), 3 days (C), and 7

days (D) of incubation. Color charts values of each 3D images correspond to A (�144 to 77lm), B (�146 to 56 lm), C (�145 to 39 lm), and D

(�110 to 23 lm). For each sample, the mean values of the surface roughness are given. [Color figure can be viewed in the online issue, which is

available at wileyonlinelibrary.com.]

FIGURE 4. Confocal laser scanning microscopy images of preosteoblastic cell line MC3T3-E1 growth on sapelli-based SiC ceramics. Cytoskeleton

organization, examined by nuclei (red) and F-actin filaments (green) stainning, along time of culture (A, D for 1 day, B, E for 3 days, and C, F for

7 days) is represented. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

180 LOPEZ-ALVAREZ ET AL. CYTOCOMPATIBILITY OF BIO-INSPIRED SiC CERAMICS

consequence of the gradual covering of the channels by thecells. When mean values of roughness were taken intoaccount, they confirmed this tendency with a decrease inthe roughness when cells spread on the whole surface, fill-ing and covering the topographic defects.

In Figure 4 the CLSM images of the preosteoblastic cellline MC3T3-E1 cytoskeleton organization throughout thetime of culture (1, 3, and 7 days) by nuclei (red) and actinfilaments (green) on sapelli-based SiC ceramics are pre-sented. Cytoskeleton organization was progressively higherthroughout the time of culture, as shown by the increasingdensification of actin filaments, observed after 7 days ofincubation (C, F). After 1 day of cell culture (A, D), imagesshowed oval nuclei, which means that cells were alreadyspreading over the SiC surface. At 3 days of cell culture (B,E), the oval nuclei of cells and F-actin fibers are clearly visi-ble (see white arrow). After 7 days of incubation, cells dis-played an organization with numerous cell-to-cell contactsand presented a complex network of actin fibers (C, F).

Alizarin red staining and quantificationThe calcium deposits by the MC3T3-E1 cells over the SiCceramics were determined by the alizarin red staining andquantification up to 1, 7, 14, 21, and 28 days of cell culture(Figure 5). The surface of the bio-inspired ceramicsappeared more stained each day, being completely mineral-

ized at 28 days of incubation. At the same time, mineraldeposits were detected at the borders of the sample at 14,21, and 28 days.

These observations were confirmed in the quantificationgraph, where the alizarin concentration increases at 7 daysof cells incubation. At 14 and 21 days its value is slightlyhigher to end with the top value at 28 days. Cells on theTCPs showed the expected behavior for this standard, whichvalidated the experiment.

The statistical analysis showed significant increases (p <

0.01) in alizarin quantification from 1 to 7 days and from21 to 28 days of incubation. The same pattern was followedby TCP.

SEM was used to evaluate the SiC surface after 28 daysof cell culture (Figure 6). The micrographs showed thewhole surface of SiC covered by a thick layer of cells (A).When the surface was analyzed closely, a premineralizedECM was observed over the cell layer (B, C). The observedcells were attached with short and numerous filopodia tothe other cells that already formed a layer (D, E).

DISCUSSION

The proliferation results of MC3T3-E1 cells growing on sapelli-based SiC along the time of culture (Figure 1) followed the gen-eral pattern found in the literature.21,22 The initial interactionof the cells with any surface involves protein adsorption to it,

FIGURE 5. Staining and quantification of calcium deposits, over the sapelli-based SiC ceramics, at 1, 7, 14, 21, and 28 days of cell culture. (Sta-

tistical significant differences: * p < 0.05, ** p < 0.01). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.

com.]

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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | OCT 2010 VOL 95B, ISSUE 1 181

contact of rounded cells, and their consequent attachment andspreading to the substrate. The cells then start to proliferate,increasing their number across the materials surface andpores. At 21 days of incubation, an expected cell proliferationdecrease was found (Figure 1). Similar effect was also reportedin the literature, using the same cell line and others, over dif-ferent materials as calcium phosphate coatings or differentsurfaces of titanium.23,24 In agreement with SEM and interfero-metric profilometry results, this fact can be because of the celllayer confluence, which means that cells have already coveredthe whole surface and the lack of space limits their attachmentand proliferation. Several authors state that at the time ofdecreasing proliferation, cells had already started to synthesizea dense ECM that could have prevented cells from a completecontent release (MTT) during the cell lysis, thereby decreasingproliferation measurements are obtained.21–25

The difference in absorbance values observed betweencells growing on SiC and on TCP could be explained by twofacts. First, TCP as negative cytotoxic control is perfectlyadapted to support cell growth and it is natural that cells pro-liferate exhibiting the highest growing rates, better than on anyother substrate. And, as second reason, because of the bio-inspired SiC porosity, cells would have been growing into allkind of pores and channels inside the substrate structure. Thediffusion of the MTT substrate to the inside of the porous andinterconnected structure is limited, and it is difficult for theMTT reagent to reach all existing cells, especially at late timesof incubation, leading to an underestimation of the amount ofgrowing cells. Similar results were found by other authors,23,25

when testing porous materials, such as titanium foams orresorbable polymeric scaffolds.

It is well-known that after the deposition of the ECM,cell mineralization occurs with calcium phosphate depositsin the crystalline hydroxyapatite (HA) form.21,22 In our case,

the different tests show that a certain level of mineralizationof preosteoblastic cell occurs on the surface of the bioSiCceramics.

In agreement with previous works,26–30 the surface micro-structure of the different materials has a great influence ontheir cellular acceptance. The structural features of the surfacemodulate the expression of phenotypic markers and influencethe way that cells respond to regulatory factors. In fact, it hasbeen shown that osteoblasts attach, spread, and proliferatemore rapidly on smooth surfaces than on rough surfaceswhereas their differentiation was enhanced by rough morphol-ogies.29,31–33 It is also important to note that pores and pocketsthat resemble pores have an effect beyond the one producedby growth factors and other signaling molecules. The protec-tive spaces in excavations or inside pores were essential in sev-eral works to stimulate differentiation of precursors to osteo-blasts.26,27,30 At the same time, it was demonstrated thatscaffolds with nanoscale architectures have larger surfaceareas to adsorb proteins, presenting many more binding sitesto cell membrane receptors, which favors an earlier bio-mineralization.28

Sapelli-based SiC ceramics present the vascular systemremaining from the original wood. This microstructure withprotective spaces could favor the mineralization of cells.There were found in the other literature cases where theporosity effects over the MC3T3-E1 cell growth was proved,such as three-dimensional titanium scaffolds with an aver-age in pore sizes from 336 to 557 lm, the most favorablepieces being the ones with the pore size of 336 lm.23

Another example with MC3T3-E1 was found over severalsurfaces with differences in roughness. The roughest surfacetested, a grit-blasted surface of biphasic calcium phosphateceramic, was proved to allow more rapid osteoblastic celldifferentiation.29 At the same time, the calcium content in

FIGURE 6. Scanning electron microscopy (SEM) images, at different magnifications, of the preosteoblastic cell line MC3T3-E1 at 28 days on

sapelli-based SiC ceramics.

182 LOPEZ-ALVAREZ ET AL. CYTOCOMPATIBILITY OF BIO-INSPIRED SiC CERAMICS

the ECM on nanophase alumina, titania, and HA wasobserved to be four, six, and two times greater than on re-spective conventional ceramics after 28 days, respectivelycompared to conventional alumina, titania, and HAformulations.28

CONCLUSION

Bio-inspired sapelli-based SiC is a ceramic material able tosupport the growth of the MC3T3-E1 cell line. The in vitrocytocompatibility of the bio-inspired sapelli-based SiCceramics was demonstrated. On this material, cells attached,spread, and proliferated properly with a gradual filling andcovering of the whole surface, including the longitudinalchannels existent within its microstructure. Moreover, a cer-tain degree of mineralization on sapelli-based SiC ceramicswas observed.

ACKNOWLEDGMENTS

The authors acknowledge the helpful contributions of Dr. J.Mendez and I. Pazos (CACTI, Universidade de Vigo) with SEMand CLSM analysis, and Dr. J. Martınez-Fernandez and Dr. A.R.de Arellano-Lopez for providing the ceramics. This work wassupported by the UE-Interreg IIIA (SP1.P151/03) Proteus andPOCTEP 0330IBEROMARE1P projects, and Xunta de Galicia(Projects 2006/12 and PGIDITO5PXIC30301PN).

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ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | OCT 2010 VOL 95B, ISSUE 1 183