Journal of Biomaterials Applications

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In vitro biocompatibility of novel biphasic calcium phosphate-mullite composites Shekhar Nath, Sushma Kalmodia and Bikramjit Basu J Biomater Appl published online 12 July 2011 DOI: 10.1177/0885328211412206 The online version of this article can be found at: http://jba.sagepub.com/content/early/2011/06/21/0885328211412206

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Article

In vitro biocompatibility of novel biphasic calcium phosphate-mullite composites

Journal of Biomaterials Applications 0(0) 1–13 ! The Author(s) 2011 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328211412206 jba.sagepub.com

Shekhar Nath, Sushma Kalmodia and Bikramjit Basu

Abstract In designing new calcium phosphate (CaP)-based composites, the improvement in physical properties (strength, toughness) without compromising the biocompatibility aspect is essential. In a recent study, it has been demonstrated that significant improvement in compressive strength as well as modest enhancement in toughness is achievable in biphasic calcium phosphate (BCP)-based composites with mullite addition (up to 30 wt%). Herein, we report the results of the in vitro cell adhesion, cell proliferation, alkaline phosphatase (ALP) activity, and osteocalcin (OC) production for a series of BCP-mullite (up to 30 wt%) composites. Mouse fibroblast (L929) cell lines were used to examine in vitro cell adhesion and cell proliferation; while osteoblast-like (osteosarcoma, MG63) cells were used for in vitro osteoblastic function study by ALP and OC expression. Much emphasis has been provided to discuss the cell viability and proliferation as well as osteoblastic differentiation marker on the investigated biocomposites in relation to the characteristics of the phase assemblage. On the basis of various observations using multiple biochemical assays, it has been suggested that BCPmullite composites would be a candidate material for orthopedic applications. Keywords BCP-mullite, composite, cell adhesion, MTT, ALP, osteocalcin

Introduction In the search of ideal bioactive bone implant materials, substantial efforts have been invested to develop hydroxyapatite (HA) or CaP-based composites with various reinforcements.1–4 As a synthetic analogue of calcified tissues of vertebrate, HA is a candidate material for bone implant applications. Also, bone matrix can directly bind to HA, which is a necessary prerequisite for implant osseointegration.5 However, its low strength and fracture toughness have reduced the field of possible applications only to those, where the implant will be subjected to very low stress.6–8 Another possible application of HA could be a coating on metallic implants. However, recent clinical studies9,10 revealed that HA coating did not produce any benefit, when implanted for long period. Keeping these information in mind, it is therefore needed to improve the performance of CaP (HA, tricalcium phosphate – TCP]), which could be used as bulk. In search of such materials, researchers used mixture of HA-TCP materials along with second phase reinforcement to develop

composites with desirable properties. To this end, the physical property enhancement without any compromise on biocompatibility aspect necessitates the use of optimal amount of reinforcement as well as tailoring the processing parameters. For this purpose, in the present study, different amounts (10–30 wt%) of mullite (3Al2O3.2SiO2) were mixed with HA and the powder mixtures were sintered under various optimal conditions. Mullite is a solid solution of alumina (Al2O3) and silica (SiO2). Mullite is chemically inert and mainly used as refractory material in high temperature furnaces.11 It has lower density

Laboratory for Biomaterials, Department Materials Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India Corresponding author: Bikramjit Basu, Laboratory for Biomaterials, Department of Materials Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India Email: [email protected]


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(3.05 g/cc) than Al2O3 (3.95 g/cc) and ZrO2 (6.1 g/ cc). It has good combination of structural properties, like high hardness of 15 GPa, high elastic modulus of 240 GPa, and moderate fracture toughness of 3 MPa m0.5.12 When mullite was mixed (up to 30 wt%) with HA, very good combinations of mechanical properties were measured (E-modulus: 70 GPa, hardness: 4–5 GPa, and fracture toughness: 1.5 MPa m0.5).13 This toughness value was 2.5 times higher than that of pure monolithic HA (0.6 MPa m0.5, measured by SEVNB technique). The compressive strength of the developed composites was 230 MPa, which is by far better than pure HA (50 MPa).13 From microstructural phase assemblage point of view, these composites contain a mixture of HA and TCP and therefore they are called as biphasic calcium phosphate (BCP)-mullite composites.14–16 Hence, it is likely that such composite could be an excellent alternative of HA, provided that the composite shows required biocompatibility. In this backdrop, an important part of research is to characterize the in vitro properties of new biomaterials. Despite various conflicts in results of in vitro and in vivo tests, the in vitro assays are considered the primary biocompatibility screening tests for a wide variety of implant materials.17 For example, the response of osteoblastic cells to a thin film of poorly crystalline calcium phosphate apatite (PCA) crystals was examined in vitro. The osteoblasts were reported to exhibit high cellular activity, such as adhesion, proliferation etc. In fact, the cells were attached more rapidly to PCA thin film than to reference dishes.18 In another study, HA was used as an additive for zirconia-alumina nanocomposite. The addition of HA was reported to increase the biocompatibility of the nanocomposites significantly, as evident from the results of the in vitro tests using MG63 osteoblast-like cells.5 In some cases, the HA-based composite materials showed better in vitro biocompatibility than pure HA. For example, Boanini et al.19 studied the interaction of osteoblast-like cells on nanocomposite of HA with aspartic acid and glutamic acid. Their results revealed that the nanocomposite of HA possessed better cell proliferation, alkaline phosphatase (ALP) activity, and osteocalcin (OC) gene expression than pure HA. Shu et al.20 studied the role of HA on the differentiation and growth of MC3T3-E1 osteoblasts cells. Their results indicated that HA enhanced osteoblast differentiation while suppressing cell growth. In contrast, Licht et al.21 reported that due to the phagocytosis of HA particles, osteoblast exhibited reduced cell growth and ALP activity. In another study, Ogata et al.22 compared the osteoblast response to HA with HA/soluble CaP (SCaP) composites. Their results revealed that HA/CaP

showed greater ability in osteogenesis than HA by increasing collagen synthesis and calcification of the extracellular matrix. Reviewing literature, it is evident that HA, TCP, and other CaP phases are biocompatible. However, there is no published data available on the biocompatibility of mullite ceramics. Therefore, the biocompatibility of BCP-mullite composite materials is needed to be examined before any biomedical application can be proposed. In this paper, we report the cell adhesion, proliferation, and differentiation behavior using mouse fibroblast (L929) as well as MG63 cell lines. The sintered HA was used as baseline material in all the in vitro experiments.

Materials and experimental procedures Synthesis of materials HA powder was synthesized in-house using commercially available chemicals, such as calcium oxide (CaO) and phosphoric acid (H3PO4), following a wellestablished suspension–precipitation route.23,24 Phase pure mullite (3Al2O3.2SiO2) powder was procured commercially (KCM Corporation, Japan). As a first step of sample preparation, the mixing of HA and mullite powders (10–30 wt% mullite) was carried out in a ball mill for 16 h. Following this, the powder was subsequently pressed to obtain pellets of 5 mm diameter. The sintering temperature was optimized based on the densification and mechanical properties results. The sintering of the composite pellets was carried out at 1350 C for 2 h, while sintering of pure HA was performed at 1200 C for 2 h, both in conventional pressureless sintering furnace. After the sintering, the diameter of the sample was around 4 mm. The sintered composites are designated by their initial mullite content (BCP M means BCP – x wt% mullite and likewise), irrespective of the phases present in the sintered materials. Therefore, throughout the text, such designation is followed for the composites.

Material characterization X-ray diffraction (Rich-Seifert, 2000 D) patterns were acquired from the sintered ceramics to identify the different phases present. Based on the X-ray peak intensity of the characteristic phases, the qualitative presence of various phases is reported in this paper. Scanning electron microscopy (SEM; model JSM-6330 F, Philips, The Netherlands) was performed on the polished and etched surface of the sintered composite to investigate various microstructural features.


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Surface topography is an important parameter which, influences the cell adhesion and, in general, the biocompatibility property of materials. In order to characterize the finer scale topography of the investigated materials, smoothly polished and thermally etched surfaces were observed under atomic force microscope (AFM; Molecular Imaging, Pico-SPM I, USA) using ‘contact mode’. A Si3N4 cantilever with a three-sided pyramidal single crystal Si3N4 tip with apex angle of 20 , a tip radius of curvature of 10 nm, and a normal stiffness of 0.6 N/m were employed.

Cell culture experiment. L929 and MG63 cell lines obtained from CCMB, Hyderabad (India), and ATCC (USA), respectably were preserved in an LN2 container. Prior to seeding the cells on biomaterials surfaces, the cells were revived. Following this, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Aldrich), supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% penicillin/streptomycin cocktail (Sigma Aldrich). The culture plate with the cell lines (gelatin-coated) was incubated for further proliferation and growth in a CO2 incubator (Thermo, USA) operated under conditions of 5% CO2, 90% humidity, and 37 C temperature. The medium was being replaced every 2 d interval and the numbers of cells in confluent monolayer was approximately 5 105/mL, in 35-mm culture plate. The confluent monolayer was detached from the culture plate using 0.50% trypsin and 0.20% EDTA solution (Sigma Aldrich).

Cell adhesion test. As described in the previous subsection (section ‘Cell culture experiment’), L929 cells were cultured. The samples used for cell adhesion experiment were pure HA, BCP10M, BCP20M, BCP30M, and control glass disc. All samples were sterilized in steam autoclave (121 C, 15 lb pressure for 15 min) and, subsequently, the cells were seeded on the samples at approximate density of 5 105/mL. In a separate experiment, the cell seeding density of L929 was reduced to 1 105/mL and the effect of cell seeding density on cell adhesion was studied. The seeded test samples were incubated in a CO2 incubator with the standard culture condition, 5% CO2, 37 C temperature, and 90% humidity. The culture medium was being aspirated after 2-d interval and fresh culture medium was being added into each wells. After the stipulated time period (1 and 3 d), the samples were washed twice with phosphate buffer saline (PBS; 1 PBS, pH 7.4) and then fixed by 2% glutaraldehyde in PBS. The cells, adhered on the material surfaces, were dehydrated using a series of ethanol solutions (30%, 50%, 70%, 95%, 100%) for 10 min twice and

then further dried using critical point drier (CPD; Quramtech, UK). The dried samples were sputtercoated (Vacuum Tech, Bangalore, India) with gold and examined under SEM. For AFM observation, the dried samples were directly used, without gold coating. The experiment was repeated for at least three times and the representative results are presented in the present article.

MTT assay L929 and MG63 cells were cultured following the previously described cell culture method. The cell proliferation was investigated on pure HA, BCP-mullite composites (4 mm diameter, 4 mm height), and control glass disc. At first, autoclaved samples were placed in the 4-well plate and then washed with PBS. Following this, 5 104 cells/mL were seeded on each sample. Subsequently, the culture plate was incubated for 2 d in the CO2 incubator. After the incubation period, the medium was aspirated and samples were washed twice with PBS. Then, 200 mL of fresh DMEM was added (without phenol red) into each well followed by 10 mL reconstitute MTT (3(4,5-dimethylthiazol-2 yl)-2-5 diphenyltetrazolium bromide: SIGMA, USA, Cat No.M5655; 5 mg/mL in DMEM culture medium and serum) per 100 mL of DMEM was added in each well and the plate was incubated for 6–8 h. In the meantime, the culture plate was viewed under the phase contrast microscope (Nikon, Eclipse 80i, Japan) to check for the formation of purple formazane crystal. After the incubation, samples were removed from the wells and placed in a new culture plate. Thereafter, 200 mL of dimethyl sulfoxide (DMSO) was added into each well, including control. The optical density of the solution was measured at 540 nm using ELISA automated microplate reader (Bio-Tek, EL 800).

ALP activities ALP is a widely recognized biochemical marker to assess osteoblast activity on biomaterial substrate. It is known that ALP plays a major role in skeletal mineralization. This enzyme is bound to the membrane of osteoblasts and functions to enhance osteogenesis by degrading pyrophosphates. For the ALP assay, the autoclaved samples were placed in the 4-well plates and MG63 cells were seeded approximately at a density of 5 104 cells/mL. Three replicates of each composition were selected for this experiment. The culture protocol and conditions were similar to that described in the earlier section. After 2 d of culture, ascorbic acid and vitamin D3 were added to activate the


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phenotypic expression of differentiated MG63 cell line. The concentration of 1,25-dihydroxy vitamin D3 solution has significant effect on the expression of differentiated MG63 cells. In an important study, Robert and Gideon25 reported that the concentration of 10 8–10 7 M, 1,25-dihydroxy vitamin D3 inhibited growth and elevated ALP as well as total cell protein. However, lower D3 concentrations (10 10 and 10 9 M) reduced ALP activity. Therefore, in the present study, the concentration of vitamin D3 was kept at 10 8–10 7 M for better ALP activity. The ALP activity experiments were conducted after 3 rd and 7th d of culture. When the cells were lysed followed by centrifuged. Therefore, the supernatant was used as the ‘sample’ for the ALP activity. The subsequent steps were followed as per the commercial kit protocol (ALP, code no. 25904, Span Diagonstics Ltd., Surat, India) in a 96-well culture plate. At the last step of the experiment, optical density was measured by ELISA reader at 405 nm.

Osteocalcin. It is known that OC, the most abundant noncollageneous protein of the bone extracellular matrix, is biologically synthesized by osteoblast-like cells. In the present study, MG63 cells were cultured for the OC assay, following the method described in an earlier section. After subconfluent, cells were trypsinized and seeded on sterilized pure HA and BCP-mullite (all samples were of 4 mm diameter) in a 4 well culture plate at a density of 5 104 cells/mL. After day 2 and 5, culture medium was replaced by differentiating medium that contain vitamin D3 (10 8 M final concentration), ascorbic acid (50 mg/mL final), and glycerol phosphate (108 mg/mL) to enhance osteogenic activity. OC production was tested at 7th d of culture and the sample preparation was similar to ALP assay. In brief, three strips of coated wells were taken from the strips supplied with the OC kit (HOSTEASIA KAP1381, Biosource Europe S.A., Belgium). Thereafter, 25 mL of each calibrator (human serum with protease inhibitors and benzamidin: supplied with kit), control (human serum with protease inhibitors, benzamidin and thymol: supplied with OC kit) and samples (Pure HA, BCP10M, BCP20M, and BCP30M) were transferred into the appropriate wells and subsequently 100 mL of anti-OST-HRP conjugate was added to all the wells, followed by 2 h shaking. Following this, the liquid from each well was aspirated and washed by 400 mL of wash solution. Then, the solution from each well was aspirated and 100 mL of chromogenic solution was added into each well. Thereafter, the well plate was incubated in a shaker at room temperature. Finally, the optical density was measured at 450 nm.

Results Phase assemblages and microstructure. In Table 1, the quantitative analysis of XRD results, obtained from the polished surface of the investigated materials, are presented. Also, a typical microstructure of BCP30M composite is provided in Figure 1. The larger grain matrix was identified as TCP/HA phase and the grain boundary phases were mullite, alumina, CaO, and gehlenite. More details of the microstructural characterization can be found elsewhere.15,16 From Table 1 it should be clear that all the HA-based composites after sintering predominantly contained TCP. In contrast, single-phase HA without any sign of dissociation to any TCP polymorph could be obtained as baseline HA. The addition of mullite as well as higher sintering temperature, in combination, caused dissociation of HA to TCP phases and it is clear that all the mullitecontaining samples were essentially BCP composites. In Fig. 2, AFM images show some representative surface topography (three-dimensional view). Figure 2(a) shows the equiaxed grains of HA of variable sizes with good grain boundary adhesion. Figure 2(b) shows the AFM image captured from BCP10M sample, sintered at 1350 C for 2 h. Here, the finer grains of reaction products were present at the boundary region of larger HA/TCP grains. Similarly, Figure 2(c) shows a coarse CaP-grain surrounded by the reaction products, that is, calcium–alumino silicate phase. Figure 2(d) presents a high magnification AFM image of BCP30M sample and the grain boundary area was almost covered by the reaction products. In all the investigated samples, the grain boundary phases appeared to be well bonded with the matrix phase, that is, HA/TCP. Also, the crystals of grain boundary phases were much smaller (less than 300 nm) compared to that of the matrix phase.

Cell adhesion. Figure 3 shows the cell adhesion behavior of BCP20M (Figure 3(a) and (b)), BCP30M (Figure 3(c) and (d)), and control (Figure 3(e) and (f)) samples, after 1 d of culture. Here, the cell seeding density was 5 105 cells/mL. In all cases, cell division, proliferation and cell–cell contacts were evident. Clearly, as far as the cell adhesion was concerned, various composites, despite compositional difference/phase assemblage, exhibited comparable behavior. Also, the cell adhesion for investigated ceramics was comparable with that of control sample. Beside major observation of cell spreading, a transient signal of cell migration was also observed through structural changes in the form of cell protrusion. More detailed observations of cell–material interaction are made with SEM and AFM. Figure 4 shows the results of similar cell adhesion experiments, but the


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Table 1. The starting powder composition and sintering conditions are mentioned as well as the sample designation of each sample used in the present investigation. Sample designation

Starting composition

Pressureless sintering conditions

HA BCP10M

Pure HA HA with 10 wt% mullite

1200 C, 2 h 1350 C, 2 h

BCP20M

HA with 20 wt% mullite

1350 C, 2 h

BCP30M

HA with 30 wt% mullite

1350 C, 2 h

Phase assemblages (after sintering) HA-ss a-TCP-ss, HA-ss, b-TCP-w, mullite-ww, CaO-ww a-TCP-s, b-TCP-ss, HA-ww, mullite-w, gehlenite-ww, CaO-ww, alumina-ww b-TCP-ss, HA-ww, mullite-s, gehleniteww, CaO-ww, alumina-ww

The phases present in the sintered ceramics are summarized based on the XRD peak intensities. Ss: very strong, s: strong, ww: very weak, w: weak.

longer distance than that was observed in the previous case (when cell seeding density was 5 105/mL) on the materials surface. Figure 4(i) shows an AFM image of a filapodia attached on pure HA sample. The branching of filapodia is clearly visible. This type of branching provided good anchorage with the material surface.

MTT assay. It is known that MTT reagent directly

Figure 1. Representative microstructure of thermally etched BCP30M ceramic. Note the uniform presence of grain boundary phases around each grain.

culture time was more (3 d) and the cell seeding density was approximately 1 105/mL. SEM images reveal adhesion of L929 cells on BCP10M (Figure 4(c) and (d)), BCP20M (Figure 4(e) and (f)), BCP30M (Figure 4(g) and (h)) samples. The cell adhesion on baseline HA sample can be seen in Figure 4(a) and (b). From the images, some interesting observations can be made, for example, Figure 4(e) reveals how the cells were connected to each other by filapodia extension on BCP20M sample. In this case, some observable differences could be found, when the cell seeding density was lower. The cells were larger in size with an approximate dimension of 40–50 mm. The major difference could be found in cell morphology. In case of cell density being 1 105/mL, most of the cells were spread on the surface and intended to enhance the cell-material contacts. The filopodia of the cells were extended over

reacts with the mitochondria (mitochondrial dehydrogenase) of living cells. Therefore, the reduction of MTT will be more if more numbers of metabolically active cells are present. In this context, MTT is widely regarded as one of the quantitative assays to determine the cytotoxicity of the materials, detecting the cell viability on the sample surface. The measured optical density, as recorded with ELISA plate reader, is directly proportional to the number of viable cells in the culture medium. Figures 5(a) and (b) plot the MTT assay results obtained using MG63 and L929 cells, respectively. In both the plots, the results are compared in reference to pure HA. Figure 5(a) shows that in all the mullite-containing composites, the numbers of metabolically active cells were comparable to pure HA. BCP10M, BCP20M, and BCP30M possessed 103%, 113%, and 101% cell viability, respectively, compared to pure HA. Similar to these results, Figure 5(b) shows the result of MTT assay using L929 cells. Here also, the result obtained with HA was considered as baseline observation (control). Again, the MTT reduction rate data revealed that both BCP10M and BCP20M, in average, possessed similar or slightly better cell viability than pure HA. Owing to the fact that error bars overlap among various MTT datasets, no statistical significance could be confirmed in terms of cell viability.

ALP activities. ALP activity is considered as a phenotypic marker of differentiated cell. The ALP produced by metabolically active MG63 cells, after 3 and 7 d of


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Figure 2. AFM images showing the surface topography of the various investigated materials: (a) pure HA sintered at 1200 C for 2 h and the composites: (b) BCP10M, (c) BCP20M, and (d) BCP30M samples, all sintered at 1350 C for 2 h. GB represents ‘grain boundary.’

experiments, is quantitatively plotted in Figure 6. After 3 d of culture, the maximum ALP activity was measured with pure HA and for composites (10–30 wt% mullite) the ALP activity was very near to pure HAp. After 7 d of culture, the ALP expression of the cultured cells was significantly higher in all mullite-containing composite with respect to pure HA. However, almost no difference in terms of ALP expression was found among various mullite-containing composites.

Osteocalcin. OC is a later stage marker of bone cell differentiation. Declercq et al.26 described calcification

as a predictor of bone mineralization capacity of biomaterials in osteoblastic cell cultures. It is normally accepted that the OC production measurement is important to substantiate the use of investigated ceramics as bone replacement materials. As part of the present study, the results of the OC assay, are plotted in Figure 7. The OC production after 7 d of culture on control, pure HA, and BCP10M showed significantly lower values in comparison with BCP20M and BCP30M samples. However, no difference in OC production could be noticed between BCP20M and BCP30M samples.


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Figure 3. Selected SEM images illustrating the adhesion of L 929 cells on various material surfaces – BCP20M (a, b), BCP30 M (c, d), and negative control sample (e, f) after in vitro culture for 1 d.


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Figure 4. SEM images of L929 cells adhered on pure HA (a, b), BCP10M (c, d), BCP20M (e, f), and BCP30M samples (g, h). AFM image revealing extensive filopodia extension on pure HA sample is also shown (i). Results were obtained after 3 d of culture. The seeded cell density was 1 105 cells/mL.

Discussion This study demonstrates that the biological response of BCP (HA and TCP) ceramics containing 20 and 30 wt% mullite is comparable (as per cell adhesion, MTT) or better (ALP and OC results) than pure HA. It is indeed an important result that the presence of mullite did not affect the cellular viability property on BCP-mullite composites, when compared to sintered HA monolith. In the following, results will be interpreted in terms of the substrate compositional differences or phase assemblage. Such interpretation will be helpful to realize the following aspects: (a) In vitro cytocompatibility property on the basis of microstructural

phase assemblages; (b) prediction of osteoconduction property based on biocompatibility with MG 63 cell line; and (c) bone-cell functionality and phenotypic marker expression based on ALP and OC expression assay.

Materials and microstructure effect on cytocompatibility. In recent years, the mixture of HA and TCP ceramics has been reported to be better than the monolithic forms of the either (HA or TCP) due to the controlled resorbability of BCP ceramics. In an interesting work, Arinzeh et al.27 studied the bone formation capabilities of BCP ceramics using human


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3.00

Days of culture 3.00 7.00

120 2.50

110 Mean optical density (405 nm)

Metabolically active cells (% Pure HA)

(a) 130

100 90 80 70 60

2.00

1.50

1.00

50 BCP10M

BCP20M

BCP30M

0.50

0.00

120

Pure HA

110

BCP10M BCP20M Samples

BCP30M

Figure 6. ALP activity of MG63 cells on pure HA, BCP10M, BCP20M, and BCP30M ceramics, after 3 and 7 d of culture in osteogenic medium.

100 90 80

60

70 60

50

50 BCP10M

BCP20M Samples

BCP30M

Figure 5. MTT assay results showing the relative number of metabolically active (a) MG63 cells (b) L929 adhered on pure HA, BCP10M, BCP20M, and BCP30M samples. In (a) and (b), the results were compared with pure HA. The results are shown after 2 d of culture.

Osteocalcin (ng/mL)

Metabolically active cells (% Pure HA)

(b) 130

40 30 20 10

mesenchymal stem cells. The results revealed that HA : TCP ratio of 20:80 was the optimum combination for better bone formation, whereas pure HA and TCP phase had much less effect on bone formation. A closer look at Table 1 further reveals that while aTCP dominated in BCP10M material, b-TCP was the major phase in BCP20M and BCP30M composites. The additional presence of CaO, Al2O3, and gehlenite, all in minor amounts (much weaker X-ray peak intensity), was also noticed. From the examples mentioned in introduction part, it is quite clear that pure HA and TCP are undoubtedly biocompatible material. However, the presence of other calcium-alumino-silicate phases may also have an influence on the biocompatibility of this material. In some earlier reported results,28,29 it was mentioned that the biocompatibility of the materials mainly depended on the leaching of

0 Control

Pure HA

BCP10M Samples

BCP20M

BCP30M

Figure 7. OC expression of cultured MG63 cells on various ceramics samples after 7 d of culture. The results were compared with control solution supplied with the kit.

ions. The adhered cells mainly proliferate and differentiate due to the activation of surface ions. In the present case, the possible leaching ions would be Ca, P, Si, and Al. The effect of Ca and P ions need not be discussed here, as these are already known marker for the biomineralization.30 The effects of other ions, therefore, need to be discussed with cited literature. For example, silicon-containing materials were already investigated as silicon-substituted HA, Si3N4, etc. Thain et al.31


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described the in vitro biocompatibility of silicon-substituted HA thin film and reported that the presence of Si did not induce any toxic effect. In another study, Kue et al.32 showed enhanced cell proliferation and OC production by human osteoblast-like MG63 cells on silicon nitride ceramic discs. Their results revealed that Si3N4 was a nontoxic biocompatible ceramic, which could be used as a potential biomaterial. This proves that the presence of Si in the composite, should not ideally degrade the biocompatibility. As far as the Alleaching is concerned, a study by Ku et al. on Ti-6Al4 V alloy suggested that the release kinetics of Al-ions could play a major role in influencing the osteoblast behavior.28 Figure 1 shows some residual porosity mainly at intragranular and few at intergranular regions. In general, porosity degrades mechanical properties, but microporosity has specific advantages during the initial period of implantation. It should be mentioned here that Hornez et al.33 reported that both mesoporosity (10–50 mm) and microporosity (1–10 mm) in HA essentially stimulated significant cell growth of MC3T3-E1 osteoblast cells. The cell viability and cell functions were better for microporous samples. The presence of micropores would therefore allow better anchorage with bone-forming cells and thus improved the mechanical attachment of these materials during initial period of implantation.34 Another parameter that is important in the context of cell adhesion was the surface roughness. In the present case, any minor difference in cell viability could be attributed to difference in surface roughness. The subgrains are clearly visible in the AFM images, while those are not distinct in SEM image (Figure 1). From Figure 2, it should also be clear that the presence of large fraction of grain boundary phase increased the local surface roughness at the nanoscale and this should enhance the cell adhesion property in mullitecontaining BCP composites. In addition, efforts were made to study the adhesion and expansion behavior of single cell in isolation and in contact with other cells on individual material surface. This is important to know how a single cell expands or attaches to material surface. The initial attachment of cells is mediated by integrins, which induces dramatic cytoskeletal changes leading to cell spreading, development of focal adhesion complexes, cell migration, etc. In fact, these are morphological signs, which are usually described as overall cell morphology changes when a cell recognizes the adsorbed adhesive proteins (and their conformation) on a given material surface. The cell adhesion and expansion of single cell on various material surfaces can be seen in Figure 4(b, d, f, h). Among various mammalian cell types, the fibroblast is one of the least-differentiated cell lines. When

fibroblast cells were seeded at lower density, it showed higher motility on the material surface. One single cell can migrate and construct cell–cell interaction with a recognizable polarity of movement.35 AFM images, presented in Figure 4(i), reveal the evidences of cell migration via the extension of cell filopodium. On the other hand, lamellipodia extends the movement toward the direction of cell travel and at the same time adhere on the materials surface.35 When the cell density was low, the cell–cell interaction was also lower and, hence, the cells interact more with material surface by spreading themselves on the surface (Figure 4(e)). The unidirectional long filopodia extension from the adhered cells often formed well-developed ‘Y’ or star junction, as can be seen in Figure 4(e). In such a situation, cells were suitable to attach to the substrate and grow in size as a part of the cell cycle in presence of some growth factors.35 In contrast, high cell density inhibits the growth of normal cells. In a cell crowding surface, cell growth is inhibited by cell-to-cell contact and as a result, it reduces spreading. The above discussion corroborates well with the difference in cell size and cell spreading, when cell seeding density was varied (Figures 3 and 4). The attachment of substrate-dependent cells, such as fibroblasts and osteoblast, to a substratum (e.g. biomaterial) is a synchronized process involving cytoskeleton reorganization, cell spreading, and formation of focal contact.36,37 The cell adhesion behavior of pure HA and composite samples showed presence of cytoplasmic extension, filopodia (Figure 4(a-h)). From Figure 4(a)– (h), it can be said qualitatively that the filopodia was more visible in case of pure HA (Figure 4(a) and (b)), BCP20M (Figure 4(e) and (f)), and BCP30M (Figure 4(g) and (h)) samples. However, the filopodia was less visible and the cells surface had minimum adhesion on BCP10M sample (Figure 4(c) and (d)). BCP20M and BCP30M samples mainly contained b-TCP (Table 1), whereas BCP10M mainly contained a-TCP. Therefore, the difference in cytocompatibility behavior could be attributed to the difference in phase assemblage.

Osteoconduction and biochemical markers of bone turnover. As mentioned earlier, the aspects of bone cell differentiation and functionality could be explained on the basis of ALP and OC assay results. The synthesis of these biochemical markers of bone cell increases with the increasing expression of osteoblasts and decreases with the maturation of osteoblasts.38 It is well known that OC and ALP are the phenotypic markers of the late and early stages of differentiation of osteoblast-like cells, respectively. An increased specific activity of ALP in a bone cell essentially reflects a shift toward a more differentiated state.


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Recalling ALP results after 7 d, it is clear that all the mullite-containing composites had comparable expression of early stage osteoblast differentiation marker, which was much higher than baseline HA. This means that osteoblast-like cells were in a better functionally differentiated state in contact with the BCPmullite composites. Furthermore, the extent of OC expression in case of BCP20M and BCP30M was much higher than both baseline HA and BCP10M. The above observations therefore confirmed that BCP20M composite could exhibit the best combination of OC and ALP expression. Our results need to be explained in the perspective of earlier literature reports. In several earlier research reports, it was mentioned that among CaP-based materials, b-TCP containing composites showed better differentiation and in vivo bone formation/mineralization capability. For example, Shiratori et al.39 studied the bone-forming ability of b-TCP, when implanted in bone defects of rat femur. Their results revealed that b-TCP was an appropriate material for the treatment of bone defects. In another study, Matsuno et al.40 compared the in vitro (ALP) as well as in vivo behavior of bTCP/collagen sponge composite with only collagen sponge (CS). In the in vitro experiments, human mesenchymal stem cell lines were used and for in vivo experiment, the samples were placed under the back skin of nude mice for a time period of up to 12 weeks. Their results revealed that the composite containing b-TCP showed better osteogenic properties and had ability to promote bone formation. In an interesting study, Arinzeh et al.27 tried to optimize the optimum HA/bTCP ratio for better stem cell–induced bone formation. For this purpose, they selected various ratios of HA : bTCP, that is, 100:1, 76:24, 63:37, 56:44, 20:80, and 1:100. In both in vitro (OC) and in vivo experiments (mouse), it was revealed that HA: b-TCP ratio of 20:80 was the best combination for new bone formation in bone remodeling process. It can be further noted that this combination showed better results compared to monolithic form of HA and b-TCP. In fact, all other combination of HA and b-TCP showed improved results compared to pure HA and b-TCP. At the molecular level, HA phase provided the required binding sites, whereas TCP, due to its higher dissolution properties, increased the concentration of Ca and P locally. All these led to the cascading of differential gene expression and positively increased the cell differentiation.41,42 In the present case, Table 1 clearly shows that both BCP20M and BCP30M predominantly contained bTCP phase, while BCP10M contained more a-TCP phase. On the basis of the above information, it should therefore be clear that in view of the dominant presence of b-TCP, both BCP20M and BCP30M exhibited better expression of osteblastic phenotypic marker

than BCP10M. The present investigation also reconfirmed that single-phase HA had inferior expression of differentiated bone cell than BCP microstructure containing varying ratio of b-TCP. Additionally, it can be stated that the additional presence of gehlenite, alumina, or CaO did not have any inhibitory effect as far as the osteoconduction property of BCP20M and BCP30M was concerned. In addition to the differences in phase assemblage, the difference in surface roughness at nanoscale due to uniform presence of grain boundary phase could also explain the observed difference in osteogenic property in the present case. In summary, it is clearly understood from MTT data and cell adhesion tests that HA and BCP-mullite substrates supported comparable number of viable cells. This, in combination with all the above observations, suggests that BCP-mullite biocomposites should be used as a substrate for bone-forming cells.

Conclusions a. The principal finding of the present study is that the pressureless sintered BCP-mullite composites favorably supported cell adhesion of L929 cells in vitro. Also, such observations were independent of mullite content (up to 30 wt%). The morphology and motility of adhered cells was dependent on cell seeding density. b. As far as the quantification of the metabolically active cells is concerned (cell viability), MTT assay results with fibroblast and osteoblast-like cells did not reveal any statistically significant difference in BCP-mullite composites in comparison with pure HA. This confirmed that mullite and additional presence of various phases (alumina, CaO, gehlenite) did not have any toxic effect in vitro and the presence of mullite did not degrade cell viability with respect to pure HA. c. The combination of ALP activity and OC expression results indicates that BCP-based composite substrates with 20% or 30% mullite supported superior osteoconduction than baseline monolithic HA ceramic. The presence of predominant b-TCP phase was found to be suitable for osteoblastic function and phenotypic expression. The presence of additional phases, like gehlenite or alumina, was not found to have any inhibitory effect in vitro. Based on all the in vitro analysis and observations, in combination, BCP-biocomposites containing 20% or 30% mullite can be considered as suitable substrate for bone cell adhesion and proliferation. d. AFM study in combination with SEM analysis revealed the uniform presence of grain boundary reaction phases, which increased the surface


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Journal of Biomaterials Applications 0(0) roughness at the nanoscale. The increased surface roughness property could explain better cell adhesion/proliferation property of BCP-mullite composites, compared to single-phase HA.

Acknowledgements The authors wish to thank Department of Biotechnology (DBT), Government of India, for the financial help. Thanks to Dr Lakshmi Nair of CCMB, Hyderabad, India, for providing us L929 cells. Thanks are also due to Dr S. Ganesh and S. Mittal of BSBE Department, IIT Kanpur, India, for their help during initial period of cell culture experiments.

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