Mesenchymal cell response to nanosized biphasic calcium phosphate composites

Colloids and Surfaces B: Biointerfaces 73 (2009) 146–151

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Mesenchymal cell response to nanosized biphasic calcium phosphate composites Avijit Kumar Guha a , Shashi Singh b , R. Kumaresan b , Suprabha Nayar a , Arvind Sinha a,∗ a b

National Metallurgical Laboratory, CSIR, Jamshedpur 831 007, India Centre for Cellular and Molecular Biology, CSIR, Hyderabad 500 007, India

a r t i c l e

i n f o

Article history: Received 19 February 2009 Received in revised form 27 April 2009 Accepted 1 May 2009 Available online 18 May 2009 Keywords: Nanocomposites Biphasic calcium phosphate Biomimetic Mesenchymal stem cells

a b s t r a c t Biphasic calcium phosphate nanoparticles comprising both hydroxyapatite (HA) and ␤ polymorph of tricalcium phosphate (␤-TCP) have been synthesized together by a polymer matrix mediated process. The process, based on in situ mineralization of poly (vinyl alcohol), exerts a good control over the morphological features of biphasic nanoparticles. By controlling the reaction chemistry (Ca:P ratios), nanobioceramic particles having three different HA/␤-TCP ratios of 50:50, 55:45 and 60:40 respectively. As the two constituents of biphasic system (HA and ␤-TCP) facilitate series of signaling cascades in osteoblast division and differentiation, the adhesion and differentiation properties of mesenchymal cells (MSCs) derived from bone marrow has been studied. © 2009 Elsevier B.V. All rights reserved.

1. Introduction In the field of bone tissue engineering, HA and ␤-TCP are the two most extensively researched biomaterials in the entire family of calcium phosphates. HA, the inorganic mineral of natural bone is osteo-conductive but has a slow resorption rate in the physiological environment while ␤-TCP has an optimum resorption rate but poor mechanical stability [1]. Hence, selecting one of them has not been a successful approach for bone tissue engineering. On the other hand, the complimentary properties of the two bioceramics, have inspired the researchers to develop a biphasic (HA/␤-TCP) system, equipped with mechanical stability and time dependent resorbability, suitable for bioactive scaffold applications in tissue engineering [2–7]. Biocompatibility of bioceramics is determined by the adherence and spreading of osteoblast (bone forming) and osteoclast (bone resorbing) cells on the surface of the scaffold as well as in the bulk [8]. Biphasic scaffolds are required to possess high surface area and three-dimensional hierarchical porosity that promotes cell attachment, proliferation and differentiation [9]. Though there are many different individual approaches for HA and ␤-TCP synthesis, very few established methods exits for biphasic nano-composite synthesis [10–18]. Initially, biphasic HA/␤-TCP powders were commonly produced as by-products of pure HA or pure TCP phase. However, to meet the demand of biphasic powder

∗ Corresponding author at: National Metallurgical Laboratory, Jamshedpur 831 007, India. E-mail address: [email protected] (A. Sinha). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.05.009

with variable HA/␤-TCP ratios, it is prepared either by mechanical mixing of HA and TCP powders or by calcination of Ca deficient HA (Ca/P = 1.5–1.6) at high temperature [19–23]. A high temperature process, generally leads to densification as well as grain growth, causing reduction in surface area and loss of porosity. Recently we have demonstrated that a polymer micro-hydrogel mediated synthesis of HA nanoparticles and its subsequent sintering at high temperature keeps the porosity and nanostructure intact [24]. The concept of matrix mediated synthesis of self-assembled nanoparticles has been derived from Nature’s process of biomineralization and known as Biomimetics [25,26]. Our group has already established this approach for the synthesis of different types of nanostructures such as polymer–HA nanocomposites functionally important inorganic nanomaterials [27–31]. In the present communication, we report here a polymer matrix mediated synthesis of calcium deficient hydroxyapatite with variable Ca/P ratio 1.62, 1.60 and 1.58 respectively. Decomposition at high temperature of the above Ca P variants directly yields macroporous aggregates of biphasic nanoparticles exhibiting three different HA/␤-TCP ratios of 60:40, 55:45 and 50:50 respectively. We have observed cell differentiation using MSC cells derived from bone marrow also reported by Cai et al. [32]. 2. Experimental procedure Reaction of Ca(NO3 )2 and (NH4 )2 HPO4 at an optimum pH is known to produce Ca deficient HA (Ca10−x (HPO4 )x (PO4 )6−x (OH)2−x ) provided 1.5 < Ca/P < 1.67. Calcination of calcium deficient HA at a temperature of 900 ◦ C leads to the simultaneous synthesis

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of HA and ␤-TCP as per the following reaction [33]:

5. Results and discussion

Ca10−x (HPO4 )x (PO4 )6−x (OH)2−x

XRD patterns of Ca deficient HA with Ca/P molar ratios of P1—1.62, P-2—1.60 and P-3—1.58 is shown in (Fig. 1a). It was observed that the diffraction patterns of the three Ca deficient HA are essentially indistinguishable from each other. All major diffraction peaks, very close to stoichiometric HA were obtained in all the three powdered samples. Obtained diffraction peaks, could be identified (0 0 2), (1 0 2), (2 1 1), (3 0 0), (2 0 2), (3 1 0), (1 1 3), (2 2 2), (2 1 3), (4 1 1), (1 0 4), (5 0 2) and (5 1 0) peaks of HA. On the other hand, XRD patterns of calcined samples established the co-existence of two crystalline phases namely, HA and ␤ TCP. Obtained diffraction peaks could be indexed as (2 0 0), (0 0 2), (2 1 1), (3 0 0), (2 0 3), (2 2 2), (2 1 3), (4 1 1), (1 0 4), (3 2 2) and (3 1 3) peaks of HA and (2 1 4), (2 1 0), (2 1 10), (2 2 6), (1 0 16), (3 0 12), (2 3 8), and (2 0 20) peaks of ␤-TCP (Fig. 1b). The XRD peak analysis based on full width at half maxima (FWHM), using Scherrer formula, yielded an average crystallite size of 20 nm for uncalcined Ca-deficient HA samples, while it was 40 nm for calcined biphasic samples. Volume fractions of two phases in biphasic samples were determined by calculating area under the peaks of the respective phases. Calculations yielded 50%, 55% and 60% HA and the remaining ␤-TCP in the samples, Ca/P molar ratios being 1.58, 1.60 and 1.62 respectively. TEM images of the calcined samples confirmed the formation of nanosized crystalline bioceramic particles of uniform shape and size range arranged in microporous aggregates (Fig. 2a–c). Samples P-1, P-2 and P-3 exhibits average size of ∼21 nm, ∼23 nm and ∼32 nm biphasic nanoparticles respectively. A closer look into TEM

→ (1 − x)Ca10 (PO4 )6 (OH)2 + 3xCa3 (PO4 )2 + xH2 O In our study, we have used the above chemistry in presence of PVA microhydrogel. A systematic variation in Ca/P molar ratio (1.62, 1.60 and 1.58) has been optimized to produce biphasic nanopowders of desired composition. 3. Synthesis of biphasic nanoparticles PVA mediated synthesis of CaP bioceramic nanoparticles carried out to produce three different biphasic compositions having HA/␤TCP ratios as described above. Calcium nitrate (Ca(NO3 )2 ·4H2 O) and di-ammonium hydrogen phosphate ((NH4 )2 HPO4 ) (both from Merck, India) taken as Ca- and P-source respectively. 300 ml of 0.4 M alkaline calcium salt solution (pH > 9.50) mixed with equal volume of aqueous solution of PVA (molecular weight 1, 25,000 and degree of hydrolysis 85–89%, from Qualigens, India) containing 3.5 g polymer. System incubated for 24 h at 30 ◦ C. Stoichiometric volume (356 ml) of 0.302 M alkaline di-ammonium salt solution (pH > 9.50) added to the above and mixed thoroughly. The entire mixture aged for one week at 30 ◦ C, the precipitate obtained washed several times with de-ionized water until pH dropped to seven and oven dried at 80 ◦ C. The powder calcined in a muffle furnace in air at a temperature 900 ◦ C for 2 h. Samples synthesized with Ca/P molar ratio of 1.62, 1.60 and 1.58 designated as P-1, P-2 and P-3 respectively. Experiments repeated three times to ensure the reproducibility of the phase formation. 4. Materials characterization To identify the phase of the synthesized materials, X-ray diffraction (XRD) was carried out using (Siemens, D500) using Cu K␣ radiation at 30 kV and 25 mA scanned for diffraction angles: 2—20–80◦ , at room temperature. To determine average crystallite size, full width at half maxima (FWHM) were calculated according to Scherrer’s equation with the shape factor K (0.9). Relative volume fractions of different crystalline phases formed were determined by the area under the diffraction peaks of the respective phases. Size and shape of biphasic nanoparticles have been characterized using transmission electron microscopy (TEM, CM 200, CX Philips at 160 kV JEOL 2100), the samples were ultrasonically dispersed in double distilled water and then one drop mounted on carbon coated copper grids. A transformation from Ca deficient HA to ␤-TCP was also ensured by Fourier transform spectroscopy (FTIR-410 JASCO). The calcium phosphate molar ratio was analyzed by inductively coupled plasma-optical emission spectrometry analysis (ICP-OES). 4.1.1. Cell adhesion studies Biphasic nanocomposites in powder form sterilized using UV and washed in Hanks’ balanced salt solution (HBSS). A suspension made in HBSS and applied as a film on cover slips coated with and without collagen, and air-dried over night. Cells were plated on these coverslips and followed for 7–10 days. Cell adherence and growth monitored everyday and at the end of 10 days, cover slips fixed with 2% glutaraldehyde. Briefly, after fixation and dehydration, samples dried in Critical Point drier (Polaron) and sputter-coated with gold in sputter coater (Quorum Technologies, UK) and examined in Scanning electron microscope (Hitachi SEM). Some samples fixed in formaldehyde for 10 min and stained for osteogenic markers and monitored for alkaline phosphatase activity using Alk. Phosphatase staining kit (Chemicon).

Fig. 1. (a) X-ray diffraction patterns of Ca-deficient HA with different Ca–P molar ratios P-1 (Ca/P = 1.62), P-2 (Ca/P = 1.60), P-3 (Ca/P = 1.58). (b) X-ray diffraction patterns of heat treated biphasic HA/TCP with different Ca–P molar ratios.

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Fig. 3. FTIR spectra of biphasic samples containing different Ca–P molar ratios.

P-2-1, 57(AU), P-3-0.94(AU) as a function of decreasing Ca/P ratio or in other terms decrease in HA/␤-TCP ratio, Absorbance band at 470 cm−1 is assigned to doubly degenerate ␯2 O–P–O bending moment, bands at 564.72 cm−1 and 606.10 cm−1 are due to PO4 3− vibration modes, 628.69 cm−1 corresponds to stretching and vibration bands of –OH group, band at 874.96 cm−1 is due to P–O(H) stretching in HPO4 −2 group. Similarly, absorbance at 960.23 cm−1 is due to 1 non-degenerate P–O symmetry stretching mode, 1041.11 cm−1 and 1097.11 cm−1 signify triply degenerate 3 anti-symmetric vibration modes and band at 1632 cm−1 correspond to –OH stretching [34–37]. Stoichiometric ratio of Ca and P in all the three samples was confirmed by ICP-OES analysis. Osteoblast commitment and differentiation are controlled by complex activities involving signal transduction and transcriptional regulation of gene expression, which can be triggered as a result of matrix, the surface chemistry of the bone mineral composite (HA/␤-TCP). We have used the mesenchymal stem cells from bone marrow in the study to show their adherence to the nanocomposites developed and their ability to differentiate into osteogenic cell types. Cells were grown in presence of the nanostructured biphasic nanocomposites in the presence and absence of collagen. It is the property of MSC cells to adhere to glass and plastic surfaces without any problem, therefore growth in the blank serves as a control. Also, since we do not have any interference, cells will grow and die a nat-

Fig. 2. (a) TEM micrograph of biphasic sample P-1. (b) TEM micrograph of biphasic sample P-2. (c) TEM micrograph of biphasic sample P-3.

images indicates the formation of neck like structures suggesting the possibility of sintering of nanosized particles forming a three dimensional porous structure with pore’s dimensions in the nano range (Fig. 2a). All the three compositions of biphasic nanoparticles with different HA/␤-TCP ratio exhibited almost similar FTIR spectrum (Fig. 3), except the fact that absorbance at 3444 cm−1 corresponding to hydroxyl groups due to presence of HA as well as moisture showed a systematic reduction in the absorbance intensity i.e., P-1-1-96(AU),

Fig. 4. Cell adhesion and growth of cells in presence of HAP/TCP scaffolds in the ratio of P-1, P-2 and P-3 with and without collagen. 5000 cells of each MSC type were plated and counted at the end of 10 days.

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ural death in the blank but in the others the respective survival rate seems to be dependent on the individual components. Since HA is not easily soluble, the cells find firm anchorage for growth and survive longer as compared to the more soluble ␤-TCP. However, in all P-1, P-2 and P-3, the one with collagen supports more cell growth, approximately about 26,000 cells survived in the blank as compared to about 24,000 in the one with highest HA and 14,200 in the one with the lowest HA (Fig. 4). SEM micrographs of all the three samples with and without collagen are shown in (Fig. 5). Sample having 50% HA and ␤-TCP seems to be best for optimal cell attachment and

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proliferation. Bernards et al. have also shown that the cells have an affinity to bind to HA [38–41]. MSCs cells were stained for the markers of differentiation after harvesting them on the 10th day, for osteogenic lineage using antibodies against osteocalcin and for alkaline phosphatase activity using Alk. Phosphatase kit. MSC cells grown with the three nanocomposite samples expressed differentiation markers like osteocalcin and increased alk phosphatase staining after 10 days (Fig. 6). Biomaterials (both natural and synthetic) like HA, polyanhydrides, polyphosphoesters, polylactic acid, and polygly-

Fig. 5. SEM micrographs of cells growth around the scaffold particles containing different Ca–P molar ratio without collagen (a–c) and with collagen (d–f).

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Fig. 6. Staining of cell grown with Scaffold for Osteocalcin. Control C does not show any osteocalcin, P-1, P-2 and P-3 manifest the presence of osteocalcin which is a marker for osteogenic differentiation.

colic acids have been shown to mimic the extracellular matrix and play a role in conduction of bone and cartilage [42–46]. It has also been previously shown that the phosphate chemistry is responsible for changes in adhesion, proliferation and cell differentiation [31,41,46–48]. Although ␤-TCP plays a lesser role in the initial cell adhesion and survival, it enhances the proliferation and differentiation. It is the surface chemistry of ␤-TCP that is responsible for the above as compared to P-1 (higher HA) in which although more cells survive, they fail to differentiate and proliferate. [32,42].

Acknowledgments Authors wish to acknowledge the financial support received from Council of Scientific and Industrial Research (CSIR) under Network Project on Nanomaterials and Devices for Health Applications (NWP 0035) and Department of Biotechnology (DBT), India to support NML-CCMB collaborative studies on Biomimetic Nanocomposites.

References 6. Conclusions A PVA mediated process has been established to synthesize biphasic nanoparticles, a systematic change in Ca:P ratio varying from 1.58 to 1.62, has resulted in the formation of biphasic nanoparticles with systematic increase in HA varying from 50 wt% to 60 wt%. Mesenchymal stem cells were compatible with the nanocomposite material synthesized. Cells were grown in presence of the nanostructured biphasic particles in the presence and absence of collagen. The nanobioceramic particles P-1 though allowed the mesenchymal cells to adhere and differentiate; proliferation was lesser P-3. ␤-TCP seems to encourage proliferation and an optimal would be a 50:50 HA:␤-TCP ratio.

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