Acta Biomaterialia xxx (2014) xxx–xxx
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Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface Alieh Aminian a, Karoline Pardun a, Eike Volkmann a, Giovanni Li Destri b, Giovanni Marletta c, Laura Treccani a,⇑, Kurosch Rezwan a a b c
Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, 28359 Bremen, Germany ESRF-The European Synchrotron, 71 Avenue des Martyrs, CS 40220, 38043 Grenoble Cedex 9, France Laboratory for Molecular Surfaces and Nanotechnology (LAMSUN), Department of Chemistry, University of Catania and CSGI, V.le A. Doria 6, 95125 Catania, Italy
a r t i c l e
i n f o
Article history: Received 21 July 2014 Received in revised form 8 October 2014 Accepted 4 November 2014 Available online xxxx Keywords: Alumina Alkaline phosphatase Surface functionalization Biomineralization Bioactivity
a b s t r a c t In this study a bioinspired approach to induce self-mineralization of bone-like material on alumina surfaces is presented. The mineralizing enzyme alkaline phosphatase (ALP) is covalently immobilized by a carbodiimide-mediated chemoligation method. The enzymatic activity of immobilized ALP and its mineralization capability are investigated under acellular conditions as well as in the presence of human bone cells. Analytical, biochemical and immunohistochemical characterization show that ALP is efficiently immobilized, retains its activity and can trigger calcium phosphate mineralization on alumina at acellular conditions. In vitro cell tests demonstrate that ALP-functionalized alumina clearly boosts and enhances bone cell mineralization. Our results underpin the great potential of ALP-functionalized alumina for the development of bioactive surfaces for applications such as orthopaedic and dental implants, enabling a fast and firm implant osseointegration. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction The immobilization of specific, active biomolecules on material surfaces (biofunctionalization) is considered as a highly effective strategy to improve the biocompatibility and bioactivity of materials [1–5]. A variety of biological molecules including carbohydrates, extracellular matrix (ECM) proteins or their components, e.g. arginine-glycine-aspartic acid (RGD) peptides and growth factors have been considered to elicit specific cellular responses, increase the interaction between the cells, material surfaces and tissues, and thereby result in integration and tissue regeneration [6–9]. The enzyme alkaline phosphatase (ALP, Enzyme Commission number EC 3.1.3.1) is known to play a central role in hard tissue formation and osteogenesis and is commonly used as marker for assessing maturity of mineralized tissue or cell phenotype in biochemical and histological assays [9–14]. ALP is present in many species, including humans. It is present in the plasma membrane of the cells and in the ECM [15] and its important role in hard tissue formation was postulated more than 80 years ago [16,17]. ALP catalyzes the hydrolysis of organic phosphate monoesters into inorganic phosphates (Pi), increases their local concentration, and directs the nucleation of hydroxyapatite crystals in so-called ⇑ Corresponding author. Tel.: +49 (0)421 218 64938; fax: +49 (0)421 218 64932. E-mail address: [email protected] (L. Treccani).
matrix vesicles [10,13,18,19]. At the same time, ALP decreases the concentration of the crystal growth inhibitors as pyrophosphates (PPi) [10,13,18]. Due to its central role in bone formation some in vitro and in vivo studies have exploited the clinical potential of ALP for ligament and bone regeneration. For instance, ALP deposited on polymer surfaces induces the attachment of collagen fibrils [15] or achieves a higher deposition of calcium phosphate (CaP) on titanium alloys [12,18], as well as on bioactive materials such as bioglasses [11,20–22] and nanofibrous fibrin scaffolds [9]. Nonetheless, due to the differently employed deposition and/or immobilization approaches for ALP, support materials and test conditions, clear evidence of the relationship between immobilized ALP activity and its effect on CaP mineralization with and without cells, and its impact on bone formation is still unclear. In this study the possibility of immobilizing biomolecules such as ALP, which can simultaneously promote cell–surface interactions, cell attachment and boost the mineralization capability and induce self-biomineralization of CaP on inert ceramic surfaces, has been considered. ALP has been immobilized to highly inert surfaces, such as alumina, to catalyze and induce CaP mineralization. Alumina (Al2O3), due to its remarkable mechanical properties, chemical stability and biocompatibility, is widely employed for permanent bone orthopaedic implants. Nonetheless due to its bioinertness, Al2O3 cannot stimulate bone formation and
http://dx.doi.org/10.1016/j.actbio.2014.11.007 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Aminian A et al. Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.007
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osseointegration, which is a fundamental requirement for longterm and overall implant success. Here, a simple and biocompatible immobilization approach based on the zero-length cross-linking system 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (EDC/NHS) was used to stably bind active ALP molecules on dense a-Al2O3 surfaces. Al2O3 surfaces were first silanized with aminopropyltriethoxysilane (APTES) to create surfacereactive conjugation sites for the covalent immobilization of ALP with the EDC/NHS linkers. To see if the immobilization can lead to a substantial loss of ALP activity and CaP formation capability, the specific activity of ALP after immobilization was investigated by enzymatic assays. Immobilized ALP-driven mineralization was assessed by in vitro soaking experiments at acellular conditions and in the presence of human osteoblasts. Additionally, in vitro cell tests were conducted to simultaneously characterize the surface biocompatibility and the effect of immobilized ALP on cell proliferation, differentiation and cellular biomineralization. Mineral formation and deposition was characterized by several analytical techniques, including electron microscopy, energy dispersive spectroscopy and X-ray diffractometry, as well as different biochemical, immunohistochemical and histological methods.
2. Materials and methods 2.1. Materials High-purity a-alumina powder (Taimei, TM-DAR), 99.99%, particle size d50 = 210 ± 15 nm, specific surface areas (BET) = 3.98 g cm2, Lot No. SY301061B) was purchased from Krahn Chemie GmbH (Germany). APTES (Lot o. BCBH2173V, 98%), EDC (Lot No. 080M14582V, 99%), NHS (Lot No. BCBD1270V, 98%), 2-(N-morpholino)ethanesulfonic acid hydrate (MES, Lot No. 051M8424, 99.5%), ammonium hydroxide (25 vol.% in water, Lot No. 1336216), fetal calf serum (FCS, Lot No. 010M3395), p-nitrophenylphosphate (pNPP, Lot No. 091K5315), trypsin/EDTA (0.25% w/v trypsin, 0.02% w/v EDTA, Lot No. SLBH0771), glutaraldehyde (Lot No. 110M5300V), sodium cacodylate trihydrate (Lot No. 110M0056V,P98%), dexamethasone (Lot No. BCBB2722,P98%), L-ascorbic acid (Lot No. 127K0099,P99.0%), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, Lot No. 021M4069V, suitable for cell culture), b-glycerolphosphate (b-GP, Lot No. 128K54204V, 99%), gentamicin (Lot No. 041K12481V), calcium chloride (CaCl22H2O, Lot No. 1448251,P99.0%), Alizarin Red S (Lot No. 069K1639), glycine (Lot No. 118K00181,P99%), magnesium chloride hexahydrate (MgCl26H2O, Lot No. 019K00381V, suitable for cell culture), sodium chloride (NaCl, Lot No. 038K00451), zinc chloride (ZnCl2, Lot No. 119K1539), sodium hydroxide (NaOH, Lot No. BCBC7310V, 98%), ethanol (Lot No. SZBD2130V, absolute 99.8%), phosphate-buffered saline (PBS, Lot No. SLBF5741V), acetic acid (C2H4O2, Lot No. SZBA2030, 99.8%), 4-nitrophenol (pNP, Lot No. 0001369092), hexamethyldisilazane (HMDS, Lot No. SHBC8538V, 99.9%) and Triton™ X-100 (Lot No. MKBL5839V) were supplied by Sigma Aldrich (Germany). Lyophilized ALP (from chicken intestine, Lot No. L12112758, 0.9 units per mg dry weight, at 25 °C, pH 8.8) was obtained from Biomol GmbH (Germany). Dulbecco’s modified Eagle’s medium (DMEM), AlexafluorÒ488phalloidin, AlexafluorÒ546-phalloidin goat anti-mouse IgG and Quant-iT™ PicoGreenÒ dsDNA assay kit (Lot No. P7589) were purchased from Invitrogen (Germany). Polyacrylic acid (molecular weight = 4000 g mol1, SyntranÒ8220, Interpolymer GmbH, Germany), anticollagen I antibody (COL-1, Lot No. 041M4784, Acris, Germany), antibiotic–antimycotic (Life Technology, Germany), 40 ,6-diamidino-2-phenylindole (DAPI, Lot No. 1242642, Fluka, Germany), Alizarin Red S solution (ARS,
pH 4.1–4.3, Sigma, St Louis, MO, USA), WST-1 cell proliferation assay (WST-1, Roche Diagnostics GmbH, Mannheim, Germany), FluitestÒ CA-CPC (AnalyticonÒ Biotechnologies AG, Germany), paraformaldehyde (PFA, Lot No. 53260, Riedel-de Haën) and hydrochloric acid (HCl, Lot No. 12G180525, 37%, VWR) were obtained from different suppliers as specified. Double-deionized water (ddH2O) with an electrical resistance of 18 MX (Synergy, Millipore, Germany) was used for all experiments. 2.2. Alumina discs Planar alumina discs were fabricated using a modified micromoulding method described in Holthaus et al. [23]. Alumina aqueous suspensions (solid content of 20 vol.%) were obtained by mixing 19.9 g particles with 20 ml ddH2O. Polyacrylic acid (1.2 vol.%) was used as binder. The pH was adjusted to 10–11 with 2 vol.% ammonium hydroxide for electrosteric stabilization and to prevent particle agglomeration. Suspensions were sonicated for 10 min with an ultrasonic horn using an output of 150 W and pulse rate 0.5 s (Sonifier 450, Branson, USA) to break agglomerates after stirring. 1 ml suspension was placed into cylindrical moulds with a diameter of 15 mm (24 non-treated well plates, Nalge NUNC, Rochester, NY), previously greased with a thin layer of vaseline. Subsequently, the discs were dried for 5 days at 25 °C and 30% relative air humidity to prevent crack formation. After demoulding, the alumina discs were sintered at 1500 °C for 2 h (LHT08/17, Nabertherm GmbH, Lilienthal, Germany). Sintered alumina discs had a final diameter of 13.42 ± 0.17 mm and height of 2.0 ± 0.21 mm. Before ALP immobilization, samples were sterilized by autoclaving. Silanization and ALP immobilization were carefully conducted under sterile conditions and using sterile solutions to avoid any possible contamination. 2.3. ALP immobilization on alumina discs ALP was covalently immobilized on alumina discs using the coupling cross-linkers EDC/NHS and using a protocol adapted from Kroll et al. [24]. Prior to immobilization, alumina discs were autoclaved (Systec 2540, Germany) and silanized with APTES to generate the surface functional amino groups necessary to bind ALP through the EDC/NHS coupling reaction. Silanization was carried out in aqueous solutions as described as described in Refs. [24,25]. Briefly, autoclaved alumina discs were immersed in a 8.5 mM APTES solution (2 vol.%) for 1 h at room temperature (RT), then cured for an additional 1 h at 70 °C. Afterwards, the samples were washed 10 times with ddH2O to remove unreacted APTES and dried for 10 h at 70 °C. APTES-silanized alumina discs were immersed in a solution of 10 mg ml1 EDC, 0.6 mg ml1 NHS, 1 mg ml1 ALP dissolved in MES buffer (0.1 M, pH 6). For optimizing the ALP immobilization procedure, incubation was carried out at three different temperatures (4, 25 and 37 °C) for 2 h. Afterwards alumina discs were rinsed 10 times with ddH2O to remove unbound ALP. Alumina discs with immobilized ALP are named ASNEA. Nontreated (A), silanized (AS) and alumina discs functionalized with APTES, EDC/NHS but without ALP (ASNE) were used as reference (Fig. 1a). 2.4. ALP activity assay The enzymatic activity of immobilized ALP was determined using pNPP as an enzymatic substrate as described in Ref. [26]. Alumina discs functionalized with ALP (ASNEA in Fig. 1a) were incubated in a 1 ml ALP substrate buffer solution consisting of 6 mM pNPP dissolved in 90 mM glycine buffer (0.1 mM glycine, 1 mM
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Fig. 1. (a) Functionalization scheme: silanization and covalent binding of ALP on alumina substrates with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). (b) Enzymatic activity of ALP immobilized on alumina discs (ASNEA) measured by pNPP assay immediately after immobilization at three different temperatures (4, 25 and 37 °C). Unfunctionalized alumina discs (A) were used as reference.
MgCl2, 1 mM ZnCl2 at pH 10) at 37 °C. After 30 min the reaction was stopped by addition of 1 ml of 1 M NaOH. The increase in the absorbance of p-nitrophenol (pNP) generated by the hydrolysis of pNPP by ALP was spectrometrically measured at 405 nm with a plate reader (Chameleon V, Hidex, Germany). Serial dilutions of ALP (from 50 ng ml1 = 0.05 mU to 1 lg ml1 = 1 mU) were used to generate standard curves. Five independent experimental runs were performed using six alumina discs for each type of sample. 2.5. ALP mineralization activity under acellular conditions (CCM soaking test) The activity and mineralization capability of immobilized ALP were initially evaluated at acellular conditions by in vitro soaking experiments as described in Ref. [18]. All experimental substrates were immersed in cell culture medium (CCM) consisting of DMEM, 50 lg ml1 L-ascorbic acid, 10 nM dexamethasone, 50 lg ml1 gentamicin, 10 mM b-GP and 1.5 mM CaCl2, for 1, 4, 7, 10, 14, 21 and 28 days under constant conditions (37C, 9.5% CO2 and 93% relative humidity). The medium was refreshed every 2 days. CCM soaking experiments were carried out using six discs for each type of sample. After each incubation time, discs were removed, rinsed thoroughly with ddH2O and dried at 70 °C for 15 h. Mineral formation and surface morphology were analysed by scanning electron microscopy (SEM, Supra 40, Zeiss, Oberkochen, Germany). Samples were sputtered with a thin gold layer (sputter coater 108 Auto, Cressington, USA). Chemical composition and Ca/P ratio were recorded by energy-dispersive X-ray spectroscopy (EDX, Gemini Supra 40, Zeiss, Oberkochen, Germany). 2.6. Immobilized ALP mineralization activity and surface biocompatibility assessment with bone cells In vitro mineralization and biocompatibility were assessed with human osteoblast cells (HOBs, passage 4, Provitro, Germany). Cells were pre-cultured in cell culture medium (DMEM, 10% FCS and 1% antibiotic–antimycotic) in a cell incubator (Labotect GmbH, Germany) at constant conditions (37 °C, 9.5% CO2 and 95% relative humidity). Cell medium was refreshed every 2 days. After 7 days cells were trypsinized with a trypsin/EDTA solution. Alumina discs
were put in 24-well multidish (Nalge NUNC, Rochester, NY) and cells were seeded with a density of 15 103 cells per sample. Cell culture medium was refreshed every 2 days up to day 7 until cells were confluent. Afterwards, an osteoinductive medium (O-CCM) consisting of DMEM, 10% FCS, 1% antibiotic–antimycotic, 10 nM dexametha sone, 5 108 M 1,25(OH)2D3, 50 lg ml1 L-ascorbic acid, 10 mM b-GP and 1.5 mM CaCl2 was used from day 7 to 28. O-CCM was refreshed every 2 days. Three independent experimental runs were performed. 2.6.1. Cell morphology Cell morphology at different time points was evaluated by immunofluorescence microscopy and SEM after 1, 4, 7, 14, 17, 21 and 28 days. Samples were rinsed with PBS buffer and cells were fixed with 4% PFA. Cell nuclei, actin filaments and collagen were stained with DAPI, AlexafluorÒ488-phalloidin and secondary antibody (AlexafluorÒ546-phalloidin goat anti-mouse IgG, Collagen type I), respectively, as described in Refs. [27,28]. Cells were imaged by fluorescence microscopy (Axio Imager M.1, Carl Zeiss GmbH, Germany). For SEM investigation, the samples were rinsed with 1 PBS buffer, fixed with 2.5 vol.% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), dehydrated in a graded series of ethanol (10%, 30%, 50%, 70%, 80%, 90%, 95% and 100%) and dried with HMDS for 1 min. Finally they were sputtered with thin layer of gold for SEM imaging. SEM analysis was performed using two discs for each type of sample. 2.6.2. Cell viability Cell viability was analyzed using a colorimetric WST-1 cell proliferation assay as described in Ref. [27]. After each sampling point, formazan produced by living cells was spectrometrically quantified (optical, OD) at 450 nm with a reference wavelength of 650 nm (Chameleon V, Hidex, Germany). Cell proliferation was measured using six specimens of each type of sample per sampling day. 2.6.3. Cell differentiation The activity of cellular ALP, as a marker for cell differentiation, was measured from cell lysates by determining the production of pNP from pNPP as previously described in Section 2.4.
Please cite this article in press as: Aminian A et al. Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.007
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The cell lysate was prepared as described in Ref. [29]. After each sampling point, cell layers were rinsed with PBS, trypsinized with trypsin/EDTA solution and centrifuged at 20,000g for 10 min to separate the cell pellet from the supernatant. The lysis buffer (1% Triton™ X-100 in 0.9% 0.15 M NaCl) was added to the cell pellets and mixed until a clear solution was obtained. 100 ll of ALP substrate buffer (6 mM pNNP in 0.1 M glycine, 1 mM MgCl2, 0.1 mM ZnCl2) were added to 100 ll of cell lysates and incubated for 30 min at 37 °C. The reaction was stopped with 100 ll of 1 M NaOH and pNP content was determined photometrically at 405 nm. Serial dilutions of pNP (0–100 lM) were used as standard curves to extrapolate ALP activity. ALP measurements were done with six samples of each type of sample per sampling day. 2.6.4. Cell mineralization Cell mineralization at days 10, 14, 17, 21 and 28 was analyzed with an ARS-based staining assay to stain calcium-rich deposits using a protocol adapted from Ref. [30]. Cells were rinsed with PBS, fixed with ice-cold ethanol, kept at 4 °C for 1 h and then incubated in 1% ARS solution for 10 min at RT with gentle shaking. After removing ARS supernatant, the samples were washed with ddH2O, and 800 ll of 10% acetic acid were added to each well. Samples were incubated for 30 min at RT with gentle shaking. The supernatant and cell layers were removed from the discs and pipetted into 2 ml polypropylene cups (EppendorfÒ safe-lock tubes, Hamburg, Germany), briefly vortexed, heated up to 85 °C for 10 min and cooled down in ice for 5 min. Samples were centrifuged at 20,000g for 15 min (Heraeus Megafuge 16R, Thermo Scientific, Germany). 500 ll of supernatant was mixed with 200 ll of 10% ammonium hydroxide and the absorbance was measured at 405 nm with plate reader. ARS content was normalized to the total amount of cellular DNA quantified using the Quant-iT™ PicoGreenÒ dsDNA Kit. An aliquot of the cell lysate was diluted with TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) prior to addition of PicoGreenÒ working solution. Sample fluorescence was measured after an incubation of 5 min with an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a plate reader. The DNA concentration was correlated with the cell number using a calibration curve with a defined cell number. For each experiments six samples for each type of sample, per sampling day were used. After trypsinization, the samples were gently washed with ddH2O, dried for 15 h and prepared for SEM. SEM imaging was performed without gold sputtering. The crystal phases of mineral was determined by grazing incidence X-ray diffraction (GIXRD, Ultima IV type III diffractometer, Rigaku, Tokyo, Japan) equipped with cross-beam optics using a Ka wavelength emitted by a Cu anode. Careful alignment of source and detector with respect to the sample was achieved by using a thin-film attachment with three degrees of freedom. To avoid beam defocusing, the measurements were carried out in parallel beam mode. Divergence of the primary beam was reduced by a 5° Soller slit, while divergence of the diffracted beam was reduced by a 0.5° horizontal Soller slit. The incident angle was kept at 0.5°. The phase composition was determined by Rietveld refinement using the database of the International Centre of Diffraction Data (Newtown Square, PA, USA). PDXL software licensed by Rigaku was employed to minimize the difference between experimental and calculated data. 2.7. Demineralization and calcium assay To quantify the calcium content in CaP mineralized on sample surface after CCM soaking experiments and cell tests, samples were demineralized with 104 N HCl (pH 4) at 37 °C for 48 h.
Calcium content in the supernatant was quantified using o-cresolphthalein complexone (FluitestÒ CA-CPC) as described in Refs. [18,31]. Briefly, 200 ll of the assay reagent was added to 10 ll of supernatant and incubated for 15 min at RT. The absorbance was measured at 570 nm with plate reader and the calcium concentration was calculated using standard curve (serial dilution of CaCl2, 0–200 lg ml1). After demineralization, the enzymatic activity of ALP was measured as described in Section 2.4. 2.8. Statistical analysis All the data were presented as mean value ± standard deviation. The statistical analysis was performed using the statistical software MinitabÒ 16.1.1 (Minitab Inc., State College, PA, USA). Non-functionalized alumina samples were used as reference and comparison was accomplished using one-way analysis of variance (ANOVA) with a post hoc Dunnett’s test. Differences at p < 0.05 were considered to be statistically significant. 3. Results 3.1. Enzymatic activity of immobilized ALP After immobilizing ALP on the samples by EDC and NHS (Fig. 1a) at three different temperatures (4, 25 and 37 °C), ALP enzymatic activity was directly measured using a pNNP-based enzymatic assay. The initial enzymatic activity of immobilized ALP, expressed in mU (1 mU corresponds to 1 lg ml1) was found to decrease by increasing the immobilization temperature (Fig. 1b). The activity was 0.685 ± 0.121 mU at 4 °C, 0.421 ± 0.073 mU at 25 °C and 0.158±.058 mU at 37 °C. For this reason an immobilization temperature of 4 °C was selected for ALP. 3.2. Mineralization under acellular conditions (CCM test) The activity and mineralization capability of immobilized ALP was characterized by in vitro soaking experiments in osteogenic medium without cells (CCM test under acellular conditions) for 1, 4, 7, 14, 21 and 28 days. The mineral layer deposited was analyzed by SEM (Fig. 2), EDX (Fig. S1) and Ca2+ content quantified with a sensitive and Ca2+-specific colorimetric assay (Fig. 3c). CaP nucleation was visible on ALP-functionalized samples (ASNEA) even after 4 days (Fig. 2d). A higher amount and larger CaP crystals were observed after 10 days (Fig. 2h, white arrow) and after 14 days the surface was homogeneously covered with dense CaP mineral layer (Fig. 2l). In contrast, on the reference samples without ALP (A, AS and ASNE) no mineral formation was detected up to 10 days (Fig. 2e–g) and only after 14 days was sporadic, spot-like formation of submicron crystals observed (Fig. 2i–k), possibly due to non-specific mineral precipitation from solutions. After 21 or even 28 days, on reference samples some CaP spheroids were detected (Fig. 2m–s), whereas on ASNE a dense, homogeneous CaP multilayer was formed (Fig. 2p, t). The composition of mineral clusters was determined by EDX analysis, which confirmed the presence of Ca and P. The CaP formed on ASNEA samples had a Ca/P ratio of between 1.5 and 1.8 (Fig. S1). The calcium content of the mineralized layer formed at different soaking times was determined using the o-cresolphthalein complexone calcium assay after completely dissolving the mineralized layer with HCl. Example images of the surface before and after HCl treatment are shown in Fig. 3a and b. In accordance with the SEM analysis, a significantly higher Ca2+ concentration in the mineral layer formed on ALP-functionalized alumina sample was found at all time points in comparison to reference samples (Fig. 3c). Ca2+ concentrations ranged from 0.52 ± 0.07 mM (day 1) to
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Fig. 2. SEM images of samples after immersion under acellular conditions for 4, 10, 14, 21 and 28 days: A, non-functionalized alumina (a, e, i, m and q); AS, silanized alumina discs (b, f, j, n and r), ASNE, alumina discs treated with NHS and EDC (c, g, k, o and s); ASNEA, ALP-functionalized alumina substrates (d, h, l, p and t). White arrows show the CaP formation on the surfaces of alumina discs. Scale bars: 1 lm.
5.77 ± 0.31 mM (day 28). The Ca2+ concentrations in the mineral layer were similar between all the reference samples ranging from 0.194 ± 0.045 mM (day 1) to 4.28 ± 0.17 mM (day 28). 3.3. In vitro cell test with human osteoblast cells: cell morphology, proliferation and differentiation The ability of immobilized ALP to support cellular growth and mineralization was determined by in vitro cell tests with HOBs. Three independent cell-culture experiments were carried out. Cells were initially cultured in normal cell medium up to confluency, which was reached after 7 days. Afterwards, the medium was exchanged with an osteconductive medium supplied with b-glycerophosphate. All experimental runs showed a similar cell response and behaviour. Representative results are summarized in the following. 3.4. Cell viability
Fig. 3. SEM images after immersion of ASNEA: ALP-functionalized alumina discs under acellular conditions for 21 days (a) before and (b) after demineralization with HCl (pH 4) for 5 h. (c) Calcium concentration after demineralization. Statistically significant difference ⁄p < 0.05.
Cell viability was evaluated by WST-1 assay. Cell number and viability increased from day 1 to day 10 to an equivalent extent and without a statistically significant difference between the different samples at every time point (Fig. 4a), except that at day 4 cell viability of the cells on ASNE and at day 10 the viability on AS were significantly less than the other samples. This was also confirmed by fluorescent microscopy and SEM analysis which showed an increasing cell number on sample surfaces over time (Figs. 5 and 6).
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Fig. 4. (a) Cell viability and proliferation of HOBs measured by WST-1 assay after 1, 4, 7, 10, 14, 17, 21 and 28 days. (b) Cellular ALP activity measured from cell lysates. A, nonfunctionalized alumina discs; AS, silanized alumina discs; ASNE, alumina discs treated with NHS and EDC; ASNEA, ALP-immobilized alumina discs; NM, normal medium; OM, osteoinductive medium supplied with b-GP. Statistically significant difference ⁄p < 0.05.
Fig. 5. Fluorescence microscopy images showing cell morphology (a–d) and collagen type I formation (e–h) of 7 day culture in normal cell medium. Green, cytoskeleton; blue, cell nuclei; red, collagen type I.
3.5. Cell differentiation To further analyze cell differentiation on the samples, the production of cellular ALP and collagen type I were determined at different cultivation times. The activity of cellular ALP quantified from cell lysates increased up to 7 days but decreased after day 10 (Fig. 4b). The activity of ALP was similar between reference samples but higher on ALP-functionalized discs from 7 to 21 days (statistically significant difference ⁄p < 0.05). 3.6. Cell morphology Cell growth and morphology was analyzed by fluorescence microscopy and SEM at different sampling points. There were no distinguishable differences in the cell morphologies of all experimental samples up to 7 days of culture in normal cell medium (NM). HOBs were able to adhere, flatten and spread over all sample surfaces. HOBs formed cell–cell contacts as indicated by the flattened and polygonal morphology. Cell confluency was reached after 7 days. In the presence of immobilized ALP (ASNEA) a more homogeneous and continuous cell layer was observed (Fig. 5d) in comparison to reference samples (Fig. 5a–c). A similar collagen type I formation was also detected on all experimental groups from day 1 as shown in some representative images (Fig. 5e–h).
After day 7 cells were further cultivated in osteoinductive medium and still no significant differences with respect to the cell morphology were observed between the different samples. HOBs featured a flat appearance and spread over the surface and a continuous growth was observed at each sampling time as shown in some representative micrographs (Fig. 6a, c, e, g, I, k, m and o). A cell multilayer was visible on the all samples after 21 days of culture (Fig. 6i–k). Multiple cell layers, a higher formation of fibrous ECM containing mineralized globules was detected on ALP-functionalized alumina after only 7 days of culture (Fig. 6c, white arrow). Relatively large mineralized nodules and clusters, probably formed by the cells, were observed up to day 28. In addition, on reference samples globule formation was visible after day 10, but clusters were sporadic and smaller and the formation of fibrillar ECM less pronounced. To determine if a mineral layer could form underneath the cells and on top of the samples, the cells were removed by trypsinization. This gentle approach was specifically used to remove the cells without altering the mineralized layer. On ALP-functionalized samples small mineralized nodules spread over the surface were detected at days 7 and 10, whereas after days 21 and 28 a dense, homogeneous mineral layer and large round spherical mineral agglomerates were visible. On reference samples up to day 10 no
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Fig. 6. SEM images of mineral formation with human osteoblast cells after 7, 10, 21 and 28 days of culture in osteoinductive medium: A, non-functionalized alumina discs; ASNEA, ALP-immobilized alumina discs; (a, c, e, g, i, k, m and o) with cells; (b, d, f, h, j, l, n and p) after cell detachment with trypsinization. Orange arrows, cell area; white arrows, mineral globules.
mineral deposition was observed, but after 21 days of culturing, mineralized globules were found. Representative images for unfunctionalized alumina (A) are shown in Fig. 6j–n (white arrows). A similar effect was observed on AS and ASNE samples (data not shown). In vitro HOB mineralization capability was further assessed by staining calcium minerals with ARS. Representative images are given in Fig. 7a. After as little as 10 days of culture CaP mineralization was observed on ALP-functionalized samples. After day 14 nodule formation was detectable with the naked eye on all experimental samples but markedly higher in ALP-functionalized substrates. On ALP-functionalized substrates a more intense red colour was observable, clearly indicating that higher and increasing amounts of mineralized nodules where present (Fig. 7a). This was additionally confirmed by quantifying the amount of ARS for all experimental samples from day 10 to 28 (Fig. 7b). The amount of ARS was found to increase with culture time, but generally a higher amount was found on ALP-functionalized substrates. No statistically significant differences were observed for the other reference samples. Similarly, the Ca2+ content of the mineralized nodules was significantly higher in the presence of immobilized ALP in comparison to reference samples (Fig. 7d). By using the o-cresolphthalein complexone assay, CaP deposition on ALP substrates could be detected as soon as 4 days of culture and constantly increased up to day 28. The crystal phase of the mineralized nodules was characterized by GIXRD which confirmed the presence of hydroxyapatite (Fig. 7c). Hydroxyapatite peaks (labelled with #) were also detected on reference samples, though these were less pronounced than on ALP-functionalized samples.
4. Discussion In this study, ALP was covalently immobilized on bioinert alumina substrates using a relatively simple carbodiimide-mediated approach. This widely applied and biocompatible immobilization route was found to be suitable for binding ALP without significantly altering ALP activity and mineralization capability as confirmed by enzymatic assays (Fig. 1b) and CCM-soaking tests (Fig. 2). To determine the optimal immobilization conditions and the highest activity after immobilization, three different temperatures were tested (4, 25 and 37 °C). Although ALP was found to be still active at all these temperatures, a decrease in ALP activity by increasing the immobilization temperature was observed. Under our experimental conditions, the highest enzymatic activity was obtained at 4 °C. This is apparently not in accordance with the previous publications which observed a better ALP activity at higher temperatures [20,32]. This suggests that a relatively low immobilization temperature might stabilize ALP or prevent its denaturation and to some extent improve the immobilization reaction. This difference can be also ascribed to the intrinsic variability of ALP, or the animal sources, different materials, experimental conditions or immobilization approach. The suitability of this immobilization approach and ALP activity was additionally confirmed by in vitro soaking tests under acellular conditions with O-CCM as shown in Fig. 2. Soaking tests with O-CCM are particularly useful for assaying ALP mineralization activity, as this medium contains organic phosphates which can be hydrolyzed into inorganic phosphates only by active ALP. On each ALP-functionalized surface a relative rapid CaP mineralization
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Fig. 7. Calcium mineral deposition by HOBs cultured with osteoinductive medium. (a) Alizarin Red S (ARS) staining. (b) Amount of ARS measured at 405 nm and normalized with total amount of cellular DNA. (c) GIXRD patterns of deposition layer after 14, 21, 28 days on ALP-functionalized and bare alumina discs. (d) Calcium concentration in mineralized nodules. Statistically significant difference ⁄p < 0.05.
and formation of submicron CaP particles was observed after only 4 days. This suggests that immobilized ALP remains active and can hydrolyze b-GP, thereby locally increasing the inorganic phosphate concentration. An increase in inorganic phosphate concentration on the nearby surface can induce the formation of CaP nuclei, which grow further into bigger crystals. The constant supply of Ca2+ and PO3 4 ions from the cell-culture medium leads to the formation of a dense CaP mineral layer (Fig. 2p, t). A similar observation has been already described by de Jonge et al., who detected CaP on electrosprayed ALP/CaP composite coatings on titanium after 4 days of incubation in O-CCM under acellular conditions [10,18]. The nucleation of CaP directed from immobilized ALP seems to mimic the natural bone mineralization process, where nucleation of apatite crystals in matrix vesicles is directed by ALP [17]. A delayed and less evident CaP nucleation was also observed on reference samples without ALP. This may be related to the nucleation of crystals and precipitation from cell-culture medium containing high amounts of various ions such as calcium, magnesium, phosphate and carbonate. We cannot exclude the possibility that crystal nucleation can be induced by other serum proteins such as serum albumin. Serum albumin, present at high concentrations in cell media, may induce the nucleation of calcium phosphates in solution, which later sediment on the surface [33]. Alternatively, crystal nucleation can be initiated by serum protein which first spontaneously adsorbs on the alumina surface, and then nucleates CaP seeds. In fact after a relative long soaking time (2 weeks) and due to its affinity for alumina [25], albumin adsorption is very likely and may induce promote CaP nucleation. This has been, for instance, suggested by Serro et al., who showed albumin preadsorbed on hydroxyapatite or on titania surfaces may promote or inhibit hydroxyapatite mineralization depending on the sorbent surface [34].
The effect of ALP on CaP formation was additionally confirmed by quantifying the calcium content in the mineral layer. Significantly higher calcium content on ALP substrates was detected after only 1 day of incubation and increased with increasing soaking time. Cell-culture experiments showed that normal cell growth is taking place on all substrate types, demonstrating that neither ALP nor the immobilizing agents such as APTES, EDC and NHS had a negative effect on HOBs, and this immobilization method is biocompatible. Cell proliferation increased up to day 7 when the cells were grown in normal cell medium. After day 10, cell proliferation decreased as HOBs started to differentiate [10,22,35]. A higher amount of cellular ALP was detected on ALP-functionalized alumina in comparison to reference samples (Fig. 4b). This indicates that ALP-functionalized substrates can support cell differentiation and osteogenic behaviour and can possibly activate cellular ALP synthesis [14]. These findings are in agreement with previous studies, with different ALP-treated materials and cell types such as ALP/CaP-coated titanium with osteoblast-like cells [10] and ALP-modified nanofibrous fibrin scaffolds with primary calvarial cells (MC3T3 cells) [9]. The osteoinductive properties of ALP-functionalized alumina were additionally demonstrated by the higher and faster calcium mineralization in cell tests. Nodule formation was detected for all experimental groups, but on ALP-functionalized surfaces a rapid and higher CaP production from day 14 was observed (Fig. 7c). Similar observations are reported by de Jonge et al., but only after longer culturing times (days 8 and 16) [10], and also by Verne et al. [22]. Ca2+ analysis (Fig. 7d) showed a significantly higher Ca2+ amount in the mineralized nodule already after 1 day of culture which can be attributed to ALP-mediated but not to cell-mediated mineralization.
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5. Conclusion The results presented in this study confirm that the immobilization of ALP onto alumina is a highly suitable approach to design material surfaces which can induce self-formation of bone-like material under acellular conditions and clearly stimulate bone cell mineralization activity. In our study we also confirmed that the widely applied EDC/NHS immobilization route is biocompatible and suitable for stably binding active ALP molecules, and that ALP activity can be enhanced by simply controlling the reaction parameters. Preliminary demineralization tests showed that this immobilization route is suitable for keeping ALP active even after treatment under acidic conditions. Such surfaces are feasible candidates for activating inert biomaterial surfaces and for designing load-bearing implants, featuring outstanding osteogenic, mechanical and chemical properties, and at the same time providing a bioactive surface in order to enhance and guide osteogenic behaviour. Acknowledgments We acknowledge the German Research Foundation (DFG) for funding this project (Project No. TR 978/3-1). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1, 6 and 7 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2014.11.007. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.11. 007. References [1] Hayashi K, Matsuguchi N, Uenoyama K, Sugioka Y. Reevaluation of the biocompatibility of bioinert ceramics in vivo. Biomaterials 1992;13:195. [2] Navarro M, Michiardi A, Castano O, Planell JA. Biomaterials in orthopaedics. J R Soc Interface 2008;5:1137. [3] Schickle K, Kaufmann R, Campos DFD, Weber M, Fischer H. Towards osseointegration of bioinert ceramics: introducing functional groups to alumina surface by tailored self assembled monolayer technique. J Eur Ceram Soc 2012;32:3063. [4] Zhao MZ, An MR, Wang QS, Liu XC, Lai WJ, Zhao XY, et al. Quantitative proteomic analysis of human osteoblast-like MG-63 cells in response to bioinert implant material titanium and polyetheretherketone. J Proteomics 2012;75:3560. [5] Treccani L, Klein TY, Meder F, Pardun K, Rezwan K. Functionalized ceramics for biomedical, biotechnological and environmental applications. Acta Biomater 2013;9:7115. [6] de Jonge LT, Leeuwenburgh SCG, Wolke JGC, Jansen JA. Organic-inorganic surface modifications for titanium implant surfaces. Pharm Res-Dordr 2008;25:2357. [7] Ferraris S, Vitale-Brovarone C, Bretcanu O, Cassinelli C, Verne E. Surface functionalization of 3D glass-ceramic porous scaffolds for enhanced mineralization in vitro. Appl Surf Sci 2013;271:412. [8] Fu HL, Rahaman MN, Brown RF, Day DE. Evaluation of bone regeneration in implants composed of hollow HA microspheres loaded with transforming growth factor beta 1 in a rat calvarial defect model. Acta Biomater 2013;9:5718. [9] Osathanon T, Giachelli CM, Somerman MJ. Immobilization of alkaline phosphatase on microporous nanofibrous fibrin scaffolds for bone tissue engineering. Biomaterials 2009;30:4513.
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[10] de Jonge LT, van den Beucken JJJP, Leeuwenburgh SCG, Hamers AAJ, Wolke JGC, Jansen JA. In vitro responses to electrosprayed alkaline phosphatase/calcium phosphate composite coatings. Acta Biomater 2009;5:2773. [11] Ehlert N, Muller PP, Stieve M, Behrens P. Immobilization of alkaline phosphatase on modified silica coatings. Microporous Mesoporous Mater 2010;131:51. [12] Ferraris S, Spriano S, Bianchi CL, Cassinelli C, Verne E. Surface modification of Ti-6Al-4 V alloy for biomineralization and specific biological response: Part II, alkaline phosphatase grafting. J Mater Sci: Mater Med 2011;22:1835. [13] Lorch IJ. Alkaline phosphatase and the mechanism of ossification. J Bone Joint Surg Br 1949;31:94. [14] Reyes CD, Petrie TA, Burns KL, Schwartz Z, Garcia AJ. Biomolecular surface coating to enhance orthopaedic tissue healing and integration. Biomaterials 2007;28:3228. [15] Berendsen AD, Smit TH, Hoeben KA, Walboomers XF, Bronckers ALJJ, Everts V. Alkaline phosphatase-induced mineral deposition to anchor collagen fibrils to a solid surface. Biomaterials 2007;28:3530. [16] Robison RSK. The possible significance of hexosephosphoric esters in ossification Part II. The phosphoric esterase of ossifying cartilage. Biochem J 1924;18:740. [17] Golub EEBK. The role of alkaline phosphatase in mineralization. Curr Opin Orthop 2007;18:444. [18] de Jonge LT, Leeuwenburgh SCG, van den Beucken JJJP, Wolke JGC, Jansen JA. Electrosprayed enzyme coatings as bioinspired alternatives to bioceramic coatings for orthopedic and oral implants. Adv Funct Mater 2009;19:755. [19] Hoffmann C, Zollfrank C, Ziegler G. Enzyme-catalysed synthesis of calcium phosphates. J Mater Sci: Mater M 2008;19:907. [20] Verne E, Ferraris S, Vitale-Brovarone C, Spriano S, Bianchi CL, Naldoni A, et al. Alkaline phosphatase grafting on bioactive glasses and glass ceramics (vol. 6, pg 229, 2010). Acta Biomater 2010;6:1206. [21] Taylor RH, Fournier SM, Simons BL, Kaplan H, Hefford MA. Covalent protein immobilization on glass surfaces: application to alkaline phosphatase. J Biotechnol 2005;118:265. [22] Verne E, Ferraris S, Vitale-Brovarone C, Cochis A, Rimondini L. Bioactive glass functionalized with alkaline phosphatase stimulates bone extracellular matrix deposition and calcification in vitro. Appl Surf Sci 2014;313:372. [23] Holthaus MG, Kropp M, Treccani L, Lang W, Rezwan K. Versatile crack-free ceramic micropatterns made by a modified molding technique. J Am Ceram Soc 2010;93:2574. [24] Kroll S, Brandes C, Wehling J, Treccani L, Grathwohl G, Rezwan K. Highly efficient enzyme-functionalized porous zirconia microtubes for bacteria filtration. Environ Sci Technol 2012;46:8739. [25] Meder F, Daberkow T, Treccani L, Wilhelm M, Schowalter M, Rosenauer A, et al. Protein adsorption on colloidal alumina particles functionalized with amino, carboxyl, sulfonate and phosphate groups. Acta Biomater 2012;8:1221. [26] Sila-Asna M, Bunyaratvej A, Maeda S, Kitaguchi H, Bunyaratavej N. Osteoblast differentiation and bone formation gene expression in strontium-inducing bone marrow mesenchymal stem cell. Kobe J Med Sci 2007;53:25–35. [27] Holthaus MG, Treccani L, Rezwan K. Osteoblast viability on hydroxyapatite with well-adjusted submicron and micron surface roughness as monitored by the proliferation reagent WST-1. J Biomater Appl 2013;27:791. [28] Kang YQ, Kim S, Bishop J, Khademhosseini A, Yang YZ. The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and beta-TCP scaffold. Biomaterials 2012;33:6998. [29] Zhu YF, Zhu M, He X, Zhang JH, Tao CL. Substitutions of strontium in mesoporous calcium silicate and their physicochemical and biological properties. Acta Biomater 2013;9:6723. [30] Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004;329:77. [31] Sikavitsas VI, Bancroft GN, Holtorf HL, Jansen JA, Mikos AG. Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces. Proc Natl Acad Sci USA 2003;100:14683. [32] Ghiaci M, Aghaei H, Soleimanian S, Sedaghat ME. Enzyme immobilization: Part 2 immobilization of alkaline phosphatase on Na-bentonite and modified bentonite. Appl Clay Sci 2009;43:308. [33] Dorozhkin SV, Dorozhkina EI, Agathopoulos S, Ferreira JMF. The influence of glucose and bovine serum albumin on the crystallization of a bone-like apatite from revised simulated body fluid. In: Bioceramics 16. Key Engineering Materials, vols. 254-256; 2004. p. 327–332. [34] Serro AP, Bastos M, Pessoa JC, Saramago B. Bovine serum albumin conformational changes upon adsorption on titania and on hydroxyapatite and their relation with biomineralization. J Biomed Mater Res, A 2004;70A:420. [35] van den Dolder J, Spauwen PHM, Jansen JA. Evaluation of various seeding techniques for culturing osteogenic cells on titanium fiber mesh. Tissue Eng 2003;9:315.
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Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface Alieh Aminian a, Karoline Pardun a, Eike Volkmann a, Giovanni Li Destri b, Giovanni Marletta c, Laura Treccani a,⇑, Kurosch Rezwan a a b c
Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, 28359 Bremen, Germany ESRF-The European Synchrotron, 71 Avenue des Martyrs, CS 40220, 38043 Grenoble Cedex 9, France Laboratory for Molecular Surfaces and Nanotechnology (LAMSUN), Department of Chemistry, University of Catania and CSGI, V.le A. Doria 6, 95125 Catania, Italy
a r t i c l e
i n f o
Article history: Received 21 July 2014 Received in revised form 8 October 2014 Accepted 4 November 2014 Available online xxxx Keywords: Alumina Alkaline phosphatase Surface functionalization Biomineralization Bioactivity
a b s t r a c t In this study a bioinspired approach to induce self-mineralization of bone-like material on alumina surfaces is presented. The mineralizing enzyme alkaline phosphatase (ALP) is covalently immobilized by a carbodiimide-mediated chemoligation method. The enzymatic activity of immobilized ALP and its mineralization capability are investigated under acellular conditions as well as in the presence of human bone cells. Analytical, biochemical and immunohistochemical characterization show that ALP is efficiently immobilized, retains its activity and can trigger calcium phosphate mineralization on alumina at acellular conditions. In vitro cell tests demonstrate that ALP-functionalized alumina clearly boosts and enhances bone cell mineralization. Our results underpin the great potential of ALP-functionalized alumina for the development of bioactive surfaces for applications such as orthopaedic and dental implants, enabling a fast and firm implant osseointegration. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction The immobilization of specific, active biomolecules on material surfaces (biofunctionalization) is considered as a highly effective strategy to improve the biocompatibility and bioactivity of materials [1–5]. A variety of biological molecules including carbohydrates, extracellular matrix (ECM) proteins or their components, e.g. arginine-glycine-aspartic acid (RGD) peptides and growth factors have been considered to elicit specific cellular responses, increase the interaction between the cells, material surfaces and tissues, and thereby result in integration and tissue regeneration [6–9]. The enzyme alkaline phosphatase (ALP, Enzyme Commission number EC 3.1.3.1) is known to play a central role in hard tissue formation and osteogenesis and is commonly used as marker for assessing maturity of mineralized tissue or cell phenotype in biochemical and histological assays [9–14]. ALP is present in many species, including humans. It is present in the plasma membrane of the cells and in the ECM [15] and its important role in hard tissue formation was postulated more than 80 years ago [16,17]. ALP catalyzes the hydrolysis of organic phosphate monoesters into inorganic phosphates (Pi), increases their local concentration, and directs the nucleation of hydroxyapatite crystals in so-called ⇑ Corresponding author. Tel.: +49 (0)421 218 64938; fax: +49 (0)421 218 64932. E-mail address: [email protected] (L. Treccani).
matrix vesicles [10,13,18,19]. At the same time, ALP decreases the concentration of the crystal growth inhibitors as pyrophosphates (PPi) [10,13,18]. Due to its central role in bone formation some in vitro and in vivo studies have exploited the clinical potential of ALP for ligament and bone regeneration. For instance, ALP deposited on polymer surfaces induces the attachment of collagen fibrils [15] or achieves a higher deposition of calcium phosphate (CaP) on titanium alloys [12,18], as well as on bioactive materials such as bioglasses [11,20–22] and nanofibrous fibrin scaffolds [9]. Nonetheless, due to the differently employed deposition and/or immobilization approaches for ALP, support materials and test conditions, clear evidence of the relationship between immobilized ALP activity and its effect on CaP mineralization with and without cells, and its impact on bone formation is still unclear. In this study the possibility of immobilizing biomolecules such as ALP, which can simultaneously promote cell–surface interactions, cell attachment and boost the mineralization capability and induce self-biomineralization of CaP on inert ceramic surfaces, has been considered. ALP has been immobilized to highly inert surfaces, such as alumina, to catalyze and induce CaP mineralization. Alumina (Al2O3), due to its remarkable mechanical properties, chemical stability and biocompatibility, is widely employed for permanent bone orthopaedic implants. Nonetheless due to its bioinertness, Al2O3 cannot stimulate bone formation and
http://dx.doi.org/10.1016/j.actbio.2014.11.007 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Aminian A et al. Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.007
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osseointegration, which is a fundamental requirement for longterm and overall implant success. Here, a simple and biocompatible immobilization approach based on the zero-length cross-linking system 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (EDC/NHS) was used to stably bind active ALP molecules on dense a-Al2O3 surfaces. Al2O3 surfaces were first silanized with aminopropyltriethoxysilane (APTES) to create surfacereactive conjugation sites for the covalent immobilization of ALP with the EDC/NHS linkers. To see if the immobilization can lead to a substantial loss of ALP activity and CaP formation capability, the specific activity of ALP after immobilization was investigated by enzymatic assays. Immobilized ALP-driven mineralization was assessed by in vitro soaking experiments at acellular conditions and in the presence of human osteoblasts. Additionally, in vitro cell tests were conducted to simultaneously characterize the surface biocompatibility and the effect of immobilized ALP on cell proliferation, differentiation and cellular biomineralization. Mineral formation and deposition was characterized by several analytical techniques, including electron microscopy, energy dispersive spectroscopy and X-ray diffractometry, as well as different biochemical, immunohistochemical and histological methods.
2. Materials and methods 2.1. Materials High-purity a-alumina powder (Taimei, TM-DAR), 99.99%, particle size d50 = 210 ± 15 nm, specific surface areas (BET) = 3.98 g cm2, Lot No. SY301061B) was purchased from Krahn Chemie GmbH (Germany). APTES (Lot o. BCBH2173V, 98%), EDC (Lot No. 080M14582V, 99%), NHS (Lot No. BCBD1270V, 98%), 2-(N-morpholino)ethanesulfonic acid hydrate (MES, Lot No. 051M8424, 99.5%), ammonium hydroxide (25 vol.% in water, Lot No. 1336216), fetal calf serum (FCS, Lot No. 010M3395), p-nitrophenylphosphate (pNPP, Lot No. 091K5315), trypsin/EDTA (0.25% w/v trypsin, 0.02% w/v EDTA, Lot No. SLBH0771), glutaraldehyde (Lot No. 110M5300V), sodium cacodylate trihydrate (Lot No. 110M0056V,P98%), dexamethasone (Lot No. BCBB2722,P98%), L-ascorbic acid (Lot No. 127K0099,P99.0%), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, Lot No. 021M4069V, suitable for cell culture), b-glycerolphosphate (b-GP, Lot No. 128K54204V, 99%), gentamicin (Lot No. 041K12481V), calcium chloride (CaCl22H2O, Lot No. 1448251,P99.0%), Alizarin Red S (Lot No. 069K1639), glycine (Lot No. 118K00181,P99%), magnesium chloride hexahydrate (MgCl26H2O, Lot No. 019K00381V, suitable for cell culture), sodium chloride (NaCl, Lot No. 038K00451), zinc chloride (ZnCl2, Lot No. 119K1539), sodium hydroxide (NaOH, Lot No. BCBC7310V, 98%), ethanol (Lot No. SZBD2130V, absolute 99.8%), phosphate-buffered saline (PBS, Lot No. SLBF5741V), acetic acid (C2H4O2, Lot No. SZBA2030, 99.8%), 4-nitrophenol (pNP, Lot No. 0001369092), hexamethyldisilazane (HMDS, Lot No. SHBC8538V, 99.9%) and Triton™ X-100 (Lot No. MKBL5839V) were supplied by Sigma Aldrich (Germany). Lyophilized ALP (from chicken intestine, Lot No. L12112758, 0.9 units per mg dry weight, at 25 °C, pH 8.8) was obtained from Biomol GmbH (Germany). Dulbecco’s modified Eagle’s medium (DMEM), AlexafluorÒ488phalloidin, AlexafluorÒ546-phalloidin goat anti-mouse IgG and Quant-iT™ PicoGreenÒ dsDNA assay kit (Lot No. P7589) were purchased from Invitrogen (Germany). Polyacrylic acid (molecular weight = 4000 g mol1, SyntranÒ8220, Interpolymer GmbH, Germany), anticollagen I antibody (COL-1, Lot No. 041M4784, Acris, Germany), antibiotic–antimycotic (Life Technology, Germany), 40 ,6-diamidino-2-phenylindole (DAPI, Lot No. 1242642, Fluka, Germany), Alizarin Red S solution (ARS,
pH 4.1–4.3, Sigma, St Louis, MO, USA), WST-1 cell proliferation assay (WST-1, Roche Diagnostics GmbH, Mannheim, Germany), FluitestÒ CA-CPC (AnalyticonÒ Biotechnologies AG, Germany), paraformaldehyde (PFA, Lot No. 53260, Riedel-de Haën) and hydrochloric acid (HCl, Lot No. 12G180525, 37%, VWR) were obtained from different suppliers as specified. Double-deionized water (ddH2O) with an electrical resistance of 18 MX (Synergy, Millipore, Germany) was used for all experiments. 2.2. Alumina discs Planar alumina discs were fabricated using a modified micromoulding method described in Holthaus et al. [23]. Alumina aqueous suspensions (solid content of 20 vol.%) were obtained by mixing 19.9 g particles with 20 ml ddH2O. Polyacrylic acid (1.2 vol.%) was used as binder. The pH was adjusted to 10–11 with 2 vol.% ammonium hydroxide for electrosteric stabilization and to prevent particle agglomeration. Suspensions were sonicated for 10 min with an ultrasonic horn using an output of 150 W and pulse rate 0.5 s (Sonifier 450, Branson, USA) to break agglomerates after stirring. 1 ml suspension was placed into cylindrical moulds with a diameter of 15 mm (24 non-treated well plates, Nalge NUNC, Rochester, NY), previously greased with a thin layer of vaseline. Subsequently, the discs were dried for 5 days at 25 °C and 30% relative air humidity to prevent crack formation. After demoulding, the alumina discs were sintered at 1500 °C for 2 h (LHT08/17, Nabertherm GmbH, Lilienthal, Germany). Sintered alumina discs had a final diameter of 13.42 ± 0.17 mm and height of 2.0 ± 0.21 mm. Before ALP immobilization, samples were sterilized by autoclaving. Silanization and ALP immobilization were carefully conducted under sterile conditions and using sterile solutions to avoid any possible contamination. 2.3. ALP immobilization on alumina discs ALP was covalently immobilized on alumina discs using the coupling cross-linkers EDC/NHS and using a protocol adapted from Kroll et al. [24]. Prior to immobilization, alumina discs were autoclaved (Systec 2540, Germany) and silanized with APTES to generate the surface functional amino groups necessary to bind ALP through the EDC/NHS coupling reaction. Silanization was carried out in aqueous solutions as described as described in Refs. [24,25]. Briefly, autoclaved alumina discs were immersed in a 8.5 mM APTES solution (2 vol.%) for 1 h at room temperature (RT), then cured for an additional 1 h at 70 °C. Afterwards, the samples were washed 10 times with ddH2O to remove unreacted APTES and dried for 10 h at 70 °C. APTES-silanized alumina discs were immersed in a solution of 10 mg ml1 EDC, 0.6 mg ml1 NHS, 1 mg ml1 ALP dissolved in MES buffer (0.1 M, pH 6). For optimizing the ALP immobilization procedure, incubation was carried out at three different temperatures (4, 25 and 37 °C) for 2 h. Afterwards alumina discs were rinsed 10 times with ddH2O to remove unbound ALP. Alumina discs with immobilized ALP are named ASNEA. Nontreated (A), silanized (AS) and alumina discs functionalized with APTES, EDC/NHS but without ALP (ASNE) were used as reference (Fig. 1a). 2.4. ALP activity assay The enzymatic activity of immobilized ALP was determined using pNPP as an enzymatic substrate as described in Ref. [26]. Alumina discs functionalized with ALP (ASNEA in Fig. 1a) were incubated in a 1 ml ALP substrate buffer solution consisting of 6 mM pNPP dissolved in 90 mM glycine buffer (0.1 mM glycine, 1 mM
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Fig. 1. (a) Functionalization scheme: silanization and covalent binding of ALP on alumina substrates with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). (b) Enzymatic activity of ALP immobilized on alumina discs (ASNEA) measured by pNPP assay immediately after immobilization at three different temperatures (4, 25 and 37 °C). Unfunctionalized alumina discs (A) were used as reference.
MgCl2, 1 mM ZnCl2 at pH 10) at 37 °C. After 30 min the reaction was stopped by addition of 1 ml of 1 M NaOH. The increase in the absorbance of p-nitrophenol (pNP) generated by the hydrolysis of pNPP by ALP was spectrometrically measured at 405 nm with a plate reader (Chameleon V, Hidex, Germany). Serial dilutions of ALP (from 50 ng ml1 = 0.05 mU to 1 lg ml1 = 1 mU) were used to generate standard curves. Five independent experimental runs were performed using six alumina discs for each type of sample. 2.5. ALP mineralization activity under acellular conditions (CCM soaking test) The activity and mineralization capability of immobilized ALP were initially evaluated at acellular conditions by in vitro soaking experiments as described in Ref. [18]. All experimental substrates were immersed in cell culture medium (CCM) consisting of DMEM, 50 lg ml1 L-ascorbic acid, 10 nM dexamethasone, 50 lg ml1 gentamicin, 10 mM b-GP and 1.5 mM CaCl2, for 1, 4, 7, 10, 14, 21 and 28 days under constant conditions (37C, 9.5% CO2 and 93% relative humidity). The medium was refreshed every 2 days. CCM soaking experiments were carried out using six discs for each type of sample. After each incubation time, discs were removed, rinsed thoroughly with ddH2O and dried at 70 °C for 15 h. Mineral formation and surface morphology were analysed by scanning electron microscopy (SEM, Supra 40, Zeiss, Oberkochen, Germany). Samples were sputtered with a thin gold layer (sputter coater 108 Auto, Cressington, USA). Chemical composition and Ca/P ratio were recorded by energy-dispersive X-ray spectroscopy (EDX, Gemini Supra 40, Zeiss, Oberkochen, Germany). 2.6. Immobilized ALP mineralization activity and surface biocompatibility assessment with bone cells In vitro mineralization and biocompatibility were assessed with human osteoblast cells (HOBs, passage 4, Provitro, Germany). Cells were pre-cultured in cell culture medium (DMEM, 10% FCS and 1% antibiotic–antimycotic) in a cell incubator (Labotect GmbH, Germany) at constant conditions (37 °C, 9.5% CO2 and 95% relative humidity). Cell medium was refreshed every 2 days. After 7 days cells were trypsinized with a trypsin/EDTA solution. Alumina discs
were put in 24-well multidish (Nalge NUNC, Rochester, NY) and cells were seeded with a density of 15 103 cells per sample. Cell culture medium was refreshed every 2 days up to day 7 until cells were confluent. Afterwards, an osteoinductive medium (O-CCM) consisting of DMEM, 10% FCS, 1% antibiotic–antimycotic, 10 nM dexametha sone, 5 108 M 1,25(OH)2D3, 50 lg ml1 L-ascorbic acid, 10 mM b-GP and 1.5 mM CaCl2 was used from day 7 to 28. O-CCM was refreshed every 2 days. Three independent experimental runs were performed. 2.6.1. Cell morphology Cell morphology at different time points was evaluated by immunofluorescence microscopy and SEM after 1, 4, 7, 14, 17, 21 and 28 days. Samples were rinsed with PBS buffer and cells were fixed with 4% PFA. Cell nuclei, actin filaments and collagen were stained with DAPI, AlexafluorÒ488-phalloidin and secondary antibody (AlexafluorÒ546-phalloidin goat anti-mouse IgG, Collagen type I), respectively, as described in Refs. [27,28]. Cells were imaged by fluorescence microscopy (Axio Imager M.1, Carl Zeiss GmbH, Germany). For SEM investigation, the samples were rinsed with 1 PBS buffer, fixed with 2.5 vol.% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), dehydrated in a graded series of ethanol (10%, 30%, 50%, 70%, 80%, 90%, 95% and 100%) and dried with HMDS for 1 min. Finally they were sputtered with thin layer of gold for SEM imaging. SEM analysis was performed using two discs for each type of sample. 2.6.2. Cell viability Cell viability was analyzed using a colorimetric WST-1 cell proliferation assay as described in Ref. [27]. After each sampling point, formazan produced by living cells was spectrometrically quantified (optical, OD) at 450 nm with a reference wavelength of 650 nm (Chameleon V, Hidex, Germany). Cell proliferation was measured using six specimens of each type of sample per sampling day. 2.6.3. Cell differentiation The activity of cellular ALP, as a marker for cell differentiation, was measured from cell lysates by determining the production of pNP from pNPP as previously described in Section 2.4.
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The cell lysate was prepared as described in Ref. [29]. After each sampling point, cell layers were rinsed with PBS, trypsinized with trypsin/EDTA solution and centrifuged at 20,000g for 10 min to separate the cell pellet from the supernatant. The lysis buffer (1% Triton™ X-100 in 0.9% 0.15 M NaCl) was added to the cell pellets and mixed until a clear solution was obtained. 100 ll of ALP substrate buffer (6 mM pNNP in 0.1 M glycine, 1 mM MgCl2, 0.1 mM ZnCl2) were added to 100 ll of cell lysates and incubated for 30 min at 37 °C. The reaction was stopped with 100 ll of 1 M NaOH and pNP content was determined photometrically at 405 nm. Serial dilutions of pNP (0–100 lM) were used as standard curves to extrapolate ALP activity. ALP measurements were done with six samples of each type of sample per sampling day. 2.6.4. Cell mineralization Cell mineralization at days 10, 14, 17, 21 and 28 was analyzed with an ARS-based staining assay to stain calcium-rich deposits using a protocol adapted from Ref. [30]. Cells were rinsed with PBS, fixed with ice-cold ethanol, kept at 4 °C for 1 h and then incubated in 1% ARS solution for 10 min at RT with gentle shaking. After removing ARS supernatant, the samples were washed with ddH2O, and 800 ll of 10% acetic acid were added to each well. Samples were incubated for 30 min at RT with gentle shaking. The supernatant and cell layers were removed from the discs and pipetted into 2 ml polypropylene cups (EppendorfÒ safe-lock tubes, Hamburg, Germany), briefly vortexed, heated up to 85 °C for 10 min and cooled down in ice for 5 min. Samples were centrifuged at 20,000g for 15 min (Heraeus Megafuge 16R, Thermo Scientific, Germany). 500 ll of supernatant was mixed with 200 ll of 10% ammonium hydroxide and the absorbance was measured at 405 nm with plate reader. ARS content was normalized to the total amount of cellular DNA quantified using the Quant-iT™ PicoGreenÒ dsDNA Kit. An aliquot of the cell lysate was diluted with TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) prior to addition of PicoGreenÒ working solution. Sample fluorescence was measured after an incubation of 5 min with an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a plate reader. The DNA concentration was correlated with the cell number using a calibration curve with a defined cell number. For each experiments six samples for each type of sample, per sampling day were used. After trypsinization, the samples were gently washed with ddH2O, dried for 15 h and prepared for SEM. SEM imaging was performed without gold sputtering. The crystal phases of mineral was determined by grazing incidence X-ray diffraction (GIXRD, Ultima IV type III diffractometer, Rigaku, Tokyo, Japan) equipped with cross-beam optics using a Ka wavelength emitted by a Cu anode. Careful alignment of source and detector with respect to the sample was achieved by using a thin-film attachment with three degrees of freedom. To avoid beam defocusing, the measurements were carried out in parallel beam mode. Divergence of the primary beam was reduced by a 5° Soller slit, while divergence of the diffracted beam was reduced by a 0.5° horizontal Soller slit. The incident angle was kept at 0.5°. The phase composition was determined by Rietveld refinement using the database of the International Centre of Diffraction Data (Newtown Square, PA, USA). PDXL software licensed by Rigaku was employed to minimize the difference between experimental and calculated data. 2.7. Demineralization and calcium assay To quantify the calcium content in CaP mineralized on sample surface after CCM soaking experiments and cell tests, samples were demineralized with 104 N HCl (pH 4) at 37 °C for 48 h.
Calcium content in the supernatant was quantified using o-cresolphthalein complexone (FluitestÒ CA-CPC) as described in Refs. [18,31]. Briefly, 200 ll of the assay reagent was added to 10 ll of supernatant and incubated for 15 min at RT. The absorbance was measured at 570 nm with plate reader and the calcium concentration was calculated using standard curve (serial dilution of CaCl2, 0–200 lg ml1). After demineralization, the enzymatic activity of ALP was measured as described in Section 2.4. 2.8. Statistical analysis All the data were presented as mean value ± standard deviation. The statistical analysis was performed using the statistical software MinitabÒ 16.1.1 (Minitab Inc., State College, PA, USA). Non-functionalized alumina samples were used as reference and comparison was accomplished using one-way analysis of variance (ANOVA) with a post hoc Dunnett’s test. Differences at p < 0.05 were considered to be statistically significant. 3. Results 3.1. Enzymatic activity of immobilized ALP After immobilizing ALP on the samples by EDC and NHS (Fig. 1a) at three different temperatures (4, 25 and 37 °C), ALP enzymatic activity was directly measured using a pNNP-based enzymatic assay. The initial enzymatic activity of immobilized ALP, expressed in mU (1 mU corresponds to 1 lg ml1) was found to decrease by increasing the immobilization temperature (Fig. 1b). The activity was 0.685 ± 0.121 mU at 4 °C, 0.421 ± 0.073 mU at 25 °C and 0.158±.058 mU at 37 °C. For this reason an immobilization temperature of 4 °C was selected for ALP. 3.2. Mineralization under acellular conditions (CCM test) The activity and mineralization capability of immobilized ALP was characterized by in vitro soaking experiments in osteogenic medium without cells (CCM test under acellular conditions) for 1, 4, 7, 14, 21 and 28 days. The mineral layer deposited was analyzed by SEM (Fig. 2), EDX (Fig. S1) and Ca2+ content quantified with a sensitive and Ca2+-specific colorimetric assay (Fig. 3c). CaP nucleation was visible on ALP-functionalized samples (ASNEA) even after 4 days (Fig. 2d). A higher amount and larger CaP crystals were observed after 10 days (Fig. 2h, white arrow) and after 14 days the surface was homogeneously covered with dense CaP mineral layer (Fig. 2l). In contrast, on the reference samples without ALP (A, AS and ASNE) no mineral formation was detected up to 10 days (Fig. 2e–g) and only after 14 days was sporadic, spot-like formation of submicron crystals observed (Fig. 2i–k), possibly due to non-specific mineral precipitation from solutions. After 21 or even 28 days, on reference samples some CaP spheroids were detected (Fig. 2m–s), whereas on ASNE a dense, homogeneous CaP multilayer was formed (Fig. 2p, t). The composition of mineral clusters was determined by EDX analysis, which confirmed the presence of Ca and P. The CaP formed on ASNEA samples had a Ca/P ratio of between 1.5 and 1.8 (Fig. S1). The calcium content of the mineralized layer formed at different soaking times was determined using the o-cresolphthalein complexone calcium assay after completely dissolving the mineralized layer with HCl. Example images of the surface before and after HCl treatment are shown in Fig. 3a and b. In accordance with the SEM analysis, a significantly higher Ca2+ concentration in the mineral layer formed on ALP-functionalized alumina sample was found at all time points in comparison to reference samples (Fig. 3c). Ca2+ concentrations ranged from 0.52 ± 0.07 mM (day 1) to
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Fig. 2. SEM images of samples after immersion under acellular conditions for 4, 10, 14, 21 and 28 days: A, non-functionalized alumina (a, e, i, m and q); AS, silanized alumina discs (b, f, j, n and r), ASNE, alumina discs treated with NHS and EDC (c, g, k, o and s); ASNEA, ALP-functionalized alumina substrates (d, h, l, p and t). White arrows show the CaP formation on the surfaces of alumina discs. Scale bars: 1 lm.
5.77 ± 0.31 mM (day 28). The Ca2+ concentrations in the mineral layer were similar between all the reference samples ranging from 0.194 ± 0.045 mM (day 1) to 4.28 ± 0.17 mM (day 28). 3.3. In vitro cell test with human osteoblast cells: cell morphology, proliferation and differentiation The ability of immobilized ALP to support cellular growth and mineralization was determined by in vitro cell tests with HOBs. Three independent cell-culture experiments were carried out. Cells were initially cultured in normal cell medium up to confluency, which was reached after 7 days. Afterwards, the medium was exchanged with an osteconductive medium supplied with b-glycerophosphate. All experimental runs showed a similar cell response and behaviour. Representative results are summarized in the following. 3.4. Cell viability
Fig. 3. SEM images after immersion of ASNEA: ALP-functionalized alumina discs under acellular conditions for 21 days (a) before and (b) after demineralization with HCl (pH 4) for 5 h. (c) Calcium concentration after demineralization. Statistically significant difference ⁄p < 0.05.
Cell viability was evaluated by WST-1 assay. Cell number and viability increased from day 1 to day 10 to an equivalent extent and without a statistically significant difference between the different samples at every time point (Fig. 4a), except that at day 4 cell viability of the cells on ASNE and at day 10 the viability on AS were significantly less than the other samples. This was also confirmed by fluorescent microscopy and SEM analysis which showed an increasing cell number on sample surfaces over time (Figs. 5 and 6).
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Fig. 4. (a) Cell viability and proliferation of HOBs measured by WST-1 assay after 1, 4, 7, 10, 14, 17, 21 and 28 days. (b) Cellular ALP activity measured from cell lysates. A, nonfunctionalized alumina discs; AS, silanized alumina discs; ASNE, alumina discs treated with NHS and EDC; ASNEA, ALP-immobilized alumina discs; NM, normal medium; OM, osteoinductive medium supplied with b-GP. Statistically significant difference ⁄p < 0.05.
Fig. 5. Fluorescence microscopy images showing cell morphology (a–d) and collagen type I formation (e–h) of 7 day culture in normal cell medium. Green, cytoskeleton; blue, cell nuclei; red, collagen type I.
3.5. Cell differentiation To further analyze cell differentiation on the samples, the production of cellular ALP and collagen type I were determined at different cultivation times. The activity of cellular ALP quantified from cell lysates increased up to 7 days but decreased after day 10 (Fig. 4b). The activity of ALP was similar between reference samples but higher on ALP-functionalized discs from 7 to 21 days (statistically significant difference ⁄p < 0.05). 3.6. Cell morphology Cell growth and morphology was analyzed by fluorescence microscopy and SEM at different sampling points. There were no distinguishable differences in the cell morphologies of all experimental samples up to 7 days of culture in normal cell medium (NM). HOBs were able to adhere, flatten and spread over all sample surfaces. HOBs formed cell–cell contacts as indicated by the flattened and polygonal morphology. Cell confluency was reached after 7 days. In the presence of immobilized ALP (ASNEA) a more homogeneous and continuous cell layer was observed (Fig. 5d) in comparison to reference samples (Fig. 5a–c). A similar collagen type I formation was also detected on all experimental groups from day 1 as shown in some representative images (Fig. 5e–h).
After day 7 cells were further cultivated in osteoinductive medium and still no significant differences with respect to the cell morphology were observed between the different samples. HOBs featured a flat appearance and spread over the surface and a continuous growth was observed at each sampling time as shown in some representative micrographs (Fig. 6a, c, e, g, I, k, m and o). A cell multilayer was visible on the all samples after 21 days of culture (Fig. 6i–k). Multiple cell layers, a higher formation of fibrous ECM containing mineralized globules was detected on ALP-functionalized alumina after only 7 days of culture (Fig. 6c, white arrow). Relatively large mineralized nodules and clusters, probably formed by the cells, were observed up to day 28. In addition, on reference samples globule formation was visible after day 10, but clusters were sporadic and smaller and the formation of fibrillar ECM less pronounced. To determine if a mineral layer could form underneath the cells and on top of the samples, the cells were removed by trypsinization. This gentle approach was specifically used to remove the cells without altering the mineralized layer. On ALP-functionalized samples small mineralized nodules spread over the surface were detected at days 7 and 10, whereas after days 21 and 28 a dense, homogeneous mineral layer and large round spherical mineral agglomerates were visible. On reference samples up to day 10 no
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Fig. 6. SEM images of mineral formation with human osteoblast cells after 7, 10, 21 and 28 days of culture in osteoinductive medium: A, non-functionalized alumina discs; ASNEA, ALP-immobilized alumina discs; (a, c, e, g, i, k, m and o) with cells; (b, d, f, h, j, l, n and p) after cell detachment with trypsinization. Orange arrows, cell area; white arrows, mineral globules.
mineral deposition was observed, but after 21 days of culturing, mineralized globules were found. Representative images for unfunctionalized alumina (A) are shown in Fig. 6j–n (white arrows). A similar effect was observed on AS and ASNE samples (data not shown). In vitro HOB mineralization capability was further assessed by staining calcium minerals with ARS. Representative images are given in Fig. 7a. After as little as 10 days of culture CaP mineralization was observed on ALP-functionalized samples. After day 14 nodule formation was detectable with the naked eye on all experimental samples but markedly higher in ALP-functionalized substrates. On ALP-functionalized substrates a more intense red colour was observable, clearly indicating that higher and increasing amounts of mineralized nodules where present (Fig. 7a). This was additionally confirmed by quantifying the amount of ARS for all experimental samples from day 10 to 28 (Fig. 7b). The amount of ARS was found to increase with culture time, but generally a higher amount was found on ALP-functionalized substrates. No statistically significant differences were observed for the other reference samples. Similarly, the Ca2+ content of the mineralized nodules was significantly higher in the presence of immobilized ALP in comparison to reference samples (Fig. 7d). By using the o-cresolphthalein complexone assay, CaP deposition on ALP substrates could be detected as soon as 4 days of culture and constantly increased up to day 28. The crystal phase of the mineralized nodules was characterized by GIXRD which confirmed the presence of hydroxyapatite (Fig. 7c). Hydroxyapatite peaks (labelled with #) were also detected on reference samples, though these were less pronounced than on ALP-functionalized samples.
4. Discussion In this study, ALP was covalently immobilized on bioinert alumina substrates using a relatively simple carbodiimide-mediated approach. This widely applied and biocompatible immobilization route was found to be suitable for binding ALP without significantly altering ALP activity and mineralization capability as confirmed by enzymatic assays (Fig. 1b) and CCM-soaking tests (Fig. 2). To determine the optimal immobilization conditions and the highest activity after immobilization, three different temperatures were tested (4, 25 and 37 °C). Although ALP was found to be still active at all these temperatures, a decrease in ALP activity by increasing the immobilization temperature was observed. Under our experimental conditions, the highest enzymatic activity was obtained at 4 °C. This is apparently not in accordance with the previous publications which observed a better ALP activity at higher temperatures [20,32]. This suggests that a relatively low immobilization temperature might stabilize ALP or prevent its denaturation and to some extent improve the immobilization reaction. This difference can be also ascribed to the intrinsic variability of ALP, or the animal sources, different materials, experimental conditions or immobilization approach. The suitability of this immobilization approach and ALP activity was additionally confirmed by in vitro soaking tests under acellular conditions with O-CCM as shown in Fig. 2. Soaking tests with O-CCM are particularly useful for assaying ALP mineralization activity, as this medium contains organic phosphates which can be hydrolyzed into inorganic phosphates only by active ALP. On each ALP-functionalized surface a relative rapid CaP mineralization
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Fig. 7. Calcium mineral deposition by HOBs cultured with osteoinductive medium. (a) Alizarin Red S (ARS) staining. (b) Amount of ARS measured at 405 nm and normalized with total amount of cellular DNA. (c) GIXRD patterns of deposition layer after 14, 21, 28 days on ALP-functionalized and bare alumina discs. (d) Calcium concentration in mineralized nodules. Statistically significant difference ⁄p < 0.05.
and formation of submicron CaP particles was observed after only 4 days. This suggests that immobilized ALP remains active and can hydrolyze b-GP, thereby locally increasing the inorganic phosphate concentration. An increase in inorganic phosphate concentration on the nearby surface can induce the formation of CaP nuclei, which grow further into bigger crystals. The constant supply of Ca2+ and PO3 4 ions from the cell-culture medium leads to the formation of a dense CaP mineral layer (Fig. 2p, t). A similar observation has been already described by de Jonge et al., who detected CaP on electrosprayed ALP/CaP composite coatings on titanium after 4 days of incubation in O-CCM under acellular conditions [10,18]. The nucleation of CaP directed from immobilized ALP seems to mimic the natural bone mineralization process, where nucleation of apatite crystals in matrix vesicles is directed by ALP [17]. A delayed and less evident CaP nucleation was also observed on reference samples without ALP. This may be related to the nucleation of crystals and precipitation from cell-culture medium containing high amounts of various ions such as calcium, magnesium, phosphate and carbonate. We cannot exclude the possibility that crystal nucleation can be induced by other serum proteins such as serum albumin. Serum albumin, present at high concentrations in cell media, may induce the nucleation of calcium phosphates in solution, which later sediment on the surface [33]. Alternatively, crystal nucleation can be initiated by serum protein which first spontaneously adsorbs on the alumina surface, and then nucleates CaP seeds. In fact after a relative long soaking time (2 weeks) and due to its affinity for alumina [25], albumin adsorption is very likely and may induce promote CaP nucleation. This has been, for instance, suggested by Serro et al., who showed albumin preadsorbed on hydroxyapatite or on titania surfaces may promote or inhibit hydroxyapatite mineralization depending on the sorbent surface [34].
The effect of ALP on CaP formation was additionally confirmed by quantifying the calcium content in the mineral layer. Significantly higher calcium content on ALP substrates was detected after only 1 day of incubation and increased with increasing soaking time. Cell-culture experiments showed that normal cell growth is taking place on all substrate types, demonstrating that neither ALP nor the immobilizing agents such as APTES, EDC and NHS had a negative effect on HOBs, and this immobilization method is biocompatible. Cell proliferation increased up to day 7 when the cells were grown in normal cell medium. After day 10, cell proliferation decreased as HOBs started to differentiate [10,22,35]. A higher amount of cellular ALP was detected on ALP-functionalized alumina in comparison to reference samples (Fig. 4b). This indicates that ALP-functionalized substrates can support cell differentiation and osteogenic behaviour and can possibly activate cellular ALP synthesis [14]. These findings are in agreement with previous studies, with different ALP-treated materials and cell types such as ALP/CaP-coated titanium with osteoblast-like cells [10] and ALP-modified nanofibrous fibrin scaffolds with primary calvarial cells (MC3T3 cells) [9]. The osteoinductive properties of ALP-functionalized alumina were additionally demonstrated by the higher and faster calcium mineralization in cell tests. Nodule formation was detected for all experimental groups, but on ALP-functionalized surfaces a rapid and higher CaP production from day 14 was observed (Fig. 7c). Similar observations are reported by de Jonge et al., but only after longer culturing times (days 8 and 16) [10], and also by Verne et al. [22]. Ca2+ analysis (Fig. 7d) showed a significantly higher Ca2+ amount in the mineralized nodule already after 1 day of culture which can be attributed to ALP-mediated but not to cell-mediated mineralization.
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5. Conclusion The results presented in this study confirm that the immobilization of ALP onto alumina is a highly suitable approach to design material surfaces which can induce self-formation of bone-like material under acellular conditions and clearly stimulate bone cell mineralization activity. In our study we also confirmed that the widely applied EDC/NHS immobilization route is biocompatible and suitable for stably binding active ALP molecules, and that ALP activity can be enhanced by simply controlling the reaction parameters. Preliminary demineralization tests showed that this immobilization route is suitable for keeping ALP active even after treatment under acidic conditions. Such surfaces are feasible candidates for activating inert biomaterial surfaces and for designing load-bearing implants, featuring outstanding osteogenic, mechanical and chemical properties, and at the same time providing a bioactive surface in order to enhance and guide osteogenic behaviour. Acknowledgments We acknowledge the German Research Foundation (DFG) for funding this project (Project No. TR 978/3-1). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1, 6 and 7 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2014.11.007. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.11. 007. References [1] Hayashi K, Matsuguchi N, Uenoyama K, Sugioka Y. Reevaluation of the biocompatibility of bioinert ceramics in vivo. Biomaterials 1992;13:195. [2] Navarro M, Michiardi A, Castano O, Planell JA. Biomaterials in orthopaedics. J R Soc Interface 2008;5:1137. [3] Schickle K, Kaufmann R, Campos DFD, Weber M, Fischer H. Towards osseointegration of bioinert ceramics: introducing functional groups to alumina surface by tailored self assembled monolayer technique. J Eur Ceram Soc 2012;32:3063. [4] Zhao MZ, An MR, Wang QS, Liu XC, Lai WJ, Zhao XY, et al. Quantitative proteomic analysis of human osteoblast-like MG-63 cells in response to bioinert implant material titanium and polyetheretherketone. J Proteomics 2012;75:3560. [5] Treccani L, Klein TY, Meder F, Pardun K, Rezwan K. Functionalized ceramics for biomedical, biotechnological and environmental applications. Acta Biomater 2013;9:7115. [6] de Jonge LT, Leeuwenburgh SCG, Wolke JGC, Jansen JA. Organic-inorganic surface modifications for titanium implant surfaces. Pharm Res-Dordr 2008;25:2357. [7] Ferraris S, Vitale-Brovarone C, Bretcanu O, Cassinelli C, Verne E. Surface functionalization of 3D glass-ceramic porous scaffolds for enhanced mineralization in vitro. Appl Surf Sci 2013;271:412. [8] Fu HL, Rahaman MN, Brown RF, Day DE. Evaluation of bone regeneration in implants composed of hollow HA microspheres loaded with transforming growth factor beta 1 in a rat calvarial defect model. Acta Biomater 2013;9:5718. [9] Osathanon T, Giachelli CM, Somerman MJ. Immobilization of alkaline phosphatase on microporous nanofibrous fibrin scaffolds for bone tissue engineering. Biomaterials 2009;30:4513.
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Please cite this article in press as: Aminian A et al. Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.007
