J. Am. Ceram. Soc., 1–10 (2015) DOI: 10.1111/jace.13626 © 2015 The American Ceramic Society
Journal
Hierarchically Designed Bioactive Glassy Nanocoatings for the Growth of Faster and Uniformly Dense Apatite
Indranee Das,‡ Samar K. Medda,‡ Goutam De,‡,† Susanne Fagerlund,§ Leena Hupa,§ Mervi A. Puska,¶ and Pekka K. Vallittu¶,k ‡
Nano-Structured Materials Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata 700032, India Akademi University, FI-20500 Abo, Inorganic Chemistry Department, Abo Finland
§ ¶
Turku Clinical Biomaterials Centre-TCBC, University of Turku, FI-20520 Turku, Finland
k
Department of Prosthetic Dentistry and Biomaterials Science, Institute of Dentistry, University of Turku, Turku, Finland tive glasses affects their bioactivity, expressed as osteoconductivity, and tissue attachment properties.11 Moreover, spherical morphologies form more effectively surface apatite layers than irregular bioactive glass particles.11,12 Bioactive glass microspheres with controlled and regular shape are reported to maintain the stability in silica dissolution kinetics, uniformity in calcium phosphate precipitation and they show low cytotoxicity.11 It is known that surfactant-template technique can be applied in sol–gel process to generate different types of morphologies for inorganic bioactive glass particles.11,12 The goal of this work is to study the influence of morphology of sol–gel-derived nanocoatings embedded with bioactive glassy nanospheres of controlled structure generated in situ through micellization process on their in vitro bioactivity. Also the cellular response on the nanocoatings embedded with nanospheres was studied. This type of bioactive glassy nanocoating with embedded nanospheres on bio-inert glass has not been reported before as a candidate for inert glass reinforced bone implants. In this work, using such surfactant assisted method, glassy nanospheres embedded hierarchically designed composite crack-free nanocoatings on glass with excellent adhesion were achieved. The complex morphologies of the nanocoatings were easily prepared by dip-coating method on bio-stable inert glass substrates followed by annealing at a relatively low temperature. The morphology, in vitro reactivity and carbonated hydroxyapatite growth, and crystallizations with respect to the silica dissolution of the coated samples were monitored. In addition, the cytotoxicity and cell growth behaviors of the nanocoatings were qualitatively studied. This work gives guidelines for developing hierarchically structured bioactive nanocoatings suitable for enhancing the attachment and bonding of inert glass reinforced implants with tissues and can be considered as pivotal toward the preclinical success of bone implants.
Crack-free bioactive nanocoatings embedded with uniformly distributed silica-rich bioactive spherical aggregates were successfully prepared in situ by controlling the micellization of a SiO2–CaO–P2O5 sol using the tri-block copolymer P123 followed by dip-coating onto a bio-inert glass substrate and calcined. These hierarchically designed nanocoatings embedded with such bioactive glassy nanospheres (BGNS) enabled to induce the deposition of a densely populated, uniform, and welldeveloped needlelike crystalline carbonated hydroxyapatite coating reminiscence of the mineral phase of natural bone within a short immersion time in simulated body fluid. The BGNS nanocoatings also supported the growth and attachment of human gingival fibroblasts. The results suggest that these newly designed composite nanocoatings are noncytotoxic, capable of supporting rapid and homogeneous calcium phosphate deposition as well as subsequent crystallization, and likely to be promising candidates for inert glass reinforced bone implants.
I.
B
Introduction
glasses are a group of materials having the capability to form crystalline apatite layer on their surfaces. This apatite layer being chemically similar with the mineral phase of the natural bone promotes bonding between the bioactive glass and the soft as well as hard tissues of the host.1,2 Accordingly, bioactive glass coatings on inert implants (e.g., inert glass reinforced bone implants) facilitate bonding between the implant and the surrounding tissue.1,3 Coatings can also help to maintain mechanical durability of the implant for longer period.4,5 Hench et al. showed for the first time that a certain range of SiO2 and CaO contents are essential for a glass to become bioactive for bone mineralization.6 Ternary silicate glasses are generally composed of SiO2 (45–60 wt%) with high CaO/P2O5 ratio.7 Whereas phosphorous does not play any direct role in bioactivity, a small amount helps the nucleation process of apatite crystals in body fluids.6 Recently, emphasis has been put on developing composite implants which compose of inert glass reinforcement, polymer, and bioactive glass.8–10 It is noteworthy here that typically cracks, poor densification and lack of adhesion can be observed in the bioactive glass coating due to the thermal stress or irregular interparticle spacing problems.3,4 The morphology of sol–gel derived bioacIOACTIVE
II.
Experimental Procedure
(1) Materials Tetraethylorthosilicate (TEOS, 98% pure) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (P123, Mw = 5800; CAS 9003-11-6) were purchased from Sigma-Aldrich (Steinheim, Germany). Triethylphosphate (TEP, 98% pure) was purchased from Spectrochem (Mumbai, India), calcium nitrate tetrahydrate (CN, 98% pure) was obtained from S.D Fine-chem. Ltd., (Mumbai, India) Ethanol (99.9% pure) and hydrochloric acid (HCl, 35%) for preparation of 0.1M HCl was purchased from Merck (Mumbai, India). Water used for hydrolysis was obtained from Milli-Q System (Millipore, Bangalore, India). Bio-inert glass substrates (3″ 9 1″) for coating purpose was purchased from Reviera
H.-E. Kim—contributing editor
Manuscript No. 35518. Received August 23, 2014; approved March 30, 2015. † Author to whom correspondence should be addressed. e-mail:
[email protected]
1
2
Journal of the American Ceramic Society—Das et al.
and the mean composition of the glass was analyzed by inductively coupled plasma-atomic emission spectroscopy (ICPAES) model Spectro Ciros Vision, Spectro Analytical Instruments GmbH, Kleve, Germany. The instrument was calibrated with the standard multicomponent glass samples (NBS 1412 and NIST-SRM 1831). For the analysis of substrate, 0.20 g of solid air dried glass powder was taken in platinum (Pt) basin and a mixture of 20 mL water, 2 mL perchloric acid (HClO4) and 5 mL hydrofluoric acid (HF) was added into it. The sample was digested and allowed to evaporate to dryness. The same procedure was repeated for three times, the final mass was digested in 1% nitric acid (HNO3) and volume was made up 100 mL. On the basis of the ICP-AES elemental analysis data, we have estimated the composition (wt%) of the Reviera glass substrate as: silicon dioxide (SiO2) 78.04%, aluminum oxide (Al2O3) 0.96%, ferric oxide (Fe2O3) 0.13%, titanium dioxide (TiO2) 0.03%, calcium oxide (CaO) 6.08%, magnesium oxide (MgO) 2.90%, sodium oxide (Na2O) 11.06%, and potassium oxide (K2O) 0.48%. It was assumed that the materials present in glass were in their oxide forms only.
(2) Preparation of Nanocoatings Ternary bioactive glass sol with equivalent composition SiO2 (58 mol%), CaO (38 mol%), phosphorus pentoxide (P2O5, 4 mol%) were prepared at room temperature (35°C) by hydrolysis and polycondensation reactions using TEOS, TEP, and CN. The sol–gel reactions were catalyzed using 0.1M HCl and required amount of ethanol was added as solvent. At first, a mixture of 12.08 g TEOS and 12 g ethanol was added to 8.35 g acid-water solution and magnetically stirred for 30 min to facilitate partial hydrolysis. Then, 1.46 g TEP was added into the hydrolyzed TEOS solution and stirred for an additional 30 min. Finally, 8.97 g CN dissolved in 5 g ethanol was mixed with the above solution and kept under stirring for 6 h to get final sol. To synthesize regular shaped nanospheres in the sol, 0.2 g P123 block copolymer was added to 10 g of the above sol and stirred for another 6 h. The homogeneous transparent sol thus obtained was used to prepare hierarchically designed bioactive glassy nanosphere embedded coatings (designated by “BGNS nanocoatings”). Another sol with same composition was prepared without P123 for the simple bioactive glass (BG) coatings as control. The clear sols were used for performing dip-coating (Dip-master 200, Chemat Corporation, Northridge, CA) on glass slides using the withdrawal velocity of 16 cm/min. As prepared films were aged at 60°C for 1 h and calcined at 500°C in air with a ramp of 2°C/min for 90 min in a silica glass tube furnace. (3) Characterizations of Nanocoatings The newly designed BGNS nanocoatings were characterized by several standard tests. Phase compositions of the samples coated on bio-inert glass were analyzed by grazing incident X-ray diffraction (GIXRD) method using a Rigaku SmartLab (Tokyo, Japan) X-ray diffractometer operating at 9 kW radiation in (200 mA, 45 kV) using CuKa (k = 1.54059 A) thin film mode at a scan rate 2° 2h s1. A grazing incident angle (x) of 0.3° was used for data collection so that the X-rays can penetrate only the uppermost layer (
