Engineering the surface functionality of 45S5 bioactive glass-based scaffolds by the heterogeneous nucleation and growth of silver particles
- First Online:
- 240 Downloads
An emerging topic in the field of biomaterials is the incorporation of silver ions, metallic silver or silver oxides into bioactive glasses to impart novel functionalities. In this work, a new approach of surface functionalization of 45S5 bioactive glass (BG) is introduced. In contrast to more common methods, which are based on the inclusion of silver during the sol–gel synthesis of BG, our method allows the surface functionalization of BG powders and BG scaffolds after their preparation. Hereby, we demonstrate the transferability of a previously reported approach on the wet chemical synthesis of cup-like and dendritic silver patches first from colloidal silica particles to BG particles and further to macroscopic highly porous BG scaffolds which were prepared by the sacrificial foam replica technique. The time-dependent silver releases of BG scaffolds with different silver loadings into simulated body fluid (SBF) were measured. Further studies were addressed to investigate the bioactivity of BG scaffolds before and after the silver coating procedure. It was found the silver deposition on 3D BG scaffolds did not affect the formation of crystalline hydroxyapatite during immersion into simulated body fluid.
Since L.L. Hench found in 1969 that a silicate glass composition (known as 45S5 Bioglass®) could bond strongly to bone , the field of BGs has been expended continuously and different types of glass substrates like BG powders [2, 3, 4], BG pellets [5, 6, 7] and macroscopic highly porous scaffolds [8, 9, 10, 11, 12] have been developed. BGs are characterized by their bioactivity, meaning the formation of crystalline hydroxyapatite (HA) on the surface of BG substrates when immersed into relevant physiological fluid like simulated body fluid (SBF), which makes BGs highly interesting biomaterials for bone regeneration [13, 14]. A current main focus in research is to include further functionalities into BG by incorporating biologically active ions , for example, lithium for cell stimulation in bone formation  or silver [17, 18, 19, 20, 21, 22, 23] and copper [24, 25, 26] for antibacterial activity. While in most cases silver-doped BGs get derived by the sol–gel method under the addition of a further silver salt like silver nitrate [17, 27, 28, 29], for non-bioactive antimicrobial substrates like glass or polymer fibers, also methods which are based on the immobilization of silver nanoparticles at their surface are reported [30, 31]. In contrast to these strategies, in the present work a new approach of surface functionalization of 45S5 BG-based scaffolds by heterogeneous nucleation of silver particles at the BG scaffolds surface is introduced. The process is based on the colloidal one-pot synthesis of silver patchy particles onto Stöber silica particles , as established in batch  and continuous-flow synthesis  by Klupp Taylor and co-workers. Within both approaches, colloidal silica particles (dp < 500 nm) were dispersed in silver nitrate and formaldehyde solution. By subsequent addition of diluted ammonia hydroxide solution, the reduction reaction between silver and formaldehyde was initiated, leading to heterogeneous nucleation and surface conformal growth of dendritic and cup-like silver patches.
Materials and methods
Preparation of bioactive glass scaffolds
BG scaffolds were prepared by the foam replica technique as established by Chen et al. . Briefly, BG powder of 45S5 composition from Schott AG was grinded using a Retsch (PM 100) zirconia ball planetary mill to adjust the median glass particle size to below 5 µm. 15.66 g of the derived powder was dispersed into a slurry containing 1.1 g polyvinyl alcohol (PVA) and 25 mL Millipore water and stirred for 30 min at 80 °C. After the slurry was allowed to cool down to room temperature, cubic 10 mm × 10 mm 45 ppi polyurethane (PU) foams (Eurofoam) were immersed into the slurry for 1 min. Afterward the excessive slurry was squeezed out of the foams. This whole procedure was repeated once to increase the loading of BG particles on the foams surface. The as-prepared soaked foams were dried overnight at 60 °C before being sintered in a high-temperature (Nabertherm P330) furnace. The temperature profile is shown in Fig. S1.
Silver coatings on bioactive glass powder
45S5 BG powder was coated by silver particles according to the method reported by Bao et al.  with minor changes in concentrations and core particles. Briefly, a suspension of 5.7 mg BG powder and 5 mL of silver nitrate at concentrations between 30 and 90 µM (see main text for details) was heated to 50 °C. Afterward, 30 µL of 37% formaldehyde solution was added. To initiate the silver reduction reaction, a total volume of 30 µL of 8% ammonia solution was added dropwise within 30 s under vigorous stirring.
Silver coatings on bioactive glass scaffolds via immersion technique
For the synthesis of silver patches on BG scaffolds, the volumes of all solutions used in the reaction described in "Silver coatings on bioactive glass powder" section were doubled. The BG scaffold, which was fixed with tweezers, was immersed into the silver nitrate solution while leaving a sufficient gap below it for vigorous stirring at 50 °C, as illustrated in Fig. 1. Following this, 60 µL of formaldehyde was added and subsequently 60 µL of 8% ammonia solution was added dropwise within 30 s under vigorous stirring. For samples which were multiply treated, the same procedure was applied up to five times, all solutions being freshly prepared for each silver coating step with a silver nitrate concentration of 90 µM.
Bioactivity and silver release of bioactive glass scaffolds
The formation of hydroxyapatite (HA) and the release of silver ions were investigated in simulated body fluid (SBF) which was prepared based on the work of Kokubo et al. . For the examination of the bioactivity and the silver release, a set of five BG scaffolds was prepared at the same conditions of the silver coating procedure. Each of these BG scaffolds was cut into four pieces of similar mass. Three of these pieces were immersed together into ~50 mL SBF by adjusting the mass concentration to 1.5 mg/mL and placed in an orbital shaker (KS 4000i control, IKA®) for up to 30 days at 37 °C. After different times, the precipitation of HA on the BG scaffolds was examined by FTIR, XRD and SEM image analysis. In addition, the silver release into the SBF supernatant above the BG scaffolds was investigated by inductively coupled plasma-optical emission spectroscope (ICP-OES).
For the investigation of the morphology and elemental composition of BG powder and BG scaffolds after silver particles synthesis, scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) analyses were performed using an Auriga FIB-SEM Instrument (Zeiss, Germany). For X-ray diffraction measurements (XRD) before and after the precipitation of HA on BG, a D8 Advance (Bruker AXS, Germany) device in Bragg–Brentano geometry with a Cu Kα radiation source (λ = 0.154058 nm) was used. XRD patterns in the 2θ range of 10°–70° were recorded. For Fourier transform infrared spectroscopic measurements (FTIR) with a Nicolet 6700 FTIR spectrophotometer (Thermo Scientific USA), KBr pellets with 1 wt% of crumbled BG scaffolds were prepared. The silver content in SBF was measured by using Perkin Elmer Optima 8300 inductively coupled plasma-optical emission spectroscope (ICP-OES).
Results and discussion
Patchy silver coating on bioactive glass powder
Patchy silver coating on bioactive glass scaffolds
Similar as presented for BG powder, the silver nitrate concentration for the silver coating on BG scaffolds was experimentally optimized within a first set of experiments at different silver nitrate concentrations ranging from 60 to 150 µM (shown in Fig. S2). It can be seen that after silver coating the BG scaffolds turned different shades of gray depending on the silver nitrate concentration. Interestingly, an increase in darkness of the scaffold was just observed with an increase in the silver nitrate concentration from 60 to 90 µM. The scaffolds treated by silver coating at higher silver nitrate concentrations of 120 and 150 µM showed a less intensive gray color, indicating a deposition of less silver. We assume the surface exposed by one scaffold and the overall undefined mixing conditions during the silver coating procedure lead to a higher nucleation rate and consequently to the occurrence of more homogeneous nucleation in the scaffold surrounding aqueous bulk phase. From this preliminary set of experiments, we conclude that a concentration of 90 µM silver nitrate leads to the optimum ratio of heterogeneous nucleation at the scaffolds surface to homogeneous nucleation in the bulk phase of silver particles.
Silver release of bioactive glass scaffolds
Bioactivity of bioactive glass scaffolds
Within this work, it could be shown that a simple technique using the principle of heterogeneous nucleation of silver particles on a silica-related surface can be transferred from a colloidal synthesis using silica particles to BG powder and further to the coating of macroscopic highly porous structures like 3D 45S5 BG scaffolds. The FTIR analysis before silver coating of a plain 45S5 BG scaffold gave an insight into the present surface chemistry which is governed by siloxane and calcium phosphate groups. We believe, in contrast to our earlier work with colloidal silica particles, whose surface is governed by hydroxyl groups , the presence of different surface groups on 45S5 BG results in no surface conformal silver patch growth. However, by iteration of the silver coating procedure the amount of deposited silver could be increased. This allowed the tailoring of the absolute and time-dependent silver release of BG scaffolds in simulated body fluid. By SEM, FTIR and XRD measurements, the independence of the bioactivity on the silver coating reaction could be demonstrated. Future work is addressed to the manipulation of the surface chemistry of the BG scaffolds and advanced process design to achieve surface conformal diffusion limited growth of dendritic silver patches. In addition, electrical conductivity measurements and cytocompatibility tests will be conducted.
The authors are grateful to Fabrizio-Zagros Sadafi for fruitful discussions and for financial support from the Cluster of Excellence “Engineering of Advanced Materials” which is funded by the German Research Foundation (DFG) within the framework of the German Federal and State Excellence Initiative. V. Miguez Pacheco and Aldo R. Boccaccini acknowledge the European Commission funding under the 7th Framework Programme (Marie Curie Initial Training Networks; Grant Number 289958, “Bioceramics for bone repair”).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 5.Kontonasaki E, Zorba T, Papadopoulou L, Pavlidou E, Chatzistavrou X, Paraskevopoulos K, Koidis P (2002) Hydroxy carbonate apatite formation on particulate bioglass in vitro as a function of time. Cryst Res Technol 37:1165–1171. doi:10.1002/1521-4079(200211)37:11<1165:AID-CRAT1165>3.0.CO;2-R CrossRefGoogle Scholar
- 7.Bretcanu O, Misra SK, Yunos DM, Boccaccini AR, Roy I, Kowalczyk T, Blonski S, Kowalewski TA (2009) Electrospun nanofibrous biodegradable polyester coatings on Bioglass®-based glass-ceramics for tissue engineering. Mater Chem Phys 118:420–426. doi:10.1016/j.matchemphys.2009.08.011 CrossRefGoogle Scholar
- 16.Miguez Pacheco V, Büttner T, Maçon A, Jones JR, Fey T, de Ligny D, Greil P, Chevalier J, Malchere A, Boccaccini AR (2016) Development and characterization of lithium-releasing silicate bioactive glasses and their scaffolds for bone repair. J Non-Cryst Solids 432:65–72. doi:10.1016/j.jnoncrysol.2015.03.027 CrossRefGoogle Scholar
- 24.Hoppe A, Meszaros R, Stähli C, Romeis S, Schmidt J, Peukert W, Marelli B, Nazhat SN, Wondraczek L, Lao J, Jallot E, Boccaccini AR (2013) In vitro reactivity of Cu doped 45S5 Bioglass® derived scaffolds for bone tissue engineering. J Mater Chem B 1:5659–5674. doi:10.1039/c3tb21007c CrossRefGoogle Scholar
- 26.Wu C, Zhou Y, Xu M, Han P, Chen L, Chang J, Xiao Y (2013) Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity. Biomaterials 34:422–433. doi:10.1016/j.biomaterials.2012.09.066 CrossRefGoogle Scholar
- 27.Bellantone M, Coleman NJ, Hench LL (2000) Bacteriostatic action of a novel four-component bioactive glass. J Biomed Mater Res 51:484–490. doi:10.1002/1097-4636(20000905)51:3<484:AID-JBM24>3.0.CO;2-4 CrossRefGoogle Scholar
- 43.Macon ALB, Kim TB, Valliant EM, Goetschius K, Brow RK, Day DE, Hoppe A, Boccaccini AR, Kim IY, Ohtsuki C, Kokubo T, Osaka A, Vallet-Regi M, Arcos D, Fraile L, Salinas AJ, Teixeira AV, Vueva Y, Almeida RM, Miola M, Vitale-Brovarone C, Verne E, Holand W, Jones JR (2015) A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants. J Mater Sci Mater Med 26:1–10. doi:10.1007/s10856-015-5403-9 CrossRefGoogle Scholar
- 44.Berzina-Cimdina L, Borodajenko N (2012) Research of calcium phosphates using Fourier transform infrared spectroscopy. In: Theophanides TM (ed) Infrared spectroscopy—materials science, engineering and technology. Rijeka, InTechGoogle Scholar