Acta Biomaterialia 7 (2011) 3606–3615

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Acta Biomaterialia

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Protein interactions with nanoporous sol–gel derived bioactive glasses

Sen Lin a, Wouter Van den Bergh a, Simon Baker b, Julian R. Jones a,⇑a Department of Materials, Imperial College London, London SW7 2AZ, UKb MedCell Bioscience Ltd., Barbraham, Cambridge CB2 4AT, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 November 2010Received in revised form 21 June 2011Accepted 22 June 2011Available online 30 June 2011

Keywords:Bioactive glassSol–gelNanoporousProtein adsorptionFibrinogen

1742-7061/$ - see front matter � 2011 Acta Materialdoi:10.1016/j.actbio.2011.06.042

⇑ Corresponding author. Tel.: +44 2075946749.E-mail address: julian.r.jones@imperial.ac.uk (J.R.

Sol–gel derived bioactive glasses are excellent candidates for bone regenerative implant materials as theybond with bone, stimulate bone growth and degrade in the body. Their interactions with proteins are crit-ical to understanding their performance after implantation. This study focuses on the interactionsbetween fibrinogen and sol–gel glass particles of the 70S30C (70 mol.% SiO2, 30 mol.% CaO composition).Sol–gel silica and melt-derived Bioglass� were also used for comparison. Fibrinogen penetration into thenanoporous glasses was observed by live tracking the fluorescent-labelled fibrinogen with confocalmicroscopy. The effect of pore size on protein penetration was investigated. Nanoporous networks withmodal pore diameters larger than 6 nm were accessible to fibrinogen. When the modal nanopore diam-eter was decreased to 2 nm or less, the penetration of fibrinogen was inhibited. The surface properties ofthe glasses, which can be modulated by media pH, glass composition and final stabilisation temperaturein the sol–gel process, have effects on fibrinogen adsorption via long-range Coulombic forces before theadsorption and via short-range interactions such as hydrogen bonding after the adsorption.

� 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Bioactive glasses are one of the most promising materials forbone regeneration scaffolds [1,2]. The glasses bond with boneand stimulate new bone growth via glass dissolution products[3]. Sol–gel derived bioactive glasses are nanoporous and havemuch larger surface areas compared to melt-derived glasses, whichenhances bioactivity compared to similar compositions of melt-derived glasses, due to the accelerated ion release and higher den-sity of exposed Si–OH groups [4,5]. The nanoporosity and Si–OHgroups provide nucleation sites for hydroxycarbonate apatite(HCA) formation, which forms on the surface of the glasses afterthey contact body fluid. The HCA layer bonds with bone [1].Another advantage of the sol–gel process is that scaffolds can beproduced with an interconnected macropore structure by foaming[6,7].

Proteins in the physiological environment interact with materi-als soon after implantation [8] and cell–material interactions aremediated by the adsorbed protein layer [9]. For example, adsorbedproteins can also delay the dissolution of a glass and HCA forma-tion [10]. The surface properties of the implanted materials deter-mine the composition and conformation of the adsorbed proteinlayer, which in turn direct cell activities via integrin–ligand inter-actions [9]. It is therefore critical to understand the interactionsbetween proteins and implanted materials.

ia Inc. Published by Elsevier Ltd. A

Jones).

Nanoporous silica is also often used for the controlled deliveryof protein molecules or anticancer drugs [11–13]. It is thus impor-tant to maintain the functions of the pre-loaded proteins withinthe implanted biomaterials. Protein delivery [14] using sol–gelderived materials have been extensively studied [15]. However,most of the studies were conducted by loading proteins beforethe drying stage of the sol–gel process. The drying stage coulddenature the proteins, and the pre-loaded proteins preclude thehigh-temperature stabilization treatment (600–700 �C) of theglasses [16]. Very few studies have focused on the protein upload-ing onto stabilized sol–gel derived bioactive glasses. Lenza et al.[17] studied the protein adsorption onto and release from stabi-lized sol–gel derived glass scaffolds, but the glass surfaces werechemically modified with amide groups, which have covalentbonds to protein molecules, which could denature the proteins.

In this study, fibrinogen (Fg) was taken as a model molecule tostudy its interactions with bioactive glasses with different porosi-ties and surface chemistries. Fg was chosen due to its key role inhaemostasis and fibrous capsule formation. There have also beensuggestions that bioactive glasses can accelerate wound healing[18,19], in which Fg plays a key role. Fg is often neglected in pro-tein adsorption studies since it is a plasma protein rather than aserum protein, but Fg concentration in plasma is much higher thanthe more often studied fibronectin and laminin. In orthopaedicoperations, the dominant adsorbed protein on implanted materialsis Fg since Fg can cause fibrous encapsulation of implants [20–23].

One aim of this study was to investigate the interactionsbetween Fg and 45S5 Bioglass, a commercial product (NovaBone�,

ll rights reserved.

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Alachua, FL), which has been demonstrated to successfully preventfibrous encapsulation in vivo [24]. The main aim was then to inves-tigate how sol–gel glasses, which have inherent nanoporosity, inter-act with Fg. One hypothesis was that the uptake of Fg into thenanoporous glasses would be dependent on nanopore size. Anobjective was therefore to determine the accessibility of the nano-pore network to Fg molecules as a function of nanopore size, i.e.whether Fg penetrates into a pores of a given modal pore diameter.A second hypothesis was that an increase in nanoporosity would en-hance protein adsorption to the surface. Therefore, a second objec-tive was to study the kinetics of Fg adsorption onto and release fromthe nanoporous glasses. Our previous work characterized the evolu-tion of atomic structure and nanopores throughout sol–gel glasssynthesis. High-resolution microscopy showed that the pores arethe interstices that result from an assembly of silica nanoparticlesduring gelation. Sintering the glasses to higher temperature reducedthe nanopore size, but also densified the glass matrix and changedthe surface chemistry [25]. Changes in nanoporosity affect glass sur-face area and therefore dissolution rates, which directly affects thepH of the medium. pH is known to strongly influence protein inter-actions. The final objective was therefore to investigate the effect ofpH without changing the surface of the material. Decoupling pHfrom changes in surface will determine whether changing the nano-structure of a glass affects the interactions with the Fg directly, orwhether it is an indirect effect of the different nanostructure chang-ing pH. These studies are important for the further research on theinteractions between adsorbed proteins and apatite growth ontosol–gel derived bioactive glasses.

2. Materials and methods

2.1. Materials

Five groups of glass particles were prepared for protein adsorp-tion tests. Three sol–gel glasses with the 70 mol.% SiO2 and30 mol.% CaO (70S30C) composition were produced with differentfinal stabilization temperatures of 600 �C (70S600), 700 �C(70S700) and 820 �C (70S820), providing three different nanopo-rosities. They were compared to sol–gel silica (100S) and commer-cial melt-derived 45S5 Bioglass� (BG). The compositions of thesamples are shown in Table 1. The 70S30C samples were synthe-sized using the standard process of 70S30C monoliths (detailedin previous studies [26]) except that they were stabilized at differ-ent temperatures (Table 1). 100S was also synthesized based onthe standard process of 70S30C monoliths without the additionof a calcium source [26]. Bioglass� was provided by NovaBoneProducts LLC (Alachua, FL, USA). To standardize the particle sizes,all of the glass samples were ground with a ball mill and sievedto below 38 lm.

2.2. Materials characterization

The surface area and nanoporosity of the sol–gel derivedsamples were characterized by using nitrogen sorption (Autosorb6, Quantachrome Instruments, UK). The BJH (Barrett–Joyner–

Table 1The compositions and stabilization temperatures of the glasses investigated.

Samples Composition (mol.%) Stabilization temperature (�C)

BG 46.1 SiO2, 26.9 CaO, 24.4Na2O, 2.5 P2O5

N/A

70S600 70 SiO2, 30 CaO 60070S700 70 SiO2, 30 CaO 70070S820 70 SiO2, 30 CaO 820100S 100 SiO2 600

Halenda) [27] and BET (Brunauer–Emmett–Teller) [28] methods,as detailed in our previous study [25], were applied to determinethe nanopore size distribution [29] and the specific surface area,respectively. The samples for nitrogen sorption were prepared bydegassing for 8 h at 150 �C. The zeta potentials of the glass particleswere measured with a Zetasizer Nano ZS (Malvern, UK).

2.3. Fibrinogen uptake into porous glasses

Fluorescent-labelled Fg (Fg from human plasma, Alexa Fluor�

488 conjugate, F13191, Invitrogen™, UK) and laser scanning confo-cal microscopy (LSCM, Zeiss LSM-510 inverted, UK) were used tostudy Fg penetration into the pore structure of the glasses. Glasspowders with various sintering temperatures (70S600, 70S700,70S820) were used; calcium-free 100S was also used for compari-son. To test the penetration of Fg molecules, 0.02 g of glass parti-cles was immersed in 0.9 ml of 0.1 mg ml�1 fluorescent-labelledFg PBS solution for 10 min before images were taken (no washingapplied). The Fg distribution was determined by characterizing thefluorescence intensity within the particle cross-sections at a rangeof levels along z-axis (‘‘stack imaging’’). The effect of nanopore sizeas a result of final sintering temperature on Fg penetration wasinvestigated.

2.4. Live and in situ tests

The effects of three individual factors on the Fg adsorption wereinvestigated: glass type (dense BG and nanoporous sol–gel);nanoporosity as a result of final sintering temperature (70S600,70S700, 70S820); and then initial Fg concentration, and mediumpH. 0.02 g of glass particles were immersed in 0.9 ml of0.1 mg ml�1 fluorescent-labelled Fg PBS solution for less than1 min; images were then taken while the glass powders were stillimmersed in the Fg solution. A range of initial Fg concentrations(0.05, 0.1, 0.2 and 0.4 mg ml�1) was used. Medium pH wasadjusted prior to immersion using HNO3 or NaOH solution. Fgadsorption was monitored in situ by tracking the fluorescent-labelled Fg under the confocal microscope. The glass/solution ratiowas identical with that in the penetration tests. All the measure-ments were carried out at room temperature.

Live release tests were also conducted by immersing 5 mg70S700 glass particles in 10 ml 1 mg ml�1 Fg solution for 10 min.The particles were then washed with PBS twice before immersionin 3 ml PBS for 2 h, during which images were taken with confocalmicroscope. The fluorescent intensity within individual particleswas quantitatively tracked. The effect of the medium pH on Fgrelease was also tested by adjusting the medium pH with NaOHafter the proteins and the glasses had been mixed.

2.5. Solution-depletion protocol

To decouple surface chemistry from pH changes as a function ofnanoporosity, a solution-depletion protocol was used to investi-gate the effects of medium pH and initial Fg concentration on Fgadsorption onto a glass of a fixed nanoporosity (70S700). HumanFg (F4883, Sigma, UK, used as received) was used as a model mol-ecule for the solution-depletion protocol. Standard phosphate buf-fer solution (pH 7.14, BP665-1, Fisher, UK), was used as a solvent toprepare the Fg solution. 9 ml of Fg solution (with various initial Fgconcentrations: 0.05, 0.1, 0.2 and 0.4 mg ml�1) was mixed with0.2 g of glasses (the identical glass/solution ratio to that used inthe penetration tests). Protein concentration was quantified byusing a UV–Vis spectrometer (Lambda 25, PerkinElmer, UK)5 min after the mixture. The effects of medium pH on the extinc-tion coefficients of Fg (the extinction coefficient of Fg is 15.1 when

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the medium pH is 7.24, as provided by the Fg manufacturer) werecharacterized. All the measurements were conducted in triplicate.

2.6. Statistical analysis

Student’s t-test (two tailed distribution and two-sampleunequal variance) was used for the statistical analysis in this work.

3. Results and discussion

3.1. Glass characterization

The nitrogen sorption results of various types of the glasses areshown in Table 2 and the nanopore size distributions of nanopor-ous samples are shown in Fig. 1. There is no pore size distributionfor the Bioglass� as it was not porous. The nanopores in 100S weremuch smaller (<2 nm) than those in the 70S30C samples but thesurface area of 100S was much larger. For the 70S30C samples,when the stabilization temperature increased from 700 to 820 �C,the modal nanopore size reduced from 17 nm (70S700) to 6 nm(70S820) and the surface area reduced from 140 to 25 m2 g�1.Reduction in pore size is due to the viscous flow, when the temper-ature approaches 800 �C, due to the temperature surpassing theglass transition temperature of the glass [25].

The zeta potentials of the glass particles and their effects on themedium pH are shown in Table 2 (0.2 g of glass particles immersedin 9 ml of PBS for 5 min). For all 70S30C samples and BG, the zetapotentials were negative and the medium pH values increased dueto cation exchange effects, i.e. cations (e.g. Ca2+) that leave theglass are replaced by H+ from the solution [30] (Eqs. (1) and (2) be-low). No metallic cations are present in 100S, only H+. Although thezeta potential of 100S was also negative, the medium pH slightlydecreased instead of increasing, which could be due to the dissolu-tion of the silica and the release of small amounts of silicic acid

Table 2Nanoporosity and zeta potential of the glasses and their effects on the medium pH.

Samples Modal pore size(nm)

Surface area(m2 g�1)

Zeta potential(mV)

MediumpH

BG NA NA �19.0 ± 1.0 10.370S600 17.4 ± 0.2 173 ± 3 �26.6 ± 1.4 10.370S700 17.6 ± 0.1 140 ± 6 �19.9 ± 2.0 10.170S820 6.1 ± 0.5 25 ± 3 �30.8 ± 1.1 9.0100S <2 548 ± 14 �19.7 ± 0.8 6.7

Fig. 1. Nanopore size distribution of the glasses, obtained by nitrogen sorption. Thevertical axis is a derivative of the volume of nitrogen adsorbed into the surface ateach pore diameter, per gram of glass.

Si—O�þCaþHþ ! Si—OHþ Ca2þ ð1Þ

Si—O�þNaþHþ ! Si—OHþ Naþ ð2Þ

As shown in Table 2, the modifying effects of the 70S30C glasseson the medium pH were reduced as the stabilization temperatureof the glasses increased. This is because the higher stabilizationtemperature of the glasses caused further condensation reactionsbetween the surface OH groups, and viscous flow (above 700 �C),which increased the network connectivity and reduced the surfacearea [25]. The increased connectivity decelerates the cationexchange and the glass dissolution. The modified medium pHwould in turn affect the zeta potential of glass since a balanceexists between the medium pH and the zeta potentials of the glassparticles. The balance is demonstrated in the reversible reactionshown in Eq. (3) below. The zeta potentials of implanted materialsare critical in terms of protein adsorption since the Coulombicforce is one of most important interactions between proteins andsolid surfaces [31]

SiOHþ OH� $ SiO� þH2O ð3Þ

3.2. Fibrinogen uptake into porous glasses

The confocal microscopy images (z stack view) in Fig. 2a–d weretaken after immersing the different nanoporous glasses (70S600,70S700, 70S820, 100S, respectively) in fluorescent-labelled Fgsolution to visualize any penetration of the Fg molecules into thenanopores. The intervals between the cross-sections were 0.76,0.38, 0.76, 1.1 and 1.6 lm in Fig. 2a–e, respectively. Identicaladsorption conditions (glass/solution ratios) were applied in allthe tests except the medium pH value in the first 70S600 test(Fig. 2a) was pre-adjusted from 10.3 to 8.6, due to the high concen-tration of calcium ions released by the 70S600 particles causing arapid increase in pH. As pore diameter was the primary factor tobe investigated, the pH was adjusted before the Fg was added.The images illustrate the fluorescent intensity within cross-sections at a range of levels along the z-axis, which indicates the3-D distribution of Fg within nanoporous glass particles.

The results indicate that Fg could penetrate into the inner nanop-ores in 70S600, 70S700 and 70S820, whereas penetration was notpossible in 100S (Fig. 2). Therefore, nanoporous networks with mod-al pore diameters of 6 nm or above allowed Fg penetration, but the2 nm pores were too small. The penetration of protein moleculeswas reported in previous studies [32–34]. The penetration of Fgproves that the nanopores within the sol–gel derived bioactiveglasses are interconnected. This is in good agreement with theconclusion drawn in a previous study, i.e. the nanopores are derivedfrom the interstitial spaces between the tertiary particles [25].

Sintering at higher temperatures reduced surface area butincreased adsorption, which may suggest that the adsorption perunit area was even stronger. However, it is more complicated inthat the ‘‘unit area’’ should be referred to the area available foradsorption of Fg molecules. Some ‘‘unit area’’ that is measured byBET is not accessible for Fg molecules, especially the surface areaof the inner tiny nanopores (smaller than the Fg molecule, whichcannot reach and adsorb onto the surface). Unfortunately, the areaavailable for Fg molecule penetration and adsorption is extremelydifficult to quantify. Therefore, the unit ‘‘adsorption/glass mass’’ isused throughout this paper.

Fg molecules have approximate dimensions of 4.5 � 47 nm. Dueto their high flexibility and long length, Fg molecules are denaturedonce adsorbed onto glass surfaces. Due to the rapid kinetics of Fgadsorption (a fraction of 1 s), it is extremely difficult to determinethe sequence of the alkaline-induced denaturing and adsorption-induced denaturing. Since this study focuses on the Fg adsorption

Fig. 2. Confocal microscope images (cross-section view at a range of levels along z-axis, ‘‘stack view’’) showing the distribution of Fg within various types of the glass samples.(a) 70S600 (the medium pH adjusted from 10.3 to 8.6), (b) 70S700, (c) 70S820, (d) 100S, (e) 70S600 immersed in Fg solution with higher initial Fg concentration (1 mg ml�1).The intervals between the cross-sections are 0.76, 0.38, 0.76, 1.1 and 1.6 lm, in (a)–(e), respectively.

S. Lin et al. / Acta Biomaterialia 7 (2011) 3606–3615 3609

and the static electric interactions between the Fg and glasses, thedenaturing effects of the highly alkaline environment or the statusof the Fg molecules before the adsorption was not considered here.

The results of the penetration tests reveal that nanoporous sol–gel derived glasses (modal pore size > 6 nm) have much largersurface areas accessible for protein adsorption per unit mass com-pared to normal solid materials. Although the inner nanopores arenot accessible for cells, the enlarged interface between the nano-porous network of the sol–gel derived glasses could significantlyenhance the interaction between the physiological environmentand implanted materials via adsorption of active molecules andrelease of ions.

3.3. Live and in situ tests

3.3.1. Adsorption testsThe confocal microscopy images (low-magnification view) in

Fig. 3a–e show the Fg adsorption on various types of the glasses(BG, 70S600, 70S700, 70S820, 100S, respectively). The left halvesof the images show the fluorescent signals from the Fg molecules,whereas the right halves show the transmitted signals from theglass particles. Identical initial Fg concentrations and glass/solu-tion ratios (the same as those used for the penetration tests) wereused in all the images. Identical magnification, contrast and bright-ness were applied in all the images. All the images in Fig. 3 were

Fig. 2 (continued)

3610 S. Lin et al. / Acta Biomaterialia 7 (2011) 3606–3615

taken within 1 min after the glasses and proteins were mixed andall images were taken while the glass powders were immersed inthe protein solution.

The images indicate that Fg adsorption on BG and 70S600 wasnot noticeable, but the amount of Fg adsorbed onto 70S700 and70S820 was much higher. Fg adsorption onto 100S was also higherthan BG and 70S series glasses and concentrated on the outer sur-face, which was consistent with the penetration tests (Fig. 2d). Theimages also indicate that Fg adsorption occurred rapidly (within1 min), which is consistent with literature [35]. The Fg adsorptiondifference between various types of the glasses can be explained bytwo reasons: Coulombic forces and the hydrophobicity of the glasssurfaces.

Coulombic forces, one of the critical interactions between Fgand the glasses, are determined by the charges of the glass surfacesand the Fg molecules. The charges are modulated by the mediumpH. Since the glasses have different effects on the medium pHvia cation exchange (Table 2), the glasses exhibit different degreesof Fg adsorption via Coulombic forces. The medium pH was consid-erably increased by 70S600 and BG (from 7.2 to 10.3), due to rapiddissolution of Ca2+ and Na+ ions, respectively, which caused build-up of negative charge on the glass surfaces (shown by the zeta po-tential data in Table 3) and on the Fg molecules (the isoelectricpoint (IEP) of Fg molecules is 5.8 [36]). The repulsive forces be-tween the negatively charged solid surfaces and the negativelycharged Fg molecules then effectively inhibit the Fg adsorption.

Fig. 2 (continued)

Fig. 3. Confocal microscopy images showing the Fg adsorption onto various types of glass samples (left halves of the images show the fluorescent signals from Fg and theright halves show the transmitted signals from the glass particles). (a) BG, (b) 70S600, (c) 70S700, (d) 70S820 and (e) 100S.

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Fig. 4. Confocal microscopy image showing the significantly enhanced Fg adsorp-tion onto BG after the medium pH was decreased from 10.3 to 8.6 by adding 0.1 mlof 0.1 M HNO3 into the suspension (left half of the image shows the fluorescentsignals from Fg and the right half shows the transmitted signals from the glassparticles).

Table 3The combined effects of 70S700 glass particles (cation exchange) and additives (acidor base solutions) on the medium pH and the zeta potentials of the glasses.

Additives pH values Zeta potential (mV)

10 vol.% 1 M NaOH 12.0 �21.9 ± 1.02 vol.% 0.1 M HNO3 10.0 �23.2 ± 0.65 vol.% 0.1 M HNO3 9.6 �24.0 ± 1.510 vol.% 0.1 M HNO3 8.6 �6.1 ± 0.4

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As dissolution rate decreased as sintering temperature increased,due to changes in the pore and glass networks reducing surfacearea, the pH increase was less, reducing the repulsion betweenthe 70S surface and the Fg, increasing adsorption. For 100S, thenegative charge on the both sides was reduced due to the reducedmedium pH (from 7.2 to 6.7) and the reduced repulsive forces en-hanced the Fg adsorption. This confirms the conclusion drawn byprevious studies on the interactions between zeta potential andprotein adsorption [37].

The zeta potential data suggests 100S was less hydrophilic thanthe 70S30C glasses due to the absence of cation exchange effects.The lower hydrophilicity enhances the Fg adsorption on 100S, asfewer water molecules have to be displaced prior to adsorptionof the Fg, which is in a good agreement with this literature [38].The adsorption on 70S700 (Fig. 3c) seems to be less homogeneousthan for 70S820 (Fig. 3d). This may be due to larger and more het-erogeneous pores in the 70S700 glass (Fig. 1). The Fg adsorptioncomparison between 70S600, 70S700 and 70S820 is discussed fur-ther in the solution-depletion section.

The confocal microscopy image in Fig. 4 shows the fluorescentintensity on the BG particles in Fg/BG suspension after reducingthe medium pH from 10.3 to 8.6 with HNO3. Since the mediumpH was still higher than 7.4 after the acid addition, the proteinwas not denatured. The image indicates the significant increaseof Fg adsorption, as compared to Fig. 3a, when the medium pHwas decreased. This confirms the role of the medium pH on Fgadsorption: it is the medium pH that modified the surface chargeson the glasses and the Fg molecules, which reduced the repulsiveinteractions (Coulombic forces) and enhanced the Fg adsorption.In the case of BG, the high release of Na+, which are weakly boundin the glass network caused the high pH rise.

3.3.2. Release testsThe confocal microscopy images in Fig. 5a show the fluorescent

signal from an individual 70S700 particle that was pre-loaded withfluorescent-labelled Fg and pre-washed with PBS twice at differenttime points (taken every 10 min). The glass particle was soaked in

fresh PBS for over 2 h, during which the images were taken. Fig. 5bshows the mean fluorescent intensity per unit surface of cross-sections in the field of view (the bottom trace, indicating thesystematic bleaching) and the cross-section of the particle (thetop trace, indicating the variation of adsorbed Fg). As the intensitywas essentially constant, there was neither noticeable Fg desorp-tion nor systematic bleaching throughout the release test. Theweak desorption is due to the irreversible contact interactionsbetween Fg and the glasses such as hydrogen bonding and hydro-phobic interactions. Each Fg molecule (MW 340 kDa) adsorbs ontosolid surface via 703 contact points [39] and the molecules canonly desorb, theoretically, when all of the contact points aredetached, which is unrealistic. The results conflict with previousstudies [17]. This could be due to the lack of washing treatment be-fore the release tests in the previous studies, resulting in the re-lease of non-adsorbed proteins which would be confused asbeing desorbed proteins.

The image in Fig. 5c illustrates the release tests conducted byincreasing the medium pH values from 10.1 to 12.0 with NaOH.The 70S700 particles in the image were pre-loaded with Fg andwashed with PBS twice before immersion in fresh PBS solution.Since the fluorescent intensity on the particles was not noticeablydecreased after the medium pH was increased to 12.0, the desorp-tion of Fg was not detectable. However, the results in solution-depletion section show that unnoticeable Fg adsorbed onto70S700 if the medium pH was pre-adjusted to 12.0. This indicatesthat Coulombic forces, which dominate Fg adsorption, do not dom-inate Fg desorption and the dominant interactions switch intoirreversible hydrophobic interaction. This is discussed in thesolution-depletion section.

3.3.3. Solution-depletion protocolFig. 6 shows the amounts of Fg adsorption onto various glasses

with a range of initial Fg concentrations. Traditionally, proteinadsorption is calculated by adsorption per surface area. Since thesurface area accessible for Fg cannot be quantified by gas sorption(N2 droplets can access where Fg molecules cannot), the Fg adsorp-tion per unit mass of the glasses was calculated. The results indi-cate that the Fg adsorption considerably increased as the initialFg concentration increased.

Fig. 6 also shows that the Fg adsorption onto 70S30C samplesincreased as the stabilization temperatures of the glassesincreased: 70S600 (negligible adsorption, not shown in the fig-ure) < 70S700 < 70S820. The Fg adsorption difference could beexplained by the two reasons discussed in the live test section,namely the Coulombic forces and surface hydrophobicity. Increas-ing the stabilization temperature decreased the surface area of the70S30C glasses (Table 2) and thus increased the medium pH to alesser extent, which resulted in less repulsive forces between70S700 and Fg (zeta potential measurements, Table 2). However,the higher temperature also increases the connectivity of the silicanetwork and decreases the surface OH density, which decreasesthe hydrophilicity. These two reasons can also explain why theFg adsorption onto 100S is higher than 70S820 since dissolutionof 100S decreased the medium pH to 6.7 (dissolution of 70S820 in-creased the pH to 9.0) and has higher hydrophobicity than 70S820.This was the reason that effects of pH change of the medium had tobe investigated for one type of glass.

To isolate the effects of the medium pH on the Fg adsorption,the medium pH values were controlled by manually adding variousamounts of acid and base solutions into the medium. 70S700 wastaken as model glass for the tests. The medium pH was modifiedbefore the mixture with Fg and the details are shown in Table 3.The results of the medium pH effects on Fg adsorption are shownin Fig. 7, which clearly indicates that the Fg adsorption on70S700 was increased as the medium pH decreased. This further

Fig. 5. (a) Confocal microscopy images showing the fluorescent signal from an individual 70S700 particle that was pre-loaded with fluorescent-labelled Fg and pre-washedwith PBS twice at different time points (taken every 10 min). The glass particle was soaked in fresh PBS for over 2 h; (b) The mean fluorescent intensity per cross-section areaof the field of view (indicating the bleaching of the system) and particle cross-section (indicating the Fg release), respectively (intensity measured every 10 min); (c) Confocalmicroscopy image showing the fluorescent signals of 70S700 particles in 70S700/Fg suspension after 0.1 ml of 1 M NaOH was added into the suspension (left half of the imageshows the fluorescent signals from Fg and the right half shows the transmitted signals from the glass particles).

S. Lin et al. / Acta Biomaterialia 7 (2011) 3606–3615 3613

confirms the effects of the Coulomb forces and the role of the med-ium pH.

As Fig. 7 shows, when the pH reached 12.0, the Fg adsorptiononto 70S700 became unnoticeable. This indicates that the repul-sive forces became so large at pH 12.0 that negligible amounts ofFg adsorbed onto 70S700 particles. However, when the pH valuewas changed into 12.0 after Fg adsorption, the proteins did notdesorb from the surfaces, as shown in Fig. 5c. This can be explained

by the irreversible Fg adsorption discussed in the live test section.The Coulomb forces are no longer dominant interactions after theprotein attachment. This is because the Coulomb forces are long-range interactions and the forces could effectively inhibit the Fgadsorption before the attachment. However, the dominant interac-tions switch to short-range interactions such as hydrogen bondingand hydrophobic interactions after the Fg attachment. The effect isconsistent for other glasses. Fig. 2e confirms this as 70S600

Fig. 6. Quantitative Fg adsorption results onto various types of glass samples(70S700, 70S820 and 100S; the Fg adsorption onto BG and 70S600 was negligibleand is not shown in the figure) at a range of initial Fg concentrations, obtained withthe solution-depletion protocol. ⁄P < 0.05; ⁄⁄P < 0.01.

Fig. 7. Quantitative Fg adsorption results onto 70S700 at a range of the medium pHvalues, obtained with solution depletion protocol. ⁄P < 0.05; ⁄⁄P < 0.01.

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particles adsorbed few Fg molecules at non-pre-adjusted mediumpH and the adsorption became significant after pH reduction from10.3 to 8.6.

In summary, protein adsorption to sol–gel glasses is reduced byhigher nanopore size and lower silica network connectivity due toincreased cation dissolution. Protein adsorption can therefore becontrolled to some extent by choosing a final sintering temperature.

4. Limitations

There are a number of limitations of this system with respect tomimicking the protein adsorption environment. Firstly, PBS solu-tion, the adsorption medium, has an insufficient buffer capacityin terms of the medium pH and Ca2+ concentration since these fac-tors in the real physiological environment are kept homeostatic viathe circulatory system. PBS instead of simulated body fluid (SBF)was used in order to avoid the interference with UV signals, whichis derived from the organic contents in SBF (Tris). Secondly, theratios of glass over buffer solution are considerably large, whichresults in a significant impact on the medium pH values. This isbecause the high ratios can result in large differences betweenthe initial and final Fg concentrations (solution-depletion proto-col), which can compensate for the detection limits of the UVsystem. Finally, protein complexation within the physiologicalenvironment was extensively simplified by using diluted mono-protein solution (fibrinogen). This is due to the difficulties involvedin separating and quantifying mixed protein solution.

5. Conclusions

The interactions between proteins and sol–gel derived bioactiveglasses can be studied with live tests and quantitative solution-depletion protocols. The penetration of Fg into nanoporous glasseswas found to depend on the nanopore sizes of the glasses. 70S30Cglass, with a modal pore size larger than 6 nm, was sufficientlyinterconnected for Fg penetration. 100S, with a modal pore sizesmaller than 2 nm, inhibited the Fg penetration. The Fg adsorbedonto glass particle surfaces rapidly (within 1 min). The Fg adsorp-tion is irreversible as Fg release was not detected. The Fg adsorptionincreased with the initial Fg concentration and decreased with themedium pH. The adsorption interactions between the glasses andthe Fg molecules are dominated by the long-range Coulombic forcesbefore the attachment, whereas the dominant desorption interac-tions, after the Fg attachment, switch to short-range interactionssuch as hydrogen bonding and hydrophobic interactions. The Cou-lombic forces were modulated by the medium pH. More Fg ad-sorbed onto the 70S30C samples at higher stabilizationtemperatures. Protein adsorption to sol–gel glasses is reduced byhigher nanopore size and lower silica network connectivity due toincreased cation dissolution. Protein adsorption can therefore becontrolled to some extent by choosing a final sintering temperature.

Declaration

S.B. was an employee of MedCell BioSciences at the time thework was carried out. None of the other authors have any financiallink with the company.

Acknowledgements

NovaThera Ltd. (a wholly owned subsidiary of MedCell BioSci-ences Ltd.) is thanked for partially funding S.L.’s studentship. J.R.J.was a Royal Academy of Engineering EPSRC research fellow, andEPSRC is thanked for additional funding work on bioactive calciumsilicates via projects EP/E057098.

Appendix A. Figures with essential colour discrimination

All the figures in this article are difficult to interpret in blackand white. The full color images can be found in the on-line ver-sion, at doi:10.1016/j.actbio.2011.06.042.

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