# Interaction of bioactive glasses with fibrinogen protein.
## Effect of porosity and surface modification

Anita Lucas, T. Berthelot, M. Lebreton, P. Jauneau, D. Brezulier, Soizic

Chevance

► To cite this version:

Anita Lucas, T. Berthelot, M. Lebreton, P. Jauneau, D. Brezulier, et al. Interaction of bioactive glasses with fibrinogen protein. Effect of porosity and surface modification. Ceramics International, 2026, 52 (1), pp.618-626. 10.1016/j.ceramint.2025.11.361. hal-05420585

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# Interaction of bioactive glasses with fibrinogen protein. Effect of porosity and surface modification

A. Lucas<sup>a,∗</sup>, T. Berthelot<sup>a,†</sup>, M. Lebreton<sup>a</sup>, P. Jauneau<sup>a,‡</sup>, D. Brezulier<sup>a,b,§</sup>, S. Chevance<sup>a</sup>

## ARTICLE INFO

### Keywords:

Sol-gel

Bioactive glass

Ordered porosity

Surface silanization

Protein interaction

Fibrinogen

## ABSTRACT

Bioactive glasses are of great interest because of their osteostimulative effects and their intimate bond with bone tissue. Their porosity structuring and surface modification also offer opportunities for controlled adsorption, and further controlled release, of active molecules. These new textural properties and chemical surface modifications undoubtedly have an impact on their interaction with blood proteins. In the present work, we investigate the influence of two parameters, together or separately, mesostructuring porosity and surface modification by silanization, on the interactions of a sol-gel derived bioactive glass (92S6) with the blood protein fibrinogen. Results indicate first that the ordered porosity significantly favors the fibrinogen adsorption at the glass surface. On the other hand, surface silanization induces systematically a delayed adsorption of fibrinogen for at least 8 h. After this period of inhibition of protein adsorption, the protein is better adsorbed on silanized glasses surfaces comparatively to unsilanized surfaces. This effect is particularly noticeable when the glass has no mesostructured porosity. In the case of mesostructured porosity, the surface silanization has a minor impact on the adsorbed quantity of protein after 48 h, the protein adsorption being more influenced by the mesostructured porosity. The study also highlights that a modification of the protein conformation occurs when its approaches the glass surface. This modification is not related to ions release in the medium but really related to surface contact. Additionally, the study shows that the strong fibrinogen adsorption is not associated to the creation of covalent bond between fibrinogen and glass surface but due to a reversible electrostatic interaction.

The mesostructured porosity and surface silanization offer new opportunities for tunable properties of the 92S6 bioactive glass.

## 1. Introduction

Bioactive glasses, a class of biomaterials for bone reconstruction and bone tissue engineering, were first introduced by L. Hench [1–4] in the early 1970s. The first melt-derived bioactive glass, 45S5 Bioglass® [4], and other melt or sol-gel derived bioactive glasses have demonstrated remarkable potential in promoting bone formation and integration. They exhibit an ability to enhance revascularization, osteoblast adhesion, enzyme activity and differentiation of mesenchymal stem cells, as well as osteoprogenitor cells [5]. Then, they are of great interest in bone reconstruction strategies alone or associated with molecules of biological interest.

To demonstrate the bioactive feature of a new glass, samples are soaked in a Simulated Body Fluid (SBF). The process associated to bioactivity involves successive steps. For silicate glasses containing glass

manifiers ions, like Na⁺ and Ca²⁺, the first step consists in a cationic exchange between the glass surface and SBF: Na⁺ and Ca²⁺ ions are released from the glass surface and H₃O⁺ ions, from the SBF solution, are adsorbed. Afterward, silanol groups (Si-OH) are formed at the glass surface. The decrease in H₃O⁺ concentration in SBF leads to a rise of pH which favors the hydrolysis of Si-O-Si bridges in glass surface and induces the formation of soluble silanol groups Si(OH)₄. These silanol groups at the glass surface then condense to form a silica gel layer SiO₂ on which Ca²⁺ and PO₄³⁻ precipitate as a carbonated apatite phase from the SBF [6]. The formation of this apatitic phase, with a composition close to that of bone mineral, is recognized to be responsible for the intimate contact between bioactive glasses and living bone tissue after implantation [6–10].

If for new glasses developed for bone reconstruction applications, the bioactivity is well documented, less studies described their interactions

<footnote><sup>a</sup> Univ Rennes, CNRS, ISCR - UMR 6226, F-35000, Rennes, France</footnote>

<footnote><sup>b</sup> Univ Rennes, CHU Rennes, Pole Odontologie, CNRS, ISCR - UMR 6226, F-35000, Rennes, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: anita.lucas@univ-rennes.fr (A. Lucas).</footnote>
with proteins. However, when bioactive glasses are implanted, they immediately interact with body fluid containing proteins. Among these proteins, fibrinogen, a 340-kDa glycoprotein soluble in blood plasma, is involved both in the blood clotting and inflammatory process [11,12]. In the context of biomaterial implantation, using a surgical procedure, blood fibrinogen interacts early with the implant. In addition, fibrinogen is highly involved in the modulation of inflammatory host response. Under physiological conditions, fibrinogen concentrations in plasma are in the range 2–4 g L⁻¹ [13]. After injury, for instance in case of vascular disruption, infection or inflammation, the blood concentration of fibrinogen increases several folds [11]. In particular, a proinflammatory role for fibrinogen has been reported in vascular wall disease, stroke, spinal cord injury, brain trauma, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, bacterial infection. This protein is easily adsorbed on biomaterials surfaces after implantation and drives the foreign body reaction [12]. Fibrinogen adsorption on the bioactive glasses surface will influence their biological features [14]. After adsorption, fibrinogen promotes platelet and monocyte/macrophage adhesion, playing a key role in blood coagulation and foreign body response. Platelet and monocyte adhesion is enabled by specific peptide segments recognized by cell adhesion receptors. Platelets have the glycoprotein GPIIb/IIIa integrin receptor, which binds fibrinogen and causes platelets aggregation [15]. Monocytes/macrophages have the Mac1 integrin adhesion receptor that links to fibrinogen and plays a key role on inflammatory response in vivo [16,17]. This strong ability of fibrinogen to bind other biological entities will have a direct impact on the glass biocompatibility by modulating the host response in vivo. The study of fibrinogen adsorption on bioactive glasses, and biomaterials in general, may be useful to predict potential further foreign body reaction.

In the present work, we studied the influence of ordered porosity and surface silanization on fibrinogen interaction and adsorption onto new bioactive glasses surfaces. The studied glasses are sol-gel silicate, sodium-free, ternary glasses named 92S6 with the composition (wt %) 84,82 % SiO₂ – 10,14 % CaO – 5,04 % P₂O₅). We previously studied this new composition and demonstrated its bioactivity [18–22]. We highlighted the difference in bioactivity and formation of mineral phases at the glass surface related to the porosity patterning using non-ionic surfactant in the synthesis medium to obtain the 92S6 P123 glass [20,22]. We also demonstrated that the surface silanization with -CH₂-CH₂-CH₂-NH₂ group, a widely used method to allow further molecule grafting, did not reduce the glass bioactivity in vitro [22]. In addition, this non cytotoxic composition can easily be associated to polymers for fabrication of 3D composite scaffolds. Particularly, it exhibits a weaker impact on lactic acid polymer's degradation than the 45S5 Bioglass® [23].

## 2. Materials and methods

### 2.1. Preparation of bioactive glass samples

The bioactive glasses (BGs) named 92S6 and 92S6 P123 were synthesized using sol-gel method according to a procedure previously described [22]. The 92S6 P123 is the notation which refers to the 92S6 glass synthesis by incorporating the surfactant pluronic P123 into the synthesis medium to induce porosity organization [20].

In short, syntheses were conducted at 40 °C in 120 mL of 2 M HNO₃ and 30 mL of distilled water. In the case of the 92S6 P123 synthesis, we added 6 g of P123, an amphiphilic block copolymer: poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-PPO-PEO, Sigma-Aldrich, Germany) used as organic structure-directing agent.

Then 3.6 g of Tetraethyl orthosilicate (TEOS) (99 % Aldrich, France), 0.16 g of TEP (99,8 %, Aldrich, France) and 0.53 g of Ca(NO₃)₂·4H₂O (≥99 %, Fluka, Germany) were then added successively at 1 h intervals to the clear solution. The mixture was stirred at 40 °C for 24 h, and then hydrothermalized at 100 °C for 48 h. Without any filtering and washing,

the resulting precipitate was directly dried at 100 °C for 24 h in air. The synthesized powders were calcined in air at 650 °C for 6 h, to remove the organic structure directing agent completely and converting all chemical species into oxide.

The obtained powders were crushed and sieved. Powders with a diameter between 40 and 63 µm were used in all experiments.

### 2.2. Surface silanization of the BGs

The silanization process of glasses was described elsewhere [22]. In short, after dehydration, the two glasses were refluxed during 24 h in a 3-aminopropyltriethoxysilane (APTES, 98 %, Alfa-Aesar) solution in dried toluene. The obtained product was centrifugated and rinsed with a mixture of diethyl ether and dichloromethane (1:1) and finally dried at 100 °C during 12 h in air.

After surface silanization we counted four materials to be studied named as follows, non-silanized 92S6 noted (92S6), silanized 92S6 noted (92S6 Sil), non-silanized 92S6 P123 noted (92S6 P123) and silanized 92S6 P123 noted (92S6 P123 Sil).

### 2.3. Zeta potential measurement

The zeta potentials of glasses and fibrinogen were measured using Malvern Zetasizer Nano ZS series. All zeta potentials were measured in physiological saline environment: 0.9 g NaCl in 100 mL of Ultra-pure MilliQ water (18.2 MΩ).

For glasses, suspensions containing 5 mg·mL⁻¹ of each glass were prepared and submitted to measurement. Additionally, similar suspensions were placed in incubator at 37.0 °C under stirring (50 rpm) for t = 15 min, 2 h, 8 h, 15 h and 48 h, to study the time dependent changes in BGs zeta potentials. Incubation times were the same as those used for incubation of BGs with fibrinogen solution (see 2.5 section).

For fibrinogen, saline solution containing 2.5 mg·mL⁻¹ of fibrinogen (from human plasma, 35–65 % protein, Sigma-Aldrich) was prepared and analyzed (see 2.4 section).

Each measurement was repeated two times. The average zeta potentials and standard deviation were then calculated. For comparison, we measured the zeta potential of a non-bioactive mesostructured (hexagonal) SiO₂ MCM 41 (Sigma-Aldrich) which porosity organization is close to that of the 92S6 P123 glass.

### 2.4. Fibrinogen soaking solution

A fibrinogen (from human plasma, 35–65 % protein, Sigma-Aldrich) solution, with a concentration of 2.5 mg·mL⁻¹, was prepared using saline (0.9 g NaCl/100 mL) solution. This concentration is close to that used in a previous study (2 mg·mL⁻¹) [14] and in the range of the concentration in human blood (2–4 mg·mL⁻¹) [13].

### 2.5. Incubation protocol of glasses in fibrinogen solution

5 mg of glass sieved powder, with a diameter between 40 and 63 µm, were placed in closable conical polypropylene flasks and immersed in 1 mL of the fibrinogen in saline solution (2.5 mg/mL of fibrinogen in 0.9 g NaCl/100 mL solution). Suspensions were placed in an incubator at a temperature of 37 °C, under continuous stirring, during 15 min, 2 h, 8 h, 15 h and 48 h. The suspensions were then centrifuged for 2 min. The supernatants were collected and pH was measured. Then, UV-Visible (UV-Vis) spectra were recorded, using a Jasco V-650 (JASCO), in the range 240–320 nm, considering the maximum absorption of proteins (due to the presence of aromatic amino acids) being at 280 nm [24,25]. Circular Dichroism (CD) spectra, in the near-UV region (200–260 nm), were recorded with a JASCO J-815 CD spectrophotometer. CD spectra were recorded on the same samples that those used for absorbance measurement, with 10 mm path length cells (quartz). Spectra were recorded with a bandwidth of 1 nm, a scanning speed of 500 nm·min⁻¹
and a data pitch of 1 nm. All spectra were measured against an appropriate reference, with ten scans being accumulated for each sample. The cell holder was thermostated at 37 ± 0.1 °C, using a circulating water bath (JASCO PTC-4235/15). Each experiment was duplicated. Solutions of fibrinogen with pH adjusted at the same pH than the glass suspensions were submitted to the same protocol. The absence of impact of centrifugation on UV-Visible absorbance and CD signal of fibrinogen was checked.

Additionally, the resulting glass powders were collected and rinsed with water. On one hand, the rinsing solution was collected for further analyses. On the other hand, glass powders were incubated with sodium dodecyl sulfate (SDS, >99.0 %, Sigma-Aldrich) solution (50 mM in water) for 20 h. After centrifugation, the SDS solution was analyzed by UV-Vis spectroscopy. A solution of fibrinogen at 2.5 mg.mL⁻¹ in the same SDS solution was used as reference.

## 2.6. Effect of ions released from glass and effect of associated pH rise on fibrinogen adsorption and conformation

When BGs are in contact with aqueous solution, ionic species are released and pH rise (see Introduction section [6,22]). We wanted to check if released ions and pH increase had no influence on UV-Vis absorbance and CD signal of fibrinogen during the duration of our experiment. For this purpose, 5 mg of sieved glass powder were incubated at 37 °C in 1 mL of saline solution, under stirring, for 48 h. The saline solution with the glass was then centrifugated. Afterward, the obtained supernatant solution (without glass) was mixed with 2.5 mg. mL⁻¹ of fibrinogen. The UV-Vis absorbance (260-340 nm range) and CD spectra (200-260 nm range) were recorded immediately (t₀) and after an incubation of 20 h (t+20h) at 37 °C.

# 3. Results and discussion

## 3.1. Zeta potentials (ZPs)

Zeta potentials (ZPs) and pH values, measured for glasses and fibrinogen in saline solutions, are reported on Table 1.

We observed that for all glasses zeta potential values are negative. This result is consistent with those obtained for BGs independently of their synthesis mode (melting-quenching or sol-gel synthesis) [26,27]. This negative zeta potential is related to the high concentration of -OH groups at the glass surfaces of BGs [28]. The presence of glass modifier (Ca, P) elements induces more non-bridging oxygens, that convert in -Si-OH at the glass surface, compared with pure Silica MCM 41. After silanization of the glass surface, ZPs remain negative but increase due to the presence of the amine group on the glass surface. The silanization process of glasses is known to convert negative surfaces to positive ones [29,30]. Even if silanized bioactive nanoparticles with positive zeta potential allowed differentiation of dental pulp stem cells [30], negative values of zeta potentials seem to have a significant favorable effect on the attachment and proliferation of bone cells [26]. Our results indicate that the silanization allows to maintain a negative zeta potential for the glasses tested.

Fibrinogen has a very slightly negative ZP value. This average value is the result of contributions from positively and negatively charged sites

<table><caption>**Table 1**
Zeta potentials and pH for glasses suspensions and fibrinogen in saline solution.</caption><thead><tr><th>Sample</th><th>Zeta potential± SD (mV)</th><th>pH</th></tr></thead><tbody><tr><td>Fibrinogen</td><td>-5.1 ± 0.7</td><td>6.9 ± 0.1</td></tr><tr><td>92S6 P123</td><td>-22.3 ± 0.5</td><td>9.9 ± 0.1</td></tr><tr><td>92S6 P123 Sil</td><td>-8.3 ± 0.4</td><td>10.9 ± 0.1</td></tr><tr><td>92S6</td><td>-21.0 ± 0.4</td><td>9.6 ± 0.1</td></tr><tr><td>92S6 Sil</td><td>-7.1 ± 0.3</td><td>10.9 ± 0.1</td></tr><tr><td>SiO<sub>2</sub> MCM 41</td><td>-1.3 ± 0.3</td><td>4.7 ± 0.1</td></tr></tbody></table>

in the protein, composed of several chains with pI values ranging from 5.4 to 8.4. The average pI of fibrinogen calculated in this way is of the order of 6.4 [31].

The evolution of zeta potentials (ZPs) values of BGs with immersion time in saline solution at 37 °C is presented in Table 2.

For unsilanized BGs, ZPs values did not significantly change until 15 h of immersion in saline solution. This fact is related to the equilibrium between the release of Ca²⁺ and the simultaneous uptake of H₃O⁺ by the glass surface [2]. After 48 h, BGs tend to form a hydroxyapatite (HA) [22] layer that makes ZPs values less negative [26,27,32]. The difference between the 92S6 and 92S6 P123 is related to slightly lower kinetics of formation of HA for 92S6 than for 92S6 P123 [22]. The porosity patterning using Pluronic® P123 surfactant in the synthesis medium of 92S6 glass induces a regular, organized and well-defined structured porosity. Specific surface area increases from 372 m²/g to 392 m²/g with a greater average pore diameter: 65 Å to 119 Å. 92S6 P123 glass also presents a lower dispersion of pores diameter, because of regular ordered porosity, compared to 92S6 synthesized without any surfactant. The structured porosity leads to a greater and easier contact with SBF by an easy diffusion and then a higher reactivity of the glass towards SBF. Consequently, it allows a rapid availability of high concentration of Ca²⁺ in the medium which precipitates afterward with PO₄³⁻ to form HA [22].

For silanized glasses, ZPs values were less negative than those of their untreated counterparts. This fact is due to the presence of the amine group. The pKa value of the grafted amine may be quite like that of n-propyl amine that has a pKa of 10.7 [33]. The amine group is not predominantly protonated at the high pH of the saline solution after glass immersion (pH 10.9), but the amine group can act as ligand for positive ions that stay close to the surface.

After 48 h, all glasses have similar values of ZPs due to the formation of apatite layer for all glasses [22].

## 3.2. Effect of ions released from glass and effect of associated pH rise on fibrinogen adsorption and conformation

UV-Vis spectra of fibrinogen solutions, recorded immediately (t₀) and after 20 h (t+20 h) of contact with the centrifugated saline solutions incubated 48 h with glasses are presented in Fig. 1A for 92S6 P123 and in Fig. 2A for 92S6 P123 Sil. CD spectra, obtained in the same conditions, are presented in Fig. 1B for 92S6 P123 and Fig. 2B for 92S6 P123 Sil.

Results indicate that there is no difference in UV-Vis spectra for fibrinogen at t₀ and after 20 h (t+20h) of contact with centrifugated saline solution containing released glasses ions. Similar results were recorded for 92S6 and 92S6 Sil (not presented here). As absorbance is proportional to protein concentration according to the Beer-Lambert law (formula (1)), it seems that no fibrinogen aggregation is induced by the presence of glass released ions.

$$Abs(\lambda) = l.e(\lambda).C \quad (1)$$

with Abs: absorbance at the wavelength λ, l: thickness of the solution, ε(λ): extinction coefficient at the wavelength λ, C: concentration of the studied species.

Looking at the CD Spectra, no significant change in conformation of fibrinogen is observed. Indeed, only a very slight increase in intensity of the band at 222 nm, typical of α-helix, is observed. This can be induced by the presence of salts. This surface-active effect of salts has already been widely described [34].

These results indicate that the presence in solution of ions released by exchange between the glass surface (silanized or not) and the saline solution, inducing an increase of 3 or 4 pH units, has no influence on the three-dimensional structure of fibrinogen and does not induce aggregation of the protein [35,36].
<table><caption>**Table 2**
Evolution of zeta potentials (mV) with immersion time in saline solution at 37 °C for different bioactive glasses.</caption><thead><tr><th>Time</th><th>0</th><th>15 min</th><th>2 h</th><th>8 h</th><th>15 h</th><th>48 h</th></tr></thead><tbody><tr><td>92S6 P123</td><td>-22.3 ± 0.5</td><td>-21.9 ± 0.4</td><td>-21.8 ± 0.5</td><td>-21.8 ± 0.5</td><td>-19.8 ± 0.2</td><td>-9.8 ± 0.7</td></tr><tr><td>92S6 P123 Sil</td><td>-8.3 ± 0.4</td><td>-9.8 ± 0.4</td><td>-10.1 ± 0.5</td><td>-8.67 ± 0.4</td><td>-9.7 ± 0.4</td><td>-10.6 ± 0.2</td></tr><tr><td>92S6</td><td>-21.0 ± 0.4</td><td>-20.8 ± 0.4</td><td>-21.5 ± 0.3</td><td>-20.5 ± 0.2</td><td>-19.2 ± 0.5</td><td>-9.2 ± 0.4</td></tr><tr><td>92S6 Sil</td><td>-7.1 ± 0.3</td><td>-9.7 ± 0.1</td><td>-9.5 ± 0.2</td><td>-9.2 ± 0.2</td><td>-6.7 ± 0.4</td><td>-9.5 ± 0.4</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** UV–Vis (A) and CD (B) spectra of fibrinogen in saline solution (2.5 mg.mL⁻¹) incubated 0 h (t₀; dashed line) or 20 h (t+20h; solid line) with the supernatant of a 92S6 P123 glass saline solution at 37 °C and pH = 9.9.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** UV–Vis (A) and CD (B) spectra of fibrinogen in saline solution (2.5 mg.mL⁻¹) incubated 0 h (t₀; dashed line) or 20 h (t+20h; solid line) with the supernatant of a 92S6 P123 Sil glass saline solution, at 37 °C and pH = 10.9.</figcaption></figure>

### 3.3. Protein adsorption at the glass surface

Initial UV–Vis spectrum of fibrinogen solution exhibited a maximum of absorption at 280 nm [24,25]. The spectra of fibrinogen solutions obtained, either after contact with glasses or with solutions containing released glasses ions, exhibited also a maximum of absorbance at the same wavelength (see Fig. 1A and 2A). We then chose this wavelength to quantify absorbance for different solutions. The evolution of UV–Vis absorbance at 280 nm of the supernatant fibrinogen solution, as a function of glass soaking time (0 min, 15 min, 2 h, 8 h, 15 h, 48 h), was then studied for the four glasses: 92S6 and 92S6 P123, with or without silanization. As an example, observed UV–Vis spectra of fibrinogen solution after incubation with 92S6 P123 are presented in Fig. SI–1. From the obtained absorbance values on UV–Vis spectra, recorded on the supernatant solution, we studied the evolution of absorbance value, recorded at 280 nm on UV–Vis spectra, as a function of incubation time. Results are presented in Fig. 3. According to the Beer-Lambert law

presented in formula (1) in section 3.2, the absorbance was linearly correlated to the concentration of fibrinogen. We deduced the remaining percentage of fibrinogen in solution, compared to the initial fibrinogen amount, (RF (t)%), calculated by formula (2).

$$RF(t) \% = (Abs \ t (280 \ nm) / Abs \ t_0 (280 \ nm)) \times 100 \quad (2)$$

with RF (t) %: the percentage of remaining protein in solution at the t time compared to the initial amount of fibrinogen in solution, Abs t (280 nm): the absorbance measured at 280 nm at the soaking time t and Abs t₀ (280 nm): the absorbance measured at 280 nm before glass soaking.

Fig. 4 presents the evolution of RF (t)% as a function of incubation time.

Results indicate that there is a significant difference in the kinetics of fibrinogen adsorption to the glass surface between 92S6 and 92S6 P123. This difference in behavior does not appear to be linked to any difference in the electrostatic interactions of fibrinogen with the two glasses, since both have similar ZP values. On the other hand, this difference in
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Evolution of absorbance at 280 nm (A<sub>280nm</sub>) of the supernatant fibrinogen solution incubated with the four glasses 92S6 (—), 92S6 Sil (---), 92S6 P123 (■—), 92S6 P123 Sil (---), as a function of incubation time: 0 h, 0.25 h (15 min), 2 h, 8 h, 15 h and 48 h.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Effect of silanization on the evolution of fibrinogen concentration in the supernatant solution compared to the initial amount in solution before soaking for 92S6 (A), and 92S6 P123 (B), with (hatched) or without (fill) silanization. Percentage of protein remaining in solution at time *t* relatively to the initial amount of fibrinogen in solution: RF (%), as a function of incubation time: 0 h, 0.25 h (15 min), 2 h, 8 h, 15 h, 48 h.</figcaption></figure>

adsorption kinetics could be related to the difference in porosity of the two glasses. Indeed, Lin et al. [37] reported that fibrinogen molecules have approximate dimensions of 4.5 × 47 nm (i.e. 45 × 470 Å) and need pores of 6 nm (60 Å) and more to penetrate, whereas pores of 2 nm (20 Å) are too small to allow protein penetration. Thus, 92S6 P123 exhibits organized and interconnected porosity [20] with a well-defined pore size and a median pore size of 119 Å [22]. This pore size is suitable for fibrinogen penetration [38]. Compared with 92S6 P123, 92S6 has a smaller median pore size (65 Å) with a greater dispersion of porosity, due to the absence of porosity patterning [22], with many pores too small to allow fibrinogen penetration. Fibrinogen is slowly adsorbed by

the porosity of 92S6, whereas fibrinogen rapidly penetrates the interconnected pores of 92S6 P123. In addition, a second phenomenon could also contribute to this difference in adsorption kinetics: as the kinetics of hydroxyapatite formation on the surface of 92S6 P123 is faster than on the surface of 92S6 [22], this could also facilitate and accelerate protein adsorption [38].

Results also indicate that the silanization process induces a delayed adsorption of the fibrinogen for the two glasses as the amount of fibrinogen in the supernatant solution did not decrease during the first 2 h and slowly between 2 and 8 h. This result could be either related to the decrease in the pore diameter for silanized glasses (median pore size of 59 Å for 92S6 Sil and 69 Å for 92S6 P123 Sil) or to the difference in surface attraction for protein due to the presence of amine group. However, the difference in porosity did not inhibit the adsorption of fibrinogen after 8, 15 or 48 h. Our previous study [22] showed that the silane coupling agent is strongly fixed to the glasses surface. Therefore, the protein adsorption registered at 8 h is not related to an increase of porosity due to the uncoupling of silane group from the surface. The difference of surface attraction due to a difference in ZPs values for silanized and unsilanized glasses will not explain the delayed protein adsorption because ZPs values did not notably change for silanized glass during 48 h. Conversely, a noteworthy difference exists between the initial glasses and silanized counterparts: a rise in pH glass environment induced by the presence of the amino group at the glass surface: 1–1.3 units of pH respectively for 92S6 P123 and 92S6 between untreated and silanized glasses. pH increase is known to have an adverse effect on fibrinogen adsorption [37]. The pH returns to less alkaline values with the incubation time due to the formation of hydroxyapatite which favors the protein adsorption [38].

Additionally, results indicate that when the adsorption begins, the silanized surface favors the fibrinogen adsorption for the 92S6 glass. This result was previously reported for silanized surfaces that favor a better anchoring of protein [38–40]. In the case of 92S6 P123 Sil, the better anchoring is not significant. In the case of 92S6 P123, the fibrinogen adsorption is mainly influenced by the ordered porosity that favors the protein adsorption.

### 3.4. Evolution of protein conformation

We have previously shown that the adsorption kinetics of fibrinogen on the BGs is dependant of the surface state (porosity, functionalization) of the BGs. CD spectra analyses allowed us to further investigate whether contacting the glasses surfaces had any effect on the conformation of fibrinogen present in solution. CD spectra of fibrinogen supernatant solutions, recorded after different incubation times with the four different glasses, are presented in Fig. 5. As shown in Fig. 5A, 92S6 does not appear to significantly alter fibrinogen conformation. A slight shift of the most negative band from 225 to 222 nm is observed, which can be interpreted as a slight enhancement of fibrinogen helicity [41,42]. More significant is the loss of signal, due to the adsorption of fibrinogen to the material over time, as previously demonstrated by UV–Vis spectroscopy. No conformational change is observed with 92S6 Sil as shown in Fig. 5B. The same study was made with 92S6 P123 and 92S6 P123 Sil (Fig. 5C–D). Silanization appears to have a strong effect on adsorption kinetics but does not deeply affect protein conformation. We can notice that the low quantity of protein in solution after 15 and/or 48 h of incubation, in the case of 92S6 Sil, 92S6 P123 and 92S6 P123 Sil, could prevent fine analysis of the recorded spectra. Finally, the most significant change in fibrinogen conformation is observed in the presence of 92S6 P123, with a probable strengthening of the helicity of the fibrinogen in contact with it over time: a shift of the most negative band from 225 to 222 nm and the concomitant appearance of a second negative band at around 208 nm were observed. This couple of bands is characteristic of helical structures [41,42]. In conclusion, glass porosity seems to be the factor with the greatest impact on the conformation of fibrinogen in the presence of these different BGs, at least in terms of
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Effect of incubation time with 92S6 (A), 92S6 Sil (B), 92S6 P123 (C) or 92S6 P123 Sil (D) at 37 °C on fibrinogen conformation studied by CD. Increasing incubation times were tested: 0.25 h (15 min) (yellow), 2 h (purple), 8 h (blue), 15 h (orange), 48 h (pink). T0 corresponds to the dotted curve. Negative CD signals at 208 and 222 nm wavelengths are typical of helical structures. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

thermodynamics stability. This effect is delayed by the functionalization of the surface.

The modification of protein conformation is regularly related to an aggregation phenomenon due to pH increase or to the Ca²⁺ ions release [35,36,43]. However, in the present study and as previously discussed, the modification of conformation of fibrinogen was not significant when the fibrinogen was incubated with the supernatant solution, containing the glass released ions and presenting a high pH (glasses were incubated 48 h in water, and eliminated by centrifugation before fibrinogen incubation) (see Fig. 1 B and 2 B). It must be noticed that the modification of conformation is few observed when the adsorption is delayed even if the pH increases and Ca²⁺ ions are released, in case of silanized surfaces. These results are consistent with a conformational change due to the protein conformational reorientation upon surface contact and the possible exchange between adsorbed and free proteins [43]. We may conclude that if no proteins are adsorbed, there is no exchange between free and adsorbed protein and no conformational change is observed on proteins in the supernatant.

### 3.5. Nature of interaction between glass and protein

In order to determine whether the interaction between fibrinogen and BGs is covalent or non-covalent (that is, proceeds by adsorption), the glasses that have been in contact with fibrinogen for 48 h were isolated by centrifugation. Once the supernatant was removed, the glasses powders were treated with an anionic detergent, sodium dodecyl sulfate (SDS). SDS is known to break low-energy interactions and is used in various studies to elute proteins adsorbed onto different surfaces [44], such as fibrinogen with silica [45,46]. Thus, we hypothesized that if fibrinogen was adsorbed onto the glass surface, the protein would be desorbed from the glass surface by SDS and released into the saline solution. Conversely, if a covalent bond was formed, SDS would not break the covalent bond, and no protein would be released into the saline

solution after contact with SDS. No signal would then be detected on the CD spectrum of the solution.

The CD spectra of these SDS solutions are shown in Fig. 6. For reference, the CD spectrum of fibrinogen in the absence and in presence of 50 mM SDS are also presented in Supporting Information (Fig. SI–2).

Due to the denaturing nature of SDS [47–49] and its strong propensity to react or precipitate in the presence of salts, this reference spectrum is provided for informational purposes only and cannot be presented as a “control.” In the case of fibrinogen, a clear change in the CD spectrum is observed, reflecting a significant change in the conformation of fibrinogen, without however observing complete denaturation. This has often been observed for other proteins for which SDS does not cause the disappearance of secondary structures but rather a loss of three-dimensional structure in favor of a molten globule structure (partially preserved secondary structures) [47,48].

On the CD spectra (Fig. 6) recorded from the BGs 92S6 P123 and 92S6 P123 Sil, incubated for 48 h with fibrinogen and then treated with SDS, a characteristic protein signal is recorded between 200 and 240 nm, with a maximum (negative) around 207 nm. This signal demonstrates a desorption of fibrinogen from the glass surfaces due to the action of SDS (no signal was observed before the addition of SDS). These signals are very different from those of the initial fibrinogen. This result is related to the well-known denaturing action of SDS [44–49]. The same results were obtained with 92S6 (data not shown). These results demonstrated that the loss of absorbance in fibrinogen solutions was caused by the non-covalent adsorption of fibrinogen onto different BGs. It should be noted that the intensity of the protein signal is lower than expected. It is likely that not all of the adsorbed fibrinogen was desorbed. This has already been described by Zembala et al. and Poumier et al.: the denaturing action of SDS induces adsorption, which competes with desorption [45,46].
<figure><img src="image_8.png" /><figcaption>Fig. 6. CD spectra of supernatant fibrinogen saline solution obtained after incubation for 48 h with 92S6 P123 (A) or 92S6 P123 Sil (B) at 37 °C. Before (—) and after (–) treatment of glasses with sodium dodecyl sulfate (SDS).</figcaption></figure>

## 4. Conclusion

We evaluated the adsorption of fibrinogen on 92S6 BGs ((wt %) 84,82 % SiO₂ – 10,14 % CaO – 5,04 % P₂O₅) exhibiting different porosity features (92S6 versus 92S6 P123) and different surface chemistry (untreated and silanized surface). Obtained results highlighted the influence of tailored porosity and surface modification on fibrinogen adsorption. The ordered porosity induced an increased speed of protein adsorption which may explain the accurate cellular response *in vitro* and *in vivo* observed for BGs presenting an ordered mesoporosity [50]. The surface silanization induces two different important results: a delayed protein adsorption associated to an increased protein adsorption, which may encourage the immunomodulation for tissue regeneration. As previously mentioned, fibrinogen favors platelets, monocytes and macrophages adhesion to biomaterials surfaces. Platelet adhesion is strongly correlated with the degree of fibrinogen unfolding induced by adsorption, and there is virtually no correlation with the amount adsorbed. Adsorption-induced unfolding exposes two distinct types of binding sites: while one induces platelet adhesion exclusively, the other induces both platelet adhesion and activation initiating thrombus formation [51]. Platelets then release their content of growth factors vital for bone healing [52]. When protein adsorption is observed, BGs tested here may induce a fibrinogen conformation modification, due to the surface contact, and may favor the growth factor release. Initial and silanized glasses 92S6 and 92S6 P123 may exhibit the same action on platelet adhesion and consequently the same impact on growth factor release because there is no correlation between the platelet adhesion and the amount of fibrinogen adsorbed.

The adhesion of monocytes and macrophages sets up the inflammatory reaction with chemotaxis of other cell populations required for bone healing. However, too extensive or sustained inflammatory reaction can be deleterious, resulting in the subsequent formation of a fibrous capsule that can lead to loss of biomaterial function [12]. With BGs, a balance is then created between two opposite activities i) a pro-inflammatory activity thus induced and ii) an anti-inflammatory activity, particularly for mesoporous BGs. This last activity allows the proliferation of macrophages without inducing their polarization towards the M1 pro-inflammatory phenotype [53]. Additionally, contact of the BGs with biological fluids generates a temporary and local rise in pH, counteracting the acidity of the inflammatory reaction, allowing osteoblasts to adhere and inducing the differentiation of mesenchymal precursors [5].

The effect of surface amine functionalization was recognized i) to increase endothelial cell growth, ii) to enhance differentiation and mineralization of osteoblast cells, iii) to induce a preferable adhesion, growth and matrix formation with fibroblasts [54], iv) to promote

osteogenesis [55]. Concomitant silanization and protein (fibronectin) adsorption on melt derived S53P4 BG also promoted the expression of relevant osteogenic markers from human adipose stem cells [55]. If previous studies showed the interest of BGs surface silanization on osteogenic cells and consequently on osteoinduction, the present study highlights an unexpected result: the delayed adsorption of fibrinogen on the glass surface during the first 8 h. The binding capacity to fibrinogen is not exclusive to eukaryotic cells, since fibrinogen harbors epitopes recognized by bacterial cells via specific integrins [12]. As an example, *Staphylococcus epidermidis* (normally found on human skin) has the ability to adhere to adsorbed fibrinogen [56,57]. This bacteria is known to be one of the main causes of biomaterial-associated infections [58]. A delayed fibrinogen adsorption may be correlated with delayed or deleted bacterial colonization, in the event of material contamination or insertion into septic sites, which represents a definite interest.

In conclusion, results of this study reinforce the interest of ordered mesoporous BG 92S6 P123 as potential reconstructive material and particularly with a silanized surface for the association of BG with organic molecules with biological interest [28,59] or to prevent bacterial contamination in the early stages of implantation. These promising properties will be investigated in further studies.

## CRediT authorship contribution statement

**A. Lucas:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**T. Berthelot:** Writing – original draft, Investigation, Data curation.
**M. Lebreton:** Investigation, Data curation.
**P. Jauneau:** Writing – original draft, Methodology, Investigation, Data curation.
**D. Brezulier:** Writing – original draft.
**S. Chevance:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors of this article would like to acknowledge the SSIM platform of Biosit (UAR 3480 CNRS- US18 Inserm, Université de Rennes) for access to UV-Vis and CD analyses, and the "Institut des Sciences
Chimiques de Rennes" (ISCR, UMR CNRS 6226 - Université de Rennes)
for financial support.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ceramint.2025.11.361.

## Data availability

The data that support the findings of this study are available from the.

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