Complementarity between Brillouin signature and Scholte wave for controlled elasticity in fibrillated collagen medium for culture cell Open Access

Collagen, making up 30% of the body’s proteins, is prevalent in bones, muscles, blood, and skin, where it constitutes 75%. It is crucial for skin elasticity and serves as an ideal matrix for joints, tendons, and ligaments. Understanding collagen fibrillation kinetics is essential in tissue engineering and cell culture, where optimal conditions for cell growth and function are paramount. Manipulating the mechanical properties of the extracellular matrix (ECM) is crucial as ECM elasticity significantly influences cellular processes, such as adhesion, migration, proliferation, and differentiation. Fibrillar collagen, with its adjustable mechanical properties, stands out due to its ability to influence cellular behavior and tissue development. By controlling collagen concentration, cross-linking density, and fiber alignment, researchers can tailor ECM stiffness and elasticity to mimic various tissue microenvironments, enhancing the relevance of in vitro models.^1–3 This modulation has significant implications for regenerative medicine, drug discovery, and disease modeling, aiding in understanding cellular responses to different conditions.

Despite its potential, ECM elasticity’s quantitative measurements remain incomplete. Brillouin spectroscopy, which is particularly well suited for investigations of semitransparent systems, has already been applied to the elastic characterization in numerous biological studies.^1–3 In this article, we report on alternative experiments on the thermoelastic excitation of acoustic waves induced using a pulsed laser in a collagen solution. Simultaneous detection of longitudinal waves and Scholte waves at the collagen/solid interface enables us to quantitatively measure the effect of the fibrillation process on sound velocity and refractive index vs the fibrillation time, providing insights into the relationship between collagen fibrillation and ECM mechanical properties.

Collagen fibrillation was performed directly on the supports, which were 1 × 1 cm² silicon wafers, for variable durations ranging from 30 minutes to 72 h. The collagen utilized was concentrated to 20 mg/ml, specifically SYMATESE collagen, which contained 17 mM acetic acid. For each sample, a mixture of 0.387 ml collagen, 0.018 ml sodium hydroxide, and 0.044 ml phosphatebuffered saline (PBS) was prepared. The mixture was stirred to ensure uniform distribution of sodium hydroxide and PBS within the collagen. Subsequently, the dish was resealed and placed in the culture room’s incubator for the required fibrillation duration.

Time resolved pump and probe experiments have been realized using a mode-locked Ti:sapphire (MAI-TAI Spectra) laser source operated at 800 nm with a pulse duration below 100 fs at the laser output and a repetition rate of 79.4 MHz. Synchronous interferometric detection is performed by modulating the pump beam at 1.8 MHz using an acousto-optic modulator. A Michelson scheme allowed measuring the perpendicular surface displacement. The pump and probe are first superimposed on the acoustic transducer to study longitudinal acoustic waves. To obtain information on surface or interface waves, the probe beam is swept around the pump excitation using a 4f configuration. In both configurations, we performed a two-color pump–probe experiment by doubling the frequency of the probe or pump beam. As the fibrillation process starts at the free surface of the collagen, we used a sufficiently thin metallic transducer, deposited on a sapphire or glass substrate, placed in contact with this free surface. The titanium or aluminum layer is thick enough to generate an acoustic burst and thin enough to allow the probe beam to follow the propagation of the acoustic pulse in the first few micrometers of the biomaterial layer.

First, let us consider longitudinal acoustic wave propagating perpendicular to the surface, using the experimental geometry described in Fig. 1(a). In this case, the advantage of sapphire, compared with a glass substrate for example, lies in the fact that the Brillouin signature of this transparent compound is in a much higher frequency range, around 100 GHz, than that expected in the collagen gel. In Fig. 1(b), reflectivity variations are measured with variable pump and probe delay. After subtraction of the photothermal background, a double Brillouin oscillation can be clearly observed. The high-frequency signature comes from the acoustic wave transmitted into the sapphire, while the low-frequency component corresponds to the collagen layer signature. In this case, the transducer should be pressed sufficiently on the collagen layer to ensure good acoustic transfer.

Consequently, knowledge of the refractive index is mandatory to determine longitudinal velocity. This parameter varies considerably in the literature and is also likely to change with fibrillation time. To overcome this limitation, we used an alternative geometry to directly measure the wave velocity in the layer, independently of the optical properties. While our previous focus was on longitudinal volume waves, we are now concentrating on waves that may exist at the interface between the metal transducer and the collagen gel.

Following this, the probe beam is scanned around the pump epicenter to be sensitive to in-plane propagation in the collagen layer. The experimental setup is briefly illustrated in Fig. 3(a). The mapping of surface displacements when the collagen layer is simply replaced by air is shown in Fig. 3(b). A set of concentric circles corresponding to the Rayleigh waves emitted by the various pump pulses can be observed.⁴ 

The isotropic nature within the plane is associated with the isotropy of the glass substrate used here. Figure 3(c) shows a similar situation when the aluminum transducer is in contact with non-fibrillated collagen. In this case, we note the presence of similar features that clearly exhibited a much lower propagation velocity. By pointing out the position of these rings and knowing the repetition rate of our laser, 12.59 ns in this case, we obtain Fig. 4, enabling us to extract the propagation velocity of these different waves.

For an air layer, a velocity of 3020 m/s is obtained, which is in perfect agreement with a Rayleigh wave propagating in a glass substrate.⁵ Here, the very thin aluminum layer has no influence on this propagation due to the low frequency of the surface acoustic wave involved.

For the glass–ethanol interface, the Scholte wave velocity is expected to be very close to the longitudinal wave velocity of ethanol, equal to 1150 m/s, which is in good agreement with the experimental speed deduced from Fig. 4, demonstrating that the concentric rings depicted in Fig. 3(b) may be associated with such Scholte waves. Scholte waves induced by laser excitation have already been demonstrated in the lower frequency range.⁹ Conversely, by introducing the Scholte velocity measured in a collagen solution into Eq. (2), we can deduce a longitudinal velocity of 1512 m s⁻¹ for the non-fibrillated collagen using a density equal to 1. Then, considering this deduced longitudinal velocity, and the Brillouin frequency measured in Fig. 2, we obtain a value for refractive index at 400 nm equal to 1.35. This new approach, which enables the refractive index to be determined using purely acoustic measurements, sheds new light on issues relating to the elasticity of living cells, where this parameter has hitherto been neglected due to a lack of quantitative measurements.^10–14 However, it should be pointed out here that this approach neglects possible viscoelastic effects that may affect the propagation velocity as a function of the frequency range considered, which here is at least one order of magnitude. As a result, the underestimation of velocity in the Brillouin frequency may lead to a slight overestimate in the optical index.

By reproducing the same acoustic mappings for fibrillated solutions, we can extract the evolution of longitudinal velocity and refractive index as a function of fibrillation time, without having to rely on the knowledge of additional parameters, except density. These evolutions are shown in Fig. 5. Consequently, the fluctuation in Brillouin frequency observed in Fig. 2 was, in fact, essentially linked to the variation in collagen elasticity, with the optical index varying by only around 3%. Now, if the density is assumed to be roughly constant, equal to 1, then the bulk modulus given by B = ρV² may change by 40% varying from 2.5 GPa up to 3.5 GPa. It should, nevertheless, be kept in mind that the treatment proposed here is based on the assumption that the longitudinal velocities in the plane and perpendicular to the layer are equal, and potential anisotropies in the propagation properties are neglected.

We have developed a new technique to determine the longitudinal velocities and refractive index in materials whose elasticity can be modified on demand, which is particularly relevant for biological systems such as biomaterials. This method has shown remarkable efficiency in studying cell growth, allowing for variations of up to 40% in the bulk elastic modulus. The application of this technique opens new perspectives for the analysis and engineering of biomaterials, providing precise and adaptable means to optimize mechanical properties according to the specific needs of biological studies. However, a major challenge remains: the precise determination of material’s density, a crucial step for complete characterization of its mechanical properties; interested readers may find some pointers in Ref. 13. Continuing research in this direction could not only enhance our understanding of biomaterials but also lead to significant advancements in their practical applications.

The authors have no conflicts to disclose.

A. Hamraoui: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). O. Sénépart: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). L. Belliard: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal).

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