1. Introduction
Recent advances in terms of stability, sensitivity, availability, and high throughput capacity make quartz crystal microbalance (QCM) highly applicable for investigating complex biological problems in advanced research areas such as nanomedicine and tissue engineering (TE)
[1].
Some of the applications of quartz crystal microbalance with dissipation monitoring (QCM-D) in biomaterials studies include interaction studies (i.e., mass and thickness measurement, the kinetics of reaction) during the formation of ultrathin single
[2] or multilayers
[3][4] of biomaterials in liquid phase on various solid surfaces, solid–liquid interactions of biological molecules such as proteins
[5][6], cells
[7], and microorganisms (e.g., antifouling)
[8] with various functional solids, enzymatic degradation of biomaterials
[9], structural rearrangement of polymers
[10], and many others. Particularly, its usefulness in studying interfacial phenomena between biomaterials and cells makes it a fascinating tool for application in TE
[11].
In a typical process, thin nanofilms of the studied biomaterials and their combinations are prepared on QCM crystals by selecting one of the numerous available methods such as spin coating, dip coating, self-assembly, flame synthesis, layer-by-layer (LbL), and others. Using nanofilms reduces material costs in the initial stage of surface interaction testing. Their ease of fabrication makes them suitable for a quick survey of a larger number of biomaterials
[12]. This is of the utmost importance in developing cellular scaffolds, especially when using natural materials or extracellular matrix (ECM) components, for example, in cartilage TE (CTE)
[13][14]. It is particularly useful when protein (e.g., ECM components) adsorption to a base biomaterial is intended
[15]. This is a common strategy in designing the scaffold for CTE
[16][17] since it is well-known that the protein adsorption and morphology of the protein layer on the biomaterial surface govern cellular adhesion, cell function, and activity, such as proliferation, survival, and gene expression
[18][19][20]. QCM is also most suitable on the nanoscale, as it becomes very difficult to determine the physical properties of the load on the microscale and impossible on the macroscale. These processes can be monitored with the QCM-D in real time, thereby providing information about protein–biomaterial interactions, hence the functionality of the protein–biomaterial composite
[21]. Once a formulation of the protein–biomaterial composite is achieved, the QCM-D can be further applied as a biosensing platform to evaluate interactions of the protein–biomaterial composite with cells
[22].
The possibility of monitoring cell biological phenomena in real time is pivotal in the early stage of TE. Therefore, it is important to evaluate whether the surfaces of engineered biomaterials can induce desirable initial cell interactions, as this affects the outcome of artificially engineered tissues
[12][23]. Cell adhesion is an essential aspect of cellular behavior since this affects other basic cellular responses, such as growth, migration, and differentiation
[24]. Accordingly, innovative methods to study cellular behavior in their native state can advance understanding their response to various biomaterials.
2. Problem of Material Choice, Nanotechnology, Surface Topography, and the Role of Nanofilms
Developing new biomaterials for biomedical applications has made tremendous progress, and there are several biomaterials at the disposal
[25]. There have been many attempts to identify suitable biomaterials for a particular biomedical application. The scaffold design for CTE is mostly based on a combination of biomaterials to achieve specific tissue requirements, including structural, physical, chemical, and biological properties
[13][26][27][28]. The selection of suitable biomaterials for a scaffold design is a key factor since it drives the cellular responses, guides the growth, and mimics the extracellular matrix (ECM) of native tissues
[29]. Nowadays, CTE’s main types of scaffolds are polymeric films, hydrogels, and fibrous scaffolds
[16][30]. Nanofiber-based scaffolds are emerging as a versatile alternative for TE and regenerative medicine applications
[31]. These structures can mimic the architecture of natural human tissue on the nanometer scale and favor cell adhesion, proliferation, migration, and differentiation
[32]. However, hydrogel scaffolds made from natural raw materials have become the most popular in CTE due to their excellent biological properties. Their physiological elasticity, smooth surface, and high water content can better simulate the ECM microenvironment of natural cartilage. In addition, significant progress has been made in the preparation of hydrogels containing nanomaterials to produce nanocomposite hydrogels for the CTE
[33].
In addition, the surface properties of scaffolds have been recognized as being of utmost importance due to the direct interface between materials, cells, and other tissue components
[14]. Therefore, the crucial part of TE is controlling the surface properties of biomaterials and adjusting cellular behavior, which affects the formation of new tissues
[34]. Chemical surface properties such as type, density, or sequence (e.g., amino acid sequence in proteins) of functional groups are pivotal in cell attachment and proliferation. It was shown that some hydrophilic functional groups (hydroxyl, amino) promote chondrocyte attachment, while others (carboxylic) inhibit it on scaffolds from polylactic acid
[35]. However, the type of functional group alone does not define attachment. For example, their protein sequence (amino acid sequence) defines the attachment and proliferation on surfaces crucially
[36][37]. Surface topography has also been identified as the crucial surface parameter affecting cell behavior by altering protein adsorption. Cells respond to the nanostructured surface by altering adhesion, organizing the cytoskeleton, and expressing the desired phenotype
[18]. The basis of these cellular responses represents focal adhesions (FA), a specific gateway for a particular cellular type called “nanoimprinting”
[38]. It is well-known that the surface properties of every biomaterial influence the adsorption of proteins, which in turn affects integrin-mediated cell adhesion. The surface nanoarchitecture may affect mechanotransduction, leading to conformational changes in the cell cytoskeleton and consequently changing the shape of the nucleus and the phenotype
[39]. QCM frequency was shown the be highly sensitive to changes in surface topography. Superhydrophobic surfaces with pillar-like microfeatures prepared by lithography showed a high-pillar-height dependence of the measured frequency. At lower heights, the adsorbed liquid caused high “in-phase” frequency shifts as the pillars were mass-loading. At a critical pillar height, however, the elastic effect of the micropillar resulted in the coupled frequency veering to the “out-phase”
[40].
At the same time, different signaling cellular mechanisms can affect proliferation, growth, and differentiation in response to surface characteristics. This simply means that the fate of each cell depends on the initial cell–biomaterial contact
[41]. Therefore, two key factors must be monitored simultaneously at an early stage in TE: protein adsorption to the base biomaterial and the following cell response to its surface.
Researchers have found that macromolecular concepts alone cannot solve all issues in biomaterial science, so nanoscale materials are gaining more attention in many biomedical applications, including TE
[42]. Interesting materials for biomedical applications are nanoscale polymer nanofilms
[14]. These offer great promise in biomedicine as they serve as the interface to explore interactions between biological objects (biomolecules or cells) and various materials
[43]. Nanofilms are thin material layers ranging from a nanometer to a few micrometers. They represent the boundary where most physicochemical processes take place. The LbL method for nanofilm preparation is most promising in TE research as it offers the possibility to fabricate layered composites of various biomaterials in situ in the QCM device, which monitors the build-up of such composites. LbL films are easy to fabricate and are susceptible to fine control of the physicochemical properties. In general, the LbL technique is used to develop biomaterials with suitable biological and mechanical properties. Such constructs are believed to represent an appropriate cellular microenvironment with suitable pore size, interconnectivity, and biological activity for inducing cell differentiation towards desired phenotypes
[44]. Thus, nanofilms are widely used for biomedical applications in orthopedics (71,72) and CTE
[45][46] to investigate different types of biomaterials and their interactions with cells.
3. QCM for Interaction Monitoring with ECM Components in CTE
Monitoring the fabrication procedure during scaffold formation is vital to ensure product quality and artificial tissue development. Various methods, including scanning electron microscopy, mercury and flow porosimetry, gas pycnometry, gas adsorption, and microcomputed tomography, are available to monitor scaffold formation and its interactions with biological components
[47]. However, most of these methods provide a limited insight into the scaffold formation, especially time-resolved solid–liquid interactions between specific scaffold components, cells, and the biological environment. A niche area for QCM offers just that—real-time monitoring of three key steps that enable systematic and thoughtful scaffold formation:
-
Formation of nanofilms (e.g., LbL) from biomaterials (important to choose the suitable materials to form desired surfaces with desired properties),
-
Solid–liquid surface interactions of ECM components with nanofilms (e.g., protein absorption) (important to provide insight into concrete interactions of base scaffold materials with ECM components, key for cell growth);
-
Solid–liquid cellular interactions (e.g., cellular adhesion, growth, cytotoxicity and so on) (important to understand cell growth dynamics on/in the scaffolds).
Monitoring of LbL nanofilms from biomaterials by QCM is well-documented. By simultaneous frequency and dissipation recording, QCM allows for the identification of several factors influencing LbL build-up, such as electrostatic charges, salt concentration, polymer conformation, and others. QCM proved to be a suitable technique to study the adsorption kinetics during the formation of a multilayer polymer film
[48]. The importance of measuring frequency and dissipation changes simultaneously in film-formation processes was well-described in Rodahl’s work where it has been shown that even very thin (few nm) biofilms dissipate a significant amount of energy. Three main contributors for the high dissipation were identified: a viscoelastic porous structure that is strained during oscillation, trapped liquid moving within the pores or in and out of them, and the load from the bulk liquid increasing the strain
[49].
The abovementioned key steps can be performed subsequently or simultaneously. For example, the formation of biomaterial nanofilms is often performed in combination with ECM components. Such an approach lays the groundwork for the use of LbL nanofilms in investigations of biomaterials in CTE
[50][51][52]. Degradable biopolymers and ECM components are preferred materials in CTE since they possess outstanding biocompatibility, low immunological response, low cytotoxicity, and excellent capability to promote cell adhesion, proliferation, and regeneration of new tissues. Unlike synthetic materials, ECM-based scaffolds allow for direct attachment of cells because they often possess unique ligands (e.g., amino acid sequences) that bind to specific cell receptors
[53]. Naturally derived biomaterials used in CTE may be protein-based (gelatin, collagen, fibroin and so on), polysaccharide-based (alginate, agarose, chitosan, cellulose, hyaluronic acid (HA), dextran), or made from decellularized tissues
[16].
Recently, the novel formulation termed proteosaccharides was introduced, a combination of polysaccharides and various proteins, to mimic the natural cartilage environment and to improve the scaffold’s physiological signaling and mechanical strength
[16]. The design of such a composite construct is complex. In addition to the correct choice of biomaterials, it is necessary to study the interactions of the materials to ensure proper protein binding and create a stable construct. It was extensively emphasized that the processes occurring between cells and materials at the nano–bio level govern subsequent cellular behavior and influence tissue formation
[54]. In situ monitoring of these phenomena has been found crucial in controlling cell functions. The analysis of interfacial interactions with protein adsorption and initial cell adhesion was well-demonstrated with the QCM-D technique
[55].
The LbL technique, in combination with QCM-D analysis, was used to prepare films from collagen (Col1)/chondroitin sulfate (CS) and Col1/Heparin (HN) with mammalian primary chondrocytes. Data generated from the QCM-D observations showed a consistent build-up of films
[56]. In addition, functional multilayer scaffolds for in vivo osteochondral tissue engineering were also developed
[57].
QCM in Measurement of Protein Adsorption on Biomaterials
ECM proteins initially adsorbed on biomaterial surfaces can mediate initial cellular interactions. Therefore, it is of the utmost importance to assess the interaction potential of ECM proteins with scaffold biomaterials (e.g., polysaccharides) and to monitor the formation and stability of the protein fiber growth and adlayers. The simplest method to study such processes is to design thin films based on raw materials meant for the subsequent scaffold formation using the LbL technique. QCM was shown to be useful for measuring the amount of protein adsorbed onto different surfaces and evaluating the rate of fibril growth
[47][58][59]. There are several other techniques available to observe fiber growth and protein ensemble, including atomic force microscopy (AFM)
[60], total internal reflection fluorescence microscopy (TIRFM)
[61], and surface plasmon resonance (SPR)
[62] or dynamic light scattering (DLS)
[63] and so on. However, they mostly do not provide real-time data recording.
Detailed QCM applications for the adlayer monitoring have already been described and reviewed in other journals
[55][64]. Protein adsorption is a key factor in cell activity, attachment, surface migration, proliferation, and differentiation towards the desired phenotype. It was reported that the monocomponent protein adsorption and conformational changes could be investigated with the QCM-D technique
[55]. For example, the conformation of bovine serum albumin when adsorbed on a silica surface was determined by applying the QCM-adsorbed mass at various pH conditions
[65].
QCM was also successfully used to study the influence of protein conformation on cell attachment
[66]. It was showed that bovine serum albumin, fibrinogen, and collagen all bind in different conformations on various solid substrates and that certain conformations that minimize the energy after adsorption favor cell attachment
[66].
In addition, QCM-D was used to investigate interfacial phenomena between cells and surfaces modified by various serum proteins such as albumin, fibronectin, and collagen with subsequent adsorption of fetal bovine serum (FBS) to form different adlayers
[55]. Investigation of the adsorption phenomena enables the control of the structural–chemical properties of nanomaterials. This is possible since various surface properties, such as wettability, free energy, charge, and roughness influence protein adsorption behavior on a solid substrate
[67]. However, fibril formation often results in a complex sensor response; therefore, additional microscopic measurements may be used. One of the most recent achievements is the development of a combined QCM-TIRF technique, which allows for the simultaneous measurement of the mass of peptide adsorbed on the sensor surface and the visualization of fibril growth by a TIRF microscope
[68][69]. Such comprehensive understanding of the adlayers’ adsorption, viscoelastic properties and surface arrangement is crucial to understanding further interaction of the biomaterials with cells.
4. QCM in the Measurement of Interactions with Cells
Cell adhesion and detachment are crucial aspects of cell function and biological processes in bioengineering applications, since these parameters affect basic cellular processes such as cellular communication, growth, migration, and differentiation. Among the most important steps in TE is the initial attachment and growth of cells to achieve sufficient biointegration
[24]. Recent literature suggests that QCM is a promising method for studying cellular behavior, as it provides data that are pivotal in understanding many biological phenomena in TE. Different aspects of cell adhesion can be evaluated, including the kinetics of cell attachment, spreading, growth, and cytoskeletal changes
[70].
Conventional analytical methods cannot follow these initial steps in cell–biomaterial interactions that QCM can monitor, making QCM a tool beyond the current state-of-the-art. In addition, the QCM system is a useful technique for the detection of cell adhesion without the need for detaching cells from the surface or using labeled molecules
[71].
The collected data of QCM measurements are unique and require accurate cellular biological phenomena interpretation. However, considering the advantage of the high sensitivity of QCM instruments in detecting subtle changes in cellular activity, it is possible to monitor even tiny changes in the cytoskeleton induced by various environmental conditions. To date, different QCM-based cell biosensors have been applied to monitor the attachment of different cell types, including fibroblasts
[72], osteoblasts
[22][73], endothelial cells
[74], and others. These studies have indicated that the QCM cell biosensor is suitable for evaluating cell attachment in the early stage of TE. QCM successfully detected mesenchymal stem cell (MCS) responses to biomaterial surfaces
[75]. Cell adhesion depends on the surface properties of the biomaterial, such as topography, wettability, charge, and protein adlayers. Once cells adhere and spread on the biomaterial surface, various cellular reactions and morphological changes occur. Thus, in situ monitoring of these phenomena on biomaterial surfaces is crucial for controlling cell behavior, function, growth, reproduction, and differentiation
[55]. QCM-D measurements provide information about different adhesion processes and interfacial viscoelastic properties depending on the surface material
[71]. In addition, the QCM-D technique was also used to detect the rearrangement of the cell cytoskeleton in monitoring cell biological phenomena such as cell migration and differentiation. Furthermore, the ability of QCM-D signals to detect cytoskeletal changes can be exploited to assess cell health or to sense cell death in response to different external conditions
[76][77].
5. Outlook and Prospects of QCM in Tissue Engineering
The main purpose of this review was to highlight the role of QCM in regenerative medicine. QCM is a practical tool for detection of a wide range of molecules and monitoring of bioprocesses in real time due to its unique characteristics such as high sensitivity, low sample quantity, simplicity, low cost, high throughput, and overall versatility
[78]. Its practicality and versatility in TE were reflected in works such as the development of QCM-based mammalian cell biosensors
[79] or monitoring of nanoscaled solid–liquid interactions in TE such as initial cell binding, focal adhesion formation, cell spreading, and complex changes in the cytoskeleton
[70][80][81], or simple monitoring of multilayer formation (e.g., LbL)
[15][73].
Looking to the future, the rapid development of biomaterials requires proper selection and preparation before using them as a potential cellular scaffold in TE. Moreover, the clinical translation of bioactive technologies for medical use should be facilitated; therefore, in situ monitoring and understanding of the interfaces are of great importance for clarifying the nature of biocompatibility
[82].
In addition, QCM can be coupled with other devices such as microscopes and ellipsometers to obtain more detailed morphological and optical properties of studied platforms. Combining the advantage of the high sensitivity of QCM in detecting nanoscale changes in cell conformation and the power of microscopical visualization, a beyond-state-of-the-art setup can be designed to analyze the cytoskeleton changes induced by different stimuli, e.g., growth factors, cell and gene therapies, immunomodulation, and other external stimuli (electrical, mechanical, or magnetic pulses and so on). In this way, QCM could be practically applied in CTE to monitor tissue growth and investigate the course of chondrogenesis of different cell types (e.g., precursor cells, MCSs, chondrocytes and so on). Moreover, the potential of QCM to monitor cellular responses in real time can be used in designing multifunctional and smart scaffolds
[83]. Hereby, QCM can act as a biosensor at the cell–tissue interface to detect any subtle changes in the tissue construct and to activate various mechanisms (bioelectric signals for appropriate physiological functions) to preserve desired tissue characteristics in the long term. Essentially, this might allow for the construction of a self-sustaining closed system mimicking native in vivo conditions.