Aesthetic medicine is a dynamic world in constant development, with the greatest challenge being obtaining new innovative products that allow for ever more accurate and precise applications. Hydrogels are a polymeric semisolid material with a three-dimensional (3D) architecture, with mesh structure and highly interconnected porosity [
1]. They show high water content and good biocompatibility, and for their physical similarities to human tissues, hydrogels have been commonly used for aesthetic purposes, as cell culture substrates and wound dressings, for drug delivery, biosensing and bioimaging in response to the complex environment of the human body, so as to carry out the accurate diagnosis and treatment of a variety of diseases [
2]. The study of a new formulation requires multiple steps to demonstrate safety, efficacy and stability in order to guarantee the protection and satisfaction of the consumer. During formulation, filling, usage and storage, products are exposed to possible external factors such as physical, microbiological and chemical influences which can lead to instability with different grades. Microbiological instabilities are caused by fungi, yeast or bacteria contaminations, while physical and chemical changes in the formulation are influenced, for example, by temperature, light and interactions of the ingredients with the packaging or with each other [
3]. Since thermodynamic influences may affect the storage stability, accelerating storage conditions with temperature variation in order to induce rapid physical and chemical alterations in the product represents a valid way to obtain stability performance prediction [
4]. The rheological characterization of the hyaluronic acid (HA) dermal filler allows us to study the physical stability, aesthetical outcome, quality and usefulness. A thermal stress can alter the product’s viscosity and storage and loss modulus, so rheological studies can grant useful insight into the material behavior under different temperature stresses [
5]. In particular, rheology allows for understanding and evaluating the response of the material to different stimuli, such as stress or deformation at various frequencies, and at different temperatures and conditions. Rheology has also become a tool to support safer developments of the product and to quickly reproduce the consumer sensation during the administration or the application of the hydrogel. The rheological characterization is also useful for evaluating the consistency of the product, as well as the effect of the stabilizers on the formulation itself [
6]. Hydrogels have rheological properties that allow them to be classified as viscoelastic, as they exhibit both elastic and viscous behavior and present physical properties between liquid and solid states. Through the frequency sweep test, it is possible to observe how the relationship between the viscous and elastic moduli evolves in function of the frequency of the applied stress. The test is performed within the Linear Viscoelastic Region (LVER), determined by a preliminary Amplitude sweep test at a constant frequency, in order to avoid sample damage. In this range, G’ (elastic modulus), G’’ (viscous modulus) and tan δ (tangent of the phase angle) do not vary with the application of deformation [
7].
The viscosity of a dermal filler is related to the concentration of not-crosslinked and crosslinked HA, to the degree of crosslinking, to the molecular weight distribution, to the average gel particle size and to the manufacturing process [
8]. HA dermal fillers need to be formulated in order to have gel particles that, at low frequencies, retain elasticity (stiffness). It is also crucial that HA hydrogels have low viscosity at high shear (100 s
−1), so they can be extruded through a small-gauge needle. High viscosity under low-shear rates is indeed comparable to the condition of the hydrogel after injection or when at rest in the packaging (shear rate 0.1 s
−1) [
9]. In our study, we use rheology as tool for the stability assessment of a HA-based dermal filler cross-linked with polyethylene glycol diglycidyl ether (PEGDE), containing 26 mg/mL HA, 0.1% of Glycine and L-Proline and 1% Calcium Hydroxiapatite (CaHA) (Matex Lab S.p.a. Brindisi, Italy). Amplitude and Frequency sweep tests were performed to verify that the rheological parameters maintain a similar trend when the samples are subjected to different thermal stimuli over time. The evaluation of the viscoelasticity trend can be used as an index of the formulation stability when subjected to external stimuli. Characterization through shear rate is instead used as a tool to investigate the ability of the hydrogel to flow with a non-Newtonian shear-thinning behavior. PEGDE is a cross-linker agent widely used to modify and improve the physical properties of the linear chain of HA, conferring a 3D more stable structure [
10]. The cross-linking reaction consists of an epoxide ring opening with the hydroxyl group of the HA in an alkaline environment that leads to the formation of a stable ether bond while, at the same time, maintaining the biocompatibility and biological activity of the native HA [
11]. The filler used in the present study also contains Glycine and L-Proline, two amino acid constituents of all the types of collagen [
12]. The use of CaHA microspheres for aesthetic purposes is closely related to its ability to provide a non-permanent volumizing effect for revolumization and tissue support. CaHA plays a key role in the rheological profile of the HA-based dermal fillers and provides a higher viscosity (η) and elastic modulus (G’) than HA fillers, conferring a scientific basis for the observed ability of CaHA in tissue support and facial revolumization [
13]. This filler has an important clinical indication and high level of biosafety, as shown in a precedent laboratory evaluation [
14].