Abstract
BACKGROUND:
Hyaluronic acid (HA) is a polysaccharide present in almost all animal tissues, in which it carries out important biological functions, among them, the protection of the joints by lubricating them and dampening the tension in them.
OBJECTIVE:
This study compares the viscoelastic properties of several commercial preparations of HA, to determine their suitability for use as viscosupplementation therapy in joint pathology (osteoarthritis).
METHODS:
4 HA hydrogels: Durolane®, Synocrom_Forte_One®, Synvisc_One® and Viscoplus_Matrix® and 4 HA solutions: Ostenil®, Ostenil_Plus®, Viscoplus_Gel® and Orthovisc® were analyzed to compare their viscoelatsic rheological parameters using an oscillatory-rotational rheometer.
RESULTS:
With respect to the 4 HA hydrogels, comparison of crossover frequencies allowed division into two main groups: Synvisc_One® and Viscoplus_Matrix®, with crossover frequencies in the order of magnitude of 10−2 Hz, while Synocrom_Forte_One® and Durolane® showed crossover frequencies on the order of 10−1 Hz. Only one of the 4 HA solutions, Viscoplus_Gel®, showed a crossover frequency on the order of 10−2, whereas Ostenil_Plus® and Orthovisc® showed crossover frequencies on the order of 10−1, and Ostenil® remained as a predominantly viscous fluid for frequencies as high as 4.8 Hz.
CONCLUSIONS:
The viscoelastic properties of the HA preparations can be ordered according to the values of G ∗ (the rigidity, or vector sum of the elastic modulus G ′ and the viscous modulus G ′′ ) at both transition points (0.5 and 2.5 Hz) as follows: Viscoplus_Matrix® > Viscoplus_Gel® > Durolane® > Synocrom_Forte_One® > Ostenil_Plus® > Synvisc_One® > Orthovisc® > Ostenil®.
Introduction
Hyaluronic acid (HA) is a unique polysaccharide naturally synthesized by integral membrane proteins in various types of cell, such as fibroblasts, and exported to the extracellular matrix. It has numerous structural and physiological functions as well as important rheological properties, for instance playing an outstanding role in the activation and modulation of the inflammatory response [1–5]. In addition to its fundamental effect on inflammatory pathologies through its ability to scavenge reactive oxygen species [6,7], this crowded molecular network fulfils the role of maintaining the visco-elastic properties required for joint mobility [8]. Simultaneously, HA augments the connective tissue and protects the adjacent tissues (cartilage and synovial cells) from potential damage through the absorption of the mechanical energy [9].
The therapeutic use of HA preparations as a visco-supplement is based on their rheological viscoealstic properties which are explicable from a physical, chemical and biomechanical viewpoint, by taking into consideration the relationship between the frequency dependencies of the elastic modulus G ′ and the viscous modulus G ′′ (in Pa). Other major rheological parameters are the crossover point (i.e. the transition from “viscous fluid” to “elastic body”), the phase angle 𝛿 (where tan 𝛿 = G ′′ ∕G ′ , measures the ratio of the energy dissipated as heat to the maximum energy stored in the material during one cycle of oscillation) and G ∗ (the rigidity, the vector sum of G ′ and G ′′ at every frequency, defined as the complex modulus) [9–12]. While the magnitude of the rigidity at low frequency relates to the overall stiffness at low deformation rate, the logarithm of rigidity (or the elasticity at high frequencies) is directly proportional to the molecular weight of the HA and to its concentration and decreases with age and illness [9,12]. Overall, the elasticity of the fluid depends on the interaction between various segments of the molecular chain of HA (i.e. on the states of order and disorder in its arrangement). Moreover, it has been demonstrated that these visco-elastic characteristics contribute to a reduction in the mechanical dislocation of the cell membranes, leading to a decrease of any perception of pain [8,9,12,13].
As an example, in the knee, HA fulfils the role of maintaining the visco-elastic properties required for joint mobility. The transition between the moduli (i.e. the crossover point) occurs at frequencies (of mechanical energy) appearing in this joint structure upon different types of movement. When the knee joint flexes without any load (in a lying position) the frequency of the mechanical energy received by the synovial fluid is low, and this liquid behaves in a viscous manner. This is due to the speed of the molecules constituting this fluid (mainly HA) in realizing configurational adjustments to keep their original conformation. When people slowly adopt a standing position, the entry frequency remains low and the fluid continues to be viscous. However, when people are walking, running or jumping, the frequency becomes greater and more rapid and the fluid behaves as an elastic body. At these strain frequencies, the configurational readjustment of the chains cannot occur between the short periods of the oscillating strain, and consequently the molecules deform sinusoidally and alternately store the mechanical energy and then release it elastically. These changes protect the neighboring tissues from mechanical damage [9,13–15].
Due to the various essential functions attributed to this compound in processes of healing and pain relief, interest in the improvement of therapies involving HA has increased over the last few years. In fact, the pharmaceutical industry is developing a wide range of HA preparations with the aim of improving their average life within joints, their biocompatibility and their rheological properties. With this objective, the possibility of provoking the polymer chains to covalently cross-link (CL) into three-dimensional networks in order to form biocompatible hydrogels with better mechanical properties is remarkable [16,17]. In any event, when HA is used in visco-surgery and visco-supplementation, it must be kept in mind that viscosity and elasticity are related to molecular size, concentration and cross-linking degree.
We aimed to establish a connection between the nature of HA preparations (which might be obtained from animal, biotechnological or other sources, have diverse molecular weights and cross-linking degree, as well as being presented in different concentrations) and their rheological properties, focusing on in biomedical applications. Thus, we performed a comparative study of relevant visco-elastic parameters shown by such polyanion products, including commercially available preparations.
Methods
HA preparations
Eight HA preparations were analyzed to compare key rheological parameters: 4 HA hydrogels (commercial preparations produced by cross-linking of linear HA molecules) and 4 HA solutions (commercially available hydrated HA solutions). The HA hydrogels analyzed were: Durolane (D, 2%, Lot 14063-1), Synocrom Forte One (SCF, 2%, Lot 802082), Synvisc One (SVO, 0.8% Lot 5RSE055) and Viscoplus Matrix (VPM, 2.5%, Lot 20170921). The HA solutions studied were: Ostenil (Ost, 1%, Lot QF0910AEA), Ostenil Plus (OP, 2%, Lot QE1002ALA), Viscoplus Gel (VPG, 2.5%, Lot VGQ13300) and Orthovisc (Ort, 1.5%, Lot NO10217).
According to the commercial suppliers, the average molecular weight of SVO is 6000 kDa. Previous publications refer to a molecular weight in the range of 1000–2900 kDa for Ort [18], 1000–2000 kDa for OP [19], 2500 kD for VPG [19], 2100 kDa for SCF [19] and 1000 kDa [19] or 90,000 kDa for D [20]. In any case, the determination of the molecular weight of HA is inexact, since there is no reported technique that allows to obtain a precise result and the existing literature is controversial on this issue. In this work we have not performed any experiment with the aim of determining this particular characteristic.
Measurment of viscoelatic parameters
The procedure employed for the visco-elastic characterization of the various solutions of HA was based on the protocol described by Balazs [9] and adapted in such a way to set identical (and consequently, comparable) conditions for every sample. With this purpose, we took into account previous measurements performed in our laboratory with similar HA preparations (both linear HA solutions and cross linking HA hydrogels) in the same equipment [21]. On this basis, 20 Pa was chosen as the constant shearing tension as it is the lowest possible tension to deliver the greatest linearity in the results. Simultaneously, 25 °C was established as constant temperature since even though the normal temperature in the knee is known to be around 32 °C in healthy people, it decreases to nearly 25 °C in pathological conditions within this joint or, for instance, after the performance of an arthroscopy [22,23]. Moreover, it is the normal warmth of the commercially available sterile HA preparations we study in this work, while the recommended temperature for joint injection is close (25–30 °C). This temperature also minimizes possible evaporation effects and AH degradation during the measurements. As a consequence, changes in the biophysical and visco-elastic properties of the samples during the analysis are avoided while the best compromise among the manageability of the sample and the linearity of the measurements and comparability for every HA preparation is achieved. A Bohlin CSR-10 rheometer was set up for a sweep through frequencies from 0.003 Hz to 20 Hz under a constant shearing tension (20 Pa) and temperature (25 °C). The technical specifications of the high-performance Bohlin CSR-10 rheometer are as follows: (1) torque range: 35 μN ⋅ m–10 mN ⋅ m; (2) torque resolution: <3 ×10−9 N ⋅ m; (3) shear stress range: 0.06–596.8 Pa; (4) shear rate range: 10−3–104 s−1; (5) resolution in angular position: 2 ×10−5 rad; (6) maximum rotational-velocity: 60 rad∕s; (7) oscillation frecuency range: 0.001–30 Hz.
A stainless steel device with a 4° cone on plate geometry and a diameter of 40 mm was employed for every measurement in order to keep the comparability among all the results while maintaining the best configuration for the great range of viscosities measured, as previous work in our laboratory had shown [21]. Since we compare HA preparations with quite different biophysical and visco-elastic properties, the optimal conditions (specially with respect to the geometry employed) were not identical for every preparation. Consequently, we decided to choose the geometry best suiting simultaneously every sample in order to obtain comparable measurements. Different geometries would have been chosen for studying each HA preparation on its own to obtain better individual results. However, the best geometry for the most elastic HA hydrogel did not give rise to acceptable results for the most viscous HA solution. We observed previous results obtained in our laboratory to reach this conclusion. Therefore we focused on the main objective of the present work: the comparison of different HA preparations. In addition, for our purpose, the differences between cone-and-plate 4° geometry and cone-and-plate minor angle geometry are minimal (only relating to the volume of sample used). The most important issue is the use of cone-and-plate geometry, but not parallel-plates geometry, in order to obtain more reproducible results. The rheological parameters (crossoverpoint, Hz and Pa), G ′ , G ′′ , and 𝛿 were monitored through a coupled computer with appropriate software for data acquisition, recording and processing.
Statistical analysis
All experimental measurements were performed in triplicate. Every set of three values from the rheological measurements obtained in three independent experiments for the 8 HA preparations was submitted, in pairs, to a two-tailed paired Student’s T-Test. The alpha significance level or p-value was set as 0.05. These values were obtained from the comparison between every two HA for the frequency (Hz) and the G ′ ∕G ′′ values at the crossover point and the G ′ , G ′′ and tan 𝛿 values at the studied frequencies (0.5 and 2.5 Hz).
Results
With respect to the 4 HA hydrogels studied, comparison of crossover frequencies (Hz, see Fig. 1) allowed their division into two main groups: the CL-HA of the first one presented crossover frequencies in the order of magnitude of 10−2 Hz (SVO and VPM), while the other HA preparations showed crossover frequencies in the order of 10−1 Hz (SFO and D). In contrast, only one of the 4 studied linear HA reached the transition point to become a mainly elastic fluid at very low frequency (in the order of magnitude of 10−2 Hz, VPG, see Fig. 2), whereas two others exhibited crossover frequencies in the order of 10−1 Hz (OP and Ort) and the other one (Ost) remained as a predominantly viscous fluid at frequencies as high as 4.8 Hz.
The lowest values of G ′ = G ′′ obtained at the crossover point corresponded to the CL-HA SVO and the linear HA solutions Ost and Ort (∼30–40 Pa). The CL-HA SFO, and linear HA VPG and OP, gave rise to values more than 3 times higher (∼100 Pa), D 7 times higher (∼200 Pa) and VPM 11 times higher (∼300).
The rheological parameters at frequencies of 0.5 Hz and 2.5 Hz are displayed in Table 1. They reveal that the HA hydrogels were more elastic (tan 𝛿 < 1) than viscous (tan 𝛿 > 1) at both frequencies [i.e. the elastic component (G ′ ) prevailed over the viscous component (G ′′ )] except for D at 0.5 Hz. In contrast, although the linear HA solutions Ort and OP at 2.5 Hz as well as VPG at both studied frequencies showed a similar behavior (tan 𝛿 < 1), a predominantly viscous behavior was shown in Ort and OP at 0.5 Hz and Ost at both frequencies.
Statistically significant differences were determined for every comparison between the 4 HA hydrogels except two: the tan 𝛿 of SVO vs. this parameter of VPG at 2.5 Hz and the tan 𝛿 of SFO vs. this parameter of D at 2.5 Hz (see Tables 2 and 3).
Regarding the 4 linear HA solutions, statistically significant differences were determined for every comparison between preparations for every parameter (see Tables 4 and 5).
Discussion
Previous reports have described comparisons of the rheological properties among different commercial HA preparations and human synovial fluid [18], and reviewed the in vivo performance of injected HA in osteoarthritis [24]. Here we compared the rheological properties of 8 different HA preparations in vitro, to support their employment for different biomedical applications.
The frequency and G ′ = G ′′ value at the crossover point of an HA preparation define its viscoelasticity. In this way, our rheological studied showed visco-elastic behavior in the 8 solutions analyzed. One product, CL-HA VPM showed a remarkably higher G ′ = G ′′ value at this point with a very low crossover frequency.
According to Balazs [9], 0.5 Hz and 2.5 Hz are the transition points from a quiet state to the beginning of a walking movement and running movement. At these frequencies, the G ′ values presented by the CL-HA VPM were particularly higher than the values observed in the other HA products. This difference was also detected in the G ′′ values at both frequencies.
The quality of HA preparations for diverse biomedical applications has widely been related to these rheological parameters, so that the suitability of the fluid in this context increased with them [25–28]. For instance, Edsman et al. [29] state that the resistance to deformation or gel strength increases with the G ∗ value (where G ∗ = ((G ′ )2 + (G ′′ )2)1∕2, the vector sum of G ′ and G ′′ or the modulus of the vector G ′ + iG ′′ [30]). In this way, the visco-elastic properties (i.e. the rheological relevance) of the HA preparations can be ordered according to the values of G ∗ at both transition points (0.5 Hz and 2.5 Hz) as follows: VPM > VPG > D > SF > OP > SVO > Ort > Ost.