Characterizing the Physical Properties of HA Dermal Fillers Using Rheology and Particle Size Measurements

Hyaluronic acid, also known as HA, is a naturally occurring polysaccharide that is often employed as a functional ingredient in subcutaneous and topical anti-ageing treatments like dermal fillers. These dermal fillers use the special viscoelastic properties of polymer for effective augmentation of soft tissues.

When HA is administered subcutaneously, it creates an elastic network inside rhytides and wrinkles to give a plumper and fuller appearance to the skin. However, a major drawback of HA is that it has less than three days of half-life, and hence it is important to increase the polymer’s durability to develop products that have an acceptable shelf life and greater clinical persistence. One way to enhance the mechanical strength and extend the degradation time is to increase the degree of cross-linking and the molecular weight (MW) of the polymer. These properties, however, also affect other properties of the HA such as viscoelasticity and viscosity.

In order to successfully formulate with HA, it is important to understand the effect of factors such as concentration, molecular structure, molecular weight, and degree of cross-linking on rheological properties such as viscoelasticity that are directly associated with aspects of product performance. Combining structural properties to product performance, through rheological properties, aids fast, smart, and effective formulation of HA. This study demonstrates how particle size and rheology measurements can be utilized to differentiate the physical characteristics of HA dermal fillers. Figure 1 shows the HA dermal fillers in syringe.

HA dermal fillers in syringe.

Figure 1. HA dermal fillers in syringe.

Experimental Framework

First, three commercial HA dermal fillers were assessed by means of laser diffraction and rotational rheometry techniques for characterization of particle size and rheological behavior, respectively. Rotational rheometer measurements were then carried out through a 40 mm parallel plate measuring system and a Kinexus rotational rheometer equipped with a Peltier plate cartridge. All rheology measurements were carried out at 25°C temperature. Next, a standard loading sequence was utilized to make sure that both specimens were subject to a reliable and controllable loading procedure.

Oscillation testing involved variable frequency and variable amplitude tests. Amplitude sweep tests at 1 Hz frequency were carried out to ascertain the critical strain and linear viscoelastic region (LVER). Then, succeeding frequency sweep tests were conducted from 0.1 to 10 Hz through a constant strain in the LVER. Steady state shear measurements were performed to check the viscosity dependence on shear rate (0.1 s-1 - 100 s-1). A stress ramp test (0 Pa - 200 Pa in 100 s) was also carried out to measure the fillers’ yield stress. The coarseness of the fillers was evaluated through axial testing on the rheometer, wherein the gap was quickly changed from 1 to 20 mm and the normal force profile was recorded. Tack was associated with the peak normal force calculated in Newtons. A Mastersizer 3000 from Malvern Panalytical was used for measuring the particle size of the gel particles in the dermal fillers. The fillers were dissolved in saline solution and the median particle size distribution and particle size was measured.

Results and Discussion

Oscillation Testing

Figure 2 shows the elastic modulus (G') curves as a function of shear strain. All samples displayed similar size LVER regions with a vital strain denoting the onset of non-linearity in the area of 20%. Elastic modulus values determined within the LVER region revealed that the lowest elastic stiffness was found in Sample A, with G' having a value of 150 Pa. Among the three samples, Sample C had the most elastic stiffness with G' having a value of 320 Pa and Sample B having a value between the two of 220 Pa.

Amplitude sweep data showing elastic modulus (G’) as a function of shear strain.

Figure 2. Amplitude sweep data showing elastic modulus (G’) as a function of shear strain.

Figure 3 shows the phase angle and elastic modulus curves as a virtue of the oscillation frequency. For all samples, the phase angle across the entire frequency range is approximately 10°, indicating that all samples are highly elastic gels. The G' values for samples A, B, and C at 1 Hz frequency are approximately 150 Pa, 220 Pa and 320 Pa respectively, which correlate with the amplitude sweep information at the same frequency. The slight slope in G' with frequency implies a slight degree of structural relaxation where stored elastic energy is dispersed with increasing time, although this is relatively small.

Frequency sweep data showing elastic modulus (G’) and phase angle (d) as a function of frequency.

Figure 3. Frequency sweep data showing elastic modulus (G’) and phase angle (d) as a function of frequency.

A number of factors impact the viscoelastic properties of HA dermal fillers, including the degree of cross-linking, HA concentration, and molecular weight. If these properties are changed, the viscoelastic properties, in particular the elastic modulus G', can be made for a specific application. Gels having high G' show greater resistance to deformation and can prove more effective as fillers; however, they could be harder to inject and may cause more pain. Thus, strong gels with high G' can be used for deeper winkles.

However, weaker gels having low G' may be suitable for light or fine wrinkles found in tear troughs or lips. Since these areas are more sensitive, weaker gels may cause less pain when injected. In addition, the lower modulus may match the properties of the local tissue. Among the three HA samples examined, Sample A is the softest and weakest gel, and sample C is the strongest and stiffest gel, based on the results shown in Figures 2 and 3.

Steady Shear Testing and Yield Stress Determination

Figure 4 shows the results of the steady state shear measurement - shear viscosity determined as a function of shear rate.

Flow curves showing steady state shear viscosity (η) as a function of shear rate.

Figure 4. Flow curves showing steady state shear viscosity (η) as a function of shear rate.

With increasing shear rate, the viscosity decreases considerably, suggesting that the materials are highly shear thinning. The fillers’ structure is also so strong that at a low shear rate, the viscosity is extremely high and steadily increases with decreasing shear rate, indicating solid-like behavior or yield stress at rest. This matches with the observations made from oscillation testing that revealed a highly elastic, gel-like structure.

A yield stress denotes that while the material behaves like a solid below critical stress, it will also flow like a liquid above this critical stress. The degree of the yield stress must be correlated with the structural strength, and hence concentration and degree of cross-linking of the gel particles, which must be reflected in G'.

Several tests can be used to determine the yield stress, but a stress ramp provides the easiest and fastest way for estimating the yield stress, where the instant viscosity is determined constantly with rising shear stress. Figure 5 shows the stress ramp data for the three HA samples.

Stress ramp data showing instantaneous shear viscosity (η) as a function of shear stress.

Figure 5. Stress ramp data showing instantaneous shear viscosity (η) as a function of shear stress.

The peak in viscosity denotes the point of yield; the stress value at which this takes place is the yield stress. The lowest yield stress (42 Pa) is seen in Sample A, and the highest (55 Pa) is seen in Sample C; Sample B has a slightly lower yield stress than C (53Pa). This same order is seen in oscillation testing with Sample A being the weakest and Sample C being the strongest of the three gels. Since these gels are present as a group of covalently cross-linked gel particles, the yield stress is correlated with the stress needed to unblock the particles, enabling them to travel past one another.

Tack testing

Figure 6 shows the standard force profiles as a function of time as the plate-plate gap is increased. The standard force value is found to be negative and this is because the sample is pulling down on the upper plate owing to cohesive and adhesive forces and decays towards zero at failure. The remaining force at extended times is attributed to the weight of the samples retained on the upper plate. For samples A, B, and C, the peak normal forces are 0.35, 0.46, and 0.54 N respectively, which again corresponds with the order of yield stress and G' measurements for all the three samples. Therefore, the highest degree of cohesivity or tack was seen in Sample C and the lowest in Sample A.

Tack tests data showing normal force profiles as a function of time during a pull-away test.

Figure 6. Tack tests data showing normal force profiles as a function of time during a pull-away test.

Particle Size

It is important to control the particle size of the gel particles so as to reduce the extrusion force and related side effects, such as bleeding and pain when gels are administered. Hence, the gels should be made to pass through needles at the proper rate with the preferred extrusion force. Figure 7 shows particle size distribution of gels as a cumulative volume percent.

Particle size distribution (Cumulative volume) for gel particles in dermal fillers.

Figure 7. Particle size distribution (Cumulative volume) for gel particles in dermal fillers.

The median size of samples A, B, and C is 480, 425, and 203µm, respectively. Strong gels having high yield stress and G' values had to be resized to tiny particles so that they are easily administered through the needles. Sample C exhibits the smallest particle size as it has the highest G' value out of the samples, while the largest particle size is seen in sample A as it is the weakest gel and can be easily injected through the needles. The resulting size will also be correlated to the molecular weight and cross-linking, because highly cross-linked polymers related to higher values of G' will be more compact.

Conclusion

Particle size and rheological properties of three HA-based dermal fillers were differentiated and compared. Using oscillation testing, the elastic modulus G' was established and these values matched with gel strength and stiffness. Also, steady state shear measurements were performed to test the viscosity dependence on the shear rate, and stress ramp tests were carried out to ascertain the force needed to break down the gel structure, that is, the yield stress. The fillers’ tackiness was determined by measuring the standard force profile when the plate-plate gap was rising, and was associated with the yield stress and oscillation data. In addition, the gels’ particle size was determined because the size impacts extrusion, and this was also found to correspond with the rheological data.

To sum up, the particle size and rheological properties of HA-based dermal fillers are key factors to consider when determining the performance and application of these products.

This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.

For more information on this source, please visit Malvern Panalytical.

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