Aesthetic demand is growing rapidly, with more people showing great concern regarding facial aging. Hyaluronic acid fillers are thus an essential tool that must be thoroughly studied. Nowadays there are few comparative studies regarding these fillers’ behaviour found in literature, although it’s necessary for clinicians to familiarize themselves with their properties, reaching further than the manufacturers’ indications.
The ART FILLER® line is a group of fillers produced with Tri-hyal technology, meaning they have long chain, very-long chain, and free non-crosslinked HA in their composition [1, 3]. The Universal filler was chosen as the control group, as it is the most used one out of the five and the most versatile.
As is shown in Fig. 4, all fillers had an increase in weight after 24 hours, meaning neither of the products were saturated prior to their use. All fillers absorbed the maximum amount of water they could, since after 24 hours there was an excess PBS inside the Eppendorf tubes [25].
According to previous studies that compared the swelling ratios of different HA fillers, those with lower cross-linking density and G’ were capable of absorbing more water and expand, even though expansion wasn’t a direct variable and was instead deduced from water absorption [8–10, 22–24, 26].
However, in this study, the Lips filler showed the highest swelling ratio (Sw= 2,6158), even though, according to the manufacturer, the filler with the lowest cross-linking density and G’ is actually Fine Lines, which absorbed the least amount of water (Sw=2,0262). Lips Soft and Universal, which have the same indications, showed the most similar swelling ratios (Sw=2,2678 and Sw=2,3062, respectively), and they also present the same cross-linking degree and the most similar G’, even though there is a difference in price, with 1 mL of Lips Soft being around 20€ more expensive than 1 mL of Universal. The properties of each filler as stated by the manufacturer are shown in table 1.
Still, aforementioned studies used different fillers and protocols from the ones used in the present study, meaning we cannot establish a straightforward comparison between them. Furthermore, hyaluronic acid’s properties act synergistically, influencing one another, meaning there are plenty more factors to take into consideration, such as cohesivity, the process used to hydrate the gel and HA concentration [9, 10, 21, 30, 37].
In fact, fillers with lower HA concentration are supposed to absorb less water [23, 26, 37], which is in line with what happened regarding the Fine Lines filler. Despite having the lowest cross-linking degree and G’, this is the filler with the lowest HA concentration, and the one that showed the lowest swelling ratio, which suggests the possibility that HA concentration might have more influence over water absorption than other properties. Aside from Fine Lines, all other fillers have the same HA concentration, and out of these, Volume is the one with both the highest cross-linking degree and G’, and the one with the lowest swelling ratio, which is in agreement with literature. However, the same can’t be said for Lips, as it absorbed the most water, whilst not having the lowest cross-linking degree or G’. This suggests that there might be another property influencing these results, that would allow the Lips filler to absorb more water and be more efficient in the treatment of deeper perioral wrinkles and the restoration of larger lip volumes, as the manufacturer’s indications advocate.
Rather than measuring filler expansion caused by water absorption in the 2D evaluation, an increase in width was determined instead, as height changes weren’t evaluated, and without surrounding tissue to sustain the fillers, they ended up quickly losing their consistency, and spreading out sideways on the coverslips. The observed increase in width is possibly caused by other properties.
When looking at Fig. 6, we can see that Volume had both the lowest initial width and the lowest width increase after adding PBS, which can be explained by the fact that this filler has the highest cohesivity according to Fillmed. Cohesivity corresponds to internal adhesion forces of a filler [30], therefore it makes sense that this filler would retain its shape and spread out less. Fine Lines, on the other hand, seemed to generally show the highest initial width and ensuing increase, while also being the least cohesive filler, which causes it to be more malleable and easily deformed [30].
Besides cohesivity, other properties like cross-linking degree or the amount of uncrosslinked HA, can also be responsible for the different initial line widths observed in Figs. 6 and 7, since lower cross-linking degrees and higher amounts of uncrosslinked HA increase fluidity [4, 13, 21, 34]. This can help explain why Volume had the lowest initial width, given its highest cross-linking degree and least amount of free HA, which would reduce its fluidity and make it spread out less on the coverslip. In fact, Volume, alongside Lips, seems to be one of the more viscous fillers. Fine Lines is the opposite, showing the largest initial width and the highest amount of free HA (alongside Lips Soft) and lowest cross-linking degree, while seemingly being the most fluid filler.
For 3D evaluation, pig skin was stored in DMEM at 25ºC, which allows the maintenance of its cellular viability up until 18 hours [14]. Still, an additional scanning at 24h was performed, to see whether there were any further volume changes.
As is shown in Fig. 9, all fillers expanded after being injected into the pig skin. This helps reenforce that clinicians should be careful when planning how much filler to inject into patients’ skin, and that an under-filling may be beneficial [24], since it’s expected that the initial volume will increase for at least up until 24 hours after the injection.
The Lips filler absorbed the most water and revealed high viscosity, which translated to an overall 3D expansion of 44%. As is shown in Fig. 9, this was the second lowest total expansion observed after Volume (39%), which was the most viscous and cohesive of the fillers. On the other hand, Fine Lines tended to absorb less water, while being the most fluid and least cohesive of the fillers, which translated to a total expansion of 92%. The Universal filler, which was highly fluid and had the second highest swelling ratio, also had the second highest total expansion (79%).
Expansion or lifting capacity, like all HA characteristics, is influenced by many other properties. A stronger gel, with higher G’, cross-linking degree and viscosity, is expected to better resist deformation and lift the tissues [8, 13, 18]. Fillers with higher HA concentration and ratio of crosslinked HA/uncrosslinked HA similarly allow for bigger expansion [24]. Since lifting capacity correlates to how much projection fillers can induce, it would be expected that a filler meant to be applied in deeper planes of the skin and restore bigger volumes (such as Volume) should expand more, and a filler meant to be applied more superficially and correct more delicate fold and wrinkles (such as Fine Lines) should expand less [18].
Our results are not in accordance with this, since Volume, which expanded the least, was the most viscous filler, while also having the highest G’, cross-linking degree and crosslinked HA/uncrosslinked HA ratio. Fine Lines, which showed both the highest total expansion and the highest volume immediately after injection, was the most fluid, while simultaneously having the lowest G’, cross-linking degree, HA concentration and crosslinked HA/uncrosslinked HA ratio.
The impact cohesivity may have on fillers’ capacity to expand is still debatable, since there isn’t a standardized protocol to measure it [10, 13]. Nonetheless, some studies suggest that more cohesive fillers will have a greater capacity to volumize the tissues and show bigger initial vertical projection, since they resist surrounding tissue pressure more easily [10, 13, 27, 30]. We aren’t able to confirm this based on our results, since it was the least cohesive filler (Fine Lines) that showed the biggest capacity to expand vertically.
Some papers mention that higher water absorption translates to a higher potential for volume increase [18, 24], but such association was not perceived in this study, as Fine Lines expanded the most, even though it absorbed the least amount of water.
The discordance between our 3D results and the data present in literature could exist due to the small sample size, scanning errors caused by skin deformation or different injection depths (filler injection in a superficial plane proved to be challenging, due to pig skin’s high thickness and strength), or the influence of other physicochemical and rheological properties and tissue-related factors, such as surrounding tissue structure and consistency (muscle tensile strength can act as a constricting barrier to filler expansion), tissue fluid balance and pH value [22, 24, 25].
Based on our results, since water absorption was determined from swelling ratio which corresponds to an increase in filler weight [22], and a filler’s lifting capacity depends on its ability to resist surrounding tissue pressure [30], we could hypothesise that fillers which absorb more water and become heavier, can’t resist as easily to tissue pressure due to their weight, thus having a lower projection. This would help explain why fillers with higher swelling ratio values had lower 3D volume values. Besides, even though these results seem to not be in accordance with literature, it’s important to remember that the chemistry of these fillers is extremely complex, and that all their properties are interconnected, making it difficult to specify exactly which ones caused Fine Lines to expand the most and Volume the least. To justify and validate these results, and achieve statistical significance, future studies with a bigger sample and the inclusion of other fillers are necessary.
It´s also important to further investigate HA fillers’ volume changes in the skin throughout time and clarify their connection with water absorption, and how each HA property can influence it, along with expansion capacity. Additionally, in vivo filler performance should be evaluated and compared to in vitro results regarding HA rheological and physicochemical properties.