Nonparticulate Hyaluronic Acid Fillers
Although the particulate nature of all cross-linked HA fillers has been demonstrated and recognized by those in the field, products are sometimes described as “homogenous gel”18–2118–2118–2118–21 “continuous gel,”22 or “nonparticulate”1,13–15,231,13–15,231,13–15,231,13–15,231,13–15,23 and “smooth”1,24–261,24–261,24–261,24–26 implying that there is only 1 single piece of gel in the syringe. It is true that when an HA filler is being formed, the cross-linking process will create 1 single bulk of material, actually 1 immense molecule of cross-linked HA. However, because of the requirement that the material is to be filled into a syringe and injected through a needle, the bulk material has to be fragmented into smaller pieces. This can be performed in different ways, for example by cutting with rotating knives or by pressing the material through a mesh of a chosen size. The resulting material is a bulk of HA gel particles that has flow properties suitable for injection. Because the HA gel particles are soft, colorless, and transparent, it is difficult to observe the separate particles. Because of the softness, the particles pack tightly and the material seems to consist of 1 single body of material. Using the dispersion and staining technique, however, the particulate nature of any cross-linked HA filler on the market can be demonstrated.
Although the particles in any cross-linked HA can be visualized using dispersion and staining, some investigators opt for direct observation of the gel after ejection from the syringe onto a microscope slide or a petri dish. In such cases, some HA fillers are said to have observable particles whereas other products do not, sometimes with a specification of “up to a magnification of x times.” Notably, such images are shown for RES and JUV Ultra in Beasley and colleagues25 whereas only RES and PER are shown using what seems to be a dispersion and staining technique similar to the one described in this study.
For the gel particles to be observable without sample preparation, the particle size needs to be large enough so that the particles are readily visible. The particles also have to possess a certain gel firmness. A very soft gel will deform from the forces of gravity and water surface tension, and therefore form a flat pool, much like an HA solution would. A firmer gel particle will be able to resist larger forces than those caused by its own weight, and will therefore retain its shape to a larger extent, and hence be more visible in this type of experiment.
As shown by several authors,17,25,27,2817,25,27,2817,25,27,2817,25,27,28 using rheometry products stated to be monophasic are generally softer than those stated to be biphasic, leading to the different appearances when tested as above.
The level of water uptake showed that all tested products absorbed water when adding saline (Figure 3). This means that they were all unsaturated, that is there is no excess water in the products.
The water-unsaturated nature of cross-linked HA fillers has been demonstrated by other investigators. For comparison, the water uptake data of Kablik and colleagues27 and Monheit and colleagues28 are shown (Table 1), after recalculating percent dilution into swelling factor.
The reason for the lower values of Kablik and colleagues27 and Monheit and colleagues28 is accounted for by the different methodology used, where the products were diluted with phosphate-buffered saline to chosen levels, followed by rheometric determination of the phase angle. The dilution at which the phase angle increased by 50% from its original value was interpreted as the maximum swelling before phase separation. This approach does not yield values for complete saturation because a complete saturation and thereby phase separation would result in a water layer between the sample and the measuring probe, making rheometric measurements impossible.
Although there are method-induced differences between the results in those publications compared with the results in this study, the water-unsaturated state of the products was clearly demonstrated, because a fair amount of water could be added before the estimated maximum swelling was achieved.
Some publications2–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,182–7,9,11–13,18 have stated that the biphasic fillers consist of gel particles suspended in liquid, implying that there is more water in the composition than can be absorbed by the gel particles. However, such a composition would be impossible to use. If there was excess water, some portions of the ejected material from the syringe would contain mostly water, whereas some portions would contain gel particles. Such a composition would also cause great difficulties during filling of the syringe.
To avoid such a situation, all HA filler products on the market are unsaturated. This means that if water is added to the product, the water will be absorbed. If water was added in a large enough quantity, a true phase separation would occur. The result would then be HA gel particles suspended in an excess of water.
Some of the confusion may be caused by the “wet” impression that HA fillers give because of their very high water content. For an HA concentration of 20 mg/mL and a buffer concentration of 1%, the content of water will be approximately 97%. Despite this very high water content, the HA fillers are not saturated and can typically absorb a fair amount of water in swelling experiments.
Extractable Hyaluronic Acid
Determination of extractable HA showed that a noticeable amount of extractable HA was found in the HA fillers studied (Figure 4). For comparison, the results of Beasley and colleagues,25 Kablik and colleagues,27 and Monheit and colleagues28 are shown (Table 2). In the Beasley and colleagues article,25 the HA gel concentration was divided by the total HA concentration, yielding the fraction of gel bound HA. By subtracting the result from 100%, the fraction of extractable HA was obtained. In addition, the results were presented in the Beasley and colleagues article25 as gel to fluid ratios. However, for the products JUV Ultra and JUV Ultra Plus, the gel to fluid ratios did not match the HA gel concentrations and the total HA concentrations given for those 2 products.
In some publications,2,4–7,11,132,4–7,11,132,4–7,11,132,4–7,11,132,4–7,11,132,4–7,11,132,4–7,11,13 the term biphasic is mentioned in conjunction with a discussion of the extractable HA, sometimes denoted free HA or native HA. Some authors5,24,295,24,295,24,29 imply that native HA is added to the RES family of products after the cross-linking as a lubricant to lower the force required to extrude the product through the needle. However, there is no need to do so because extractable HA is a natural part of all cross-linked HA products, including RES for reasons described below.
Ideally, the cross-linking process would result in 1 single molecule consisting of all the HA strands of the added HA raw material. However, many HA filler manufacturers keep the amount of cross-linker added to a reasonably low level, resulting in less than 10% of the HA disaccharides being bound to a cross-linker molecule.17 Inevitably, some HA strands will have more cross-linkers attached, some will have fewer, whereas some HA strands will not be cross-linked at all. Because non–cross-linked HA strands will not be connected to the HA network, they will be extractable.
Another source of extractable HA is the heat sterilization that all HA filler manufacturers use to ensure sterility of the products. Because HA is very sensitive to heat, the sterilization process will cause a large number of breaks in the glycosidic bonds of the HA chains. The chemical crosslinks formed during the cross-linking procedure are much more stable and consequently not affected by sterilization. Again it is inevitable that some of the chain breaks will cause some strands of HA to disconnect from the cross-linked HA network.
The chain breaks during cross-linking and sterilization, and during storage, will lead to a certain amount of extractable HA, and most likely, there will be no HA filler completely without extractable HA.
The term extractable HA does not specify what size a fragment should be to be defined as extractable. Therefore, the term should preferably be used only in combination with a description of how to discriminate extractable HA from the HA connected to the HA network in the gel particles. The method for determination of the amount of extractable HA presented in this study is based on extraction of extractable HA in saline and filtration through a 0.22-μm filter. In this case, any HA fragment passing through the filter is termed extractable. Some fragments may contain crosslinks, but still be small enough to pass through the filter, and some fragments passing through the filter may be single HA strands without any crosslinks.
Because the gel particles in the syringe are tightly packed together, there is no space that is not occupied by gel particles. Because there is no space outside the particles, the extractable HA will disperse homogenously through the body of gel particles in the syringe. Because the extractable HA will be found in equal concentration throughout the body of gel particles in the syringe, the extractable HA cannot be considered another phase, merely another component. This also applies to the salts making up the buffer. Because they are dissolved in equal concentration all over the product, the term component is suitable, whereas the term phase is not.
True Biphasic Fillers
Outside the world of HA fillers, true biphasic fillers exist. As stated by Elson and colleagues,28 biphasic fillers are defined as “injectable fillers that use 2 different materials in the syringe”. Examples of true biphasic filler products are those based on polymethyl-methacrylate, poly-lactide, or calcium hydroxyl apatite, where solid particles of 1 composition are dispersed in a carrier solution of a different composition. In those cases, the composition is different in both chemical composition and physical state in different locations in the syringe, and the term biphasic is appropriate.
In this study, the categorization of HA fillers into biphasic and monophasic was shown to be scientifically incorrect. The use of the term should therefore be avoided. Analytical measurement of the properties related to this misinterpretation of the terms, however, can provide information to discriminate and categorize HA fillers on a sound scientific base.
These analytically measurable properties vary gradually across the range of products on the market, which makes it possible to discriminate products in much finer detail, helping the practitioner to select a suitable product for the patient, indication, and injection technique. The coarse division into monophasic and biphasic, based on whatever reasoning, does not aid a detailed comparison. It is the authors' hope that the terms biphasic and monophasic will soon be “phased” out from any scientific discussions on HA fillers.
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