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The Myth of the “Biphasic” Hyaluronic Acid Filler

Öhrlund, J. Åke MSc; Edsman, Katarina L. M. PhD

doi: 10.1097/DSS.0000000000000545
Original Article
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BACKGROUND The terms “biphasic” and “monophasic” have been used frequently as a means of differentiating hyaluronic acid (HA) fillers. This type of categorization is based on misinterpretations of the term “phase” and provides no help to the practitioner when selecting the most appropriate product for each indication, patient, and injection technique.

OBJECTIVE The purpose of this study was to analyze the properties of 2 HA filler families; Juvederm (JUV) (Allergan), often stated to be monophasic and Restylane (RES) (Galderma), often stated to be biphasic, and discuss what properties may have led to the use of the terms monophasic and biphasic.

MATERIALS AND METHODS Three different methods were used for JUV and RES: determination of extractable HA; determination of water uptake; and microscopy.

RESULTS The analyzed products were shown to contain both observable gel particles and extractable HA and have the ability to absorb added water.

CONCLUSION The categorization of HA fillers as biphasic or monophasic was shown to be scientifically incorrect and should therefore be avoided. Further analytical measurement of the properties leading to this misinterpretation can provide information to discriminate and categorize HA fillers on a sounder scientific basis.

Galderma, Uppsala, Sweden

Address correspondence and reprint requests to: J. Åke Öhrlund, MSc, Galderma, Seminariegatan 21, Uppsala SE-752 28, Sweden, or e-mail: ake.ohrlund@galderma.com

Both authors are employed by Galderma.

The hyaluronic acid (HA) filler business is accelerating rapidly with a number of new products being introduced each year. Today the European market includes more than a hundred different HA fillers. With the increased number of fillers on the market, the need for differentiation between them has led to an array of descriptors being used by the different vendors.

Hyaluronic filler descriptors that are scientifically backed up by measurements using well-accepted techniques, such as rheometry, generate precise numerical data that are valuable to enable comparison between them. Terms such as malleability, cohesivity, plasticity, etc are easy to comprehend but more challenging to measure. Especially confusing is the categorization of HA fillers as either monophasic or biphasic. This nomenclature can be found in a number of publications.1–151–151–151–151–151–151–151–151–151–151–151–151–151–151–15 A search for literature on biphasic HA fillers will yield numerous results, but despite this, scientific definitions on how to determine whether a filler is monophasic or biphasic is typically omitted.

It is possible that some of the properties typical for HA fillers may be the source of the confusion about the term biphasic. These include the particulate nature of HA fillers in general, the gel particle size distribution, the level of water saturation, and the amount of extractable HA and other soluble components in the product. These properties are analyzed and discussed in relation to the term phase.

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The term Phase

As a basis for interpreting the results of the analysis in relation to the terms monophasic and biphasic, the term phase needs to be understood in a materials context. The definition of phase according to the International Union of Pure and Applied Chemistry Gold Book16 is as follows: “An entity of a material system which is uniform in chemical composition and physical state.” From this definition it follows that a biphasic material must be inhomogenous regarding chemical composition and/or physical state.

A biphasic material can consist of 2 or more substances in a blend where it can be observed that different regions have different chemical compositions. An everyday example of such a mix is salad dressing, where oil and water is mixed. Because oil and water are not soluble in each other, they will always occupy different regions in the vessel. In one spot there will be only oil, in one spot there will be only water. A biphasic liquid/liquid system may be termed emulsion.

A biphasic material can also consist of 1 material in 2 different physical states, although having the same chemical composition throughout. An example is a glass of water with ice. The liquid water and the ice can be readily observed, existing in separate locations. A biphasic solid/liquid system may be termed suspension.

A monophasic material can consist of 1 material, but it can also consist of a mix of 2 or more components. In for example saline, there is both water and salt, but because the salt is uniformly distributed in the water, the composition is the same at all locations, and therefore, saline should be described as a monophasic material. In fact, all materials where the composition in different locations cannot be demonstrated to be different should be regarded as monophasic.

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Materials and Methods

Microscopy

Microscopy was performed by adding a small amount of gel into a 9-cm petri dish containing 10 mL of water and 30 μL of a 1% toluidine blue solution. Approximately 0.1 mL of gel typically yields a particle concentration suitable for imaging. The petri dish was put on a gentle shaker allowing the gel particles time to disperse and absorb water and staining color to equilibrium. The samples were imaged at 10× magnification using a MZ-16 A stereo microscope supplying transmitted light from a CLS 150 X cold light source (Leica Microsystems GmbH, Wetzlar, Germany).

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Water Uptake

The water uptake test was performed as described for swelling factor in Edsman and colleagues.17 Approximately 0.5 g of product was weighed into a 10-mL measuring glass. Approximately 10 mL of saline was added and then mixed thoroughly until the gel particles were fully dispersed. Softer gels required a longer dispersion time than firmer gels. The gel particles were allowed time to settle and the volume of the gel column was read. The swelling factor was calculated as V/V0, where V0 is the initial volume of the gel, and V is the volume of the fully swollen gel in 0.9% NaCl. All gels were assumed to have a density of 1 g/cm3. Typical precision of the method is 2% relative standard deviation.

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Extractable Hyaluronic Acid

The amount of extractable HA was analyzed as described for gel content in Edsman and colleagues.17 The gel content was determined by adding an excess of saline to a known amount of product and dispersing the gel thoroughly to form a dilute suspension. The dilute suspension of the gel was filtered through a 0.22-μm filter, and the concentration of HA in the filtrate (the extractable part) was determined using the carbazole method. In Edsman and colleagues17 the gel content was calculated as the fraction of HA in the filler that could not pass through the 0.22-μm filter when filtering the diluted suspension of the product. In this study, the fraction of HA in the filler that did pass through the 0.22-μm filter was evaluated, to demonstrate the fraction of extractable HA rather than the fraction of actual gel bound HA. Typical precision of the method is 2% relative standard deviation.

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Results and Discussion

Microscopy

After dispersion in water and staining with toluidine blue, the gel particles are easily discernible in products from both product families (Figures 1 and 2). The most obvious difference between the product families is the particle size distribution resulting from the different types of particle sizing processes used. The Restylane (RES) products each have their own specific particle size, whereas the Juvederm (JUV) products contain a wider array of particle sizes.

Figure 1

Figure 1

Figure 2

Figure 2

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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.

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Particle Firmness

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.

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Water Uptake

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.

Figure 3

Figure 3

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.

TABLE 1

TABLE 1

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.

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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.

Figure 4

Figure 4

TABLE 2

TABLE 2

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.

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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.

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Conclusion

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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References

1. Ahn CS, Rao BK. The life cycles and biological end pathways of dermal fillers. J Cosmet Dermatol 2014;13:212–23.
2. Andre P. New trends in face rejuvenation by hyaluronic acid injections. J Cosmet Dermatol 2008;7:251–8.
3. Attenello NH, Maas CS. Injectable fillers: review of material and properties. Facial Plast Surg 2015;31:29–34.
4. Buntrock H, Reuther T, Prager W, Kerscher M. Efficacy, safety, and patient satisfaction of a monophasic cohesive polydensified matrix versus a biphasic nonanimal stabilized hyaluronic acid filler after single injection in nasolabial folds. Dermatol Surg 2013;39:1097–105.
5. Flynn TC, Sarazin D, Bezzola A, Terrani C, et al.. Comparative histology of intradermal implantation of mono and biphasic hyaluronic acid fillers. Dermatol Surg 2011;37:637–43.
6. Gilber E, Hui A, Walldorf HA. The basic science of dermal fillers: past and present Part I: background and mechanisms of action. J Drugs Dermatol 2012;11:1059–68.
7. Goh AS, Kohn JC, Rootman DB, Lin JL, et al.. Hyaluronic acid gel distribution pattern in periocular area with high-resolution ultrasound imaging. Aesthet Surg J 2014;34:510–5.
8. Huang X, Liang Y, Li Q. Safety and efficacy of hyaluronic acid for the correction of nasolabial folds: a meta-analysis. Eur J Dermatol 2013;23:592–9.
9. Kontis TC. Contemporary review of injectable facial fillers. JAMA Facial Plast Surg 2013;15:58–64.
10. Kühne U, Imhof M. Treatment of the ageing hand with dermal fillers. J Cutan Aesthet Surg 2012;5:163–9.
11. Kulichova D, Borovaya A, Ruzicka T, Thomas P, et al.. Understanding the safety and tolerability of facial filling therapeutics. Expert Opin Drug Saf 2014;13:1215–26.
12. Leonardis M, Palange A. New-generation filler based on cross-linked carboxymethylcellulose: study of 350 patients with 3-year follow-up. Clin Interv Aging 2015;10:147–55.
13. Park KY, Kim HK, Kim BJ. Comparative study of hyaluronic acid fillers by in vitro and in vivo testing. J Eur Acad Dermatol Venereol 2014;28:565–8.
14. Gold MH. Soft tissue augmentation in dermatology—2009 update. J Cutan Aesthet Surg 2010;3:2–10.
15. Pavicic T. Efficacy and tolerability of a new monophasic, double-crosslinked hyaluronic acid filler for correction of deep lines and wrinkles. J Drugs Dermatol 2011;10:134–9.
16. Clarke JB, Hastie JW, Kihlborg LHE, Metselaar R, et al.. Definitions of terms relating to phase transitions of the solid state (IUPAC recommendations 1994). Pure Appl Chem 1994;66:577–94.
17. Edsman K, Nord L, Ohrlund A, Lärkner H, et al.. Gel properties of hyaluronic acid dermal fillers. Dermatol Surg 2012;38:1170–9.
18. Pierre S, Liew S, Bernardin A. Basics of dermal filler rheology. Dermatol Surg 2015;41(Suppl 1):S120–6.
19. Lupo MP, Smith SR, Thomas JA, Murphy DK, et al.. Effectiveness of juvederm ultra plus dermal filler in the treatment of severe nasolabial folds. Plast Reconstr Surg 2008;121:289–97.
20. Monheit GD. Nonsurgical facial rejuvenation. Facial Plast Surg 2014;30:462–7.
21. Markarian MK, Hovsepian RV. The interface of cosmetic medicine and surgery: working from the inside and the outside. Clin Plast Surg 2011;38:335–45.
22. Jones D. Volumizing the face with soft tissue fillers. Clin Plast Surg 2011;38:379–90.
23. Patel U, Fitzgerald R. Facial shaping: beyond lines and folds with fillers. J Drugs Dermatol 2010;9(Suppl 8):S129–37.
24. Flynn TC, Thompson DH, Hyun SH. Molecular weight analyses and enzymatic degradation profiles of the soft-tissue fillers belotero balance, restylane, and juvéderm ultra. Plast Reconstr Surg 2013;132(4 Suppl 2):22S–32S.
25. Beasley KL, Weiss MA, Weiss RA. Hyaluronic acid fillers: a comprehensive review. Facial Plast Surg 2009;25:86–94.
26. Baumann LS, Shamban AT, Lupo MP, Monheit GD, et al.. Comparison of smooth-gel hyaluronic acid dermal fillers with cross-linked bovine collagen: a multicenter, double-masked, randomized, within-subject study. Dermatol Surg 2007;33:S128–35.
27. Kablik J, Monheit GD, Yu L, Chang G, et al.. Comparative physical properties of hyaluronic acid dermal fillers. Dermatol Surg 2009;35(Suppl 1):302–12.
28. Monheit GD, Baumann LS, Gold MH, Goldberg DJ, et al.. Novel hyaluronic acid dermal filler: dermal gel extra physical properties and clinical outcomes. Dermatol Surg 2010;36(Suppl 3):1833–41.
29. Sundaram H, Cassuto D. Biophysical characteristics of hyaluronic acid soft-tissue fillers and their relevance to aesthetic applications. Plast Reconstr Surg 2013;132(4 Suppl 2):5S–21S.
30. Elson ML. Facial contouring with injectable biphasic filler materials. Cosmet Dermatol 2010;23:304–7.
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