The interaction between hyaluronidase and hyaluronic acid gel fillers - a review of the literature and comparative analysis

Varieties of hyaluronic acid filler

At the time of this writing, 18 individual formulations of HA have been approved for use as dermal fillers by the FDA^[21]. The subtypes and properties of three major filler families, Restylane, Juvéderm, and Belotero Balance, are described below and summarized in Table 1.

Restylane is manufactured by Q-Med AB (Uppsala, Sweden) and produced from HA generated via fermentation^[22]. Produced entirely from non-animal sources, it is classified as a non-animal stabilized hyaluronic acid (NASHA) and is stabilized though a crosslinking process to the compound 1,4-butanediol diglycidyl ether (BDDE)^[22,23]. It has a HA concentration of 20 mg/mL and a biphasic formulation with a gel particulate size of 330-430 micrometers^[22,24]. The degree of crosslinking is relatively low at 1%^[24]. Subsequently introduced members of the Restylane family include Restylane Lyft (formerly known as Perlane), which contains fewer, larger gel particles per milliliter (8,000 per mL vs. 100,000 per mL); Restylane-L, which contains lidocaine; and Restylane Silk, which contains lidocaine, is less viscous, and was formulated specifically for lip augmentation^[22,24]. Restylane Refyne, Restylane Defyne, and Restylane Kysse are manufactured with XpresHAn Technology, which varies the degree of crosslinking and gel particle size to create softer gels^[25,26].

Juvéderm, initially produced by Lea Derm (Paris, France) and subsequently manufactured and distributed by Allergan (Irvine, CA), is also a BDDE-crosslinked NASHA produced from equine streptococci^[24]. In contrast to Restylane, it is a monophasic gel without distinct particles, originally produced through Hylacross technology that allows for a high concentration of crosslinking. First approved by the FDA in 2006, the Juvéderm family of fillers has expanded to many formulations that vary in concentration of HA and crosslinking density. The two main varieties available for facial wrinkles and folds in the US market are Juvéderm Ultra (Juvéderm 24 HV) and Juvéderm Ultra Plus (Juvéderm 30 HV), which both have a concentration of 24 mg/mL but differ in their crosslinking percentages (9% and 11%, respectively)^[27].

The more recent formulations of Juvéderm are manufactured using Vycross technology, which combines low- and high-weight HA molecules to improve crosslinking efficiency^[24]. Juvéderm Voluma XC (20 mg/mL) is a formulation with high cohesivity and viscosity used for deep injections and cheek augmentation. Juvéderm Vollure XC (17.5 mg/mL) is slightly less concentrated and designed for volumizing nasolabial folds^[28]. Even denser crosslinking is present in Juvéderm Volbella XC (15 mg/mL), which is manufactured with a higher proportion of low molecular weight HA and designed for lip augmentation and perioral rhytids^[29]. Juvéderm Volite, the least concentrated variety at 12 mg/mL, is for superficial cutaneous depressions and fine lines^[30].

Members of the Belotero family, in contrast to the above monodensified varieties, are polydensfied compounds that contain continuously crosslinked HA in a single phase^[31]. Manufactured by Anteis S.A. (Geneva, Switzerland), fillers of the Belotero family are made with a cohesive polydensified matrix (CPM) technology that creates a gel with nonuniform crosslinking^[15]. This creates a low-viscosity filler that exhibits homogenous intradermal distribution as compared to other fillers, theoretically allowing for increased injection precision^[24,32]. Like Juvéderm, the Belotero family has expanded into many different formulations that vary in concentration^[31]. However, the only one currently available in the US is Belotero Balance, which has a concentration of 22.5 mg/mL and is approved for facial wrinkles and folds^[31].

Several other filler varieties, including RHA 2, RHA 3, RHA 4 (Teoxane SA), Revanesse Versa, and Revanesse Versa Plus, are also available in the US, but are less commonly studied and used clinically. Hydrelle (previously known as Elevess) is manufactured by Anika Therapeutics (Bedford, MA) and marketed through Coapt Systems (Palo Alto, California). Though also sourced from equine streptococci, it is crosslinked with p-phenylene bisethyl carbodiimide (BCDI)^[22]. With a concentration of 28 mg/mL, it has amongst the highest available content of HA^[22]. Prevelle Silk, the second generation of now-unavailable Captique, is manufactured by Genzyme Corporation (Cambridge, MA) and marketed by Johnson & Johnson (Skillman, NJ); it has a concentration ranging from 4.5-6 mg/mL and is cross-linked with di-vinyl sulfone^[22]. Hylaform (manufactured by Genzyme Biosurgery in Ridgefield, NJ), was an avian-sourced formulation with concentration ranging from 4.5-6 mg/mL of HA. Notably, it is no longer available on the market in the US^[21,27].

Varieties of hyaluronidase

There are four varieties of hyaluronidase currently available in the United States: two derived from purified bovine testicular hyaluronidase, one derived from purified ovine testicular hyaluronidase, and one manufactured from recombinant human DNA^[19].

Vitrase, manufactured by STA Pharmaceutical (Irvine, CA), is derived from purified ovine testicular hyaluronidase. Amphadase, manufactured by Amphastar Pharmaceuticals, Inc. (Rancho Cucamonga, CA), and Hydase, manufactured by PrimaPharm, Inc. (San Diego, CA), are derived from purified bovine testicular hyaluronidase. Hylenex, manufactured by Halozyme Therapeutics (San Diego, CA), is made from recombinant human DNA^[33]. Though one experimental study has suggested these varieties are equally potent and can be used interchangeably^[6], subsequent tests and prevailing clinical perceptions have questioned this assumption^[16,34]. Clinically, it is noteworthy that animal-derived enzymes, though less expensive and more readily available, generally have a less favorable immunogenic profile than those that are recombinantly produced^[34].

Greater concentrations of hyaluronic acid are associated with greater resistance to degradation

The concentration of HA in currently available fillers range from 4.5 mg/mL to 30 mg/mL, with the most commonly injected varieties falling between 20-24 mg/mL^[13]. A higher concentration of HA generally corresponds to increased stiffness and longevity^[27]. In nearly every study of HA-hyaluronidase interaction, filler varieties with a lower concentration of HA tended to dissolve more quickly than fillers with higher HA concentrations. For example, in comparing three filler varieties with different HA concentrations (24 mg/mL, 20 mg/mL, and 5.5 mg/mL), Jones et al.^[2] measured the generation of soluble HA to demonstrate that a lower concentration of HA correlated with increased susceptibility to dissolution by ovine hyaluronidase. Rao et al.^[6] observed similar findings in an in vitro study with ovine-derived hyaluronidase and validated this finding for recombinant hyaluronidase. Cavallini et al.^[14] studied the response of six fillers to bovine-derived hyaluronidase and observed the most rapid and homogenous dissolution in the two varieties with the lowest concentrations of HA, Juvederm Volite (12 mg/mL) and Teosyal RHA 1 (Teoxane Laboratories, Geneva, Switzerland; monophasic; 15 mg/mL). In vivo, using a rat model, Shumate et al.^[16] demonstrated faster degradation by ovine hyaluronidase of Restylane-L (20 mg/mL) when compared to two filler varieties from the Juvederm family (20-24 mg/mL). Altogether, the improved dissolution of lower concentration HA by hyaluronidase was validated across all major HA filler varieties and with all three sources of hyaluronidase, in both in vivo and in vitro studies.

Greater degree of crosslinking is associated with greater resistance to degradation

HA is stabilized by crosslinking individual particles to each other with covalent bonds in all HA-containing fillers^[12]. Across all studies of HA-hyaluronidase interactions, a higher degree of crosslinking generally correlated with greater resistance to degradation by hyaluronidase. Sall et al.^[12] examined the response of 11 individual formulations of HA filler to bovine hyaluronidase and found that the slowest to dissolve, Surgiderm 30 (Allergan, Irvine, California; monophasic; 24 mg/mL) had the greatest degree of crosslinking. The fastest to dissolve, Restylane and Perlane, had the lowest degree of crosslinking. These findings were further validated by comparisons of fillers within the same family, which eliminated possible confounders such as the monophasic/biphasic status that differ between filler classes such as in Surgiderm and Restylane. Within the Juvéderm family, for example, less-crosslinked fillers such as Juvéderm 18 dissolved more quickly than their more-crosslinked counterparts.

These findings were validated in additional in vitro experiments of bovine hyaluronidase^[14,17], ovine hyaluronidase^[2,6,15], and recombinant hyaluronidase^[6]. In vivo, these findings were validated in human subjects by Juhasz et al.^[18], who found less dissolution of Juvéderm Ultra Plus than Juvéderm Ultra. Although this difference was small and the sample size was too small to achieve significance, these two filler varieties are ideal for comparing this variable as both have the same concentration of HA (24 mg/mL), but Juvederm Ultra Plus has an increased density of crosslinking^[27]. The decreased dissolution observed makes scientific sense, as a higher density of crosslinking decreases the access hyaluronidase has to its enzymatic substrate^[12].

Monophasic hyaluronic acid formulations are more resistant to degradation than biphasic formulations

HA fillers are classified as monophasic (cohesive gels without distinct particles) or biphasic (particles suspended in gel)^[35]. Because the individual particles of a biphasic filler create a greater surface area for enzymatic attack, these formulations, e.g., Restylane, have been predicted to exhibit increased susceptibility to degradation compared to their monophasic counterparts^[12]. Although this and other principles may have origins in manufacturer claims or manufacturer-sponsored studies, this trend is upheld by the reviewed articles on HA-hyaluronidase interactions. Sall et al.^[12] studied both major biphasic compounds (Restylane and Perlane) and found these fillers significantly more susceptible to degradation by bovine hyaluronidase than monophasic varieties of Juvéderm. In other studies, Restylane was routinely found to be more dissolvable than comparably concentrated monophasic fillers with all three varieties of hyaluronidase^[2,6,15,17], in both in vitro and in vivo experiments^[16,18].

Restylane

Restylane varieties were studied in all ten^[1] (8 studies, 2 of which examined 2 different hyaluronidase types) reported experiments of HA-hyaluronidase interactions and were observed to be the most responsive to hyaluronidase in six studies and the least responsive to hyaluronidase in one. When compared directly to Belotero in the same experiment, Restylane was found to be more susceptible to degradation 60% of the time. When compared directly to any member of the Juvéderm family, it was found to be more susceptible to degradation 90% of the time.

A biphasic formulation with relatively little crosslinking, Restylane and its derivatives were unsurprisingly almost always found to be more susceptible to degradation than the Juvéderm family of fillers. In an outlier study, Cavallini et al.^[14] qualitatively found Restylane (20 mg/mL) to be the least responsive of six fillers to bovine hyaluronidase. Using a ratio of 30 U hyaluronidase per 0.1 mL filler (15 U hyaluronidase per mg Restylane), which exceeds established recommendations of 5 U hyaluronidase per 0.1 mL Restylane for ischemic complications^[34], they noted that Restylane needed both additional time and enzyme to reach complete liquefaction, as compared to other fillers, including Juvéderm Volite (12 mg/mL), which dissolved instantly. The results of this study contrast with other studies of similar design. Rao et al.^[6], using a qualitative in vitro model, also found Restylane to be the most susceptible compound to ovine and recombinant hyaluronidase using lower ratios of 5 U per 1 mg and 3.75 U per 1 mg, respectively. In addition, other studies of bovine hyaluronidase also found that Restylane dissolved more rapidly than comparably concentrated varieties of Juvéderm^[12,17]. The reason for Cavallini’s experimental discrepancy is unclear, but may be attributable to subjective errors in qualitative interpretation of filler gel consistencies. Interestingly, Restylane is the only one of the six tested fillers to not be pictured in this study’s photographic figure of its experimental results.

With regard to Restylane’s response to different types of hyaluronidase, there is no evidence that any one enzyme is best at dissolution. Again, using Juvéderm as a comparison, Restylane was found to be more degradable in 2/2 studies of recombinant hyaluronidase, 5/5 studies of ovine hyaluronidase, and 2/3 studies of bovine hyaluronidase. While this might superficially suggest a relatively poor response to bovine hyaluronidase, this single anomaly (Cavallini et al.^[14]) should be considered in the context outlined above. The slight predilection of Restylane for ovine hyaluronidase over recombinant hyaluronidase reported by Shumate et al.^[16] is not clearly replicated across all studies considered together, though the small number of studies examining recombinant hyaluronidase limit the conclusions that may be drawn.

Juvéderm

Juvéderm varieties were also studied in all ten reported experiments and were observed to be the least responsive to hyaluronidase in five and the most responsive to hyaluronidase in one. When compared directly to Belotero, Juvéderm was found to be more resistant to degradation 40% of the time. When compared directly to Restylane, Juvéderm was found to be more resistant to degradation 90% of the time.

Overall, the Juvéderm family exhibits many of the qualities described to be associated with resistance to degradation by hyaluronidase: it is a monophasic compound and its formulations generally have higher concentrations of HA and more crosslinking than other HA fillers^[22]. It is not unexpected then that it resists degradation more effectively than Restylane in a wide variety of experiments, including both in vivo and in vitro designs and with all three different sources of hyaluronidase.

In the single study in which a Juvéderm filler was observed to exhibit the most degradation of its studied subset^[14], the specific variety was Juvéderm Volite (12 mg/mL), a formulation with significantly lower HA concentration than the majority of the Juvéderm family of fillers. Juvéderm Voluma (20 mg/mL) demonstrated significantly more resistance to degradation in this experiment. No other studies examined the Volite member of the Juvéderm family.

When studies looking for differences in Juvéderm’s response to different varieties of hyaluronidase are compared, no variety of hyaluronidase is clearly better at dissolving Juvéderm than others. In the recombinant hyaluronidase experiments where direct comparison is possible, Juvéderm fillers are universally more resistant than Restylane (2/2) and less resistant than Belotero (1/1). In ovine experiments, Juvéderm fillers are more resistant than Restylane (5/5) and again, generally less resistant than Belotero (2/3). In bovine experiments, Juvéderm fillers are generally more resistant to dissolution than Restylane (2/3) and, interestingly, more resistant than Belotero (1/1). Though this last result may appear to suggest that bovine hyaluronidase is less effective against Juvéderm (relative to Belotero), it is also notable that the one instance in which Juvéderm dissolved more than Restylane also occurred with bovine hyaluronidase, which would suggest the opposite.

When comparing studies that examined both Juvéderm and Belotero, the single in vitro study in which Juvéderm was less resistant utilized bovine hyaluronidase to compare the fillers^[17]. However, no effective degradation at all was seen in Juvéderm Ultra 3 in this experiment, despite degradation of this formulation being observed with bovine hyaluronidase in other experiments^[12]. This discrepancy makes it difficult to draw any specific conclusions about Juvéderm’s response to bovine hyaluronidase, and no additional data from other studies is available as a point of comparison.

Belotero balance

Belotero balance was studied in 5 experiments and was observed to be the least responsive to hyaluronidase in 3 and the most responsive to hyaluronidase in 2. In 3 out of 4 in vitro experiments^[6,15], including 2 with ovine hyaluronidase and 1 with recombinant hyaluronidase, Belotero was observed to be the most resistant to degradation, likely in part due to its monophasic status, more extensive CPM crosslinking, and higher molecular weight than other common fillers like Juvéderm Ultra and Restylane^[15].

In an experiment on human subjects^[18], with ovine hyaluronidase, Belotero was observed to be the least resistant to degradation as compared to Restylane and Juvéderm Ultra Plus. This discrepancy might be attributable to factors introduced by the in vivo system or the experimental design of the study, in which HA degradation was measured clinically via a standardized palpation scale following hyaluronidase injection. Belotero, with its unique polydensified matrix structure, has a lower viscosity than other HA fillers and thus a greater homogenous intradermal distribution of injection^[31]. Per the authors’ own acknowledgement, these properties may have confounded measurements of Belotero relative to its more viscous counterparts.

Interestingly, the in vitro experiment in which Belotero was found to be the least resistant to degradation (Buhren et al.^[17]) was the only experiment that studied Belotero (22.5 mg/mL) with bovine hyaluronidase. In this experiment of Belotero, Emervel (Restylane, 20 mg/mL) and Juvéderm Ultra 3 (24 mg/mL), 50 microliters of filler was combined with a fluorescent linker dye and exposed to 10 units (U/ml) of hyaluronidase, with degradation measured by recorded changes in fluorescence intensity. This can be contrasted with the work of Flynn et al.^[15], who used a different experimental design (chromatography) but the same ratio of hyaluronidase to filler (16 U per 0.08 mg) to show Belotero was more resistant to ovine hyaluronidase than Juvéderm Ultra 3. When compared directly, these studies may suggest that Belotero is more susceptible than Juvéderm to ovine hyaluronidase than bovine hyaluronidase.

However, Buhren et al.^[17] notably observed no effective degradation of Juvéderm Ultra 3 by bovine hyaluronidase, even after 24 h. Sall et al.^[12] did observe degradation of Juvéderm Ultra 3 at a lower concentration of bovine hyaluronidase than Burhen et al. Though Sall et al. did not study Belotero, this difference in results with the same Juvéderm variety using the same enzyme suggests that the Burhen experiment may have had some confounding feature either in measurement methodology or other experimental factors such as pH or temperature. More studies are needed to clarify whether Belotero is less responsive to bovine hyaluronidase than ovine hyaluronidase.