Comparison of Hyaluronidase-Mediated Degradation Kinetics of Commercially Available Hyaluronic Acid Fillers In Vitro

Abstract

Background

The ability to degrade hyaluronic acid (HA)-based fillers with hyaluronidase allows for better management of adverse effects and reversal of suboptimal treatment outcomes.

Objectives

The aim of this study was to compare the enzymatic degradation kinetics of 16 commercially available HA-based fillers, representing 6 manufacturing technologies.

Methods

In this nonclinical study, a recently developed in vitro multidose hyaluronidase administration protocol was used to induce degradation of HA-based fillers, enabling real-time evaluation of viscoelastic properties under near-static conditions. Each filler was exposed to repeated doses of hyaluronidase at intervals of 5 minutes to reach the degradation threshold of G' ≤ 30 Pa.

Results

Noticeable differences in degradation characteristics were observed based on the design and technology of different filler classes. Vycross fillers were the most difficult to degrade and the Cohesive Polydensified Matrix filler was the least difficult to degrade. Preserved Network Technology products demonstrated proportional increases in gel degradation time and enzyme volume required for degradation across the individual resilient hyaluronic acid (RHA) products and indication categories. No obvious relationship was observed between gel degradation characteristics and the individual parameters of HA concentration, HA chain length, or the degree of modification of each filler when analyzed separately; however, a general correlation was identified with certain physicochemical properties.

Conclusions

Manufacturing technology was the most important factor influencing the reversibility of an HA product. An understanding of the differential degradation profiles of commercially available fillers will allow clinicians to select products that offer a higher margin of safety due to their preferential reversibility.

Level of Evidence: 4

Hyaluronic acid (HA)-based dermal fillers are injectable viscoelastic gels commonly used in cosmetic medicine to create a more youthful appearance, restore lost volume, or augment areas of structural deficiency. The primary goal of treatment with HA-based fillers is to achieve aesthetically pleasing enhancement or correction of soft tissue aging while optimizing durability and safety.^1,2 The composition (HA chain length, HA concentration, crosslinker content) of HA-based fillers can be designed to achieve different physicochemical properties, which can affect the clinical durability, degradation time, and optimal anatomic location for injection of the filler. Exogenous hyaluronidase enzymes are routinely used to reverse adverse events and other undesired outcomes that can be caused by HA-based fillers.¹ Fillers that require longer time frames for degradation and higher volumes of enzyme may impact the efficiency of resolving an adverse event or undesired outcome, which may become a critical consideration in the event of a medical emergency.^3-5 However, few studies have comprehensively analyzed the characteristics of hyaluronidase-induced degradation of commercially available HA formulations, and there are even fewer comprehensive analyses reflective of real-world clinical practice.^6-8

HA, a glycosaminoglycan found in high abundance in the extracellular matrix of the skin, has the unique ability to bind and retain water molecules, making it a key molecule for retaining skin moisture and providing plumpness.⁹ HA consists of repeating disaccharide units (D-gluconic acid and D-N-acetylglucosamine) linked by a β-1,4 glycosidic bond (Figure 1)¹⁰ and, in addition to hyaluronidase, is degraded by a number of factors including mechanical stress, reactive oxygen species, temperature, pH, and ultrasonic stimulation.^11,12 Because HA has a short half-life of <1 to 4 days in the skin, most HA fillers are chemically modified to improve stability and maintain cosmetic results.^13,14 Modification is most commonly achieved by crosslinking HA chains using 1,4-butanediol diglycidyl ether (BDDE),¹⁵ which reacts with the hydroxyl groups in HA to form an ether bond.¹² This crosslinking method does not hinder the availability of the β-1,4 glycosidic bond for cleavage by hyaluronidase (Figure 1).¹²

Figure 1.

Cleavage sites for hyaluronidase on HA chains crosslinked by BDDE. BDDE, 1,4-butanediol diglycidyl ether; HA, hyaluronic acid.

Previous in vitro and in vivo comparisons of enzymatic degradation of HA-based fillers have used a variety of methods to assess efficacy, including visual assessment, quantification of HA fragment release, palpation, and imaging.^6-8,16-20 Of the factors analyzed, a higher concentration of HA, higher degree of modification, and a monophasic formulation have been associated with higher resistance of HA-based fillers to degradation.¹⁶ Despite this, the lack of standard methodology and assessment of physicochemical properties in these studies makes the provision of comprehensive guidelines for the reversal of HA-based fillers with hyaluronidase challenging.

We have recently developed an in vitro rheological method to analyze the change in physicochemical properties of HA-based fillers after administering hyaluronidase.²¹ This method provides a platform for comparing HA-based filler degradation in real time by assessing rheological characteristics that are known to affect filler characteristics in vivo. Here, we use this method to compare enzymatic degradation kinetics of 16 HA-based fillers that are commercially available in the United States, reflecting the market landscape. The range of fillers represents 6 manufacturing technologies (Table), with indications ranging from superficial line correction to deep plane volumizers.

Table.

Characteristics of the HA-based Fillers in the Present Study

Abbreviation	Filler	MoD (%)	HA (mg/mL)	Static G' (Pa)a	Mean time to degrade (min)	Mean enzyme volume required for degradation (μL)
Technology: Cohesive Polydensified Matrix (CPM; Merz, Frankfurt, Germany)
CPMBB	Belotero Balance	4.7	22.5	44	3.6	50
Technology: Preserved Network Technology (PNT; Teoxane, Geneva, Switzerland)
RHARb	RHA Redensity	1.9	15	59	4.2	50
RHA2	RHA 2	2.9	23	139	16.8	175
RHA3	RHA 3	3.6	23	146	20.1	200
RHA4	RHA 4	4.1	23	263	28.3	300
Technology: Optimal Balance Technology (XpresHAn/OBT; Galderma, Lausanne, Switzerland)
XPRESRR	Restylane Refyne	5.7	20	74	8.8	100
XPRESRC	Restylane Contour	6.9	20	172	17.3	175
XPRESRD	Restylane Defyne	8.4	20	221	18.2	200
Technology: Hylacross (HYL; AbbVie, Allergan, Irvine, CA)
HYLJU	Juvéderm Ultra XC	5.9	24	102	14.3	150
HYLJUP	Juvéderm Ultra Plus XC	6.9	24	137	17.2	175
Technology: Non-animal stabilized hyaluronic acid (NASHA; Galderma)
NASHR	Restylane	1.1	20	792	22.3	200
NASHLYF	Restylane Lyft	1.2	20	807	22.3	200
Technology: Vycross (VYC; AbbVie, Allergan)
VYCVOLB	Juvéderm Volbella	5.3	15	220	29.9	300
VYCVOLL	Juvéderm Vollure	5.6	18	275	26.0	250
VYCVOLU	Juvéderm Voluma XC	5.9	20	305	29.9	300
VYCVOLX	Juvéderm Volux	9.4	25	439	45.1	475

HA, hyaluronic acid; MoD, degree of modification; RHA, resilient hyaluronic acid. ^aMeasured previously under near-static conditions.^22bRHA_R results apply for RHA₁ outside the US market.

METHODS

A total of 16 HA-based fillers marketed in the United States were evaluated, covering the full spectrum of product indications and representing the following crosslinking technologies: Preserved Network Technology (PNT; Teoxane SA, Geneva, Switzerland), Cohesive Polydensified Matrix (CPM; Belotero Balance, Merz, Frankfurt, Germany), Vycross (Allergan, Irvine, CA), Hylacross (Allergan), non-animal stabilized hyaluronic acid (NASHA; Restylane, Galderma, Lausanne, Switzerland), and XpresHAn/Optimal Balance Technology (XpresHAn/OBT; Galderma) (Table). Hylenex, a commercially available recombinant human hyaluronidase (150 USP U/mL; Halozyme Therapeutics, San Diego, CA), was used to degrade the HA-based fillers (Figure 1). The study started in November 2020 and finished in January 2021.

A multidose hyaluronidase administration protocol was used to induce degradation of HA-based fillers.²¹ This multidose protocol was selected over a single dose of hyaluronidase because more effective degradation of the HA gel is achieved when multiple doses are used (Figure 2). A rheometer (HR-20; TA Instruments, New Castle, DE) was set up with a cone-plate geometry (anodized aluminum, 40 mm, 1°) and measurements were performed at 37°C. A time-sweep protocol at an oscillatory strain of 0.1% at 1 Hz was used. At the start of the experiment, 300 μL of the HA-based filler was placed onto the Peltier plate of the device and allowed to equilibrate to 37°C for 1 minute. Before starting the experiment, 50 μL of hyaluronidase was added onto the gel. Another 50 μL of hyaluronidase was added every 5 minutes. The experiment continued until the elastic modulus (G') reached the degradation threshold of 30 Pa. The 30 Pa threshold was chosen as the lowest reliable measurement of G'. Values lower than this cannot be monitored accurately with cone-plate geometry. A solvent trap was used to prevent solvent evaporation and gel drying. All experiments were performed at least twice. Calculations for mean time to degradation, mean volume of hyaluronidase required for degradation, and standard deviation (SD) were performed with Microsoft Excel (Microsoft, Redmond, WA). The relative impact of physicochemical parameters (HA concentrations, HA chain length, degrees of modification, G', and cohesivity) on the degradation pattern of evaluated fillers was analyzed based on previously reported values from a comparative study.²²

Figure 2.

Change in G' with time of a commercially available HA gel (product A). Either 1 × 50 μL or 5 × 50 μL (every 5 minutes) of hyaluronidase was added and G' measured by rheological time sweep to determine the enzymatic degradation of product A over time. Data are shown as values over time from 2 independent samples. G', elastic modulus; G'₀, elastic modulus at 0; HA, hyaluronic acid.

RESULTS

Gel degradation was compared by exposure to repeated doses of hyaluronidase at intervals of 5 minutes to reach the degradation threshold of G' ≤ 30 Pa. Overall, noticeable differences were observed based on the design and technology of different filler classes (Figure 3).

Figure 3.

Change in G' with time of commercially available HA-based more superficial (A), utility (B), and deeper plane (C) fillers after repeated hyaluronidase dosing. A dose of hyaluronidase (7 U/g) was administered every 5 minutes and G' measured by rheological time sweep to determine the enzymatic degradation of the product over time, down to the degradation threshold of 30 Pa. Data are shown as values over time from 2 independent samples. RHA_R results apply for RHA_/1 outside the US market. Cohesive polydensified matrix (CPM), Merz: CPM_BB, Belotero Balance. Hylacross (HYL), AbbVie (Allergan): HYL_JU, Juvéderm Ultra XC; HYL_JUP, Juvéderm Ultra Plus XC. Non-animal stabilized hyaluronic acid (NASHA), Galderma: NASH_LYF, Restylane Lyft; NASH_R, Restylane. Optimal Balance Technology (XpresHAn/OBT), Galderma: XPRES_RC, Restylane Contour; XPRES_RD, Restylane Defyne; XPRES_RR, Restylane Refyne. Preserved Network Technology (PNT), Teoxane: RHA_R, RHA Redensity; RHA₂, RHA 2; RHA₃, RHA 3; RHA₄, RHA 4. Vycross (VYC), AbbVie (Allergan): VYC_VOLB, Juvéderm Volbella; VYC_VOLL, Juvéderm Vollure; VYC_VOLU, Juvéderm Voluma XC; VYC_VOLX, Juvéderm Volux. G', elastic modulus; HA, hyaluronic acid; RHA, resilient hyaluronic acid.

HA manufacturing technologies vary parameters of HA concentration and chain length, and degree of modification, which together ultimately determine the resultant physicochemical/rheological properties of the fillers. In our comprehensive analysis, we investigated the relative impact of these manufacturing parameters individually in addition to the classic rheological properties of G' and cohesivity. No obvious relationship was observed between gel degradation characteristics and HA concentration, measured HA chain length in the final products, or the degree of modification of each filler when analyzed separately (Figure 4). However, when we looked at these in aggregate, as proprietary manufacturing technologies, there was a clear association, with Vycross fillers being the most difficult to degrade followed by NASHA, PNT, Hylacross, and XpresHAn. The CPM filler was the least difficult to degrade (Figure 5).

Figure 4.

Relationship between HA concentration, HA chain length, and degree of modification on cumulative volume of hyaluronidase required for gel degradation and gel degradation time. (A) Enzyme volume required for degradation vs HA concentration. (B) Gel degradation time vs HA concentration. (C) Enzyme volume required for degradation vs HA chain length. (D) Gel degradation time vs chain length. (E) Enzyme volume required for degradation vs degree of modification. (F) Gel degradation time vs degree of modification. RHA_R results apply for RHA₁ outside the US market. Cohesive Polydensified Matrix (CPM), Merz: CPM_BB, Belotero Balance. Hylacross (HYL), AbbVie (Allergan): HYL_JU, Juvéderm Ultra XC; HYL_JUP, Juvéderm Ultra Plus XC. Non-animal stabilized hyaluronic acid (NASHA), Galderma: NASH_LYF, Restylane Lyft; NASH_R, Restylane. Preserved Network Technology (PNT), Teoxane: RHA_R, RHA Redensity; RHA₂, RHA 2; RHA₃, RHA 3; RHA₄, RHA 4. Vycross (VYC), AbbVie (Allergan): VYC_VOLB, Juvéderm Volbella; VYC_VOLL, Juvéderm Vollure; VYC_VOLU, Juvéderm Voluma XC; VYC_VOLX, Juvéderm Volux. HA, hyaluronic acid; MoD, degree of modification; RHA, resilient hyaluronic acid.

Figure 5.

Cumulative volume of hyaluronidase required to degrade commercially available HA-based fillers (A) and gel degradation time (B) by product indication. (A) A dose of hyaluronidase was administered every 5 minutes and G' measured by rheological time sweep to determine enzymatic degradation of the product over time. Data are shown as mean ± SD. RHA_R results apply for RHA₁ outside the US market. Cohesive Polydensified Matrix (CPM), Merz: CPM_BB, Belotero Balance. Hylacross (HYL), AbbVie (Allergan): HYL_JU, Juvéderm Ultra XC; HYL_JUP, Juvéderm Ultra Plus XC. Non-animal stabilized hyaluronic acid (NASHA), Galderma: NASH_LYF, Restylane Lyft; NASH_R, Restylane. Optimal Balance Technology (XpresHAn/OBT), Galderma: XPRES_RC, Restylane Contour; XPRES_RD, Restylane Defyne; XPRES_RR, Restylane Refyne. Preserved Network Technology (PNT), Teoxane: RHA_R, RHA Redensity; RHA₂, RHA 2; RHA₃, RHA 3; RHA₄, RHA 4. Vycross (VYC), AbbVie (Allergan): VYC_VOLB, Juvéderm Volbella; VYC_VOLL, Juvéderm Vollure; VYC_VOLU, Juvéderm Voluma XC; VYC_VOLX, Juvéderm Volux. G', elastic modulus; HA, hyaluronic acid; RHA, resilient hyaluronic acid.

When assessing rheological properties, increasing G' was associated with an increasing volume of enzyme and time required for gel degradation for the monophasic fillers; however, this relationship was not observed for the biphasic NASHA fillers (Figure 6A,B). Increasing cohesivity was generally associated with an increasing volume of enzyme and time required for degradation with the exception of Vycross and NASHA products (Figure 6C,D).

Figure 6.

Relationship between elastic modulus, cohesivity and technology on cumulative volume of hyaluronidase required for gel degradation and gel degradation time. (A) Enzyme volume required for degradation vs elastic modulus. (B) Degradation time vs elastic modulus. (C) Enzyme volume required for degradation vs cohesivity. (D) Degradation time vs cohesivity. RHA_R results apply for RHA₁ outside the US market. Cohesive Polydensified Matrix (CPM), Merz: CPM_BB, Belotero Balance. Hylacross (HYL), AbbVie (Allergan): HYL_JU, Juvéderm Ultra XC; HYL_JUP, Juvéderm Ultra Plus XC. Non-animal stabilized hyaluronic acid (NASHA), Galderma: NASH_LYF, Restylane Lyft; NASH_R, Restylane. Optimal Balance Technology (XpresHAn/OBT), Galderma: XPRES_RC, Restylane Contour; XPRES_RD, Restylane Defyne; XPRES_RR, Restylane Refyne. Preserved Network Technology (PNT), Teoxane: RHA_R, RHA Redensity; RHA₂, RHA 2; RHA₃, RHA 3; RHA₄, RHA 4. Vycross (VYC), AbbVie (Allergan): VYC_VOLB, Juvéderm Volbella; VYC_VOLL, Juvéderm Vollure; VYC_VOLU, Juvéderm Voluma XC; VYC_VOLX, Juvéderm Volux. RHA, resilient hyaluronic acid.

DISCUSSION

This is the first study to compare the gel degradation characteristics of a variety of commercially available HA-based fillers using an objective assessment of hyaluronidase-mediated degradation. Specifically, because this method allows real-time evaluation of viscoelastic properties under near-static conditions, we were able to characterize and compare gel degradation profiles in terms of filler technology and physicochemical properties. HA-based gels have a significant advantage over other soft tissue fillers in that they are degradable with hyaluronidase, which allows treatment outcomes to be reversed and contributes to immediate and better management of adverse effects and complications.¹ Overall, we observed that filler technology/manufacturing process had the greatest influence on gel degradation when compared with individual manufacturing parameters or physicochemical characteristics (Table). Vycross products were the most resistant to degradation by exogenous hyaluronidase and CPM was the least resistant to degradation. PNT fillers had a balanced degradation profile and PNT was the only family of fillers that had a broad range of degradation profiles within its class, consistent with their progressive degree of crosslinking and HA concentration.

For NASHA and Hylacross products, gel degradation was similar among fillers within each class. A lack of clear differentiation was also seen within the Vycross products and to a lesser extent with XpresHAn products. For Vycross, the exception was with VYC_VOLX which required considerably more hyaluronidase to reach the degradation threshold, likely related to its higher HA concentration and greater degree of crosslinking than other product family members. For the XpresHAn products, the exception was XPRES_RR, which required less hyaluronidase than XPRES_RD and XPRES_RC. In contrast, PNT products demonstrated proportional increases in gel degradation time and enzyme volume across the individual products and indication categories. RHA_R, which has a lower HA concentration and is intended for the most superficial injection depth, had the shortest gel degradation time and smallest enzyme volume required for degradation, while RHA₄, which is intended for deeper placement, had the longest gel degradation time and required the highest volume of enzyme for degradation within its family (Figure 3). Whereas conventional technologies rely on high heat (eg, heating HA at 40-50°C) during the crosslinking reaction with BDDE, PNT products have been manufactured with a disruptive technology that does not involve heating to crosslink HA. Therefore, these products were specifically designed to preserve HA chain integrity during the manufacturing process, thus requiring low amounts of crosslinker to achieve clinically desirable mechanical properties and durability. These less rigidly crosslinked HA chains are presumed to allow fillers to better accompany and adapt to mechanical deformation of the skin and subcutaneous tissues driven by the muscles of facial expression. The proportional increase in gel degradation time and volume observed across the PNT family of products may be reflective of the increasing degree of crosslinking and differences in HA concentration across this product line, and potentially other unassessed manufacturing parameters. This finding did not hold true for other manufacturing technologies such as for the Hylacross products.

Consistent with the findings from other studies, HA fillers manufactured with Vycross technology were more resistant to degradation than other fillers,^{6,16,19,20,23-26} with the highest degree of degradation difficulty occurring with the longest times and greatest volumes needed for degradation in each category. The resistance may be due to the fact that these products have shorter HA chains, and therefore are described as having more tightly packed HA and a more dense crosslinking than other products.¹⁵ For NASHA fillers, the particulate design and a consistent degree of modification and HA concentration (Table) are reflected in the uniform degradation profile, suggesting that particle size is not an important consideration in the degradation of these products by hyaluronidase. Additionally, NASHA fillers were not the most resistant to degradation, despite having the highest G' among all fillers tested. This is likely a function of the biphasic formulation that allows for easier hyaluronidase penetration and more efficient degradation. The rapid degradation profile of CPM_BB in the current study is consistent with an in vivo study showing that CPM_BB degraded rapidly in human participants⁸ and a clinical study showing reduced durability of a CPM filler compared with a NASHA filler.²⁷ Although trends were observed in the relationship between G' and gel degradation, it should be noted that these are resultant characteristics derived from the unique interplay of the manufacturing parameters.

The main strength of this study is that we used a rheological method at fixed strain and oscillation frequency, which enables real-time evaluation of gel degradation and allows for accurate and consistent evaluation of a variety of gel products.²¹ This method is a notable improvement over previously used methodologies,^{3,6,7,16,17,19,20,23-26,28} which did not provide an assessment of the time required for gel degradation. In addition, by using an objective method, we eliminated the individual bias or variability in interpretation that can occur with visual assessments of gel degradation.^7,17,28

This study used an ex vivo method, enabling consistent comparative assessment, but extrapolation of these results to clinical settings should be avoided. Nonetheless, these data are of interest because they may help practitioners optimize the use of hyaluronidase to dissolve HA fillers.

CONCLUSIONS

This study has evaluated the gel degradation characteristics of 16 commercially available HA-based fillers using a rigorous in vitro method that we believe is more reflective of hyaluronidase use in real-world clinical practice. We have found, for the first time, that manufacturing technology is the most important factor influencing the reversibility of an HA product, with less relevance of HA concentration and classic rheological parameters such as G' and cohesivity. For higher-risk areas, understanding the differential degradation profiles and technologies of currently marketed HA fillers will allow clinicians to make informed clinical choices based on preferential reversibility. For certain technologies (such as PNT) in which the manufacturing process is consistent, a higher degree of crosslinking was associated with a greater resistance to degradation by hyaluronidase. Understanding the differential degradation profiles of currently marketed HA gels will allow clinicians to select products that offer a higher margin of safety due to their preferential reversibility.

Disclosures

Dr Faivre, Ms Gallet, and Mr Bourdon are employees of Teoxane SA (Geneva, Switzerland). Dr Wu, Dr Sparrow, and Dr Gallagher are employees of, and hold stock/stock options in, Revance Therapeutics, Inc. (Nashville, TN).

Funding

This analysis was supported by Revance Therapeutics, Inc. (Nashville, TN) and Teoxane SA (Geneva, Switzerland), manufacturer of TEOSYAL RHA fillers. Teoxane and Revance were involved in the study design, data collection, data analysis, and preparation of the manuscript. Writing and editorial assistance, provided to the authors by Serina Stretton, PhD, CMPP, of Envision Pharma Group, was funded by Revance.