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Plastic & Reconstructive Surgery:
doi: 10.1097/PRS.0b013e31829e88a3
Cosmetic: Original Articles

Molecular Weight Analyses and Enzymatic Degradation Profiles of the Soft-Tissue Fillers Belotero Balance, Restylane, and Juvéderm Ultra

Flynn, Timothy Corcoran M.D.; Thompson, David H. Ph.D.; Hyun, Seok-Hee Ph.D.

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Author Information

Cary and Chapel Hill, N.C.; and West Lafayette, Ind.

From the Cary Skin Center; the Department of Dermatology, University of North Carolina at Chapel Hill; the Department of Chemistry, Purdue University; and Pandion Laboratories, LLC.

Received for publication April 23, 2013; accepted May 7, 2013.

Disclosure: Dr. Flynn is a consultant for Merz and Canfield Scientific; he has received research support from Merz. Dr. Thompson is a National Institutes of Health–supported organic materials chemist who has several research projects involving hyaluronic acid. He is also president and chief scientific officer of Pandion Laboratories, LLC. Dr. Hyun is a senior research scientist for Pandion Laboratories, which has received research support from Merz. The authors were assisted in development of this manuscript by David J. Howell, Ph.D. (San Francisco). Dr. Howell was compensated by Merz for his editorial assistance.

Timothy Corcoran Flynn, M.D., Cary Skin Center, 200 Wellesley Trade Lane, Cary, N.C. 27519, flynn@caryskincenter.com

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Abstract

Background: In this study, the authors sought to determine the molecular weight distribution of three hyaluronic acids—Belotero Balance, Restylane, and Juvéderm Ultra—and their rates of degradation following exposure to hyaluronidase. Lot consistency of Belotero Balance also was analyzed.

Methods: Three lots of Belotero Balance were analyzed using liquid chromatography techniques. The product was found to have high-molecular-weight and low-molecular-weight species. One lot of Belotero Balance was compared to one lot each of Juvéderm Ultra and Restylane. Molecular weights of the species were analyzed. The hyaluronic acids were exposed to ovine testicular hyaluronidase at six time points—baseline and 0.5, 1, 2, 6, and 24 hours—to determine degradation rates.

Results: Belotero Balance lots were remarkably consistent. Belotero Balance had the largest high-molecular-weight species, followed by Juvéderm Ultra and Restylane (p < 0.001). Low-molecular-weight differences among all three hyaluronic acids were not statistically significant. Percentages of high-molecular-weight polymer differ among the three materials, with Belotero Balance having the highest fraction of high-molecular-weight polymer. Degradation of the high-molecular-weight species over time showed different molecular weights of the high-molecular-weight fraction. Rates of degradation of the hyaluronic acids following exposure to ovine testicular hyaluronidase were similar. All hyaluronic acids were fully degraded at 24 hours.

Conclusions: Fractions of high-molecular-weight polymer differ across the hyaluronic acids tested. The low-molecular-weight differences are not statistically significant. The high-molecular-weight products have different molecular weights at the 0.5- and 2-hour time points when exposed to ovine testicular hyaluronidase and are not statistically different at 24 hours.

Soft-tissue fillers such as hyaluronic acids constitute a large portion of nonsurgical aesthetic treatments such that the American Society for Aesthetic Plastic Surgery has a separate category for tabulating the number of procedures performed annually. In 2012, a total of 1,429,705 soft-tissue filling procedures were performed by physicians; hyaluronic acid injections are second only to treatments with botulinum toxin type A in popularity of nonsurgical aesthetic procedures.1,2 These data were confirmed by the 2012 survey conducted by the American Society of Dermatologic Surgery, which reported that their members in 2012 performed 916,000 soft-tissue filling procedures and 1.5 million neurotoxin injections.3

A number of hyaluronic acid products now appear in the U.S. and European aesthetic marketplace.4 One of the newest hyaluronic acid products available in the United States is manufactured to create a cohesive polydensified matrix (Belotero Balance; Merz Aesthetics, Greensboro, N.C.). Belotero Balance (also known as Belotero Basic in Europe) is an injectable hyaluronic acid approved in 2012 by the U.S. Food and Drug Administration for use as a soft-tissue filler for wrinkles and lines of the face and other areas. A 2011 study by Flynn et al.5 demonstrated comprehensive integration of Belotero Balance when injected into the human dermis, with an even distribution of the hyaluronic acid in and among collagen bundles.

Questions have arisen among practitioners about the amount of hyaluronic acid contained in each syringe. Total hyaluronic acid content is usually reported. However, the polymer length, or starting molecular weight of the hyaluronic acid, is not consistent among approved hyaluronic acid fillers. Finished hyaluronic acid fillers contain both high-molecular-weight and low-molecular-weight species. In Restylane (Valeant Pharmaceuticals International, Inc., Montreal, Quebec, Canada), the high-molecular-weight polymers are manufactured by cross-linking hyaluronic acid precursors of approximately 200 to 300 kDa, producing a firm matrix that is then sized to smaller uniform particles by means of a sieving technology. These sieved particles are then suspended in lower molecular weight non–cross-linked hyaluronic acid “lubricant” to allow for extrusion and deformation as the particles flow through the needle. After injection into the skin, the non–cross-linked hyaluronic acid is rapidly metabolized, leaving the cross-linked larger molecular weight polymers behind.

Many clinicians have expressed interest in understanding the higher molecular weight cross-linked material, believing that these larger polymers are responsible for the long-lasting aesthetic effect. Studies herein are designed to address the molecular weight species of the commercially available hyaluronic acid contained in each product. The relative amounts of high-molecular-weight and low-molecular-weight material are investigated using gel permeation chromatography techniques.

As is the case with most soft-tissue fillers, postinjection complications can occur, and many of the adverse events can be managed with the use of hyaluronidase. A recent thorough review article by Lee et al. on the use of hyaluronidase following complications identifies at least five cases of “undesirable side effects”—excessive use of product, too superficial injection, granulomatous foreign body reaction, and impending or real tissue necrosis.6–11 Complications such as overcorrection, vascular occlusion, and soft-tissue loss have been reported with hyaluronic acids12–15 and with non–hyaluronic acid dermal fillers.16 Fortunately, the hyaluronic acid soft-tissue fillers are amenable to degradation by hyaluronidase. Referring to hyaluronic acids, Lee et al. observed that “management of these undesirable events with hyaluronidase is gaining popularity because of its safety, efficacy, and ease of use.”6

A study by Jones and colleagues in 2010 explored the in vitro resistance to ovine testicular hyaluronidase (Vitrase; ISTA Pharmaceuticals, Inc., Irvine, Calif.) of a smooth gel containing 24 mg/ml of hyaluronic acid (Juvéderm Ultra), a 20 mg/ml of hyaluronic acid particulate gel (Restylane), and a 5.5 mg/ml of hyaluronic acid particulate with 0.3% lidocaine (Prevelle Silk; Mentor Worldwide, LLC, Santa Barbara, Calif.).17 Investigators examined a “dose-response relationship between commercially available ovine testicular hyaluronidase” and the three hyaluronic acids described earlier.

We sought to further examine the degradation of Belotero Balance in comparison with Restylane and Juvéderm Ultra. The study being reported in this Supplement differed from the study by Jones and colleagues in several important ways. Studies were completed by analyzing multiple time points over a 24-hour period, with multiple samples collected and evaluated at each time point. We used techniques they reported in the medical literature17 and in the realm of polymer structural analysis.18–20 We also sought to evaluate the products directly by incubating the material contained in the syringes directly with ovine testicular hyaluronidase to profile the degradation patterns.

In this study, investigators sought to answer several questions: What is the molecular weight species contained in each syringe of Belotero Balance, Restylane, and Juvéderm Ultra? Do the products degrade differently following incubation with hyaluronidase? What is the rate of degradation of each product? What is the lot-to-lot consistency of Belotero Balance?

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MATERIALS AND METHODS

Size Analysis of the Hyaluronic Acid Polymers Contained in a Syringe of Belotero Balance

Analysis began by performing a bulk centrifugation experiment to determine whether a significant fraction of the Belotero Balance sample contained large-particle molecular weight species. This was evaluated by diluting several syringe samples of Belotero Balance 10-fold into phosphate-buffered saline, followed by ultracentrifugation at 100,000 g for 30 minutes in a Sorvall Model RC-M150GX ultracentrifuge (Thermo Fisher Scientific, Waltham, Mass.) fitted with an S100ATS rotor. The lower 10 percent of the sample volume was removed by pipet and analyzed for particulate matter by means of bulk dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, United Kingdom). Volume distribution analysis indicated the presence of species with diameters in the 8- to 20-nm range, a range that allowed for further analysis using chromatographic techniques.

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Evaluation of Polymer Molecular Weight Contained within the Syringes of Belotero Balance, Restylane, and Juvéderm Ultra

A gel permeation chromatography–multiple angle light scattering refractive index instrument was used to evaluate polymer sizes within the syringes of three commercially available hyaluronic acids. The instrument configuration comprises an Agilent Series 1200 (Agilent Technologies, Santa Clara, Calif.) high-pressure liquid chromatograph equipped with a Model G 1332A solvent degasser, a Model G 1311A pump, a Model 1329A auto-injector, a Shodex SB-Guard column (Showa Denko, Tokyo, Japan), a 300 × 8-mm Shodex OHpak SB-804 HQ, and a 300 × 8-mm Shodex OHpak SB-803 HQ analytical column connected in series, and a Model G 1364C fraction collector. This instrument was equipped with a Wyatt DAWN HELEOS-II multiangle light scattering detector, a Wyatt Optilab rEX refractive index detector (both from Wyatt Technology Corp., Santa Barbara, Calif.), and an Agilent Model G 1315D Diode Array Detector (Agilent). The hyaluronic acids were diluted by a factor of 10 using phosphate-buffered saline and samples were thoroughly mixed. Injection volumes of 100 µl (0.015 to 1% weight/volume) of the hyaluronic acid gel solutions were applied to the gel permeation chromatography–multiple angle light scattering instrument, with time-dependent changes in the hyaluronic acid and product concentrations recorded, using both the multiple angle light scattering and the refractive index detector channels. The mobile phase was phosphate-buffered saline (pH 7.4) containing 0.02% sodium azide, at a flow rate of 0.4 ml/minute at 60°C. Values of differential index of refraction (dn/dc) = 0.167 ml/g were used for hyaluronic acid molecular weight determinations,21 whereas dn/dc = 0.138 ml/g and 0.137 ml/g were used for the pullulan and dextran calibration standards, respectively.22 The multiple angle light scattering and refractive index data were collected and processed using ASTRA software (Wyatt Technology Corp.).

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Evaluation of Lot Consistency Regarding Molecular Weight of Hyaluronic Acid across Samples of Belotero Balance

Lot analysis focusing on evaluation of the molecular weight composition and consistency of the hyaluronic acid components present in Belotero Balance was performed using the gel permeation chromatography–multiple angle light scattering refractive index instrument. Three different Belotero Balance lots were analyzed. Nine samples of each lot were evaluated, resulting in a total of 27 independent molecular weight analyses.

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Extensive Data Collection of Time Points for Determination of Hyaluronic Acidase Digestive Susceptibility (Degradation) across All Three Soft-Tissue Filler Hyaluronic Acids

Hyaluronic acid samples were evaluated by the gel permeation chromatography–multiple angle light scattering refractive index method described above at each of six time points (i.e., 0, 0.5, 1, 2, 6, and 24 hours) after incubation with ovine testicular hyaluronidase using Belotero Balance, Restylane, and Juvéderm Ultra. Incubation was conducted as follows: 80 mg of hyaluronic acid was placed in an Eppendorf tube and temperature equilibrated with a separate tube containing 80 μl of ovine testicular hyaluronidase solution (200 U/ml) for 10 minutes at 37°C. The Vitrase and hyaluronic acid samples were combined and mixed using a vortex mixer for 10 seconds. The mixture was then continued at incubation temperature of 37°C and the enzymatic reaction sampled at 0, 30, 60, 120, 360, and 1440 minutes. The reaction was stopped by the addition of 80 μl of potassium tetraborate solution (0.8 M), followed by immediate stirring on a vortex mixer and heating for 10 minutes at 100°C. The sample was diluted with 640 μl of phosphate-buffered saline (pH 7.4) and filtered using a 0.2-μm cellulose acetate membrane syringe filter. Samples (100 μl) were then injected into the gel permeation chromatography–multiple angle light scattering system for molecular weight analysis.

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Statistical Analysis

An independent statistician using JMP statistical discovery software (SAS Institute, Inc., Cary, N.C.) assisted in data analysis. Appropriate statistical tests were applied to the data set.

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RESULTS

Ultracentrifugation was initially undertaken to see whether there were any very large particles or high-molecular-weight polymers contained within each of the three commercially available hyaluronic acids. No obvious signs of pellet formation were observed by the unaided eye for any of the three products following dilution and high-speed centrifugation. Furthermore, the dynamic light scattering data indicated that the solution in the bottom 10 percent of the centrifuge tube displayed the same volume distribution scattering profile as the noncentrifuged samples. Therefore, it was concluded that there were no very large particles of hyaluronic acid in the samples, clearing the way for gel permeation chromatography–multiple angle light scattering refractive index analysis.

All gel permeation chromatography–multiple angle light scattering instruments use a solvent filter (0.2 µm) downstream from the injection port to exclude contaminants such as dust that can accumulate in the injection port and/or sample. This guards the column from failing and any undesirable pressure buildup. Initial experiments using direct product injections into the gel permeation chromatography–multiple angle light scattering refractive index instrument produced a significant increase in system pressure. As further experimentation to determine whether any polymers with physical sizes larger than 0.2 μm existed in the products, the diluted products were passed through a 0.2-μm filter and the molecular weight profiles of the filtrate were analyzed by dynamic light scattering. The filters were then back-flow washed and the retained material on the filter was analyzed by the same technique. There were no apparent differences between the diluted hyaluronic acid that passed through the filter and that which was retained. Thus, we attribute these findings to a concentration polarization phenomenon, wherein adventitious dust particles and/or a mass of aggregated hyaluronic acid material partially occludes the filter, resulting in an accumulation of additional hyaluronic acid polymer behind it in a “logjam” process that causes pressure buildup in the instrument. The absence of any apparent difference in the samples before and after processing with a 0.2-μm filter led us to incorporate this step in all subsequent analyses.

Analysis of the gel permeation chromatography–multiple angle light scattering profiles of the three Belotero Balance lots indicated the presence of two peaks (Fig. 1). These two species, one high-molecular-weight and one low-molecular-weight, were observed in all three lots. Lot 1 was observed to have an average high molecular weight of 1287 ± 313 kDa; lot 2 and lot 3 were found to have average high molecular weights of 1231 ± 284 kDa and 1069 ± 272 kDa, respectively. Thus, the average size of the high-molecular-weight polymer contained in Belotero Balance is 1196 ± 297 kDa. The low-molecular-weight species was found to be 290 ± 117 kDa for lot 1, with this component measuring 281 ± 73 kDa and 320 ± 94 kDa for lots 2 and 3, respectively. The lots had a mean low molecular weight of 297 ± 95 kDa.

Fig. 1
Fig. 1
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The profile of the high-molecular-weight and low-molecular-weight components of the three hyaluronic acid fillers showed interesting product profiles. A single new lot analysis of Belotero Balance had the highest apparent molecular weight of the high-molecular-weight polymer fraction (i.e., 842 ± 183 kDa), whereas Juvéderm Ultra had a measured high-molecular-weight average of 491 ± 297 kDa and Restylane had a high-molecular-weight average of 249 ± 111 kDa. These results were statistically different when the Belotero Balance and Juvéderm Ultra samples (p = 0.001) were compared and when the Belotero Balance and Restylane samples (p < 0.02) were compared using a Kruskal-Wallis test. Tukey-Kramer analysis showed that Belotero Balance is significantly different from both Restylane and Juvéderm Ultra. The Restylane–Belotero Balance differences are significant at p < 0.001 and the Belotero Balance–Juvéderm Ultra comparison showed p = 0.36. Juvéderm Ultra and Restylane were not statistically different. The low-molecular-weight components were 162 ± 69 kDa for Belotero Balance, 134 ± 95 kDa for Juvéderm Ultra, and 102 ± 50 kDa for Restylane. The Kruskal-Wallis test comparing all means of the low-molecular-weight components showed no significant differences in the low-molecular-weight component.

Differences in the amount of high-molecular-weight and low-molecular-weight material in these hyaluronic acids also were observed. Analysis of the Belotero Balance samples revealed that they consisted of 56 ± 25 percent high-molecular-weight and 44 ± 16 percent low-molecular-weight fractions. Juvéderm Ultra had the second largest percentage of high-molecular-weight polymer at 38 ± 20 percent and Restylane had the lowest percentage of high-molecular-weight polymer at 35 ± 17 percent; the low-molecular-weight fractions of these products were found to be 62 ± 20 percent and 65 ± 17 percent, respectively. Table 1 profiles the differences.

Table 1
Table 1
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Experiments designed to compare the susceptibility to degradation by ovine testicular hyaluronidase also yielded interesting findings (Figs. 2 through 4). The apparent molecular weight of the high-molecular-weight species increased on addition of ovine testicular hyaluronidase in all samples analyzed at an incubation time of 0.5 hour. Specifically, the high-molecular-weight species increased from 842 kDa to 1100 kDa for Belotero Balance, from 491 kDa to 650 kDa for Juvéderm Ultra, and from 249 kDa to 490 kDa for Restylane. Experiments that used a 66-kDa inert protein (bovine serum albumin) as a control protein incubated with the hyaluronic acids at 0.5 hour failed to show a similar increase in the apparent molecular weight of the high-molecular-weight component. Because the hydrolysis mechanism of hyaluronidase enzymes (including ovine testicular hyaluronidase) are thought to proceed by means of a covalently-attached glycosyl-enzyme hydrolysis intermediate as it randomly binds to the hyaluronic acid chains and processively cleaves the β1,4-glycosidic linkages of the biopolymer,23 we infer from these observations that multiple copies of hyaluronidase become covalently attached to the hyaluronic acid polymer during the first 30 minutes of hydrolytic degradation, producing an enzyme-substrate complex that initially exhibits a significantly higher molecular weight. Ovine testicular hyaluronidase has a reported molecular weight of approximately 55 kDa. Our data would suggest that between three and six copies of ovine testicular hyaluronidase on average are bound on each strand of high-molecular-weight hyaluronic acid (i.e., approximately three for Juvéderm Ultra, approximately four for Restylane, and approximately six for Belotero Balance). Ovine testicular hyaluronidase is, no doubt, also bound to the low-molecular-weight species. However, we cannot easily discriminate using only the gel permeation chromatography–multiple angle light scattering refractive index method between these putative enzyme–low-molecular-weight substrate complexes, the low-molecular-weight material that existed at 0 hour, and the low-molecular-weight material that is continuously being generated by the enzymatic reaction.

Fig. 2
Fig. 2
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Fig. 3
Fig. 3
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Fig. 4
Fig. 4
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Measurements taken at subsequent intervals following addition of ovine testicular hyaluronidase demonstrated a time-dependent decrease of the high-molecular-weight fraction concomitant with an increase in the low-molecular-weight fraction. At the 24-hour time point, nearly all of the high-molecular-weight fraction of the hyaluronic acids had degraded as a result of the enzymatic action, and the measured molecular weight of the low-molecular-weight fraction had further decreased as a result of enzyme degradation of the low-molecular-weight components (Figs. 5 and 6).

Fig. 5
Fig. 5
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Fig. 6
Fig. 6
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Of interest is the fact that the high-molecular-weight fractions degrade into different molecular weight species but follow similar time-dependent reaction kinetics regardless of the overall molecular weight size of the hyaluronic acid tested. At 0.5 hour, the measured molecular weight increases, because of the coupling of the enzyme with the high-molecular-weight polymer—before the polymer begins to be broken down. At all given time points before 24 hours, the molecular weight of the high-molecular-weight fraction follows the same pattern: Belotero Balance is greater than Juvéderm Ultra is greater than Restylane. Statistical analysis is summarized in Table 2, which shows that the molecular weight of the degraded high-molecular-weight Belotero Balance polymer is different from one or both of the other hyaluronic acids until 24 hours, at which point the molecular weights have fallen so low that no statistically significant differences are seen between them.

Table 2
Table 2
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In spite of the substantial differences in the molecular weight and proportions of high-molecular-weight versus low-molecular-weight species in the three hyaluronic acid products, the rate of degradation was largely the same for each soft-tissue hyaluronic acid polymer. Figure 7 illustrates the percentage of high-molecular-weight polymer that is retained following ovine testicular hyaluronidase incubation. There were no statistically significant differences between the materials at any of the time points sampled. This suggests that the polymer chain length and degree of branching do not appear to have a significant influence on the processing speed of the enzyme. This is perhaps not surprising, given that hyaluronidase enzymes bind randomly to the substrate to initiate hydrolysis and can cut the polymer chains into lower molecular weight fragments regardless of whether the parent hyaluronic acid chains are linear or more cross-linked.

Fig. 7
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DISCUSSION

This research project yielded information about the nature of each commercially available hyaluronic acid in the United States. Researchers are mindful that in vitro experimentation is not necessarily analogous to what may be seen with in vivo studies. Recent studies by Kablik et al., Sundaram et al., and Edsman et al., for example, are in vitro studies but have added immeasurably to the body of knowledge.24–26 Currently commercially available hyaluronic acids contain two species of polymers—a high-molecular-weight fraction and a low-molecular-weight fraction. The high-molecular-weight polymers most likely represent cross-linked moieties, whereas the low-molecular-weight polymers represents non–cross-linked or modestly cross-linked polymers.

As shown in Table 1, there is a statistically significant difference in the molecular weight profiles of each of the products. Interestingly, Belotero Balance has the highest molecular weight of the high-molecular-weight fraction when compared with Juvéderm Ultra and Restylane (842, 491, and 249 kDa, respectively). There is no significant difference in the low-molecular-weight component when each of the three is compared with the other two. Company manufacturing data (Anteis, Geneva, Switzerland) suggest that the starting molecular weight of the hyaluronic acid chains is different, with Belotero Balance demonstrating longer—or high-molecular-weight—chains compared with Juvéderm Ultra and Restylane. Our data reported here are consistent with this information.

Table 1 further indicates differences between the amount of high-molecular-weight polymer contained in the syringe. This is important because non–cross-linked hyaluronic acids are thought to be rapidly metabolized by the body, leaving only the cross-linked hyaluronic acid behind, which lasts for months or more. Therefore, our studies show that Belotero Balance contains 56 percent of the large polymer, followed by Juvéderm Ultra with 38 percent and Restylane with 35 percent.

The degradation experiments performed using ovine testicular hyaluronidase show differences in the size of the degraded starting cross-linked polymer. Table 2 indicates that there are statistically significant differences in the size of the polymer at 0.5-, 1.0-, 2.0-, and 6.0-hour time points. Figure 5 shows that Belotero Balance retains statistically higher molecular weight polymers compared with the other products at 0.5, 1.0, and 2.0 hours, and the Belotero Balance degradation is significantly different from Restylane but not from Juvéderm Ultra at the 6-hour time point. There are no significant differences in the degradation profiles at 24 hours, which confirms what we observe in the clinic when patients are injected with hyaluronidase: the products are completely degraded at 24 hours. This complete dissolution is a benefit of hyaluronic acid fillers because the hyaluronic acid can be removed. Should the physician detect impending vascular compromise and/or threatened—or real—tissue necrosis, hyaluronidase can be used to dissolve the hyaluronic acid injected near or into vessels.

The rates of degradation of the products are not statistically different, as shown in Figure 7. After 0.5 hour of incubation, 70 percent of the high-molecular-weight products remains. At 1 hour, 64 percent remains, and at 2 hours, 44 percent remains. In addition, 25 percent of the high-molecular-weight polymers remains at 6 hours, and they are essentially gone by 24 hours.

The work reported in this article and that described by Jones et al. share some important similarities but also some significant differences. Both studies report (1) degradation of hyaluronic acid gels by ovine testicular hyaluronidase over a 2-hour time period, (2) the presence of two different hyaluronic acid components in the gels, and (3) an apparent increase in average molecular weight within 0.5 hour of ovine testicular hyaluronidase addition to the gel.

There are several key differences in the data reported for these two studies, however. The most important difference is in the reported molecular weight for the hyaluronic acid gels. Jones et al. report masses in the 234- to 355-kDa range, whereas our study reports significantly lower masses for this fraction (i.e., between 102 and 163 kDa). We also report the molecular weight of the higher mass fraction, with this parameter observed in the 249- to 761-kDa range; however, similar information does not exist in the previous study.

An equally important difference is in the observed ratios of the high-molecular-weight and low-molecular-weight fractions. We find that the low-molecular-weight component is significant in all hyaluronic acid gels, with the proportion of low-molecular-weight material ranging from a low of 44.0 percent for Belotero to the higher levels observed for Juvéderm and Restylane (61.6 percent and 65.1 percent, respectively). The Jones et al. study also reports a dose-response relationship between the amount of ovine testicular hyaluronidase added to the gels and their rates of “free hyaluronic acid” formation. However, their rate studies evaluate all three gels for only 2 hours, with endpoint analysis of a single product at high enzyme concentration out to 24 hours. Although the present study did not explore the dose-response relationship, it did compare all three hyaluronic acid gels for 24 hours, with a sampling of the products between the 2-hour and 24-hour time points reported previously. This additional information reveals that all three gels are more than 90 percent degraded within 6 hours of exposure to the ovine testicular hyaluronidase concentrations used in our experiments.

We attribute the differences in the observed molecular weight and high-molecular-weight–to–low-molecular-weight ratios to the likely differences in the gel permeation chromatography–multiple angle light scattering setup used in the two studies. Although it is not possible to precisely pinpoint the nature of the difference, we surmise that our use of two gel permeation chromatography columns in series that were selected to optimally separate hyaluronic acid in the 2-MDa to 20-kDa mass range conferred a better capacity to reveal the sample composition than reported by Jones et al.

In the clinical setting, physicians have understood that each hyaluronic acid has its own distinctive properties and is not simply interchangeable with another one. The experiential understanding of the differences between products leads to the applicative level, in which physicians have learned not only which products are most appropriate for which patients but, moreover, which products are appropriate for which areas of treatment of these patients—and at which depths. (This notion of distinctiveness is also addressed in the article “Clinical Application and Assessment of Belotero: A Roundtable Discussion” elsewhere is this Supplement.)

Clinicians may wonder about what effects, if any, the role of the high-molecular-weight fraction has on product retention in tissue. Belotero contains the largest proportion of high-molecular-weight (cross-linked) polymers of the three hyaluronic acids. As hyaluronidase acts on the high-molecular-weight polymer contained in the syringe, there are larger molecular weight degradation products in Belotero than there are in the other two hyaluronic acids at time points 0.5, 1.0, and 2.0 hours. This may mean that more of the high-molecular-weight product remains longer in the tissue than in the tissue of the other two hyaluronic acids with smaller proportions of high-molecular-weight polymers, as the polymer is acted on by the body’s own naturally occurring hyaluronidase. The rates of degradation are fairly uniform, but the larger starting high-molecular-weight polymer may account for the longer time to reach final low-molecular-weight fractions. At the dosage ratio used in this study, 16 units of ovine testicular hyaluronidase per 0.08 ml of hyaluronic acid, complete degradation occurred at 24 hours, demonstrating complete reversibility of these hyaluronic acid soft-tissue fillers.

The effect of smaller ratios of ovine testicular hyaluronidase to a given volume of hyaluronic acid may result in different degradation rates, but our study was not designed to address the effects of various ratios on degradation rates. Instead, we looked at degradation at multiple time points, with one ratio. Well-designed in vivo studies could elucidate the ratio to degradation question, allowing the clinician to inject a given overtreated area with a small amount of ovine testicular hyaluronidase, dissolving just that small area and allowing for careful detailed correction.

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CONCLUSIONS

Belotero Balance, Restylane, and Juvéderm Ultra are three commercially available soft-tissue fillers with varying polymeric molecular weights. Belotero Balance has the highest high-molecular-weight polymer, followed by Juvéderm Ultra, and Restylane. There is a large proportion of high-molecular-weight polymer in a syringe of Belotero Balance. The significant differences in the molecular weight reduction of the high-molecular-weight polymer suggest that Belotero Balance, with its significantly greater high-molecular-weight products, may retain larger molecular weight polymers as it is exposed to the body’s own natural hyaluronidase. Low-molecular-weight differences among the three soft-tissue fillers are not significant. Although degradation rates are largely the same when hyaluronic acids are exposed to ovine testicular hyaluronidase, the large cross-linked polymer retains a higher molecular weight longer with Belotero Balance than with Juvéderm Ultra, which in turn retains its molecular weight longer than Restylane. All three hyaluronic acids are essentially fully degraded by 24 hours.

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ACKNOWLEDGMENT

The authors sincerely appreciate the editorial contributions of David J. Howell, Ph.D., R.R.T. (San Francisco, Calif.), in support of development and submission of the manuscript.

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