Crosslinked hyaluronic acid dermal fillers: a comparison of rheological properties Samuel J. Falcone, Richard A. Berg FzioMed Inc., 231 Bonetti Drive, San Luis Obispo, California 93401 Received 30 November 2006; revised 20 April 2007; accepted 31 July 2007 Published online 15 January 2008 in Wiley InterScience (www. interscience.wiley.com). DOK: 10.1002/forn.a.31675 Abstract. Temporary dermal fillers composed of cross- linked hyaluronic acid (XLHA) are space filling gels that are readily available in the United States and Europe. Sev’ feral families of dermal fillers based on XLHA are now available and here we compare the physical and theologi- cal properties of these fillers to the clinical effectiveness. illers are prepared with different crosslinkers, using HA isolated {rom different sources, have different particle sizes, and differ substantially in rheological prop- frties. For these fillers, the magnitude of the complex vis- cesity, I'l, varies by a factor of 20, the magnitude of the complex rigidity modulus, IG*1, and the magnitude of the complex compliance, 1J*! vary by a factor of 10, the per- ‘cent elasticity. varies from 58% to 89.9%, and the tan 8 varies from 0.11 to 0.70. The available clinical data cannot be correlated with either the oscillatory dynamic or steady flow rotational rheological properties of the various fillers. However, the clinical data appear to correlate strongly with the total concentration of XLHA in the products and to a lesser extent with percent elasticity. Hence, our date suggest the following correlation: dermal filler persistence [polymer] X [% elasticity] and the clinical persistence of a dermal filler composed of XLHA is dominated by the mass and elasticity of the material implanted. This work predicts that the development of future XLHA dermal fil- ler formulations should focus on increasing the polymer concentration and elasticity to improve the clinical persist ence, © 2008 Wiley Periodicals, Inc. J Biomed Mater Res B7A: 264-271, 2008 Key words: dermal fillers; hyaluronic acid; soft tissue aug, mentation; oscillatory dynamic properties INTRODUCTION Dermal fillers for cosmesis of the face were intro- duced many years ago and received a large follow- ing with the introduction of injectable bovine colla- gen? As the procedures for wrinkle correction with dermal fillers became more popular, the search began for safer, longer lasting fillers. The next gener- ation filler after bovine collagen was crosslinked hy- aluronic acid (XLHA), which was an improvement over bovine collagen in that it did not require a skin test for hypersensitivity and appeared to be longer lasting The search for safer and more effective der- mal fillers has continued because patients desire temporary fillers that are safe and predictably last longer than 6 months.** In spite of a plethora of new temporary fillers composed of XLHA that are available in Europe and are undergoing clinical test- ing in the United States, it is not understood what parameters control the performance of these fillers. Although several studies have demonstrated that Correspondence to: R.A. Berg; e-mail: raberg@fziomed.com © 2008 Wiley Periodicals, Ine XLHA fillers are more persistent than bovine colla- gen, the differences among the various XLHA fillers ave not demonstrated obvious improvements. in performance. One recent study compared two XLHA fillers, Perlane and Hylaform,* and additional stud- ies are expected as more fillers become available” Comparison of dermal fillers’ effectiveness is com- pounded by the injection techniques,* and the bio: logical response of tissue to an implant in terms of degree of inflammation? Here we compare the rheological propertics of several commercial XLHA dermal fillers to under- stand the differences between them in terms of their physical properties and to attempt to correlate physi: cal properties with performance. One feature of hy- aluronic acid (HA) that has been useful in some medical device applications is its ability to form a cohesive gel.” Cohesiveness is a function of con- centration and molecular weight. The property of cohesiveness, although an advantage for certain applications, is generally not an advantage as a dermal filler? because highly cohesive (HA) materi- als are generally dilute solutions of noncrosslinked HA with low elasticity and short persistence in use Dermal fillers are injected into the connective tissue(CROSSLINKED HYALURONIC ACID DERMAL FILLERS. of the dermis and therefore must be elastic in a low- shear environment. It is hypothesized that elasticity of a dermal filler leads to increased persistence but comparisons have not been performed. For noncros- slinked HA, dynamic rheological analysis demon- strated that as the frequency decreases the elastic properties decrease and hence, at low frequencies, the material becomes less elastic, and more viscous. ‘Therefore, HA used in dermal fillers is always cross- linked to form gel particles that have high elasticity at lower frequencies? Dermal fillers prepared from XLHA are predominately elastic at low-shear envi- ronments and must have low viscosity under high shear to be able to be delivered through a small-bore needle, These unusual requirements have prompted us to compare commercially available dermal fillers prepared from XLHA in terms of their rheological properties. Clinical data comparing different XLHA dermal fillers are only starting to become available in the lit- erature. This study was undertaken to compare the physical properties of currently marketed XLHA fill: ers to determine which physical properties of XLHA dermal fillers are responsible for effectiveness when injected intradermally for soft tissue augmentation. Since the clinical data directly comparing commer- ial fillers to each other in the same study are not available and most studies have compared a given filler to bovine collagen in nasolabial folds in a split- face design, we have confined our clinical data set to using the wrinkle severity rating scale (WSRS) scor ing system for fillers used in clinical studies reported to the FDA. MATERIALS AND METHODS, Materials The dermal fillers were obtained from commercial sour ces. Restylane, Restylane SubQ, Restylane Perlane, Resty: lane Touch, and Restylane LIPP were obtained from Q- ‘Med AB, (Uppsala, Sweden), or Medicis, (Scottsdale, AZ). Hylaform, Hylaform Plus, Juvederm 24, Juvederm 24HV, Juvederm 30, and Juvederm 30HV were obtained from Allergan (Inamed) (Irvine, CA) or LEA Derm (Paris, France). Puragen was obtained from Mentor (Edinburgh, UK), and Esthelis Basic was obtained from Anteis S.A (Geneva, Switzerland). Rheological measurements Small deformation oscillation dynamic rheological meas urements were carried out with a Thermo Haake RS300 Rheometer, Newington, NH, fitted in the cone and plate geometry. All measurements were performed with a 35-mm/1" titanium cone sensor at 25°C. Oscillation mea- surements were made over a frequency range of 0.628-198, (rad/s). Percent elasticity is calculated as: Percent elasticity = (100 x Gry/(G" + G") 3932 RESULTS ‘The HA dermal filler formulations evaluated in this study are all crosslinked. The exact nature of the crosslinking reaction conditions, crosslinker type, crosslink density, resultant particle size, and particle shape, all affect the physical properties of the XLHA formulation. While noncrosslinked HA forms a vis- cous solution when dissolved in aqueous solvents, chemically XLHA produces a material that swells in aqueous solution but does not dissolve. Hence, the XLHA dermal fillers are not solutions of XLHA but suspensions of swollen XLHA particles in aqueous solution. The properties of the XLHA products are influenced by the XLHA particle size and amount of polymer per unit volume. These materials do not hhave a smooth appearance and depending on the injection technique used can be lumpy. Also, suspen- sions of crosslinked polymers require larger gauge needles for injection into the dermis compared with the solutions of noncrosslinked polymers. Table I lists some properties of several commer- cially available dermal fillers containing XLHA. The source of HA is from bacterial fermentation except for the Hylaform products where the source of HA is animal (Avian). The crosslinkers used include vinyl sulfone, for the Hylaform products, 1,4-butane diol diglycidyl ether (BDDE), for the Restylane and Juvederm series as well as Esthelis Basic. Puragen is, crosslinked with 1,2,7,8-diepoxyoctane (DEO). Much has been written concerning the nature of the cross- linking reactions of XLHA dermal fillers. The differ- ences in the products from different manufacturers are generally ascribed to the physical state of the swollen gel after crosslinking HA. Products are described as being single or double crosslinked; par- ticulate or nonparticulate; monophasic, or biphasic. The Restylane, Juvederm, and Esthelis Basic prod- ucts all use BDDE as the crosslinking agent for HA. In the Restylane series, the crosslinking reaction pro- duces particles of crosslinked HA that are swollen in the aqueous phase. The number of particles/ml and the size of the particles differentiate the prod- ucts. As the size of the particles increases the num- ber of particles/ml. decreases, and the products are advertised for use in the correction of deeper facial defects. The number of particles/ml. for some of the Restylane family of products is listed in Table 1 ‘The crosslinking reaction for the Juvederm family of products is a patented process that produces a single phase, nonparticulate, crosslinked HA gel according to the manufacturer. This crosslinking technology is reported by the manufacturer to give Journal of Biomed266 FALCONE AND BERG Juvederm a softer feel when injected into the dermis and good persistence without the stiffness of other XLHA dermal fillers. Esthelis Basic products use a proprietary technology, cohesive polydensified ma- trix (CPM) to produce a single-phase nonparticulate XLHA dermal filler according to the manufacturer. Puragen uses DEO in a double crosslinking reaction that results in both ether and ester bonds crosslink- ing HA chains. According to the manufacturer, this highly crosslinked material is expected to improve the persistence of the Puragen products. The concen- trations of XLHA in these formulations varies from ~ 5 mg/ml. to 24 mg/ml. Increasing the concentra- tion of XLHA above ~ 25 mg/mL becomes problem- atic because the products become too difficult to be injected through a small-bore needle Table II lists some rheological properties, at 0.628 (rad/s), of several commercially available dermal fillers containing XLHA. The magnitude of the com- plex viscosity (In*!), at 0.628 (rad/s) of the dermal filler formulations, Figure 1, varies widely from 58 to 1199 Pa s, almost 20 fold. Restylane LIPP has the highest magnitude of complex viscosity, 1199 Pa s and Esthelis Basic, using the CPM technology, the lowest. The XLHA products have a wide range of complex viscosities at low frequency. ‘The magnitude of the complex viscosity, |n*l, for the Restylane family of products varies from 330 Pa s for Restylane SubQ to 1199 Pa s for Restylane LIPP. For the Restylane family of products, the rheo- logical properties could be affected by the number of XLHA particles contained per milliliter in the prod- uct. The rheological properties of Restylane SubQ, Restylane Perlane, Restylane, and Restylane Touch were measured and the results indicate that although the number of particles/ml. changes and the products all have different particle sizes, and indications for use of these products are different, Table I, the magnitude of the complex viscosities for all of these products are similar. Restylane Touch hhas 500,000 particles and a magnitude of complex viscosity of 422.5 Pa s, Restylane has 100,000 par- ticles/mL and a magnitude of complex viscosity of 582.4 Pa s, and Restylane Perlane has 10,000 parti- cle/mL. with a magnitude of complex viscosity of 4864 Pa s, The Intl, at 0.628 (rad/s), for these Restylane family of products does not relate to the number of XLHA particles contained per milliliter. Figure 2 demonstrates that for the Restylane family products, Perlane, Restylane and Restylane Touch, all have similar percent clasticity that is not a func~ tion of the number of particles/mL. A change in number of particles/mL from 10° to 10* per mL is not associated with a significant change in the per cent elasticity indicating that the percent elasticity for the product is independent of number of par- ticles/mL and hence particle size Needle Size (ge) 7 30 30 2 Lg. bore 7 7 7 30 2 7 7 Indication (EU) CE Mack Medium to deep wrinkles and lips Mid or deep dermis and lips ‘Mid dermis and lips “Moderate to severe wrinkles Deep dermis and lips SubQ, add volume ‘Medium to deep wrinkles ‘Mid dermis and lips Lips Mid dermis and lips g No. of Pavticles/mL. Crosslinker crosslinked TABLE DEO, double The Physical Properties of XL HA Dermal od matrix technology results in monophasic double crosslinked HA, DOE: 1,2,7,8-diepoxyoctane— king reaction, ND: not determined. Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria (mg/mL) ks formed during crossl named Tamed Med AB Med AB Med AB Med AB (Med AB TEA Derm LEA Derm LEA Derm ‘A Derm Anteis Mentor ‘glycidyl ether, CPM: cohesive polydensi *Qamed product literature, BDDE: 14 butanedi double crosslinked—both ether and este Juvederm 2411 Juvederm 30 Javederm 30HV Esthelis Basic Restylane LIPP® Juvederm 24 Puragen Journal of Biomedical Materials Research Part A(CROSSLINKED HYALURONIC ACID DERMAL FILLERS 267 TABLET ‘The Rheological Properties, a 0.628 (rad/s) of HA-Based Dermal Fillers Product Intl (Pas) Ter Pap WPI aa) ‘an (8) Hlasticty Paragen one 317 0017 oat 304 Hiylaform 1364 357 oou7 ot 880 Hylaform Plus 108.2 68.0 oo on 399 Restylane LIPP 1199 7535 9.0013, ois. 349 Restylane 5224 3u5 9.0030 028. 782 Restylane Perlane 4864 3055 .0033 030 m2 Restylane Touch 4225 2655 0.0038 032 6 Restylane Sub 3304 2076 1004s 039 78 Juvederm 30HV 8189 51.46 oor 027 787 Juvederm 241 1515 952 0105) 031 762 Juvederm 30 39 59.0 o170 035: 74 Juvederm 24 584 367 0272 053 652 Esthelis Basic 516 387 0.0258 07 588 For the Juvederm family of products, the In*l, at 0.628 (rad/s), varies from 152 to 58 Pa s, Figure 1 ‘The magnitude of the low frequency complex viscos- ity increases for Juvederm 24, to Juvederm 30HV, next is Juvederm 30, with Juvederm 24H1V having the highest magnitude of In*!, and the products are intended for different indications. However, Juve- derm 24HV and Juvederm 30HV are stated to have higher viscosities and perceived to be the most persistent of the Juvederm products even though the magnitude of the complex viscosity, In*l, of Juvederm 30HV is no higher than Juvederm. 30. Although the magnitude of the complex viscosity of Juvederm 24 HV is higher than Juvederm 24, the magnitude of the complex viscosity, I?! at 0.628 (rad/s), are all below 200 Pa s for this family of products. Again, the magnitude of the low frequency complex viscosity does not seem to correlate to per- sistence or indicated use for the Juvederm family of products, sashes) @0ert mde ro (982 40 oo Figure 1 ‘A plot of the magnitude of the complex viscos- ity, Intl (Pa 5), at 0.628 (rad/s) for the XLHA dermal fill- crs listed in Table II. The magnitude of |*| varies widely from 1199 to 58 Pa ¢ for these products. Puragen and the Restylane series have the highest magnitudes of complex viscosities while Hylaform, Juvederm, and Esthelis Basic have much Tower magnitudes of complex viscosity. The magnitude of the complex rigidity modulus, IG*l, at low frequency, 0.628 (rad/s), for all prod- ucts is listed in Table Il. The magnitude of 1G*I at low frequency relates to the overall stiffness of the dermal filler at low deformation rate. The magnitude of the complex rigidity modulus, IG*I, versus fre- quency, for the dermal fillers is shown in Figure 3, and the higher the magnitude of the complex modu- lus, the stiffer the material. There is a large range, ~ 10-fold, in the magnitude of the complex modulus versus frequency response of the XLHA dermal fill- ers studied here. Puragen has the highest magnitude of stiffness and Juvederm 24 or Esthelis Basic has the lowest magnitude of stiffness. The Restylane family of products has higher magnitudes of com plex modulus than the Juvederm family of products For the Restylane and Juvederm product families, Flac reno) Release Ren tne Toh ° ° m0} Resin S80 10 ce ‘oe 900000 cpio. Figure 2. A plot of the percent elasticity at 0.628 (rad/s), (100 x G'/(G" |G), versus the number of particles con’ tained per milliliter for Restylane Touch, Restylane, Resty- Tane Perlane, and Restylane SubQ. The data indicate that there is no correlation between the percent elasticity and the number of particles/ml. and hence, the particle size in this Restylane product series. For this Restylane family of products, the number of particles/mL. do not correlate to Any of the low frequency (0.628 rad/s) rheological parame- ters listed in Table IL Journal of Biomedical Materials Res268 2 Pargen 4 Restylane * Reyne Prone + Restylane Touch * Restylane Sub Q Juvederm 2681V Myler + hwvedem 3 *swveterm HV 4 athe Basic vader 24 [orl a 10000 1000 a1 1 0 1001000 10000 Frequency (rad/see) Figure 3. A plot of the magnitude of complex modulus, IG*I (Pa), versus frequency for the dermal filer listed in Table Il In this figure, Hylaform Plus and Restylane LIPP have been omitted: In this figure, the legend order is in de- scending magnitude of 1G*I at 0.628 (rad/s). Puragen has the highest magnitude of 1G*lat 0.628 (rad/s) and Juve- derm 24 has the lowest magnitude of 1G" lat 0.628 rad/s. For this set of products, the magnitude of 1G*l varies ‘over 10 fold from the stffest material, Puragen, to the least stiff, Juvederm 24, The magnitude of the modulus or over- all stiffness for these products is probably due to the cross- link density. It is also of interest to note that although they. have different magnitudes, the slope of the IG"! versus frequency curves is very similar for all of the XLHA prod- ucts described here. This is not surprising since the struc ture of the crosslinked polymer swollen in the matrix is very similar for all XLHAS, the magnitude of the complex rigidity modulus is more similar within each family than between families Several studies concerning XLHA products have referred to a rheological property called the percent elasticity."°""* In these studies percent elasticity is calculated as (100 x G')/(G' + G") and is reported as the proportion of elasticity in an XLHA formula- tion. The percent elasticity versus frequency, for the XLHA dermal filler materials, is shown in Figure 4 The XLHAs have percent elasticity that range from 60 to 90% with the Restylane series more closely grouped than the others. There is a considerable range in the percent elasticity of the XLHA products with Juvederm 24 and Esthelis being the least elastic and Hylaform the most elastic. The magnitude of the complex compliance, I/*1, versus frequency, for these materials, is shown in Figure 5. The compliance is the inverse of the modu- lus, and is a measure of how easy it is to deform a material. Again, the data indicate that the XLHA dermal fillers have a wide range of magnitudes of complex compliance of over 10 fold. Puragen has the lowest magnitude of complex compliance of all the dermal fillers studied here. Materials Resench Part A FALCONE AND BERG say ooretooy = Hylafom Frog a) Figure 4. A plot of the percent elasticity, (100 G'/(G! + G°), versus frequency of the dermal fillers listed in Table I. In this figure, Hylaform Plus and Restylane LIPP have been omitted and the legend order is in descending per cent elasticity at 0.628 (rad/s). Hylaform has the highest percent elasticity at 0.628 (rad/s) and Esthelis Basic has the lowest percent elasticity at 0.628 (rad/s). The percent elasticity for these XLHA’ products varies widely from ~ 60 to 90%, Since Hylaform has the highest percent elas- ticity but the lowest &month improvement WSRS scores, (see Fig. 6), percent elasticity of the XLHA dermal filler itself does not correlate to product persistence and concen. tration must be taken into effect. bei aor) ° 01 001 . o 1 0 cn) Frequency (ec) Figure 5. A plot of the magnitude of the complex compli- ance, || (1/Pa), versus frequency of the dermal fillers listed in Table I. In this figure, Hylaform Plas and Resty lane LIPP have been omitted. The magnitude of IJ*I is a measure of the overall ease of deformation of a material and hence, a material with a lower magnitude of complex compliance is harder to deform than 2 material with 2 hhigher magnitude of complex compliance. In this figure, the legend order is in descending complex compliance at 0.628 (rad/s). Puragen has the lowest compliance and is the most difficult to deform, at 0.628 (rad/s). Juvederm 24 thas the highest magnitude of 1/*1 and is easiest to deform, at 0,628 (rad/s). The magnitude of I*| for these XLHA products varies widely (~ 10 fold). Puragen is the least compliant dermal filler followed by the Restylane products all of which have a magnitude of IJ"! below 001, The remaining products all have a magnitude of |*! above 0.01 at 0.528 (rad/s)(CROSSLINKED HYALURONIC ACID DERMAL FILLERS. TABLE I Effectiveness Data for Dermal Fillers at ‘&Month Post-Treatment ‘Month Improvement Product Score WSRS Reference Zyplast 036 Restylane control Restylane 093 Ret. Zyplast, 05 Juvederm 30 control Juvederm 30 14 Ref. 7 Zyplast 03 Juvederm 26HV control Juvederm 24HV 3 Ret. 7 Zyplast, 04 Juvederm 30H control Juvederm 30H 14 Ref. 7 In these studies all of the XLHA products were com- pared to Zyplast as a control In attempting to account for any_ relationship between rheological properties and clinical benefit or persistence, the available data are quite limited. This is because few studies compare one filler to another. Some fillers like Esthelis and Puragen have no published data on performance. The available data ‘on fillers where one product was compared with another is summarized in Tables III-V. The data set is only for the clinical studies involving filling the nasolabial folds in studies submitted to the FDA in support of approval of a Premarket Application, ‘Only the 6-month data point was evaluated in cach of the studies. If one compares the persistence of the fillers for which there are data with the measured values given in Table II, the persistence, as measured by the 6-month WSRS improvement score, correlates TABLE IV [Effectiveness Data for Dermal Fillers ‘at Months Post-Treatment Month Improvement Product Score WSRS Reference Zyplast 12 Hiylaform control Hylatorm* ul Ref 15, Zsplast. 10 Juvederm 30 control Juvederm 30 16 Ret. 7 Zyplast, 10 Juvederm 24H1V control Juvederm 24H1V a7 Ref. 7 Zyplast. 08 Juvederm 30HV control Juvederm 30H 16 Ret. 7 Hylaform* os. Control for Hylaform Plus Hylaform Plus® 09, Ref 15 “To compare Hiylaform and Hyleform Plus to the products in Table Il, studies comparing Hylaform and Hylaform Plus to Zyplast are compared. In these studies, 2 &point scale with a live evaluator was used to score the 3-month time point in the 3-month study demonstrating that Zyplast, Hylaform, Hylaform Plus are all similar. Since Zyplast was used as a control in the Restylane and. Juvederm studies in Table Ill, Juvederm, Restylane, and Hylaform can all be used to estimate effectiveness. 269 TABLE V Correlations from Tables III and IV WSRS, Improvement Score Concentration % Complex Complex: (6 months) (% Solids) Elasticity Viscosity Modulus ba 05 a9 136 6-85 09. 20 Ti-7S* 330-530" 207-334" 14 24 6576 58151 3695 Excluding Restylane LIPP. inversely to the percent elasticity as shown in Figure 6, Hence, as the percent elasticity increases, the per- sistence decreases. From the previous discussion, this result is counterintuitive and indicates that high elasticity alone cannot account for the persistence of XLHA dermal fillers; however, the concentration of polymer was not taken into effect. The effectiveness of the dermal fillers listed in Table V does correlate to the concentration of polymer in the formulation as shown in Figure 7. Here, the persistence of der- ‘mal filler is directly related to the mass of polymer in the formulation. Intuitively, it would be reasona- ble that both polymer concentration and. elasticity would relate to the effectiveness of dermal filler. This relationship is shown in Figure 8, where effec- tiveness for a series of dermal fillers is plotted versus XX dete sede 207 (© sredem0ty A totes 0 sm Figure 6. Described in this plot is the relationship of the s-month improvement score WSRS to the XLHA polymer percent elasticity at 0.628 (rad/s), for Hylaform, Restylane, Juvederm 24HV, Juvederm 30, and Juvederm 30HV. The improvement scores are from Tables Ill and IV. ‘The WSRS score is a measure of the relative effectiveness of these XLHA dermal filers after § months. For these XLHA der- mal fillers, the WSRS improvement score increases as the percent elasticity decreases, The WSRS improvement score correlates. in_a linear manner to the percent elasticity at 0.628 (rad/s). Since the prevailing theory suggests. that higher percent elasticity ‘results in dermal filers with higher persistence, this result is somewhat counterintui- tive. These data suggest that high elasticity at low fre- guency is not the only factor affecting persistence for XLHA dermal filer. Journal of Biomedical Materials Res270 6 Moai Inprovemeat “WSRS Sore uu 2 1 06 os 02) Hylton Come) Figure 7. A plot of the 6-month improvement score WSRS versus XLHA polymer concentration (mg/mL) for Hylaform, Restylane, Juvederm 21HV, Juvederm 30, and. Juvederm 30H. The improvement scores are from Tables MIE and TV. The WSRS score is a measure of the relative effectiveness of these XLHA dermal fillers after 6 months For the data shown hete, the Hylaform &-month WSRS score was assumed to be equivalent to the 6-month WSRS Zyplast score (Table IM), because when Hylaform and Zyplast were compared in a 3-month study, they had v similar WSRS scores. Zyplast was also used as the conteol in the &month study comparing Restylane to Juvederm. Tor these data, the 6-month WSRS improvement score cor- relates very well to the concentration of XLHA contained in the product, the product of the polymer concentration in solution and the percent elasticity, at 0.628 rad/s, of the for- mulation. All of the XLHA fillers for which there are clinical data correlate linearly with the polymer con- centration and elasticity. DISCUSSION ‘The clinical persistence and characteristics of HA- based dermal fillers may be influenced by their physical properties.’ When HA is formulated as a biomaterial for a specific indication, it is frequently crosslinked."” HA is subject to degradation by hyalu- ronidase in the body and crosslinking is expected to reduce susceptibility to enzymatic degradation. ‘The dynamic rheological properties of HA need to be improved for the polymer to perform its intended use as dermal filler. Crosslinking the polymer increases the elastic modulus of the polymer, at low frequency, making the XLHA more elastic than non- crosslinked HA. Hence, for dermal filler formula- tions, HA is chemically crosslinked to increase the elastoviscous properties and increase the residence time at the site of injection. ‘The advantage of comparing a series of dermal fillers manufactured from the same material, that is Journal of Biomedical Materials Reseach Part A FALCONE AND BERG HA, is that one can assume that the biological or tis sue response is similar for each filler in the series Many different filler materials have been tested and the differences in tissue responses and injection tech- niques make it very difficult to correlate the clinical persistence of these different materials to physical properties (for reviews, see Refs. 1, 17-20). Attempts to compare the available XLHA dermal fillers have been limited. Two products Hylaform and Restylane hhave been compared in terms of some chemical properties" but since these produets differ in con- centration and particle size, the data set is too lim- ited to draw conclusions. Rheologically, all of the XLHA products generally have high elastic to loss modulus characteristics exemplified by low tan (6) ve uues throughout the entire frequency range. Attempts have been made in the past to estimate the elastic nature of XLHAs by calculating the percent elasticity from the viscoelastic modulii. This approach is lim- ited by the effect of concentration of the polymer. Hylaform is highly elastic but is dilute compared with other products and also is less persistent than Restylane and Juvederm (Table V). Another possibil- ity is that the magnitude of the complex viscosity or the magnitude of the complex rigidity modulus is the controlling factor, Juvederm has magnitudes of complex viscosity and complex modulus that are considerably lower than Restylane (Table 11), and it appears to be more persistent (Table V). ‘Other suggestions have been that the persistence is proportional to particle size. The only product family where particle size is estimated is the Resty- Jane family of products, and for this family there ‘Moth egret i so X mentee : . Daan 1 sar © pavsder 308 2 A teste be © ses Clty % Bay 0604 ae Figure 8. This figure describes the correlation of the & month WSRS improvement score to the product of the concentration (mg/ml) and percent elasticity for XLHIA dermal filers Hylaform, Restylane, Juvederm 24HV, Juve derm 30, and Juvederm 30H. There is a linear correlation that indicates that both properties (concentration and elas- ticity) are factors influencing the persistence of XLHA der- mal filers,(CROSSLINKED HYALURONIC ACID DERMAL FILLERS appears to be no correlation between particle size and percent elasticity (Fig. 1), the magnitude of the complex viscosity (Fig. 4), the magnitude of the com- plex modulus (Fig. 5), or percent elasticity (Fig. 6). The major difference between members of the Resty- lane family of products with different particle sizes is the injectability of the products. Restylane and Restylane Touch are administered through 30 gauge needles whereas Perlane requires a 27-gauge needle and SubQ requires a larger cannula. Unfortunately, comparisons of persistence among the Restylane products are not available. In conclusion, the oscillatory dynamic rheological properties of XLHA dermal fillers have been deter- mined and it was found that they are quite variable with respect to the magnitude of the complex rigid ity modulus, overall stiffness (IG*1), the magnitude of the complex viscosity (In*l), and percent elastic- ity. Although there are limited controlled clinical data, the most straightforward correlation is between persistence of the product and polymer concentra- tion, when the products are compared in a typical clinical study of paired bilaterally placed fillers in the nasolabial folds. Within the same concentrations, persistence appears to be related to elasticity. A lin ear correlation exists between persistence and poly- mer concentration and also between persistence and the polymer concentration and clasticity. The data presented here indicate that the persistence of XLHA dermal fillers appears to be a function of the concen- tration of polymer in solution and to a lesser extent the elasticity of the material. The maximum concen- tration of XLHA polymers in the current XLHA der- mal filler formulations appear to be limited to ~ 25 mg/mL. Finding new methods to increase the XLHA concentration in dermal filler formulations may be the important hurdle to overcome for new filler candidates prepared from XLIIA. References 1. Klein AW, Flson ML. The history of substances for soft tissue ugmentation. Dermatol Surg 2000,26:1096-1108, 2. Fernandez EM, Mackey CL. Soft tissue augmentation: A review. J Drage Dermatol 20065630681, 2. 1. 4, 15. 16. v. 20 2 Lempetle G, Mothenn V, Chartier U. Human histology and persistence of various injectable filer substances for soft liseue augmentation, Aesthetic Plast Surg 200327354355; discussion 367, Bosniak S, Cantisano-Zilkha. Restylane and Perlane: A six ‘year clinical experience. Operative Tech Oculoplastic Orbital Reconstr Surg 200148893 "Nazins RS, Brandt F, Leyden J, Lorene ZP, Rubin M, Smith S. A randomized, double-blind, multicenter comparison of the celfeacy and tolerability of Restylane versus Zyplact for the correction of nasolabial folds. Dermatol Sung 2005,29588-585, Carruthers A, Carey W, De Lorenst C, Remington. K, Schachter D, Sapra S. Randomized, double-blind comparison fof the efficacy of two hyaluronic acid derivatives, restylane perlane and hylaform, in the treatment of nazolabial folds ‘Dermatol Surg 2005331 (11 Pt 2):1591-1595; discussion 1598 FDA. Summary of Safety and Effectiveness of Juvederm, PMA number POS0047; 2006 Bosniak §, Cantisano Zillha M, Glavas IP. Nonanimal stabi lized hyaluronic acid for ip augmentation and facial Phytid ablation. Arch Facial Plast Surg 20046:379-383 Falcone $j, Palmeri DM, Berg RA. 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