Elemental distribution confirms visualization of the HA network by SEM. Scanning electron microscope–EDX images of each HA filler are shown in Figure 4. The top row of images are unmapped cryo-SEM micrographs of the 3 different HA fillers. The second, third, and fourth rows are images of carbon (C), oxygen (O), and sodium (Na) elemental distribution maps, respectively. The column of images on the left are of BEL, the middle are of RES, and the right are of JUV. The unmapped images of the fractured HA gel material reveal the presence of a dense 3-dimensional structure with irregularly spaced fibrous or slab-like structures having an interconnected network appearance consistent with interconnected HA molecules. Elemental mapping experiments indicate that the irregular structures are rich in C and Na. Oxygen mapping shows that the dark gray zones in the unmapped images that are present between the lighter gray HA network structures are rich in oxygen. We infer from the elemental maps, and the overlays with the unmapped cryo-SEM images from the same sample area (Figure 4, bottom row), that the walls in the network are comprised of HA that is suspended in aqueous media. We attribute the sodium signal enrichment within the HA network structure to electrostatic attraction of Na counterions to the glucuronic acid carboxylate (CO2−) units within the HA polymer.
Differences in morphologic architecture are evident among BEL, RES, and JUV. As can be seen in Figure 5, a more uniform network having a finer consistency (i.e., smaller average pore dimensions—pore being the term used to describe the small spaces in between the HA polymeric chains) was found in BEL when compared with JUV and RES. Juvéderm Ultra exhibits a network structure that is more heterogeneous, with some regions of finer porosity and other zones that are of larger average pore size. Restylane images show multiple pore sizes, some of which are quite dense and others that are of the lowest density observed in any of the samples. There was no evidence of discrete particles in BEL or JUV. However, the RES samples contained many highly irregular particles with very small pores that were surrounded by a more diffuse network of HA material with larger pores.
Lidocaine addition shows no effect on HA gel morphology. In clinical practice, small amounts of anesthetic solution are frequently mixed with HA gels. We sought to determine the effect of lidocaine addition on the ultrastructure of the HA gels. As shown in Figure 6, little change is noted in the network structure appearance after addition of lidocaine to each of the 3 soft-tissue fillers. As noted in the unmodified samples (Figure 5), the appearance of BEL and JUV is more uniform before and after lidocaine addition than the RES samples that still possessed particulate matter after lidocaine dilution.
Extrusion of the HA gels through a 30-G needle has no effect on gel architecture. Each of the 3 HAs was extruded in 2 ways: (1) through a 30-G needle and (2) simply releasing it through the Luer-Lok opening of the syringe. The images in the top row of Figure 7 show that the effect of extrusion through the needle is negligible compared with the network structure apparent when the sample was extruded through the much larger diameter Luer-Lok port of the manufacturer's syringe. This suggests that the architecture of the HA networks in each soft-tissue filler remains fundamentally intact during an operation that mimics the deposition of filler product in the patient through a 30-G needle.
BEL HA gel is consistent throughout the BEL syringe and across multiple lots. Using a single syringe of each BEL gel, HA filler samples were applied by extrusion through the Luer-Lok syringe tip onto cryo-SEM stubs at 3 different stages of sample extrusion. Specifically, we applied material from the first 0.3 mL, the middle 0.3 mL, and the final 0.3 mL of the syringe onto the SEM sample holders in an effort to determine whether the sample ultrastructure changes as one proceeds from the front, mid-section, and back-section of the syringe. Comparison of each row of images in Figure 8 suggests that there is no appreciable change in sample ultrastructure as one draws material from the front, mid-section, or back of the syringe. Regarding lot consistency, note the relatively uniform network structure of BEL, when the columns of data are compared, showing a substantial uniformity of the HA gel across 3 separate lots of the soft-tissue filler. The findings show consistency in a narrow range of network porosities ranging from 1 to 10 μm both across the different BEL lots and within different positions in the syringe.
In the clinical setting, physicians have long acknowledged that 1 HA is not simply interchangeable with another. One HA has applicability and usefulness for certain areas of the face, and for one purpose, whereas another has usefulness in other areas. Examples would be the use in the tear trough or lips versus the use for deeper subdermal volume replacement. The professional literature is useful for highlighting distinctions between and among soft-tissue fillers but, ultimately, the decisions rest with the treating health care provider.
The HA fillers have different qualities with respect to tissue integration when placed intradermally.8,10 Our first study, in 2011, noted differences in diffusion throughout the skin in both biphasic and monophasic soft-tissue fillers. The HA with the cohesive polydensified filler (BEL) appeared “evenly placed in and between the collagen fibers throughout the reticular dermis,” whereas JUV showed “material throughout the dermis but in large clumps and channels.” RES showed “large pools of HA distributed as clumps or beads of material located at the lower part of the dermis.” Another study was published in 2013 that has helped better understand integration of HA fillers into the dermis, in this case, into the superficial dermis.11 Using ultrasound and histologic techniques, investigators found that the HA with the cohesive polydensified matrix, that is, BEL, was useful for the superficial dermis “because of its high degree of integration into the dermis.” In comparison, JUV showed a heterogenous appearance with a cone of shadow at the edge of the papule on intradermal injection, and RES showed a granular appearance, with a cone of shadow underneath.
In 2013, our research group published a follow-on study about the high–molecular weight and low–molecular weight components of BEL, RES, and JUV.8 Belotero Balance had the largest high–molecular weight species, followed by JUV and RES (p < .0001). Differences among the low–molecular weight species in the 3 HAs were not significant. In that study, the 3 HAs were exposed to ovine testicular hyaluronidase at 6 time points, to determine degradation rates of the 3 HAs. Degradation rates of the 3 HAs were similar, with full degradation at 24 hours. However, the proportion of high–molecular weight components in BEL remained high at early time points.
In the study reported here, we used SEM techniques to further understand the nature of these HA polymers. All of the polymers were rich in oxygen, carbon, and sodium; these elements were used to identify the localization and ultrastructure of the HA polymers in the images. The HA polymers seem to form a fibrous network structure that is suspended in an aqueous medium. In terms of appearances under SEM imaging, BEL presented as the HA with the most uniform distribution, followed by JUV. The appearance of Restylane was the least uniform of the 3, presenting with highly irregularly sized particles in a diffuse network. The findings of BEL and RES support 2 earlier findings about evenness of the 2 products after dermal injection.12,13
Much research has been conducted to investigate the rheological properties of HA and other fillers, in an effort to correlate measurements such as elastic modulus (G′) and viscosity with how the filler acts within tissue after injection.4,14,15 For example, it has been postulated that HA gels demonstrating a high G′ provide more structural support and tissue “lifting,” whereas those with a low G′ are best suited for areas of thin skin and/or superficial placement.
Our work here correlates rheologic and histologic findings with the overall ultrastructural morphology shown in the SEM images. Belotero Balance exhibits the lowest G′ and a high degree of histologic dermal integration, followed by JUV, and then RES. Our findings here show that BEL has the highest degree of uniform interlocking HA strands compared with JUV. Restylane is shown to be formed of denser particles suspended in HA strands. The findings of this study, combined with previous work, may explain why BEL is more ideally suited for superficial use and intradermal placement, whereas JUV is used subdermally and deeper, with RES also suitable as a deeper HA filler.
We also investigated how the addition of lidocaine and extrusion force might affect overall morphology. We did not find noticeable differences in the gel's structure with the addition of lidocaine in any of the 3 HAs examined. The distinctions noted above in the nonlidocaine images remained present in the ones with the lidocaine added. Similarly, no marked changes were observed in the 3 HA gels when delivered through a 30-G needle compared with simple delivery through the Luer-Lok opening of the syringe. In addition, the consistency of the HAs remained intact in both the needle and syringe aperture sampling. This is an interesting finding when one considers speculation among physicians that injection of HA through small-bore needles (30–32 G) would have a shearing force effect, altering the overall architecture of the gel. Our results show no effect on gel morphology when passed through a 30-G needle.
Our last area of analysis was lot-to-lot consistency of BEL. Our findings in this area mirrored our findings in the across-HA analyses, namely, consistency within the individual syringe and across multiple lots.
Differences were observed in terms of size and uniformity across all 3 HAs investigated. In descending order of uniformity, BEL presented more uniformly than JUV, which presented more uniformly than RES. Restylane has more dense particles of HA suspended in looser HA strands. Neither lidocaine nor extrusion through a 30-G needle appeared to influence inherent properties in the 3 HAs. In terms of consistency both within portions of the syringe and across lots of BEL, little variability was noted. These findings are useful for physicians to help them better understand HAs' structural correlates with patients' needs and appropriate product selection.
We sincerely appreciate the contributions of Matt Chansky (Research Triangle Park, NC) for his illustrations and of Dale Murphy (San Francisco, CA) for his graphics.
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© 2015 by the American Society for Dermatologic Surgery, Inc. Published by Wolters Kluwer Health, Inc. All rights reserved.
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