Three-Dimensional Imaging as a Novel Method of Evaluating the Longevity of Hyaluronic Acid Fillers in a Mouse Model

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Hyaluronic acid (HA) is a biocompatible material that can be removed by enzyme (hyaluronidase) degradation and reabsorbed over a period of 6 to 12 months. Therefore, injection of HA filler for facial volume augmentation should be repeated every few months.1 Thus, physicians and manufacturers are actively seeking new HA fillers with longer-lasting efficacy. To ensure this longevity, resistance to degradation and preservation of the 3-dimensional (3D) shape are required.2
The long-lasting efficacy of HA filler injections is usually assessed visually by physicians according to grading system. In general, researchers have used invasive methods, such as gross morphology, weight of extracted HA implants, and histological analysis, to evaluate the effects of injected HA materials in animal models. In this study, the authors aimed to evaluate the longevity of an HA filler in a hairless mouse model using noninvasive, 3D imaging methods.
All animal experiments were approved by Chung-Ang University's Institutional Animal Care and Use Committee (approval No. 2014-0033). Female SKH-1 hairless mice (6 weeks of age) were purchased from Orientbio Inc. (Gyeonggi, Korea), and 30 mice were bred for this study. Filler samples were manufactured by Across Inc., and provided by Hugel Inc. (The Chaeum; Sub-Q, Seoul, Korea) and consisted of a transparent, viscoelastic gel containing cross-linked HA at a concentration approved by the Korea Food and Drug Administration (KFDA). This filler was subcutaneously injected into the back of anesthetized mice. A total of 30 mice were divided into 3 groups (10 mice/group). Group 1 was injected with a total HA concentration of 16 mg/mL, Group 2 with 20 mg/mL, and Group 3 with 24 mg/mL. All samples were 200 μL in volume.
We evaluated the weight of the injected HA fillers in the extracted skin tissue of the killed mice. Mice from each of the 3 groups were killed at the time points of evaluation (0, 30, 60, and 180 days after injection). The HA filler that had been implanted into the subcutis of the dorsal skin was extracted and weighed using a balance (EPG213C; OHAUS Corp., Parsippany-Troy Hills, NJ). Regardless of the HA concentration, the extracted weight of implanted HA filler was highest between days 10 and 30. The extracted weight between days 10 and 30 was about twice that of baseline and then decreased over time (Table 1). Figure 1 shows the gross extracted HA filler materials using a folliscope and digital photography at Day 180.
The volume of the implants was evaluated using 2 types of 3D imaging methods: magnetic resonance imaging (MRI) and computed tomography (CT). A 3.0 T MRI system (Archieva; Philips Healthcare, Best, the Netherlands) using a T2-weighted image (T2WI) pulse sequence with a wrist coil (SENSE wrist 4 channel; Philips Healthcare) was used to obtain the magnetic resonance (MR) images. The sequence parameters were as follows: T2WI (turbo spin echo; TR, 2,863.3 ms; TE, 100.0 ms; flip angle, 90°; FOV, 80 × 80 mm; slice thickness, 2 mm; matrix size, 248 × 208; resolution, 0.32 × 0.38 × 2.0 mm). An experienced radiologist evaluated the images to identify and distinguish the injected HA filler from surrounding tissues. Computed tomography was performed with 3D CT (Brilliance iCT; Philips Healthcare), and the images were analyzed by Philips' Clinical Workflow Solution (Extended Brilliance Workspace V 4.5; Philips Healthcare) to determine the volume of the implants. Three-Dimensional volumetric measurements were performed to calculate the volume of the injected HA filler. Before the volume measurement, 3D reconstruction of MR images was carried out. All MR source data (17 sectional images were obtained by MRI for each mouse) were sent to Philips' Clinical Workflow Solution.
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