Facial rejuvenation with the new hybrid filler HArmonyCa™: Clinical and aesthetic outcomes assessed by 2D and 3D photographs, ultrasound, and elastography

Objective: Facial aging is a complex process that involves genetic, biological, and environmental factors. This paper aimed to present the first aesthetic and safety outcomes of a hybrid filler that combines hyaluronic acid (HA) (20 mg/mL) and calcium hydroxyapatite (HA/CaHa). Methods: Prospective and non-randomized interventional study conducted on consecutive healthy patients who attended the clinic for an aesthetic facial rejuvenation procedure. HA/CaHa was injected with 23G cannula (retrograde threads, 1.25 mL per side) in the preauricular region. Ultrasound examinations, elastography images, and 2D and 3D photographs were performed before and after treatment. The primary endpoint were the volumetric changes at Day 180. Results: Fifteen patients were included in the study. At Day 180 after treatment, median (interquartile-range


| INTRODUC TI ON
Facial aging is a complex process that involves genetic, biological, and environmental factors. 1,2 This process is characterized by an imbalanced distribution of soft tissue, alterations in the texture and tone of the skin, and the presence of wrinkles and folds. [1][2][3] In addition, facial aging has been associated with various histological and molecular changes, including a reduction in the physiologic hyaluronic acid (HA), dermal collagen, and elastin, which causes a gradual thinning of the skin and a loss of elasticity. [1][2][3][4][5] In addition to all these aesthetic changes, facial aging negatively influences different aspects of the subject's life, affecting interpersonal relationships or causing psychological alterations. 1,6,7 Aesthetic procedures have had to face the complex situation represented by the COVID-19 pandemic. Nevertheless, according to the International Society of Aesthetic Plastic Surgery survey 2020, nonsurgical aesthetic procedures have increased by 5.7% compared to 2019. 8 Moreover, minimally invasive procedures for total facial rejuvenation increased by 13.9% as compared to 2019. 8 In 2020, the injection of HA or calcium hydroxyapatite (CaHa) accounted for almost 4.3 million procedures. 8 Since skin aging is associated with a gradual loss of HA (sometimes it increases but in a disorganized way) and a gradual loss a dermal collagen (with fiber disorganization) and elastin (with network degradation), 4,5 it would be desirable to have aesthetic products that can address a comprehensive approach of these different mechanisms.
HArmonyCa™ (Allergan Aesthetics an AbbVie Company) is a hybrid filler that combines HA (20 mg/mL) and CaHa 55.7% (microspheres 25-45 μm). 9 It has been designed for volume restoration in preauricular areas and ascending and descending branches of the lower jaw in patients with elastosis and loss of volume. 9 Due to its physicochemical properties, HA is specially indicated to create and restore facial volume. 10,11 On the other hand, CaHa fillers have been associated with collagen genesis surrounding filler microspheres, which significantly improves the appearance of treated rhytids. 12,13 Despite the large amount of evidence available about the aesthetic use of both HA and HaCa fillers, to our knowledge, there is not published evidence assessing the efficacy and safety of the standardized combination of both fillers in a single syringe.
This study aimed to present the first aesthetic and safety outcomes obtained with HA/CaHa in a clinical setting.

| Study design
Prospective and non-randomized interventional study conducted on consecutive healthy patients who attended the clinic for an aesthetic facial rejuvenation procedure.
The study was conducted in accordance to the Declaration of Helsinki and the Good Clinical Practice/International Council for Harmonization Guidelines. The study protocol was approved by an independent ethics committee. All the subjects provided written informed consent was obtained before enrolment.

| Patients
The study sample consisted of healthy women who had undergone previous minimally invasive aesthetic procedures and attended the clinic to perform a facial rejuvenation treatment. HA/CaHa was injected with 23G cannula (retrograde threads, 1.25 mL per side) in the preauricular region (ascending and descending mandibular branches). The filler was injected into the reticular dermis, at its junction with the subcutaneous cellular tissue. Ultrasound examinations were performed before, 48 h after injections, and 30 days, 90 days, and 180 days after treatment.

| Ultrasounds
In addition, it was used to evaluate the skin and subcutaneous cellular tissue changes were assessed.

| Elastography
Strain elastography of the skin in the right and left preauricular region was assessed before the treatment and 60, 90, and 180 days after treatment by using the ElastoScan™ (HS30/XH30, Samsung Healthcare Global).

| Aesthetic outcomes
Aesthetic outcomes have been evaluated by using 2D and 3D photographs before treatment and 48 h, 30 days, 60 days, 90 days, and 180 days after treatment.
Skin tightening has been evaluated by measuring the changes in the facial tension vectors 60, 90, and 180 days after treatment.

| 3D photographs
Vectra® H2 (Canfield Scientific, Inc.) photogrammetry scanning system was used to assesses the volumetric changes. The operator took consecutive photographs at each time-point measurement (as required by the device instruction), which were subsequently combined into a 3D photo.
Additionally, Vectra® H2 was used to assess the skin tightening by measuring the changes, from pretreatment values, in the facial tension vectors.

| Endpoints
The primary endpoint were the volumetric changes at Day 180.
Secondary endpoints included the changes in the facial tension vectors, the treatment efficacy (assessed by 2D photographs), safety profile, and time to filler tissue integration.
Primary study variable was the ultrasound pattern observed at the different study zones. Friedman's two-way analysis test was used to assess the changes in volume and facial tension vectors. Post hoc analysis for pairwise comparisons were done with the Conover method.

| Volumetric changes
In the right side, median (IqR) volume increased by 1.

F I G U R E 1
Mean volumetric changes in the preauricular region from pretreatment values in the right side (dark blue) and left side (dark green). Vertical bars represent 95% confidence interval. (Statistical significance was determined using the Friedman test with the Conover method.) *p < 0.0001 as compared to pretreatment values. †p < 0.01 as compared to the other time point measurements. No significant differences were observed at any of the time points measured between the right and left sides (p > 0.05 each, respectively).

| Lifting effect
As compared to pretreatment values, in the right side, FTV significantly increased by 1.

| Ultrasonography
Pre-treatment ultrasound images showed the typical heterogeneous pattern characteristic of subcutaneous cellular tissue, with alternation of soft anechoic and hyperechoic images. Immediately after treatment, besides a globular and poorly defined ultrasound pattern; an incipient pattern of "coarse snow grain" can be observed.
Additionally, there was a subcutaneous cellular tissue edema.
Forty-eight hours after treatment, ultrasound images were still showing a globular pattern, with well-defined anechoic areas (indicating an increase in volume), as well as incipient pattern of "coarse snow grain." Thirty days after treatment, there was total integration of the HA into the tissue; while a "coarse snow grain" pattern (Characteristic of the CaHa filler) was observed. Interestingly, there was a neocollagenesis.
At 180 days after treatment, ultrasound images showed full integration of the HA, the presence of a "coarse snow grain" pattern, and an increase in collagen fibers was evident.  Aesthetic outcomes, assessed by mean 2D photographs, showed a significant improvement (Figure 9).

| Safety
There were no either unexpected or serious treatment-related adverse events. Most patients experienced a mild redness and inflammation that resolved within the first 48 h without treatment. Three (20%) patients had an ecchymosis, which was successfully solved with topical treatment (Arnica+Vit K cream). Four (26.7%) patients reported discomfort immediately after treatment, which resolved satisfactorily without treatment.
All the reported adverse events were mild in severity and all were fully recovered with topical treatment.

| DISCUSS ION
Facial attractiveness has a significant impact on social interactions. Furthermore, the physical attractiveness of a person has an important effect on the behavior and attitude and of people around us. 15 Additionally, current evidence suggests that aging, particularly facialaging, has negative consequences on perceived attractiveness. 16,17 Aesthetic procedures, particularly minimally-invasive procedures, have become increasingly popular over the last decade. 9 Despite the COVID-19 pandemic impacting negatively on different aspects of our lives, compared to 2016, the number of nonsurgical aesthetic procedures increased by 22.7% in 2020. 9 This has been  [18][19][20][21] Since skin aging is a multifactorial process, with different underlying causes, different products are needed to address all contributing factors. 22 The current study aimed to assess the aesthetic and safety outcomes of a new and innovative dermal filler that combines HA (20 mg/mL) and CaHa 55.7% (microspheres 25-45 μm).
HA-based injectable fillers are currently considered the goldstandard for volumization procedures in facial rejuvenation, with CaHa as the second most used filler. 9,23 While the effect of HA fillers is based on their physicochemical properties (HA is one of the most hygroscopic molecules in nature 11 ), which results in volumization of the different facial regions where the filler is deposited; the effect of CaHA is mainly mediated by neocollagenesis, inducing indirect volumization, tissue-lifting, and skintightening. 23 However, CaHA-treated areas may sometimes undergo unexpected early volume loss due to rapid absorption of the carboxymethylcellulose gel carrier before the CaHA particle-induced neocollagenesis has taken effect. 24 The rational of combining HA with CaHa assumes that a high elastic modulus (G') and high viscosity HA filler provides early and sustained volumization, while waiting for the CaHA particle-induced neocollagenesis process. Regarding the lifting and tightening effect of the skin in the treated area, Vectra H2 scan images showed a significant increase in the FTV of approximately 2 mm, which was maintained throughout the study's follow-up.
One of the major concerns when administering a premixed combination of HA and CaHa, mainly in those cases in which the mixture has not been carried out in perfect and standardized manufacturing conditions, is that there may be changes or alterations in the rheological properties of the products.
Over the last several years, ultrasound examinations have become increasingly popular in dermatology. They allow high-resolution visualization of the skin layers with sufficient depth to capture the full thickness of the skin. 26 Besides the diagnosis of different dermatology diseases, it has demonstrated its usefulness for planning and control of aesthetic procedures and in the treatment of complications. [27][28][29][30][31] Moreover, according to a review published recently, ultrasound provides objective data about the role of the different tissues (skin, adipose tissue, muscles, etc.) in the age-related changes in the face. 31 Evaluating the ultrasound images of our study, immediately after treatment, in addition to the globular and poorly defined pattern (characteristic of the HA filler), it was possible to observe an incipient pattern of "coarse snow grain" (characteristic of the CaHa filler). 29,30 A total tissue integration of HA was evident at Day 30, while observing a welldefined "coarse snow grain" pattern (corresponding to the typical CaHa pattern). At Month 6, in addition to total integration of the HA filler and the well-defined "coarse snow grain" pattern, the presence of numerous bands of ordered and structured collagen fibers was observed.
These findings clearly showed that the physical and rheological properties of the premixed and standardized HA/HaCa formula are similar to those of its individually administered products.
The beneficial effects of administering HA and CaHA fillers, due mainly to their complementary mechanisms, have been suggested by different publications. [22][23][24]32,33 However, it is almost impossible to compare our results with theirs, since there are insurmountable differences in the treated areas, products used, and methods for assessing outcomes.
It should be considered that volumization of the treated area is very important in "full face" strategies, due mainly to its aesthetic F I G U R E 7 Ultrasound images throughout study follow-up. Ultrasound images corresponded to preauricular region. 1: Epidermis; 2: Dermis; 3: Cellular subcutaneous tissue; 4: Collagen fibrillar area; 5: Edema/inflammation; 6: "coarse snow grain" incipient pattern; 7: Anechoic/hypoechoic areas; 8: Neocollagen; 9: "coarse snow grain" pattern. effects go beyond the temple. 34 Indeed, increasing the volume of the posterior superior temple was associated with a volume reduction of the midface and accentuates the jawline contour. 35 In addition to the subjective aesthetic improvements, which are not trivial, having measurement instruments that provide objective information on the properties of the skin may be crucial for the assessment of the aesthetic outcomes.
Elastography, first implemented in clinical practice in the 1990s, 36 has emerged as a candidate for objectively assessing facial skin conditions. It measures the deformability of tissues caused by an external force, typically compression with an ultrasonographic transducer, or the velocity of shear wave propagation within the tissue. [37][38][39] Although there is evidence showing that elastography is a reliable method for the evaluation of facial skin elasticity, 38,39 to our knowledge this is the first study that has used elastography to objectively quantify the results of a dermal filler.
In our study, elastography images have shown an increase in the viscoelasticity, both at the level of the reticular dermis and the subcutaneous cellular tissue, which might be indicative of the formation of new collagen fibers. The increase in collagen fibers begins to be observed 60 days after treatment, although it has reached its peak between Days 90 and 180 post-treatment.
Regarding safety, there were no either unexpected or serious treatment-related adverse events. All the reported adverse events were mild in severity and all were fully recovered with topical treatment. The most reported adverse event was a mild redness and inflammation, which occurred in most patients, but was resolved without treatment within the first 48 h.
This study has several limitations that should be addressed. The first one is its limited sample size. Nevertheless, according to the results of the study the power for detecting volume and FTV changes, between pretreatment and Day 180, was 99% each, respectively.
The second limitation is its limited follow-up time. Finally, other limitation was the lack of a control group. It would have been interesting to conduct a direct comparison, preferable randomized clinical trial, between HA/CaHa and other fillers.

F I G U R E 8
Ultrasound and elastography images performed before treatment and 60, 90, and 180 days after treatment of a 62-year-old woman who underwent treatment with HArmonyCA 1.25 cc. In the pretreatment images, both the ultrasound (1) and the elastography (2) show a paucity of hyperechoic areas suggestive of the absence of collagen and elastin fibers. At Day 60, a significant increase in the number of hyperechoic images suggestive of an increase in tissue density at the level of the reticular dermis and subcutaneous cell tissue (3) is observed. These hyperechoic images on ultrasound correspond to images of increased density, viscoelasticity, and firmness of the tissue on elastography, (2) suggestive of neocollagenesis in the treated areas. On Day 90, both in the ultrasound and elastography images, the increase in the density, viscoelasticity and firmness of the tissue was confirmed. At 180 days after treatment, ultrasound and elastography images confirmed the increase in collagen in the treated areas. 1: Hypo-anechoic images; 2: Collagen fibers; 3: Hyperechoic images.
As strengths of this study, it is worth highlighting the use of an objective measurement method, such as elastography, to evaluate the treatment outcomes on the skin; as well as the use of ultrasound to analyze the tissue integration of the product.

ACK N OWLED G M ENTS
Medical writing and editorial assistant services have been provided by Ciencia y Deporte S.L.

FU N D I N G I N FO R M ATI O N
The study finally did not receive any funding.

F I G U R E 9
Ultrasound and elastography images (A) and 2D photographs (B) taken before treatment (A1) and 180 days after treatment (A2) of a 62-year-old woman who underwent treatment with HArmonyCA 1.25 cc. (A1) Before treatment: Heterogeneous ultrasound pattern with greater presence of hypoechoic and anechoic images (1) and scarcity of collagen fibers in elastography (2) This image corresponds to microphotographic image B1. (A2) Day 180 after treatment: Heterogeneous ultrasound pattern with a predominance of hyperechoic images (4) and increased areas of greater density, firmness, and viscoelasticity on elastography (2), suggestive of neocollagenization and increased tissue collagen density. This image corresponds to microphotographic image B2 in which we see an increase in the quality of the skin (5). 1: Hypo-anechoic images; 2: Collagen fibers; 3: Skin not very smooth; 4: Hyperechoic images; 5: Increase in skin quality.

CO N FLI C T O F I NTER E S T S TATEM ENT
Dr. Urdiales-Gálvez has received grants from Allergan Aesthetics, an AbbVie company and from ALMA Laser, for covering medical writing services. Dr. Urdiales-Gálvez has received payments for lectures from AbbVie and ALMA Laser. Dr. André Braz has received payments as international medical consultant for Allergan Aesthetics, an AbbVie Company. Dr. Mauricio Cavallini has received payments as international medical consultant for Allergan Aesthetics, an AbbVie Company. None of the authors have received any payment for the current paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

CO M M ERCI A L I NTER E S TS
None.

I N FO R M ED CO N S ENT
All patients were fully informed about the details of the study protocol. The need of informed consent was waived for this study.