Introduction

Fillers play a prominent role in clinical practice in the correction of signs of ageing of the skin and subcutaneous tissue. Among the temporary fillers, those based on hyaluronic acid (HAF) are the most popular, whereas collagen was used more frequently in the past. HAFs typically consist of chemically cross-linked HA molecules with different molecular weights, different HA concentrations and different degrees of cross-linking. The resulting hydrogels are enzymatically more stable against hyaluronidase and have optimised rheological qualities compared to non-crosslinked HA [1,2]. HA fillers are apparently able to stimulate stem cells of the subcutaneous adipose tissue, which may contribute to the duration of the volume build-up effect [3].

In addition, biostimulatory, semi-permanent fillers such as poly-L-lactic acid (PLLA) or calcium hydroxyapatite (CaHA) are available, whose duration of action exceeds that of pure HA fillers. CaHA is a bioceramic active ingredient with very good tissue compatibility. Radiesse® (Merz Pharmaceuticals GmbH, Frankfurt/M.) is a CaHA filler in which 20-45 µm round, smooth microspheres of CaHA are incorporated into a carboxymethyl cellulose gel. After implantation, a tissue reaction occurs with increased formation of collagen I and III, elastin and proteoglycans. CaHA does not trigger a chronic inflammatory reaction [4]. However, CaHA depots show a temporary reduction in volume as a result of the degradation of the cellulose matrix. This loss is compensated with a time delay by the formation of new connective tissue. In addition, tissue tightening occurs [5].

PLLA is a biocompatible, biodegradable synthetic polymer. PLLA microparticles lead to a subclinical inflammatory reaction in the body, which stimulates the formation of new collagen.

PLLA is supplied as a lyophilised powder for volume build-up. The filler is reconstituted with sterile water or physiological saline solution. After an appropriate hydration time, an injectable suspension is obtained. The injection treatment should be carried out several times (usually 2 to 3 times) in order to achieve an optimal result.

Artefill® (Suneva Medical Inc., San Diego, CA, USA) consists of microparticles between 40 µm and 63 µm, carboxymethylcellulose and non-pyrogenic mannitol [6]. Sculptra® Aesthetics (Galderma, Dallas, TX, USA) consists of 10-200 µm PLLA microparticles of different morphologies. The phagocytic content (<20 µm) is 46.3 % ± 11.3 % [7]. Clinical results with PLLA fillers indicate that in up to 80% of patients the volume effect is still fully maintained after 24 months [8].

For optimal results, the sequential application of different fillers was propagated [9, 10].

Hybrid fillers are a new development in the filler sector. The dictionary defines the term hybrid as “composed of different things, of two different origins”. Hybrid fillers based on HA currently exist, which combine low and high molecular weight HA, as well as fillers based on HA and CaHA. New developments are on the horizon.

Hybrid filler made from low and high molecular weight HA

Hybrid complexes of low and high molecular weight hyaluronic acid (HA) promote adipogenic differentiation and proliferation of somatic stem cells of the subcutaneous adipose tissue more strongly than cross-linked HA or linear low or high molecular weight HA [11].

This principle is followed by the filler Profhilo® (IBSA Farmaceutici Italia srl, Italy), which consists of stable, hybrid and co-operative HA complexes (HyCoCos). The thermal NAHYCO® hybrid technology is used for this purpose. The HA content is 32 mg/mL. High-molecular HA (1,100-1,400 kDa) and low-molecular HA (80-100 kDa) are each contained at 32 mg in 2 mL in a buffered sodium chloride solution. The filler is characterised by very good biocompatibility and low viscosity. The Profhilo Structura® filler has a higher HA concentration of 45 mg/mL, half high molecular weight (1400 ± 200 kDa) and half low molecular weight (100 ± 20 kDa) HA. The enzymatic cleavage and the stability of the filler implants in the tissue (200 µl) were investigated in vitro and in the murine animal model. Profhilo® was detectable in the subcutaneous tissue over 10 weeks using high-resolution ultrasound, Profhilo Structura® up to week 27 [12].

Both fillers were analysed in vitro with regard to enzymatic resistance. For this purpose, 1 g of the respective filler was mixed with 20 mL of a hyaluronidase solution (8 U/mL in PBS) at 37°C in a stirrer. Bovine hyaluronidase (type I-S, Sigma-Aldrich) was used. Profhilo Structura® was completely cleaved within 3 h [12]. This property is of particular relevance in the event of accidental intravascular injection. Here, the time to complete dissolution of the filler is critical to avoid permanent tissue damage [13].

An open prospective monocentric study included 64 women aged 30-60 years (mean 53 years). Two therapy sessions were carried out 4 weeks apart for facial rejuvenation. The follow-up lasted 16 weeks. The following methods were used to quantify the success of the treatment: Wrinkle Severity Rating Scale (WSRS) – a 5-point scale from 1 (no wrinkles) to 5 (very severe wrinkles); Facial Volume Loss Scale (FVLS), – a 5-point scale, and the Beagley and Gibson Scale (BGS), a 4-point scale to assess the evenness of the skin’s microrelief.

Furthermore, objective measurement methods were used such as optical colourimetry (Chromameter CR-200 Minolta, Osaka, Japan), corneometry (Corneometer CM825, Courage-Khazaka, Cologne, Germany), skin hydrometry (MoistureMeterD, Delfin Technologies, Kuopio, Finland), torsiometry (Dermal Torque Meter, Dia-Stron Ltd, Andover, UK), profilometry of nasolabial folds and marionette lines (Primos Software) and 3D photo documentation (Vectra H1, Canfield, Parsippany, NJ, USA). Based on this, the facial volume was also analysed before treatment, at weeks 8 and 16.

The BGS improved by 12.9%, the FVLS decreased by 18.2% and the WSRS by 14.7% by week 16. Hydration and surface relief improved, wrinkle depth decreased and facial volume increased slightly.

Side effects were reported by 9 participants (14%). These concerned slight haemorrhages at the injection site or slight oedema, which disappeared between 3 and 10 days after injection [14].

Profhilo^® Structura was used to build up the midface in a retrospective half-side study of 22 patients aged 36-60 years (mean 53 years). Two injections of 2 mL of the filler were administered. The follow-up period was 6 months. The filler showed pseudoplastic properties immediately after injection and was integrated into the subcutaneous fatty tissue. The volume increase was stable over 6 months. Patient satisfaction was high. No side effects were observed [15].

In a post-marketing study, > 40,000 patients who had been treated with Profhilo® were analysed. In the years 2015-2018, 12 adverse reactions were reported worldwide. Early reactions at the injection site were swelling, oedema, erythema, ecchymosis. Local late reactions (< 72 h) involved swelling and nodules, which indicates a very good safety profile [16].

In an open study, 10 patients with skin laxity on the neck (≥ type 3 according to the Glogau Wrinkle Scale; GAIS score) aged 35 to 65 were included. Two therapy sessions were carried out. In the first session, plasma ablation was combined with an HA injection. After 30 days, the second treatment was performed with HA alone. After a further 30 days, the success of the treatment was assessed. A GAIS score of 1 or 2 was found in 90% of cases (patient view and investigator). Pain during treatment was reported as a median of 2.4 out of 10 on the VAS scale. Minimal side effects such as erythema and oedema were only temporary. There were no serious side effects [17]. Table 1 provides an overview of possible indications [18].

Hybrid filler made from HA and CaHA

HArmonyCa^® (Allergan Aesthetics, an AbbVie Company) is a hybrid filler containing hyaluronic acid HA (20 mg/mL) and calcium hydroxyapatite (CaHA; 55.7%; microspheres with a diameter of 25-45 µm) as well as 0.3% lidocaine in a 1.25-ml prefilled syringe.

In a monocentric, prospective split-face study, 14 female patients and one male patient aged between 32 and 63 years were treated for cheek ptosis. The Fitzpatrick skin phototype was between II and IV. The Physician Global Aesthetic Improvement Scale, the Subject Global Aesthetic Improvement Scale scores and a validated 5-point scale for the lower third of the face were used to quantify treatment success [19]. Patients with “mild sagging” and “moderate sagging” were included.

The injection was made via two points in a retrograde linear fan style. The first injection was placed subzygomatically 5 cm below the lateral orbital rim, the second above the mandible on the anterior edge of the masseter muscle. One prefilled syringe of 1.25 ml was used on each side of the face. The patients were followed up for 120 days. Photo documentation was carried out using the Vectra 3D Imaging System from Canfield.

At the end of the follow-up period, all participants showed an improvement in the Physician Global Aesthetic Improvement Scale, 40% of whom had above-average scores. The Subject Global Aesthetic Improvement Scale scores were very significantly improved in 14 of 15 patients, 1 x significantly improved. The skin thickness had increased on average from 1.47 ± 0.08 mm to 1.68 ± 0.08 mm after 120 days. Tolerance was very good. Pain, mild oedema and ecchymosis were observed in some cases during and immediately after injection. One patient developed a small papule on the lower half of the face. There were no serious side effects [20].

A prospective intervention study on facial rejuvenation with 15 patients used the hybrid filler in the preauricular region. 1.25 ml per side was injected retrogradely. The participants underwent a sonographic examination, elastography and photographs using 2D and 3D techniques. Follow-up was performed for 180 days after treatment.

The median volume had increased by 2.1 cm per side³ . The tension vectors increased by 2.2 mm and 2.0 mm respectively. The elastography measurements indicated an increase in collagen fibres from day 60 onwards. Minor redness and inflammation were observed as side effects, which disappeared again within 48 hours. Severe side effects did not occur [21].

In a retrospective study, 243 patients who had received at least one treatment with HArmonyCa^®  and were followed up for at least 12 months (median 15.4 months) were analysed: Of these, 94.0% were women with a median age of 50.1 years. The predominant Fitzpatrick skin phototypes were II and III with 80.1%. An average of 2.2 mL (0.5-8.9 mL) of filler was injected per treatment session. The cheeks (71.2%) and the mandibular region (69.7%) were favoured for treatment. 11 patients (12 treatments) experienced adverse events as a result of the hybrid filler, most of which were mild. Specifically, these were tissue hardening (3, 0.7%), oedema (3, 0.7%) and non-inflammatory lump formation at the injection site (5, 1.2%). In the long-term follow-up, there were 15 (6.2%) patients with 16 documented adverse reactions (6 x oedema, 5 x injection site nodules, 1 x inflammation, 3x skin induration, 1 x hypersensitivity reaction). No serious side effects were observed [22]. Table 2 provides information on possible indications [20-25].

The combination of (CaHA) and (HAF) filler in one cannula represents a hybrid filler. Radiesse^® and Belotero^® (Merz Pharmaceuticals GmbH, Frankfurt/M.) were used in 2112 patients with this concept. The follow-up was at least 12 months. Various mixing ratios were used. Only 5 minor side effects (0.24%) were registered. These were non-inflammatory nodules (n = 4) and one transient oedema. Two of the nodules disappeared completely after hyaluronidase injection [26].

In a prospective monocentric study, 25 patients were included who achieved a score of 1-2 on the Merz 5-point scale for the nasolabial fold and mandibular contour. A mixture of 1 mL HAF, 0.5 mL lidocaine and 1.5 mL CaHA was used. A total of 3 mL of filler was injected. The results were recorded using the visual analogue scale (VAS) and the 5-point Global Satisfaction Scale (GSS) from the examiner’s and patient’s perspective. In some of the patients, an additional 0.1 mL of CaHA or the CaHA/HAF mixture was injected retroauricularly and removed after 6 months for histological examination. The median VAS and GSS scores improved. Histological examination showed increased collagen without inflammation. Patient satisfaction was high. The CaHA/HAF mixture compensated for the temporary early volume loss of the CaHA filler [27].

A retrospective evaluation of CaH/HAF therapy for the middle and lower third of the face utilised the Merz Aesthetics Scale for the Jawline^® (MASJ). Forty-one patients were included. The median age was 47.5 years (21-63 years). The median MASJ score improved from 2.12 before therapy to 0.68 after 3 months and 1.27 after 12 months. All participants experienced a ≥1 score point improvement after 3 months compared to 85% after 12 months. No undesirable side effects were observed [28]. The combination with botulinum toxin A (incobotulinum toxin A; Xeomin^® ; Merz) in a syringe is known as “The gold protocol” and has been propagated for the rejuvenation of the soft tissues of the neck [29].

In a double-blind prospective study, CaH/HAF was used in a 1:1 ratio with CaH alone for rejuvenation of the back of the hand. The results were assessed using the Global Aesthetic Improvement Scale (GAIS) and the Manchester Hand Grading System (MHGS) and objectively recorded using cutometry, corneometry and ultrasound. GAIS and MHGS did not differ significantly in both groups. Hydration, skin elasticity and skin thickness improved in both treatment arms. Higher echogenicity was found in the hybrid arm, which was interpreted as increased tissue biostimulation. No side effects were observed, so that the methods were classified as safe [30].

Stimulate^® (MatexLab SA, Lugano, Switzerland) contains 26 mg/mL HA, 1% CaHA and the amino acids L-proline and glycine. Polyethylene glycol serves as the cross-linking molecule, which is why it is also referred to as pegylated HA. Stimulate^® One has 28 mg/mL HA. The average half-life of the filler after implantation is given as 6 months.

In vitro studies showed no evidence of cytotoxicity (HaCaT keratinocytes), changes in cell morphology and viability and intracellular F-actin microfilaments

[31]. The filler stimulates collagen production in vitro in fibroblast culture and in vivo in human skin [32, 33]. The filler has an anti-inflammatory effect on neutrophil granulocytes [34].

In a prospective, monocentric real-life study, 70 patients were included, 60 of whom completed the study after 6 months. The median age was 59 years (range: 26-70 years). The filler was injected once with a maximum of 1 mL per side to fill volume deficits in the cheek region. The following measurements were taken: The 6-point Mid-Face Volume Deficit Scale (MFVDS), the Global Aesthetic Improvement Scale (GAIS) and the Patient Satisfaction Score (PSS) on a 10-point VAS scale. Over 80% of the patients achieved 3 score points in the MFVDS before the start of treatment, the remaining patients had a higher volume average. Immediately before and after treatment, the MFVDS averaged 3.20 and 1.77. After 3 and 6 months, the values were 1.40 and 2.62. The GAIS averaged 2.27 before treatment and improved to 1.72 and 1.95 in months 1 and 2. The VAS values for patients and investors were in the same range between 93% (3 months) and 100% (immediately after injection). They fell to around 70% at month 6. No severe side effects were observed [35].

Table 3 provides an overview of possible indications.

Poly-L-lactic acid and hyaluronic acid

Poly-L-lactic acid (PLLA) is a biostimulatory filler and is considered the first long-acting collagen stimulator [36].

A hybrid filler consisting of HA, PLLA and polynucleotides (PN) was presented by Oh et al. (2021). PN is considered a growth factor for fibroblasts and osteoblasts. The PLLA microspheres in this filler are smaller and rounder than in the classic PLLA fillers. HA-PN/PLLA has a higher viscosity and elasticity (G’) with comparable osmolality to HA and PN fillers. In animal experiments, this new filler showed persistence in the tissue for 24 weeks [37].

The filler Reversal^® PLA+HA (Koru Pharma; Seoul, South Korea) contains 30 mg HA and 170 mg poly-D, L-lactic acid per 200 mg vial. The viscosity is stated as 44.6 Dl/g. Juvelook^® and Juvelook^® Volume are also HA hybrid fillers with poly-D, L-lactic acid (LV Plastic Surgery, Seoul, South Korea). Juvelook^® Volume contains 170 mg of polylactic acid microparticles with a diameter of 50 µm to 60 µm and 30 mg of non-crosslinked HA. AestheFill^® (Regen Bio Global Inc., Seoul, South Korea) – a hybrid filler of poly-L, D-lactic acid and HA was tested in a multicentre controlled study against HA in the correction of nasolabial folds. A total of 260 patients were included. After a single injection, follow-up was carried out over 52 weeks. The authors used the Wrinkle Severity Rating Scale (WSRS) and the Global Aesthetic Improvement Scale (GAIS) to quantify the treatment results.

At week 24, 67.6% of patients in the AestheFill^® group and 60.9% in the HA group showed an improvement in WSRS of at least one degree. The hybrid filler was superior to the HA filler at all time points. There were no serious side effects [33]. For possible indications, see Table 4, where different dilutions of the lyophilisate are used [38, 39].

PLLA in an HA suspension (PLLA-b-PEG) utilised round, smooth PLLA microspheres (diameter 20 µm – 45 μm). In animal experiments, the volume increased in the first 4 weeks after implantation of the filler and then gradually decreased. Fibrous tissue, vascular density and collagen fibre formation increased over 26 weeks after injection. Studies of patients seeking facial contouring showed significant improvements in the Global Aesthetic Improvement Scale 3 and 12 months after injection. Even after 12 months, 90% of patients still showed good contouring with minimal side effects [40, 41].

New hybrid fillers

A hybrid hydrogel of HA and pullulan was analysed in vitro. Pullulan is a natural, water-soluble linear polysaccharide consisting of maltotriose units. Biomimetic hydroxyapatite spheres were incorporated into this gel. Silane couplers were used to improve the physicochemical, mechanical and biological properties. This hybrid gel was tested in body fluids. The hydroxyapatite spheres showed a uniform distribution in the gel. The elastic modulus G’, viscosity, resistance and stability could be improved by the spheres. The gel showed a high resistance to enzymatic biodegradation. In cell cultures with L-929 fibroblasts, their adhesion and distribution improved, especially on the spheres. Cell viability was increased [42].

Another new approach is the use of lactose-modified chitosan (CTL; CHITLAC^® ) to save crosslinker molecules – HA-CTL filler. CTL can be enzymatically degraded completely by lysozyme and partially by hyaluronidase. HA was crosslinked with different amounts of 1,4-butanediol diglycidyl ether (BDDE) (degree of modification 3.5% – 8.8%) and CTL was added: 20-25 mg/mL and 5 mg/mL for HA and CTL, respectively. Due to its anionic nature, CTL is able to interact with the cationic HA. This increases the elastic modulus G’. The loss factor tan δ (quotient of G”/G’) decreases. This type of filler appears to be more resistant to shear forces and pressure. By varying the HA modification (MoD%) with BDDE, higher-molecular HA and increasing the HA concentration from 20 to 25 mg/mL, the elasticity performance could be increased to 94%. For the practical use of the filler, however, it must also be noted that it must remain injectable. Therefore, a product with a slightly lower MoD% is preferable for clinical use. Cohesiveness, hydrolysis resistance and swelling of HA-CTL were comparable with commercial HA fillers. The HA-CTL filler with the lowest degree of cross-linking showed a swelling of 89%, which was significantly lower than that of commercial fillers (100-300). This enables more precise work, e.g. on the lips.

HA-CTL fillers can be adapted to specific requirements depending on the indication by changing the degree of cross-linking in the viscosity and cohesiveness. In the in vitro tests, this filler group showed significantly higher values of the normal force F_N  under static pressure than commercial HA fillers, which indicates very good lifting properties [43].

Summary and outlook

Hybrid fillers expand the armamentarium of minimally invasive options for aesthetic medicine. Rheological properties, biostimulatory effects and safety profiles can be optimised in this way. At the same time, possible disadvantages of existing filler products are overcome or reduced. The application possibilities are extended. The safety profile is very good.