Abstract
Cutaneous aging is characterized by gradual structural and functional alterations, including collagen breakdown and reduced elasticity. In recent years, several energy-based modalities have been introduced to address these changes. The aim of this study was to assess the effectiveness of a 675 nm laser and microfocused ultrasound (MFU), applied individually or in combination, for the treatment of facial aging. This retrospective analysis included 115 patients, allocated into three groups: Group A (675 nm laser), Group B (MFU), and Group C (combined protocol). Efficacy was evaluated through the Fitzpatrick Wrinkle Scale (FWS), the Baker Gravitational Ptosis Classification (BGP), and standardized photographic assessments at 1, 3, and 6 months following treatment. At the 6-month evaluation, individuals treated with the combined protocol exhibited a more pronounced improvement in wrinkle reduction (FWS: from 1.88 to 1.13) and laxity reduction (BGP: from 2.78 to 1.55) compared with either single-treatment group. No major side effects were observed. The combined application of the 675 nm laser and MFU appears to potentiate collagen remodeling and skin tightening more effectively than monotherapy. Further prospective studies are needed to validate these results and further clarify the molecular pathways involved.
Keywords: 675 nm laser, High frequency ultrasound, Facial aging, Collagen stimulation
Introduction
Humans have always sought to counteract the effects of aging, particularly its visible signs on the body. In recent decades in Western countries the pursuit of anti-aging treatments has intensified, largely due to the visual-centric nature of social media, where aesthetics play a crucial role. Moreover, in the post-pandemic era, with increased reliance on virtual communication for work and social interactions, individuals have become more conscious of facial aging, given its constant exposure in digital media. The progressive reduction of the regenerative capacity of the tissues is a determining cause of ageing. Facial aging involves a progressive involution of all its structural components, including the epidermis, dermis, hypodermis, muscles, ligaments, and bones [1–4]. In the hypodermis, the primary consequence of skin aging is the reduction in adipocyte volume and overall adipose tissue, leading to thinning. The epidermis, the outermost layer of the skin, visibly manifests aging-related changes, although structural alterations in the dermis contribute more significantly to skin aging. Over the years there is a decrease in the proliferation capacity of cells. The result is a thinner and less elastic epidermis.
The stratum corneum and the epidermis in general are thus gradually increasingly dry and dehydrated and wrinkles become more evident. The effects of skin aging are more evident and significant in the dermis, because most of the fibroblasts, the cells that have the task of producing collagen and elastin, are found here [5–7].
Over time, the number of fibroblasts declines, leading to reduced production of collagen (mainly type I and III) and elastin, the key proteins responsible for skin firmness and elasticity. At the molecular level, aging skin exhibits increased oxidative stress and chronic low-grade inflammation (a phenomenon known as ‘inflammaging’), which disrupts normal tissue homeostasis. Key signaling pathways, including the transforming growth factor-beta (TGF-β) pathway—crucial for collagen synthesis—undergo significant alterations. Additionally, matrix metalloproteinases (MMPs), particularly MMP-1 and MMP-9, become overexpressed, leading to accelerated degradation of extracellular matrix components, further contributing to dermal atrophy and loss of skin elasticity [8–10]. Furthermore, the degradation rate of pre-existing elastic and collagen fibers accelerates, leading to structural weakening of the skin. This results in increased sagging and the formation of wrinkles and laxity-prone areas.
Microfocused ultrasound (MFU) technology, though similar to that used in medical imaging, differs in its highly convergent nature and specific energy frequencies.
By employing different transducers, energy can be precisely delivered to the superficial and deep dermis, as well as the platysmal layer. MFU heat the tissues to a temperature above 60 °C producing a small thermal coagulation [11–14].
The 675 nm laser (Redtouch, DEKA M.E.L.A. srl, Calenzano, Italy) is a non-ablative laser, which is based on the emission of a red light with a 675 nm wavelength.
Through a 15 × 15 mm scanning system capable of generating selective thermal damage on the skin with an average depth of 300 microns, this device may easily target the dermis. At this level, heat is transferred directly to the collagen fibers without affecting other chromophores. The laser-induced thermal effect promotes collagen denaturation and subsequent neosynthesis, facilitating extracellular matrix remodeling and the reorganization of elastic fibers. A built-in 5 °C contact cooling system protects the epidermis from heat-induced damage [15–21].
Combining these 2 devices may be useful to achieve a better stimulation of both components. Recent studies indicate that the 675 nm laser primarily stimulates fibroblast activity, enhancing the synthesis of collagen types I and III. Additionally, it modulates the expression of heat-shock proteins, particularly HSP47, which is essential for correct collagen folding and stabilization [17, 21]. Microfocused ultrasound (MFU) generates precise thermal coagulation points in the dermis, triggering neocollagenesis and elastogenesis. This occurs through the activation of heat-responsive genes and growth factors, particularly fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) [12, 22]. Given the complementary mechanisms of action of the 675 nm laser—targeting fibroblast stimulation and collagen remodeling—and MFU—inducing controlled thermal coagulation and neocollagenesis—their combined application may enhance clinical outcomes by addressing multiple aspects of skin aging simultaneously.
Materials and methods
115 patients showing signs of aging and with II/III level photoaging according to Rubin were retrospectively enrolled, at Skin Center Avezzano-Pescara, at Villa Donatello Clinic of Florence and at Magna Graecia University of Catanzaro. Informed consent on the procedure’s risks was obtained from all patients. These patients were divided into three groups according to the treatment performed:
Group A included patients treated for facial aging only with 675 nm laser for three sessions with standard settings (power: 10 W, dwell time: 300–400 ms, spacing: 1–1.5 mm, cooling: 5 °C).
Group B included patients treated for facial aging only with MFU, performing one treatment only with standard settings (the choice of transducers to use was guided by ultrasound visualization. The standard number of lines delivered was selected according to the indications of the machine for each district).
Group C included patients treated for facial aging that underwent a single session of MFU, followed by 3 sessions of 675 nm laser standard settings.
Exclusion criteria included: the presence of pacemakers or metal implants; ongoing systemic anticoagulant therapy; disorders affecting scarring, coagulation, or vascular function; previous aesthetic treatments in the last six months; pregnancy; hypersensitivity to red and near-infrared light; medications that increase photosensitivity; immunosuppressive therapy; seizure disorders triggered by light; personal or family history of skin cancer; prolonged sun exposure in the three weeks prior to treatment, and the presence of tattoos or dermatological conditions in the treatment area.
To further evaluate the biological response induced by each treatment, we analyzed key molecular markers associated with skin rejuvenation. Specifically, we investigated changes in the expression of procollagen type I (COL1A1), elastin (ELN), and matrix metalloproteinase-1 (MMP-1) via immunohistochemical staining in biopsy samples taken from a subset of patients. These markers provide insight into the remodeling of the extracellular matrix and the overall efficacy of the treatments at the molecular level [23, 24].
Treatment outcomes were assessed through digital clinical images taken at baseline and at 1, 3, and 6 months post-treatment, and a clinician-modified Fitzpatrick Wrinkle Scale (0 no wrinkle,; 0.5 very superficial but visible wrinkle; 1 fine wrinkle; 1.5 wrinkles visible up to 1 mm deep; 2 visible wrinkles 1 to 2 mm deep; 2.5 wrinkles 2 to 3 mm deep; 3 deep wrinkles >3 mm deep) [24–26], Baker Gravitational Ptosis Classification [27, 28], and a 10 point Visual Analogue Scale to assess pain during procedures [29–31]. SPSS 26.0 (IBM Corp., New York, NY, USA) was used to perform statistical analysis, such as means, standard deviations and unpaired Student’s t-test.
Results
115 patients were enrolled in the study, 105 females (age 35 to 65, mean 49 y.o.) and 10 males (age 40 to 63, mean age 52 y.o.). All patients had a Fitzpatrick phototype ranging from type 2 to type 4. Group A included 35 patients (32 females, 3 males); Group B included 40 patients (37 females, 3 males); Group C included 40 patients (36 females, 4 males).
Group A (675 nm laser)
6 months after the last treatment, the 35 patients treated with the 675 nm laser only had a visible improvement according to the FWS, BGP and the photographic evaluation.
Scores significantly decreased from a baseline of 1.96 on the FWS scale to 1.73 at the 3-month follow-up and further to 1.43 at the last follow-up.
Laxity measured with the BGP scale decreased from baseline 2.6 to 2.45 at control 6 months after treatment.
Pain, assessed using the VAS, was minimal (VAS score: 1.17) for most subjects, who reported good satisfaction.
A slight erythema was observed in 25% of subjects (9/35), which appeared at the end of treatment and resolved spontaneously within approximately 2 h (Table 1; Fig. 1).
Table 1.
Group A (675 nm laser) patients’ characteristics
| Patient Id | Sex | Age | Side effect | VAS | FWS | FWS (3 months) | FWS (6 months) | BGP | BGP(6 months) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | F | 35 | None | 0 | 0.5 | 0.5 | 0.5 | 1 | 1 |
| 2 | F | 35 | None | 1 | 0.5 | 0.5 | 0.5 | 1 | 1 |
| 3 | F | 36 | None | 1 | 1 | 1 | 0.5 | 1 | 1 |
| 4 | F | 37 | None | 1 | 1 | 1 | 1 | 1 | 1 |
| 5 | F | 37 | None | 2 | 1 | 1 | 1 | 1 | 1 |
| 6 | F | 38 | None | 2 | 1 | 1 | 1 | 1 | 1 |
| 7 | F | 40 | None | 1 | 1 | 0.5 | 0.5 | 2 | 2 |
| 8 | F | 40 | None | 3 | 2.5 | 2 | 2 | 3 | 3 |
| 9 | F | 42 | Mild Erythema | 1 | 1.5 | 1.5 | 1.5 | 2 | 2 |
| 10 | F | 42 | None | 1 | 1 | 1 | 1 | 2 | 1 |
| 11 | F | 43 | None | 1 | 1 | 1 | 1 | 2 | 2 |
| 12 | F | 44 | None | 0 | 1 | 1 | 1 | 2 | 2 |
| 13 | F | 44 | Mild Erythema | 0 | 1.5 | 1.5 | 1 | 2 | 2 |
| 14 | F | 45 | None | 3 | 2.5 | 2.5 | 2.5 | 2 | 2 |
| 15 | F | 46 | None | 1 | 2 | 2 | 2 | 3 | 3 |
| 16 | F | 49 | None | 2 | 2.5 | 2 | 1.5 | 3 | 3 |
| 17 | F | 49 | None | 1 | 3 | 2.5 | 1.5 | 3 | 3 |
| 18 | M | 50 | Mild Erythema | 1 | 2.5 | 2 | 1 | 2 | 1 |
| 19 | F | 51 | None | 1 | 2 | 2 | 1 | 3 | 3 |
| 20 | F | 51 | None | 0 | 2 | 2 | 1.5 | 3 | 3 |
| 21 | M | 52 | None | 3 | 2 | 1.5 | 1 | 3 | 3 |
| 22 | F | 53 | Mild Erythema | 2 | 1.5 | 1.5 | 1.5 | 3 | 3 |
| 23 | F | 54 | None | 1 | 2 | 1.5 | 1.5 | 3 | 2 |
| 24 | M | 54 | None | 1 | 2 | 2 | 1.5 | 3 | 3 |
| 25 | F | 55 | None | 0 | 2 | 2 | 2 | 3 | 3 |
| 26 | F | 56 | Mild Erythema | 0 | 2.5 | 2 | 2 | 4 | 4 |
| 27 | F | 57 | Mild Erythema | 1 | 2.5 | 2 | 2 | 3 | 3 |
| 28 | F | 58 | Mild Erythema | 1 | 2.5 | 1.5 | 2 | 3 | 2 |
| 29 | F | 59 | None | 2 | 2.5 | 1.5 | 1 | 4 | 3 |
| 30 | F | 60 | Mild Erythema | 2 | 3 | 2.5 | 1.5 | 4 | 4 |
| 31 | F | 61 | None | 1 | 3 | 2.5 | 2 | 3 | 3 |
| 32 | F | 62 | None | 1 | 3 | 3 | 2.5 | 3 | 3 |
| 33 | F | 63 | None | 1 | 3 | 2.5 | 1.5 | 4 | 4 |
| 34 | F | 64 | Mild Erythema | 1 | 3 | 2.5 | 2.5 | 4 | 4 |
| 35 | F | 64 | None | 1 | 3 | 3 | 2.5 | 4 | 4 |
Fig. 1.
Patient before (left) and 6 months after (right) 675 nm laser treatment
Group B (MFU)
In group B, consisting of 40 patients subjected to treatment with MFU (Ultherapy®), a significant improvement was observed in tissue laxity (BG scale), skin texture, and facial wrinkle reduction (FWS scale), going from an average of 1.95 on the FWS scale to 1.675 at the 3-month follow-up after the last treatment, up to the value of 1.3625 at the last checkup. Laxity measured with the BGP scale decreased from baseline 2.68 to 1.7 at control 6 months after treatment.
Pain, assessed using the VAS, was moderate (VAS score: 3.075) for most subjects, who reported good satisfaction. As side effects, we found a slight erythema in only 7.5% of the subjects (2/40), which appeared at the end of the treatment and resolved within approximately 3 h (Table 2; Fig. 2).
Table 2.
Group B MFU patients’ characteristics
| Patient ID | Sex | Age | Side effect | VAS | PWS | PWS (3 months) | PWS (6 months) | BCP | BCP (6 months) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | F | 37 | None | 5 | 0.5 | 0.5 | 0.5 | 1 | 0 |
| 2 | F | 37 | None | 3 | 0.5 | 0.5 | 0.5 | 1 | 0 |
| 3 | F | 38 | Mild Erythema | 6 | 0.5 | 0.5 | 0.5 | 1 | 0 |
| 4 | F | 39 | None | 4 | 0.5 | 0.5 | 0.5 | 1 | 1 |
| 5 | F | 39 | None | 4 | 1 | 0.5 | 0.5 | 1 | 0 |
| 6 | F | 40 | None | 3 | 1 | 0.5 | 0.5 | 1 | 1 |
| 7 | F | 41 | None | 3 | 1 | 0.5 | 0.5 | 1 | 1 |
| 8 | F | 42 | None | 3 | 1 | 1 | 0.5 | 2 | 1 |
| 9 | F | 42 | None | 3 | 1 | 1 | 0.5 | 2 | 1 |
| 10 | F | 44 | None | 4 | 1 | 1 | 0.5 | 2 | 1 |
| 11 | F | 45 | None | 2 | 1 | 1 | 0.5 | 2 | 1 |
| 12 | F | 46 | None | 2 | 1 | 1 | 1 | 2 | 1 |
| 13 | F | 46 | None | 2 | 1 | 1 | 1 | 2 | 1 |
| 14 | F | 48 | None | 3 | 1.5 | 1.5 | 1.5 | 2 | 1 |
| 15 | F | 49 | None | 2 | 2 | 1.5 | 1.5 | 2 | 1 |
| 16 | F | 49 | None | 3 | 2 | 2 | 1.5 | 2 | 1 |
| 17 | F | 49 | None | 3 | 2 | 2 | 1.5 | 3 | 2 |
| 18 | F | 49 | None | 3 | 2 | 1.5 | 1.5 | 2 | 1 |
| 19 | F | 50 | None | 3 | 1.5 | 1.5 | 1.5 | 1 | 2 |
| 20 | F | 51 | None | 4 | 2.5 | 2 | 1.5 | 3 | 2 |
| 21 | F | 52 | None | 2 | 2.5 | 2 | 1.5 | 3 | 2 |
| 22 | F | 52 | None | 2 | 2 | 2 | 1.5 | 3 | 3 |
| 23 | F | 52 | None | 3 | 2 | 1.5 | 1.5 | 3 | 2 |
| 24 | F | 53 | None | 3 | 2.5 | 2 | 1.5 | 3 | 2 |
| 25 | F | 55 | None | 3 | 2 | 1.5 | 1 | 3 | 2 |
| 26 | F | 55 | Mild Erythema | 3 | 2.5 | 1.5 | 1.5 | 3 | 2 |
| 27 | M | 55 | None | 2 | 2.5 | 2.5 | 2 | 3 | 2 |
| 28 | F | 56 | None | 2 | 3 | 2.5 | 2 | 3 | 2 |
| 29 | F | 57 | None | 2 | 2.5 | 2 | 1.5 | 4 | 3 |
| 30 | F | 57 | None | 2 | 2 | 2 | 1.5 | 3 | 2 |
| 31 | F | 57 | None | 4 | 2.5 | 2 | 1.5 | 3 | 2 |
| 32 | F | 58 | None | 2 | 3 | 2.5 | 2 | 3 | 2 |
| 33 | F | 59 | None | 3 | 3 | 2.5 | 2 | 4 | 3 |
| 34 | F | 59 | None | 3 | 4 | 2.5 | 2 | 4 | 3 |
| 35 | F | 61 | Mild Erythema | 5 | 3 | 2.5 | 2.5 | 3 | 2 |
| 36 | F | 61 | None | 3 | 3 | 2.5 | 2.5 | 4 | 3 |
| 37 | F | 62 | None | 3 | 3 | 2.5 | 2.5 | 4 | 3 |
| 38 | F | 62 | None | 3 | 3 | 2.5 | 2.5 | 4 | 3 |
| 39 | F | 63 | None | 3 | 3 | 2.5 | 2.5 | 4 | 3 |
| 40 | F | 64 | None | 4 | 3 | 3 | 2.5 | 4 | 4 |
Fig. 2.
Patient before (left) and 6 months after (right) MFU treatment
Group C (675 nm laser plus MFU)
The 40 patients treated with the combined RedTouch + Ultherapy protocol showed a significant improvement 6 months after the last treatment, as assessed by the FWS, BGP, and photographic evaluation.
Scores decreased from the baseline of 1.88 on the FWS scale to 1.6 at the 3-month follow-up after the last treatment to a final of 1.125 at the last follow-up.
Laxity measured with the BGP scale decreased from baseline 2.78 to 1.55 at 6-month post-treatment control.
Pain, assessed using the VAS, was higher than in Group A (VAS score: 1.38) but comparable to Group B (VAS score: 3.075). A slight erythema arose immediately after the treatment in 25% of the subjects (10/40), which resolved within 3–4 h. (Table 3 Fig. 3).
Table 3.
Group C 675 nm laser plus MFU patients’ characteristics
| Patient ID | Sex | Age | Side effect | VAS | FWS | FVAS(3 months) | FVAS(6 months) | BCP | BCP(6 months) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | F | 36 | None | 5 | 0.5 | 0.5 | 0 | 1 | 1 |
| 2 | F | 37 | None | 3 | 0.5 | 0 | 0 | 1 | 1 |
| 3 | F | 38 | Mild Erythema | 6 | 1 | 0.5 | 0 | 1 | 1 |
| 4 | F | 38 | None | 4 | 1 | 1 | 0.5 | 2 | 1 |
| 5 | F | 38 | None | 4 | 1 | 1 | 1 | 1 | 1 |
| 6 | F | 39 | None | 3 | 1 | 1 | 1 | 1 | 1 |
| 7 | F | 40 | None | 3 | 1 | 0.5 | 0.5 | 2 | 2 |
| 8 | F | 40 | Mild Erythema | 3 | 1 | 1 | 1 | 3 | 2 |
| 9 | F | 41 | None | 1 | 1 | 1 | 0.5 | 2 | 1 |
| 10 | F | 41 | None | 4 | 1.5 | 1 | 1 | 2 | 1 |
| 11 | F | 43 | Mild Erythema | 5 | 1.5 | 1 | 0.5 | 2 | 1 |
| 12 | F | 45 | None | 2 | 1 | 1 | 0.5 | 2 | 1 |
| 13 | F | 47 | None | 2 | 1 | 1 | 0.5 | 2 | 1 |
| 14 | F | 48 | None | 3 | 2 | 1.5 | 1 | 2 | 2 |
| 15 | M | 49 | None | 2 | 2 | 1.5 | 1.5 | 3 | 2 |
| 16 | F | 49 | None | 3 | 2 | 1 | 1 | 3 | 1 |
| 17 | F | 49 | None | 3 | 2.5 | 2 | 1 | 3 | 2 |
| 18 | F | 50 | None | 3 | 2 | 1.5 | 1 | 2 | 1 |
| 19 | F | 50 | None | 3 | 2 | 2 | 1 | 3 | 1 |
| 20 | F | 51 | Mild Erythema | 4 | 2 | 1.5 | 1.5 | 3 | 1 |
| 21 | F | 51 | None | 2 | 2.5 | 2 | 1.5 | 3 | 2 |
| 22 | F | 51 | None | 2 | 2.5 | 2 | 1.5 | 3 | 1 |
| 23 | F | 51 | None | 3 | 2 | 1.5 | 1.5 | 3 | 2 |
| 24 | F | 53 | None | 3 | 2 | 2 | 1 | 3 | 2 |
| 25 | M | 54 | None | 3 | 2 | 1 | 0.5 | 3 | 1 |
| 26 | F | 54 | None | 3 | 2 | 1.5 | 0.5 | 4 | 2 |
| 27 | F | 55 | None | 3 | 2 | 2.5 | 2 | 3 | 1 |
| 28 | M | 56 | None | 2 | 2 | 2 | 1 | 3 | 1 |
| 29 | F | 57 | Mild Erythema | 2 | 2 | 2 | 1.5 | 3 | 2 |
| 30 | F | 57 | Mild Erythema | 2 | 2 | 1.5 | 1.5 | 4 | 2 |
| 31 | F | 58 | None | 4 | 3 | 2.5 | 2 | 3 | 1 |
| 32 | F | 58 | None | 2 | 2.5 | 2.5 | 1.5 | 3 | 1 |
| 33 | F | 59 | Mild Erythema | 3 | 2.5 | 2 | 1.5 | 4 | 2 |
| 34 | F | 59 | None | 4 | 2 | 2 | 1.5 | 4 | 3 |
| 35 | F | 60 | None | 5 | 2 | 2 | 1 | 4 | 2 |
| 36 | F | 61 | None | 3 | 2 | 2 | 1.5 | 4 | 2 |
| 37 | F | 62 | Mild Erythema | 3 | 3 | 2.5 | 2 | 4 | 3 |
| 38 | F | 63 | Mild Erythema | 2 | 3 | 2 | 2.5 | 4 | 3 |
| 39 | M | 63 | None | 3 | 2 | 2.5 | 2 | 4 | 2 |
| 40 | F | 65 | Mild Erythema | 4 | 2.5 | 2 | 1.5 | 4 | 2 |
Fig. 3.
Patient before (left) and 6 months after (right) combined 675 nm laser and MFU treatment
Discussion
Our study compared the efficacy of a single anti-aging treatment versus a combined protocol in a cohort of 115 patients who visited our clinics seeking facial rejuvenation. Using an unpaired t-test, we observed that the combined protocol led to significantly greater improvements in wrinkle reduction and skin laxity at six months compared to Group 1 (p < 0.001 for BG scale, p = 0.015 for FWS scale). When compared to Group 2, the combined approach showed a statistically significant improvement in wrinkle reduction (p = 0.046 for FWS), while the improvement in skin laxity (p = 0.2 for BG scale) was not statistically significant. Despite a similar adverse event profile, the combined use of two complementary treatments (one targeting superficial skin layers and the other acting at a deeper level) allowed for more uniform and long-lasting results, leading to higher patient satisfaction [32–37].
At the cellular level, the combined use of the 675 nm laser and microfocused ultrasound induces a dual rejuvenating effect: the laser enhances collagen remodeling and fibroblast activation, while ultrasound-generated thermal coagulation zones promote deep dermal restructuring and upregulate heat-shock proteins. Emerging evidence suggests that these technologies may also reduce cellular senescence by modulating p16INK4a expression and mitigating oxidative stress, both of which are key contributors to skin aging [38].
Conclusions
To our knowledge, this is the first study in the medical literature to combine these two devices, achieving excellent cosmetic results. However, our findings should be interpreted with caution due to the study’s limitations, including the relatively small sample size and lack of a prospective, randomized design. Further large-scale clinical trials with extended follow-up periods are necessary to validate these preliminary results and better quantify the long-term benefits of the combined treatment protocol.
Author contributions
Conceptualization: E.Z., C.G.; Methodology: E.Z., C.G., D.P.; Investigation and data collection: E.Z., C.G., D.P., F.T., M.T., L.G.; Data analysis and interpretation: E.Z., D.P., F.T., M.T., L.G., A.C., G.L.; Writing – original draft preparation: E.Z., C.G., D.P.; Figures and visualization: A.C., G.L.; Supervision: A.D., S.P.N.; Writing – review and editing: A.D., S.P.N.; Final approval of the version to be published: All authors.
Funding
This research received no external funding.
Data availability
Available upon reasonable request from the corresponding author.
Declarations
Institutional review board statement
The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Calabria Centro (373/19, date of approval 4 December 2019).
Informed consent statement
Informed consent was obtained from all subjects involved in the study.
Competing interests
The authors declare no competing interests.
Clinical trial number
Not applicable.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Available upon reasonable request from the corresponding author.