Photoaging is classically described as the consequence of chronic exposure to ultraviolet (UV) radiation on the skin. Dermatology visits for the prevention and treatment of photoaged skin are rapidly increasing. The clinical sequelae including wrinkling, pigmentary changes, roughness, laxity, and telangiectasia can all result in the appearance of aging skin, affecting the quality of life 1,2.
Underlying these visible signs are various histological and cytological changes induced by acute or chronic UV exposure. The histological and ultrastructural hallmark of photodamaged skin is the accumulation of elastotic material in the papillary and mid-dermis, a process known as solar elastosis. This is accompanied by quantitative changes in collagen biosynthesis; the collagen fiber architecture becomes disorganized, with enhanced breakdown and reduced network formation 2. The loss of collagen fibers, which normally provides structural stability, and the degradation of elastic fibers, which gives skin its natural elasticity, result in a general breakdown of the skin’s fibrous matrix. This creates an inelastic, thin, dull-appearing skin 3. In addition to the structural damage of the dermal extracellular matrix, chronic exposure to the sun’s UV rays causes a generalized dysplasia of a variety of epidermal cell types, including keratinocytes and melanocytes. This cellular damage contributes to the mottled and/or hyperpigmented appearance of photodamaged skin, the severity of which increases with age 4,5.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent and calcium-dependent endopeptidases that can degrade almost all molecules in the cutaneous extracellular matrix – in particular, structural molecules of collagen and elastic fibers 6. It was reported that premature collagen damage and degradation in photoaged skin is credited to cleavage by MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) induced by UV light 7 as was previously shown by experiments using human fibroblasts 8 and in human skin 9.
Mesotherapy has been used to rejuvenate the skin by means of a transdermal injection of a multivitamin solution and natural plant extracts that are thought to improve the signs of skin aging. US Food and Drug Administration has approved most of the ingredients used in mesotherapy injection. These components are being applied for indications for which they are unapproved. The efficacy, treatment protocols, pharmacokinetics, and safety of mesotherapy are still of concern and under debate 1. Improvements in wrinkles, increased elasticity, and enhanced skin texture have been attributed to mesotherapy injection but have not been sufficiently proven 10,11.
The aim of this work was to evaluate skin remodeling induced by mesotherapy in photodamaged skin by clinical, histological, and immunohistochemical assessment.
Patients and methods
This clinical trial was conducted on 10 female volunteers with Fitzpatrick skin type III–ΙV and Glogau’s class I–II wrinkles 12. Signs of photoaging included fine lines, wrinkles, facial dyschromia (signs of uneven pigmentation), and altered skin tone (lack of elasticity, radiance, resilience, tactile roughness, pores). Patients were attending the Dermatology Outpatient Clinic at Ain Shams University Hospital, Cairo, Egypt. Informed consent was provided before participation in the study that was approved by the Ethical Committee of Scientific Research, Faculty of medicine, Ain Shams University.
Patient ages ranged from 30 to 50 years. The study participants had stopped undergoing cosmetic treatment (topical or systemic products known to affect skin aging or dyschromia) at least 6 months before the study entry. Treatment and study details were fully explained to the participants and all signed informed consent forms. The clinicians marked the treatment areas on both sides and applied a topical anesthetic cream (lidocaine 2.5%+prilocaine 2.5%) as a thick coating, which was left under occlusion for 90 min. The cream was then gently removed, and the participant was positioned for treatment.
Volunteers were subjected to a total of 3 months of treatment (six sessions at 2-week intervals). The sterile solutions used for injection were provided in two separate vials in commercial packaging (Mesologica, Madrid, Spain); the first vial contained 5 ml of a multivitamin solution of nicotinamide 20 mg, ascorbic acid 100 mg, tocopherol 4 mg, and water for injectable preparation, and the second vial contained 4 ml of non-cross-linked hyaluronic acid at 1% biotechnological origin and water for injectable preparation. The patients and investigators were not blinded to the materials, which were stored in a refrigerator, mixed immediately before injection, and transferred to 1-ml syringes to accommodate the high viscosity of the hyaluronic acid. For each volunteer, the injection material was prepared and mixed as a diluted suspension of multivitamin solution for injection and non-cross-linked, high-viscosity hyaluronic acid at a ratio of 8 : 2. Facial injection with a 30 G/4 mm needle was performed at treatment sites (forehead, perioccular, jaws) on both sides by both intradermal and nappage techniques of mesotherapy (∼0.1 ml/each point). The total amount of solution injected per session on both sides of the face was 2 ml.
Clinical efficacy parameters were assessed by a blinded dermatologist who was not an investigator in the study and was totally blinded toward treatment allocation. Assessments were made while facing the patients, at baseline and at the end of the study (2 weeks after the last session), using visual grading of facial skin 13 for the presence of fine lines, coarse wrinkles, sign of uneven pigmentation and overall skin tone (lack of elasticity, resilience, radiance, tactile roughness, and pores) using a four-point scale where 0=none, 1–3=mild, 4–6=moderate, and 7–9=severe.
Three images (Canon IXUS 950IS; Canon, Tokyo, Japan) were taken of each patient’s face (right, left, and center full face) at baseline, before each session, and at the end of the study to document visible changes in the skin.
Subjective volunteer assessments were taken using questionnaires, in which the volunteers were asked to give their degree of satisfaction in terms of overall skin appearance (facial lines, wrinkles; the appearance of brown spots, facial discoloration; and overall skin tone based on a five-point scale from 0 to 4 (0=worse, 1=little satisfaction or not satisfied, 2=fairly satisfied, 3=satisfied, and 4=very satisfied). Questionnaires were given 2 weeks after the final treatment. Signs and symptoms of irritation were evaluated as regards erythema and edema, burning, stinging, and itching using a four-point scale: 0=none, 1=mild, 2=moderate, and 3=severe.
Punch biopsies (3 mm) were obtained from facial skin at baseline and at the end of the study (2 weeks after the last session). Biopsies after treatment were taken from a hidden site near the pretreatment ones. Tissues were fixed in 10% buffered formalin, embedded in paraffin, and sectioned into 4-μm-thick sections. Sections were subjected to hematoxylin and eosin, orcein stains, and immunohistochemical staining using MMP-1 and MMP-9. The sections were analyzed and photographed under an Olympus BX50 microscope (Olympus, Tokyo, Japan) to assess the effect of UV rays on the epidermis, such as epidermal hyperplasia and hyperpigmentation, and on the dermis, such as inflammation, collagen degeneration, and breakdown of blood vessels, and to compare the quality, quantity, and arrangement of collagen and elastic fibers. Elastic densities with orcein stain were assessed semiquantitatively as follows: 0=none; +=mild; ++=moderate; and +++=extensive.
Immunohistochemical staining and evaluation
Immunohistochemical staining of matrix metalloproteinase 1 and matrix metalloproteinase 9
Paraffin sections were fixed on poly-L-lysine-coated slides and dried overnight in an oven at 60°C. They were then deparaffinized and dehydrated. The slides were treated in a microwave oven in ready-to-use antigen retrieval citrate buffer for 10 min and left to cool at room temperature for 20 min. The slides were stuck to cover plates using PBS (pH 7.6) and placed in a sequenza center for immunostaining. Endogenous peroxidase activity was blocked by adding 2–3 drops of hydrogen peroxide blocking serum and leaving the slides undisturbed for 5 min at room temperature. The sections were then rinsed well with PBS for 5 min. Two drops of protein blocking serum were added and the slides kept undisturbed for 10 min. There was no rinsing after this step. The primary antibody was applied by adding three drops to each section [MMP-1: a polyclonal rabbit antibody (Lab Vision; catalog no. #RB-1536-R7; Sacramento, California, USA) supplied as 1 ml of ready-to-use antibody 1; and MMP-9: a polyclonal rabbit antibody (Lab Vision; catalog no. #RB-9234-R7) supplied as 1 ml of ready-to-use antibody] and then incubating for 2 h at room temperature, followed by rinsing in PBS (pH 7.6). The secondary antibody was applied by adding two drops of biotinylated secondary antibody to each section and incubating in a supersensitive immunodetection system (Bigenex; catalog no. AD 000-SL) for 30 min at room temperature. The slides were then rinsed in PBS (pH 7.6). Two drops of peroxidase-labeled streptavidin were added for 20 min at room temperature, and then the slides were rinsed with PBS (pH 7.6). This was followed by incubation for 10 min with substrate chromogen (DAB) mixture and then rinsing with distilled water. The slides were immersed in Harris hematoxylin for 3 s, rinsed in tap water, and dehydrated in absolute alcohol. Finally, they were cleared in xylene and mounted with Canada balsam and covered with a glass cover. The tissue of breast carcinoma was regarded as positive control for MMP-1 and MMP-9. The tissue samples were stained in each run to judge the effectiveness of the technique. Negative control slides were processed following the previous immunostaining procedure, but the primary antibody was omitted from the steps and PBS was used instead. The pattern of staining of MMP-1 and MMP-9 was cytoplasmic or in the cell membranes.
Immunohistochemical assessment of matrix metalloproteinase 1 and matrix metalloproteinase 9
Sections were scored on the basis of the percentage of stained cells relative to the background 14 as follows: negative, less than 10% of cells stained positive; weak, 10–50% of cells stained positive; moderate, 51–75% of cells stained positive; and strong, 75% of cells stained positive.
The differences between pretreatment and post-treatment values were examined for statistical significance. Quantitative evaluations of histological measurements were analyzed using the statistical package for the social sciences (SPSS, version 16; SPSS Inc., Chicago, Illinois, USA) for Windows. Data were expressed as mean±SD and range for numerical data and as frequency and percentage for non-numerical data. Statistical analysis was carried out using one-way analysis of variance, Wilcoxon-matched pairs signed ranks, Student’s t-test, correlation analysis, Fisher’s exact test, and χ2-test. Statistical significance was defined as follows: P value more than 0.05, not significant; P value less than 0.05, significant; and P value less than 0.01, highly significant.
Ten women aged 30–50 years (mean 44.5±5.1 years) were enrolled into the study with mild-to-moderate photodamaged facial skin, evaluated as per the modified Glogau’s classification and Fitzpatrick’s classification. According to the modified Glogau’s classification, four (40%) patients were of class I and six (60%) patients were of class II. According to Fitzpatrick’s classification, five (50%) patients were of class III and five (50%) patients were of class IV.
There was a clinically detectable improvement in the appearance of fine lines, uneven skin pigmentation, and overall skin tone after 14 weeks. Significant improvement was seen as regards the percentage change in clinical grading parameters. There was a highly significant reduction in the appearance of fine lines and coarse wrinkles (43%, P=0.008; 33.4%, P=0.05, respectively) (Fig. 1) and significant decrease in uneven pigmentation (15%, P=0.014). However, an overall change in skin tone after mesotherapy sessions was detected but was not statistically significant (P=0.059). There was an inverse relationship between age and overall skin appearance (improved skin firmness/elasticity, resilience, skin radiance, and smoothness). As age increased the global facial skin appearance showed less improvement (r=−0.207, P=0.05).
Patient satisfaction was higher for all aspects of skin condition after treatment. Eighty percent of patients reported a 60% improvement in overall facial skin appearance and stated that their skin tone was more even and clearer, that fine lines and wrinkles appeared minimized, and signs of aging had reduced significantly at week 14 (P=0.009). There was mild facial erythema and edema secondary to the traumatic action of the needle and injected substances, which were reported by patients on the first day of every session with spontaneous resolution within 48 h. The treatment caused minimum discomfort, without any post-treatment pain. All patients returned to their daily activities immediately after the treatment.
Microscopic examination of hematoxylin and eosin-stained sections before treatment revealed hyperkeratosis, acanthosis, perivascular basement membrane thickening, and increased dermal melanin and melanophages. In addition, dermal blood vessel breakdown, dermal mononuclear inflammatory infiltrates, solar elastosis, and homogenization of collagen were observed (Fig. 2a).
At the end of the study, quantitative and qualitative changes in elastic and collagen fibers were observed. There were overall morphological and architectural improvements of the epidermis, with marked undulations of the dermoepidermal junction. Epidermal hyperplasia and hyperpigmentation showed a milder change. A decrease in dermal inflammatory infiltrate was evident, and there were newly formed collagen fibers that had a uniform distribution following a parallel axis to the surface of the epidermis, involving the papillary and reticular dermis (Fig. 2b). Compared with baseline, there was a nonsignificant decrease in the epidermal hyperplasia (21.2%, P=0.213) and in hyperpigmentation (24.3%, P=0.317). The dermis showed a highly significant decrease in the mean inflammation score (65.1%, P=0.005), a significant decrease in mean dermal blood vessel breakdown (63.1%, P=0.011), and significant decrease in the mean amount of degenerated and disorganized collagen fibers (53.2%, P=0.034).
To demonstrate elastic fibers, specimens were pretreated with orcein stain, which revealed dermal elastosis consisting largely of a gradual accumulation of thickened, tangled, and ultimately granular amorphous elastic structures noticeable in the papillary and mid-dermis (Fig. 3a). The decline in elastin content was associated with a translocation of the solar elastotic material away from the epidermis, accompanied by the restoration of normal-appearing elastic fibers within the papillary and upper reticular dermis. There was a 43.2% mean increase in elastic fiber content in the papillary dermis of participants. The range of increase was from 32.2 to 54% and this increase was statistically significant (P=0.048). Elastic fibers increased in number and density with a linear and regular orientation (Fig. 3b).
Tissue expression of MMP-1 and MMP-9 proteins was mainly expressed in the cytoplasm or membranes of cells. Diffuse epidermal and/or dermal expressions of MMP-1 and MMP-9 were seen as positive cases with variable staining intensities.
At baseline, positive epidermal immunostaining of MMP-1 was weak in one (10%) patient, moderate in three (30%), and strong in six (60%) patients, which changed after treatment to negative staining in four (40%) and weak in six (60%) patients at the end of the study. Positive MMP-1 dermal immunostaining was weak in one (10%) patient, moderate in three (30%), and strong in six (60%) patients at baseline, which changed to negative in two (20%) and weak in eight (80%) patients at the end of the study. There was a highly significant change in the quantitative evaluation of the percentage of epidermal (P=0.005) and dermal (P=0.006) immunostaining of MMP-1 (Fig. 4).
Positive immunostaining of epidermal MMP-9 was moderate in 30% patients and strong in 70% at baseline, whereas it changed to negative staining in 20% patients, weak in 60%, and moderate in 20% at the end of the study. Dermal immunostaining of MMP-9 showed moderate staining in 40% patients and strong staining in 60% at baseline, which changed to negative in 10% patients, weak in 80%, and moderate staining in 10% at the end of the study. There was a highly significant change in the percentage of epidermal (P=0.007) and dermal (P=0.004) MMP-9 immunostaining (Fig. 4).
There was a positive correlation between collagen fiber fragmentation/disorganization and elastic fiber degradation and the expression levels of dermal MMP-1 and MMP-9 (r=0.617, P=0.037). There was an inverse relationship between the clinical scoring of overall skin changes and dermal immunostaining pattern of MMP-1 and MMP-9 [when skin expressions of MMP-1 and MMP-9 decrease, the overall skin tone improves (r=−0.813 P=0.004)].
The results of this clinical trial support the suggestion that mesotherapy might induce facial rejuvenation of photoaged skin in individuals having Fitzpatrick’s skin type III–ΙV and Glogau’s class I–II wrinkles. This was based on finding a statistically significant clinically detectable improvement in facial skin appearance. Histopathological evaluation revealed marked increase in collagen and elastic fibers. The immunohistochemical staining pattern of MMP-1 and MMP-9 revealed a highly significant change in the epidermis and dermis. The treatment was tolerable and patient satisfaction was higher for all aspects of skin condition after 14 weeks of treatment, including greater elasticity, resilience, firmness, tightness, hydration, smoothness, brightness, and suppleness.
Several investigators have recommended mesotherapy as an antiaging strategy that helps to maintain a globally firm and bright skin, protecting it from the environmental contributors to aging; however, controlled studies required to validate their findings or elucidate whether and how the cellular and molecular processes are involved in facial skin rejuvenation are lacking 13,15. It has been proposed that skin rejuvenation might be promoted by intradermal injection of multivitamins, which stimulate fibroblasts to produce more collagen and elastin 7,15. In addition, hyaluronic acid injected into the skin can stimulate fibroblasts to express collagen type 1, MMP-1, and tissue inhibitor of MMP-1 11. Mesotherapy increases the biosynthetic capacity of fibroblasts and the reconstruction of an optimal physiologic environment, as well as the enhancement of cell activity and the synthesis of collagen, elastin, and hyaluronic acid 16. In this study, we investigated the quantitative variations of MMP-1 and MMP-9 and collagen/elastic fibers by comparing their expression levels before and after treatment to evaluate the possible effect of mesotherapy in skin remodeling of photoaged skin. It is worth mentioning that collagenase MMP-1, which is the most powerful collagenase that preferably cleaves intact collagen I, and gelatinase B (MMP-9) are essential for the degradation of collagen IV and degenerated collagen I 17. Several investigators have shown strong dermal MMP-1 and MMP-9 expressions that were associated with severe disorganization of collagen and degradation of elastic fibers 18. It was reported that MMP-1 initiates matrix degradation by cleaving fibrillar native triple helical collagen and making it susceptible to final breakdown by gelatinases MMP-9 and MMP-3 19.
Our patients showed a clinically detectable improvement in the appearance of fine lines, uneven skin pigmentation, and overall skin tone after 14 weeks. Significant improvements were seen as regards the percentage change in clinical grading parameters. There was a highly significant reduction in the appearance of fine lines and coarse wrinkles (43%) and significant decrease in uneven pigmentation (15%). However, an overall change in skin tone after mesotherapy sessions was detected but not significant. Histopathological evaluation revealed marked increase in collagen and elastic fiber density and organization with a uniform distribution following a parallel axis to the surface of the epidermis, involving the superficial, mid, and deep reticular dermis. Immunohistochemical staining pattern of dermal MMP-1 and MMP-9 revealed a highly significant change. The damage to collagen and elastic fibers showed a positive correlation with the reduced expression levels of dermal MMP-1 and MMP-9.
Our findings disagree with those of Amin et al. 20, who evaluated 10 patients who had underwent four sessions of mesotherapy conducted at 4 monthly intervals with multiple injections of a multivitamin and hyaluronic acid solution. The authors noticed no overall clinical benefit from treatment, with no significant changes in epidermal thickness, vessel size and density, solar elastosis, elastin content, mucin content, dermal thickness, and collagen fiber thickness and concluded that facial mesotherapy injection with a multivitamin and hyaluronic acid solution did not provide any significant clinically observed benefit. A different treatment protocol involving only four sessions of mesotherapy with a different dosing combination might explain the different outcome 20.
Another study evaluated the clinical effect of mesotherapy applied to periorbital wrinkles and quantitatively assessed the histological changes in the skin occurring in response to the same treatment in six volunteers with Fitzpatrick skin types III–IV and Glogau’s class I–III wrinkles following a 3-month course of treatment. Their clinical evaluation did not reveal any statistically significant effect, with a 10–15% improvement in skin tightening, improvement in wrinkles ranging from none to mild (0–5%), and no significant improvement in skin texture compared with baseline at the end of treatment. Histological and immunostaining analysis of collagen and elastin showed no statistically significant changes. Thus, they indicated that mesotherapy for skin rejuvenation did not result in statistically significant histological changes or clinical improvement 21. The discrepancy might be attributed to different recruitment criteria as most of our patients were younger (<40 years) and had mild-to-moderate photoaging.
Although previous studies refuted the clinical or histological efficacy of facial rejuvenation by mesotherapy, in our study the products used did induce an effect in tissue remodeling. Patients showed clinical improvement in their photodamaged skin that was most probably related to the increased and organized collagen and elastic fibers in the reticular dermis: in other words, decreased breakdown and increased collagen synthesis and reduced solar elastosis as a result of decreased MMP-1 and MMP-9 activity, which promoted dermal remodeling with an overall ‘skin tightening’ effect after mesotherapy injection.
These results are in accordance with those of Lacarrubba et al. 22, who evaluated the effects of mesotherapy with multiple injections of hyaluronic acid in 20 women with physical signs of moderate skin photoaging with subepidermal low-echogenic band on ultrasonography of skin. This study suggested that mesotherapy with hyaluronic acid may be an effective treatment for photoaging, as there was a statistically significant increase in subepidermal low-echogenic band echogenecity in 15/19 patients who completed the study 22. The results were also almost in agreement with those of Savoia et al.23, who carried out a study on 32 patients of both sexes aged between 50 and 60 years (older patients) and used nearly the same treatment protocol for 8 weeks. They reported significant rejuvenation effects with an improvement in epidermal texture and an increase in elasticity and brightness of the skin on the basis of a significant difference in scores on the Global Aesthetic Improvement Scale after four treatments. Using immunohistochemical analysis, the authors observed a significant decrease in the expression levels of interleukin-6, interleukin-1β, and MMP-1, and significant increase in collagen-1, at the end of treatment. They suggested that minimally invasive mesotherapy techniques would improve the clinical appearance of the skin with a restructuring action resulting in antiaging processes in patients with severe photoaging 23.
A point of special interest is that younger female patients (≤40 years) had high scores of clinical improvement, as there was an inverse relationship between age and global improvement of fine lines and skin tone. Similarly, when age increased, the histological and immunostaining pattern changes in the epidermis and dermis showed fewer changes. This indicates that younger patients would have more benefit from mesotherapy as collagen/elastin neogenesis and remodeling would be more pronounced.
Limitations of the study include the small number of patients enrolled and the short duration of observation. Therefore, it cannot be excluded that part of the amelioration effectively registered could disappear after several months.
Our study indicates that mesotherapy might be an effective treatment for facial rejuvenation of photoaged skin as it can probably interfere with the effects on collagen production in damaged skin by increasing the expression of collagen and elastin. Nevertheless, the downregulation of MMP-1 and MMP-9 could interfere with the underlying inflammatory processes involved in aging skin.
However, further controlled studies on a larger scale, dose standardization, and follow-up of improved cases over long periods for tracking sustainability of these effects are recommended before considering mesotherapy as an effective procedure in the rejuvenation of photoaged skin.
Conflicts of interest
There are no conflicts of interest.
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