Current Understanding of Genetic and Epigenetic Factors in Personalized Skin Rejuvenation

Skin ageing is a multifactorial process mediated by both intrinsic factors (such as time, genetics, hormones) and extrinsic environmental factors (i.e., lifestyle, nutrition, pollution, drugs, UV exposure) which accelerate the chronological (intrinsic/calendar) ageing [1,2]. Types of ageing including those referring to skin are presented in Table 1.

The main physiological and histological changes in aged skin phenotype might be described as follows: reduced elasticity and hydration; formation of wrinkles; sagging; itching; significant thinning and atrophy of the dermis and epidermis; altered barrier function; increased vulnerability to injury and disease; delayed wound healing; subcutaneous fat loss; uneven pigmentation; flattening of the epidermis–dermal connection; declining proliferative potential of the epidermal stem cells; collagen fragmentation and loss (approximately 1% yearly decline in collagen content in the dermis caused by parallel reduction in synthesis and increased degradation of existing collagen); disruption of the elastic fibre network; reduction in glycosaminoglycans and proteoglycans; impaired cell division of the melanocytes, of the cells in the germinative layer and those located in epidermis [2,10,11,12,13,14]. Similar genetic, biochemical and cellular disfunctions are involved in the visible signs of both chronologically aged and photoaged skin.

Intrinsic ageing and photoageing both share mechanisms like oxidative stress, senescence, and extracellular matrix (ECM) degradation, but photoageing accelerates these processes via UV-specific pathways involving stimulation of the matrix metalloproteases (MMP) activation and inflammation. Chronic localized inflammation in photoaged skin, with elevated cytokine expression (interleukin IL-6, tumour necrotic factor TNF α) and reactive oxygen species (ROS), contrasts with the low-grade, systemic inflammageing observed in chronological ageing; in addition, deoxyribonucleic acid (DNA) damage and cellular senescence accumulate more rapidly in UV-exposed skin areas [15]. Photoaged skin exhibits severe clinical signs (deep wrinkles, pigmentation, vascular changes) and histological alterations (solar elastosis, collagen fragmentation and degradation, faster ECM decline) that exceed those seen in chronological ageing [15,16,17]. Comparative insights on various features of the intrinsic aged and photoaged skin are illustrated in Table 2.

The main hallmarks of accelerated skin ageing are: (a) cellular senescence and the emergence of the Senescence-Associated Secretory Phenotype (SASP) associated with the accumulation of the senescent fibroblasts in the dermis, which are responsible for collagen degradation by the secreted matrix metalloproteases (MMP) and for secretion of pro-inflammatory factors; (b) genomic instability, due to the increased accumulation of DNA damage, mutations and genomic rearrangements; (c) telomere attrition (the gradual shortening of telomeres limiting the regeneration capacity); (d) epigenetic changes, like: DNA methylation, histone modifications, non-coding ribonucleic acid (RNA) regulation and heterochromatin-modifying gene expression patterns; (e) stem cell exhaustion, affecting tissue repair and renewal; (f) dysregulation of nutrient-sensing pathways, like mammalian target of rapamycin, (mTOR), insulin-like growth factor 1 (IGF-1) and IGF-1 signalling (IIS) pathway, AMP-activated protein kinase (AMPK, an energy-sensing enzyme), which alter metabolism and energy balance; (g) mitochondrial dysfunction, leading to impaired energy production and increased oxidative stress; (h) altered proteostasis, with the accumulation of misfolded proteins and Advanced Glycation End Products (AGEs), which is highly correlated to cellular senescence and plays a role in photoageing due to abnormal aggregation of glycated elastin fibres; (i) inflammageing; (j) altered intercellular communication and disconnectivity of the transcriptional networks; (k) microbiome disturbance; (l) compromised macro-autophagy [10,18,19,20,21,22].

Personalized and effective topical and systemic skin anti-ageing and rejuvenation strategies should target the above mentioned cellular, metabolic and (epi)genomic hallmarks, combined with minimally invasive procedures and nanotechnology, and are currently focused especially on: senotherapeutics (senolytics and senomorphics); regenerative medicine in order to restore skin-resided stem cells, based on mesenchymal stem cells or cell-free products (platelet-rich fibrin, autologous conditioned serum); epigenetic partial reprogramming; various epigenetic drugs (DNA methyltransferase inhibitors, histone deacetylase inhibitors, microRNAs, modulators of sirtuins and of long-non-coding RNA HOTAIR, telomeric repeat binding factor 2 TRF2); antioxidants (resveratrol, topical or oral use of various polyphenol-rich plants); pharmacological activation of autophagy; restoration of mitochondrial integrity essential for metabolism homeostasis [11,19,20,23,24,25].

The aim of this review is to concisely discuss: the clinical relevance of the most important clocks and biomarkers used in skin anti-ageing strategy evaluation, the fundamentals, the main illustrating examples preclinically and clinically tested, the critical insights on knowledge gaps and future research perspectives concerning the most relevant skin anti-ageing and rejuvenation strategies based on novel epigenomic and genomic acquisitions. Thus, the review dedicates distinct sections to senotherapeutics; stimulators of the DNA repair enzymes and of the proteins essential for telomeres' recovery and stability; regenerative medicine based on stem-cells exosomes, genetically and chemically induced skin epigenetic reprogramming, modulators of microRNA and long-non-coding RNA (HOTAIR's modulators) assisted or not by CRISPR-gene editing technology (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats), small molecule modulators of DNA methylases, histone acetylases, telomerases or of sirtuins, as well as modulators of the most relevant altered nutrient-sensing pathways and of mitochondrial dysfunctions and oxidative stress levels. The possibilities to positively influence skin inflammageing, proteostasis, (macro)autophagy and intercellular connections or skin microbiome, are also very briefly presented. Moreover, this review points out a comparative analysis among the newer genomic/epigenomic-based skin anti-ageing strategies vs. classical skin rejuvenation treatments from various perspectives: efficacy, safety, mechanism of action, price range, current limitations, evidence level in preclinical and clinical data and regulatory status. In these regards, a concise overview on senolytic/senomorphic agents, topical nutrigenomic pathways' modulators and DNA repair enzymes, epigenetic small molecules agents, microRNAs and HOTAIRS's modulators, assisted or not by CRISPR gene-editing technology, is illustrated in comparison to classical approaches such as tretinoin and peptide-based cosmeceuticals, topical serum with growth factors, intense pulsed light, laser and microneedling combinations, chemical peels, botulinum toxin injections, dermal fillers. Finally, this review emphasizes the future research directions in order to accelerate the clinical translation of the (epi)genomic-advanced knowledge towards personalization of the skin anti-ageing strategies by integration of individual genomic and epigenomic profiles to customize/tailor skin rejuvenation therapies.

Chronological age is fixed and does not reflect skin health; biological age is more insightful for assessing rejuvenation [3]. The main biomarkers for estimating the biological age and assessment of anti-ageing interventions efficacy are integrated into epigenetic and non-epigenetic clocks, comprising DNA methylation-based biomarkers (epigenetic clock); analysis of key age-associated genes; cellular ageing hallmarks (SASP, altered macromolecules, genomic instability and shortened telomeres; senescence-associated heterochromatin foci SAHF) [26,27,28].

Among the skin-ageing biomarkers, DNA methylation-based (DNAm) epigenetic clocks, developed by regression models based on the increasing relationship with age of chosen groups of CpGs, are considered the gold standard in primary and accurate candidate metric at the molecular level and are highly reproducible and validated by most studies, both in ageing prediction and in skin rejuvenation research [29,30,31]. Such "epigenetic clocks" based on CpGs profile in skin fibroblasts, keratinocytes, endothelial cells, as well as in blood samples, would enable a better prediction and personalization of the skin rejuvenating topical and systemic senotherapeutics [29,32,33,34].

Based on a large-scale epigenome-wide association study (EWAS), altered DNA methylation patterns at CpG sites near ECM genes, such as elastin (ELN), lysyl oxidase (LOX), collagen type VIII alpha 1 chain (COL8A1), and matrix metallopeptidase 3 (MMP3) genes, have been associated with perceived/visibly aged skin [35]. These genes are interconnected within phenotypic/biological ageing of skin. The ELN gene encodes tropoelastin, an essential protein of the elastin fibres in the connective tissue. LOX encodes the enzyme responsible for crosslinking elastin and collagen fibres which strengthen ECM and play a key role in skin remodelling and wound healing. COL8A1 contributes to collagen synthesis and thus slows down the ageing process, whilst MMP3 catalyzes collagen degradation [34,36]. Boroni M. et al. have calculated skin-specific DNAm age based on the 2266 CpG sites within hundreds of cultured cells and human skin biopsies. Their model has enabled a highly accurate chronological age prediction, which is also sensitive to skin disorders, cell passage, or therapy with senotherapeutics. The tissue-specific DNAm age predictors are endowed with superior prediction power over the similar pan- or multi-tissue tests [36]. What remains to be further established is either the causal or the consequential relationship between DNAm profile and the skin ageing process/status. Initially regarded as a valuable tool to estimate chronological age at the molecular level, DNAm of biological samples has been recently reconsidered as a powerful tool to assess also the lifespan, longevity, the overall health status and mortality risk, due to its dependence both on time and additional factors, such as lifestyle and some genetic, inflammatory, metabolic and infectious disorders [34,35,36].

The epigenetic clocks are classified into generations, the first generation being mainly represented by Horvath clock, which is a universal clock detecting DNAm at 353 CpGs across tissues, without optimization for skin anti-ageing or short-term interventions [6]. Systemic epigenetic clocks (like GrimAge) are less useful for localized skin ageing evaluation but remain valuable in broader ageing-related drug development where skin is one of multiple targeted tissues [37]. The skin-optimized epigenetic clocks from second generation (e.g., Skin and Blood Clock, GrimAge, PhenoAge) and third generation (e.g., DunedinPACE, miRNA Skin Clock, Rayan Clock) provide measurable and sensitive biomarkers for skin-targeted interventions and they are most clinically valuable in dermatology and regenerative applications [4,5,6,26,27,28]. The Skin and Blood Clock is currently the most accurate skin-specific biological age tool, it provides the most clinically relevant and accurate measure of biological skin age, making it ideal for assessing the efficacy of cosmeceuticals, laser-based treatments, and cellular therapies targeting dermal rejuvenation; so, it is regarded as the benchmark for evaluating dermatological and regenerative therapies [6,26].

Pace-of-ageing clocks (e.g., DunedinPACE) enable monitoring of responsive treatment effects over short periods. DunedinPACE complements skin strategies by offering real-time insights into ageing velocity, especially in trials involving nicotinamide adenine dinucleotide NAD⁺ boosters or gene therapies; it also enables short-term tracking of intervention response [7]. Micro-ribonucleic acid (miRNA)-based Skin clocks show promise for non-invasive, patient-compliant diagnostics and testing, particularly in cosmetic dermatology [27]. Thus, emerging third generation clocks such as DunedinPACE and miRNA-based skin clocks offer novel value in the efficacy's evaluation of skin rejuvenation strategies.

The last developed skin epigenetic clock by Rayan and collaborators represents a significant advancement by providing a tissue-specific biomarker that accurately reflects biological ageing and rejuvenation in human facial skin through DNA methylation profiling of epidermal and dermal fibroblasts. Its strength lies in sensitivity to reversible ageing changes following esthetic interventions, offering an objective measure for evaluating skin anti-ageing therapies. However, its reliance on invasive skin biopsies, limited commercial availability, and current validation primarily in lighter skin phototypes pose challenges for broader clinical application [26,28].

Mean Absolute Error (MAE) in skin epigenetic clocks quantifies the average difference between predicted skin biological age and the actual chronological age, serving as a key metric to evaluate the clock's accuracy and reliability in measuring skin ageing. Among skin clocks and related ageing biomarkers, the Rayan Skin clock ranks highest with the lowest MAE (~3–4 years), outperforming earlier models like the Horvath Skin and Blood clock, thus reflecting its superior precision in assessing biological skin age. Within the MAE hierarchy, the Rayan clock is followed by the Horvath Skin and Blood clock (~5–7 years) and the blood-based clocks (like PhenoAge and GrimAge) which typically have higher MAEs when applied to skin (often >8 years) due to tissue specificity limitations. miRNA-based skin ageing clocks, still emerging, show moderate accuracy with MAEs around 8–10.9 years, but require further validation. This hierarchy highlights the importance of tissue-specific design for precise skin ageing measurement. Epigenetic biomarkers of (skin) ageing require further studies on inter-individual variability and also validation across diverse populations [26,28,34,35].

Besides epigenetic clock estimating cellular ageing by DNA methylation variations, non-epigenetic biomarkers, like telomeres' length, senescence-associated proteins or inflammageing biomarkers, serve as mechanistic or histological adjuncts, not standalone age clocks, and they lack quantitative replication across patient cohorts, and the sensitivity and specificity needed for precision monitoring of clinical skin rejuvenation outcomes [8,9]. Among non-epigenetic senescent biomarkers might be considered: (a) p21, p53 (biomarkers of cell cycle arrest); (b) p16^INK4A—its enhanced expression significantly correlates to chronological human epidermis ageing, older facial perception and to the age-changed elastin morphology in the dermal papilla (for instance, higher proportion of p16^INK4A- positive melanocytes is associated with more facial wrinkles); (c) senescence-associated β-galactosidase (SAβGAL)—the mostly used histochemical biomarker in human dermal fibroblasts; (d) nuclear matrix protein lamin B1—a quantitative biomarker of skin senescence, decreasing with age; (e) acH4 (acetylated histone H4) and H4K20me1 (histone H4 monomethylated on lysine 20), which are altered histone biomarkers during skin senescence (correlated to delayed differentiation of keratinocytes and epidermal hyperplasia). Histones are DNA bound proteins with an essential role in chromatin organization, differentiation of epidermal stem cell niche into sebocytes and interfollicular epidermis, as well as in skin homeostasis [20,32,38,39]. In addition, the GlycanAge analysis measures changes in glycosylation patterns of immunoglobulins (IgG) which could trigger systemic chronic inflammageing and age-associated disorders [40,41].

Comparative insights on various epigenetic and non-epigenetic clocks/ biomarkers relevant to skin rejuvenation strategies' evaluation are summarized in Table 3.

In conclusion, although photoageing remains a critical clinical concern with distinct histopathological markers which molecular clocks do not yet fully encompass, the integration of the biological age clocks, particularly epigenetic clocks, has revolutionized the evaluation of skin rejuvenation strategies, from topical interventions and energy-based devices to regenerative medicine and epigenetic drugs. A combined approach using validated epigenetic clocks with skin-specific calibration and complementary biomarkers offers the most powerful and practical framework for quantifying and validating the biological effects of anti-ageing interventions on the skin. These tools will be essential in personalizing dermatologic care, evaluating anti-ageing compounds, and guiding regenerative therapies in both clinical trials and esthetic medicine [26,28,34,35]. Precise biomarkers of ageing and rejuvenation for whole-body, at cellular and molecular levels, are prospecting the current advanced spatiotemporal multi-omics approaches, including transcriptomics, proteomics, metabolomics, gene signatures, and epigenomics, which can offer in-depth understanding of the complexities and relationships regarding molecular dynamics, cellular interactions and signalling pathways, inherent in ageing and regeneration, thus supporting new potential therapeutic innovations [20,30,43].

Nuclear genome instability is mainly caused by the accumulation of DNA mutations and by the decline in the efficiency of the DNA damage repair (DDR) mechanisms. Nuclear genome instability is significanty correlated to an accelerated progression of age-related changes, as noticed also in progeroid Werner and Cockayne syndromes [84,85]. Telomeres are repetitive DNA sequences acting as protective caps at the extremities of linear eukaryotic chromosomes with a crucial role in genomic stability. During ageing and consecutive of multiple cell divisions, telomeres become progressively shorter; beyond a critical length of the telomeres, the cell cycle will be irreversibly stopped, thus triggering cell apoptosis. The parallel processes of telomeres' shortening and of cell senescence, respectively, represent an essential hallmark of ageing called "life clock" [34,86]. Besides genomic instability, telomere attrition is viewed as a main contributor to fibroblast senescence in aged skin and disease-related ageing skin. Excessive telomere attrition in the progenitor cells of the highly proliferative tissues (haematopoietic system, gastrointestinal tract, skin) ultimately triggers DDR such as cell cycle arrest, apoptosis, differentiation disorders and senescence, while in hypoproliferative tissues (heart, brain and liver) oxidative stress might further increase telomere sequence damage and attrition [87]. DDR signalling pathways are activated in response to few critically short telomeres and comprise the following steps: the overexpression of p53 and p21 (cell cycle inhibitory biomarkers); the altered secretome SASP, which modify the ECM composition and propagates the senescent phenotype to surrounding cells; finally, systemic chronic inflammation [88,89].

The protective approaches to overcome telomere attrition tested so far could be illustrated by:stimulation of reverse transcriptase telomerase, an enzyme responsible for biosynthesis of new telomeric DNA based on RNA template in highly proliferative skin cells like stem cells, using for instance as telomerase activator the compound TA-65 [8,42]. Moreover, liposomes with xenogenic DNA repair enzymes like photolyase isolated from microalgae Anacystis nidulans and T4 endonuclease from Micrococcus luteus have proven efficient due to a significant decrease in telomere shortening rates [11,33]; as well as liposomes with 8-oxoguanine glycosylase photolyase (OGG1) which have demonstrated an essential role in reducing the biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine of oxidative DNA damage and mitochondrial dysfunctions [58,84];recovering of the multi-protein shelterin complex that is essential for stabilization of chromosomes and for their protection against being detected as double-stranded breaks. The TRF2 protein of shelterin complex is involved in telomere capping and DDR inhibition; TRF2 deficiency triggers the activation of p53 signalling pathway and cellular apoptosis. In this regard, topical application of TRF2 might have a protective role in telomeric DNA [58,85,87];nicotinamide adenine dinucleotide NAD⁺ boosters (such as NAD⁺ precursors: nicotinamide riboside and nicotinamide mononucleotide), which, like telomerase activators (e.g., compound TA-65), support DNA integrity and cellular longevity, help slow DNA damage accumulation; however, direct evidence in human skin is limited and their effects may be systemic [59].

Mesenchimal stem cells (MSCs) have an essential role in tissue repair and homeostasis, due to their multipotency and self-renewal ability. Aged skin is marked by stem cells' pool declining, correlated with skin atrophy, fragility and hyperpigmentation [86,90]. Regenerative therapeutic approaches in skin rejuvenation are designed upon the current understanding of the main mechanisms and molecules involved in the physiological regeneration of skin. Using either endogenous stem cells, such as adipose-derived mesenchymal stem cells (AD-MSCs), amniotic membrane stem cells (AMSCs), human umbilical cord mesenchymal stem cells (hUC-MSCs), tissue-induced pluripotent stem cells (iPSCs), or extracellular vesicles/exosomes derived from endogenous MSCs, the following beneficial anti-ageing effects were reported: stimulation of mitosis, proliferation and differentiation, especially of human dermal fibroblasts; up-regulation of local immunological defence mechanisms; release of the angiogenesis-modulatory cytokines, antibacterial peptides and anti-inflammatory molecules; release of the growth factors and cytokines essential for skin trophicity, tissue re-epithelization, wound healing, biosynthesis of elastin and collagen fibres in the ECM; inhibition of proteins' glycation; inhibition of oxidative stress [38,45,84,91]. The stem cells have the advantage of being easily obtained and abundant. Other advantages, especially of AMSCs, rely on: multipotency, low immunogenicity, facil isolation from the placenta, unapplicable ethical issues like those imposed to embryonic stem cells [18,92].

Regenerative medicine-based skin therapies used alone or in combination to conventional treatments might offer more promising, efficient and safe solutions in facial rejuvenation, in comparison to conventional approaches (such as cosmeceuticals, microneedle, dermal fillers, injectables, fractional laser) [12,92,93]. For instance, topical application of AMSC with vitamin E to photoaged human subjects have significantly decreased wrinkles, ultraviolet spots and pores. Injected into the dermis, ADSCs have improved skin vascularization, hydration and density. In topical use, hUC-MSCs-conditioned media associated with microneedling significantly improved skin brightness and texture in comparison to microneedling alone [18,93].

On the other hand, as an alternative to stem cell-based skin rejuvenation, regenerative cell-free approaches might use: (1) topical platelet concentrates, such as: platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and injectable platelet-rich fibrin (i-PRF); and (2) blood cell secretome (BCS). PRP, an autologous and highly concentrated preparation of platelets, is extensively applied in skin anti-ageing because it has shown to boost: (a) skin rejuvenation through the great proportion of growth factors (i.e., platelet-derived growth factor PDGF, transforming growth factor TGF, vascular endothelial growth factor VEGF, and IGF-1), which are essential for fibroblasts' proliferation and for biosynthesis of the dermal collagen and elastine fibres; (b) thickening of epidermal layers and of dermal–epidermal junction; (c) wound healing; (d) recovery of oxidative homeostasis [22,38,53]. Regarded as second- and third-generation platelet concentrates, PRF and i-PRF have been so far applied in limited clinical trials to establish their effectiveness in skin rejuvenation [85]. BCS, also called autologous conditioned serum (ACS), is obtained by the enrichment of blood serum with anti-inflammatory cytokines, growth factors, lipid mediators, and exosomes. In a clinical study, topical application during 12–24 weeks of BCS significantly enhanced skin hydration and firmness [31,91].

In preclinical and clinical studies, stem-cells-derived exosomes (extracellular vesicles, EVs) as rejuvenation therapies have demonstrated the ability to modulate the expression of some longevity-associated genes (i.e., Klotho, FOXO3, FGF23) or collagen-related genes (COL1A1); to stimulate ECM remodelling, to increase collagen synthesis and elastin fibre density, thus enhancing dermal thickness; to modulate inflammation and to promote angiogenesis via PI3K/AKT, Notch pathways; to improve skin hydration, elasticity, tone and pigmentation (usually within 6–12 weeks) and to reduce wrinkles. Adipose-derived mesenchymal stem cell (AD-MSC) secretome or other stem-cells-derived exosomes investigated for facial skin rejuvenation have gained more effective transdermal delivery when coupled to microneedling, intradermal injections or fractional laser; moreover, these adjunctive methods have enabled an increased collagen production, ECM remodelling and more profound biological impact than topical-only applications. The choice among these delivery methods is dependent on the side effects' risks, patient preferences and costs. Stem-cells-derived exosomes are well-tolerated, with minimal erythema or petechiae from microneedling or laser. For instance, microneedling is associated with fewer reported adverse reactions (transient erythema, edema, pain and discomfort for several hours post-treatment), is cheaper and more accessible than fractional laser [94,95,96]. All these above-mentioned efficacy and tolerability data are confirmed and validated mainly from a cosmetic-dermatologic perspective. In addition, most clinical trials enrolled small cohorts (n < 60 participants), with short-term follow-up (≤12 weeks) and lacked robust controls (e.g., placebo, double-blind, randomized large size arms), therefore restricting their generalizability and statistical power of results [94,95,97]. Moreover, few clinical trials directly assess biomarkers specific for epigenetic age (e.g., DNA methylation), therefore the epigenetic profiling still remains an unmet opportunity in human skin rejuvenation trials [95,97].

The main aspects of the clinical trials on stem-cells derived exosomes (EV) as skin anti-ageing strategy are described in Table 6, comprising study design, the principal results and limitations. analyzing the preliminary results of the clinical interventions for stem-cell-based exosomes used alone or in combination with traditional methods, we can conclude that stem cell-derived EVs are the most clinically advanced biologic interventions in skin anti-ageing and rejuvenation, demonstrating improvements in skin texture, pigmentation and acne-scars' healing. Their regenerative and therapeutic potential in skin anti-ageing or skin repair is worthing further investigations in larger-scale RCTs, with better standardization of the study's design and optimized formulations. Current obstacles in regenerative therapies translation into clinic are related to tissue sources, isolation technology, production control over batches, limited large clinical trials for efficacy and potential adverse reactions, lack of standardized protocols, as well as ethical legislation [92,98,99,100,101,102,103,104,105,106,107,108].

Nutrient-sensing pathways comprising sirtuins, mTOR, IGF-1 and IGF-1 signalling (IIS) pathway, and AMPK, have a crucial role in mediating skin ageing [25,88]. Sirtuins, a family of signalling proteins essential in regulating metabolism, decline in aged subjects in parallel to reduced proliferation of dermal fibroblasts. Sirtuins have become especially activated during calorie/diet restriction and by the water-soluble form of vitamin B3—NAD. Applied topically, NAD has induced the expression of sirtuins and increased mitochondrial energetic chain, clinically reflected in improvement of skin ECM barrier and skin pigmentation [88,122].

Resveratrol at concentrations < 10 μM acts as antioxidant and senomorphic, preventing cellular senescence and diminishing SASP, either by activation of sirtuin SIRT1, PI3K-Akt signalling pathway and telomerase, or by inhibition of NF-κB pathway, depending on the cell type. At concentrations > 25 μM, resveratrol acts as pro-oxidant and stimulator of senescence and/or apoptotic cell death in many cell types. This biphasic effect seems to be dependent not only on concentrations, but also on administration schedule, cell types, animals' sex, genetic profile and diet composition [55,122,148]. Multiple sirtuin-activating compounds, especially SIRT1 activators, have proven superior stability and bioavailability than resveratrol, such as the compound SRT1720 that has 1000-fold increased potency, as well as the compound SRT2104 that is well tolerated and has good bioavailability in clinical trials for age-related diseases [58,149]. Topical creams with 0.01% capsaicin, applied for over 7 days, have stimulated the production of dermal IGF-1 through the activation of sensory neurons and the most noticeable clinical sign reported was the significant improvement of cheek skin elasticity [14,44]. Rapamycin, an FDA-approved drug that inhibits the mTOR complex, has improved both the appearance and histology of skin in numerous participants [23,62,63,122].

As skin ageing proceeds, the increasing accumulation of mitDNA mutations reduces mitochondrial integrity with significant mitochondrial fragmentation and clustering, impairs the oxidative phosphorylation in human skin fibroblasts and keratinocytes, and triggers metabolic conversion from oxidative phosphorylation to anaerobic glycolysis [24,150]. For instance, in chronological age, there are reported: an increased deletion rate of the 4977 base pair mitDNA, a marked decrease in mitochondrial membrane potential, a decrease in coenzyme Q10 (CoQ10) and also an increase in the ROS levels in fibroblasts. In photoageing skin, studies have revealed a 10-fold increased accumulation of mitDNA mutations in skin fibroblasts than in protected skin, increased levels of collagen-degrading metalloproteases and down-regulation of genes involved in collagen biosynthesis [29,151]. Antioxidants, such as: resveratrol, quercetin, vitamin C and E, curcumin, CoQ10, melatonin, epicatechins, phytoestrogens, epitalon (a synthetic tetrapeptide composed of Ala-Glu-Asp-Gly), or synthetic antioxidants nanoparticles, have been tested to alleviate mitochondrial oxidative stress [112,150]. Topical CoQ10 application have improved by 44% the mitochondrial membrane potential and skin smoothness by reducing wrinkles [31,148]. Elamipretide, a mitochondrial-targeted antioxidant, might also improve chronic wound healing [24,151].

Anti-ageing nanomedicines based on nanozymes with artificial enzymes mimicking superoxide dismutase, peroxidase, glutathione peroxidase, catalase, NADH oxidase or cytochrome c oxidase, have been developed by various structural modulations of active centres (i.e., metal oxide, noble metal, metal–organic-framework, single-atom catalysts) in order to regulate the mitochondrial respiratory chain, to generate NAD, to alleviate oxidative stress, as well as to improve structural stability, functional catalytic diversity, and to be easily and cost-effectively mass-produced [152,153]. For instance, Pd-based single-atom nanozyme Pd@PPy/GO mimicking peroxidase and glutathione peroxidase have been endowed with precise coordination metal-support solid bonds and significantly higher quantum size interactions and catalytic activity, thus making it a promising mitochondrial anti-ageing nanozyme. Moreover, C60(C(COOH)2)3 superoxide dismutase like nanozyme, as well as Au, gallic acid and isoflavone based nanozymes, delivered inside mitochondria have diminished age-related oxidative stress in mice skin, while ZnO-nanozymes decreased global DNA methyltransferase as age-related epigenetic alteration in cultured fibroblasts [153,154]. However, nanozymes are currently limited by some disadvantages in comparison to natural enzymes, such as lower substrate selectivity and specificity, potential systemic toxicity, the risk of cytotoxic byproducts, restrained simultaneous catalytic activities [152].

In Table 11, there is a comparative analysis among the newer (epi)genomic skin anti-ageing strategies and classical ones, from various perspectives: efficacy, safety, mechanism of action, evidence level in preclinical and clinical data, price range, and current limitations. (Epi)genomic-based skin anti-ageing strategies target fundamental molecular ageing processes and mechanisms and therefore they could enable more profound and long-lasting rejuvenation effects, than symptom-focused classical approaches. A comparative analysis on the efficacy and safety of the (epi)genomic-based skin anti-ageing strategies tested so far is quite difficult to be clearly assessed, because there is a great heterogeneity in studies' design and standardization concerning the intervention type, transdermal and targeted delivery methods, mechanistic and clinical endpoints and outcome measurement tools. However, tretinoin remains the gold standard with well-documented gene modulation effects, but side effects limit patient adherence, while newer genomic-based strategies are very promising, but they still lack validation through large-scale and long-term follow-up randomized controlled clinical trials [22,53,59,61,62,63,77,78,89,90,118,119,135,136,142,144,145,147,155,156,157,158,159,160,161,162,163].

Progresses in epigenomics and genomics underlying the accelerated ageing pathophysiology have deciphered and enabled new perspectives on the complexity, relationships and intricate pathways which should be addressed. Thus, various strategies specifically designed to target more accurately the main hallmarks of skin photoageing have already achieved promising results either in animal studies or even in clinical trials. In the future, some current limitations especially regarding methodological optimization, toxicological risks, or ethical issues, require further investigation and validation on larger cohorts, in order to translate all these achievements into approved strategies [28,97,163].

The design of future clinical trials would have to:✓prioritize the integration of epigenomic clock biomarkers (DNAm, Horvath's, GrimAge) as well as the multi-omics platforms (epi/genomics-transcriptomics-metabolomics-proteomics) in order to quantitatively assess epigenetic and biological age reversal;✓expand the correlation among epigenomic and genomic biomarkers, histologic assessment and clinical outcomes;✓elucidate the relationship between the dose and administration period and the (epi)genomic response;✓define the long-term safety implications of genomic modulation;✓reinforce the clinical validation of efficacy and toxicological profile by enrollment of larger and well-defined cohorts, increase the statistical power (at least over 100 participants), by application of placebo control and blinding (especially with half-face designs) in longer-term RCTs, by prolongation of the follow-up (at least 1 year) period for the evaluation of the desired and unwanted effects' persistence and possible regression [44,63,118,119,135,136,144,147,155].

Formulation challenges of the products containing epigenetic and genetic bioactive molecules would have to address physico-chemical and in vivo stability, skin penetration enhancement, as well as targeted delivery within deep dermal layers and bioavailability of genomic/ epigenomic agents by using nanotechnology, advanced carrier systems, optimized targeted genomics-informed delivery vehicles (e.g., smart hydrogels, nanoparticles, exosomes, niosomes, nanosomes delivery, etc.) and synergistic potential of combination therapies between classical and genomic/epigenomic strategies.

Future research in this field should also be dedicated to continuous development and implementation of harmonized regulatory guidelines for approval of senescence-targeting therapies for skin ageing: senolytics/senomorphics, epigenetic reprogramming, epigenetic drugs, miRNAs, modulators of nutrient-sensing pathways, and of skin microbiota [28,44,104,119,144]. New perspectives should also focus on personalization of the skin anti-ageing strategies by integration of the individual genomic and epigenomic profiles to customize/tailor skin rejuvenation therapies [119,144,147]. Future research perspectives in (epi)genomic-based skin rejuvenation are depicted in Figure 3.

Regarding translational potential and market accessibility of these emerging (epi)genomic-based skin antiageing strategies from niche, premium procedures, to widespreadly adopted ones, manufacturing feasibility and economic scalability remain the major determinants. In this respect, their cost-modelling analysis would enable an estimative stratification of them into near-term, mid-term, and long-term adoption strategies.

Among the strategies with the highest near-term adoption (≈5–10 years) likelihood could be mentioned: senotherapeutics, epigenetic modulators, and nano-delivery systems, based on their strong balance of biological rationale and clinical evidence, lower manufacturing cost, better scalability, and regulatory tractability [59,165]. Senotherapeutics show high/rapid translational and near future accessibility rate due to cost-effective production and supporting evidence in skin models, since many of them are repurposed active small molecules with already proved clinical and safety profile (e.g., topical rapamycin, peptide senotherapeutics in ex vivo skins), although their specificity for skin senescent cells remains to be further demonstrated in clinical studies without impairing wound healing. Senotherapeutics offer one of the best cost–benefit ratios, with cheap synthetic molecules (costing at scale USD 1–5 per dose; USD 5–50 per doses at administration), while peptide senomorphics cost more (USD 5–60) but still remain much more affordable than other biologics [50,165,166].

The experts consider epigenetic modulators (small molecules, topical epigenetic agents, topical NAD⁺/sirtuin-modulators, NAD⁺ boosters, HDAC inhibitors) translationally tractable in near-term/ early mainstream integration, since they are already inexpensive and scalable, and their widespread adoption is primarily gated by clinical evidence for meaningful skin rejuvenation, not by manufacturing constraints. Epigenetic small-molecule modulators are low-cost because of simple standard synthetic chemistry and mass production, typically under USD 1 manufacturing cost and administration costs below <USD 20 per topical-equivalent dose (some targeted chromatin-modifier drugs are more expensive) [51,59].

The adoption of nanotechnologies (nanocarriers for topical delivery, lipid and polymeric NP) is also expected to be rapid and broad, because they are industrially scalable (especially lipid NP) and they have already clearly proven long-term safety profiles. In general, nanotechnologies (e.g., nanoformulated antioxidants, peptides) scale efficiently and fall into mid-range pricing, depending on NP type and encapsulation [167]. NP are characterized by low–moderate overall cost barrier, high manufacturing scalability, and cost-efficiency; since the industrial-scale NP synthesis is mature and scalable, NP have modest costs per clinical dose, depending on route (topical NP $1–15/dose; injectable forms $20–150/dose) [168].

Moderate adoption is expected for microRNA therapeutics because they are more economically tractable, have moderate costs due to scalable chemical oligonucleotide synthesis (estimate administration costs USD 50–500 per dose equivalents), mature industrial NP formulation processes, and lower quality control (QC) burden. The major bottlenecks in their wide clinical adoption are target validation and optimized delivery specificity, stability and clear regulatory pathways [116,118,132,169].

Despite its promising, high biological and translational potential, the exosomes' therapy is appreciated by experts as a mid-term, slower/wider-risk adoption strategy, because of the regulatory complexity, manufacturing and standardization gaps (due to complex upstream production, stringent and costly QC requirements), and uncertain clinical evidence for many anti-ageing interventions. Exosomes have still the highest cost among the regenerative approaches, therefore they will likely expand first in well-funded esthetic or clinical trial settings. The EV's main GMP manufacturing cost drivers are expensive bioreactor culture, purification, potency testing, and donor-source dependence, as well as modest production yields; all these are reflected in very high costs estimated to USD 8000–20,000 per manufacturing batch and ~USD 150–550 per dose. In addition, the administration/clinical-level pricing is much higher (estimated to USD 300–1500 per clinical dose), since it carries a significant markup above raw production cost, including regulatory risk, marketing, clinician time, facility overhead, and perceived value (anti-ageing demand); however, these are estimative costs which vary a lot among peer-reviewed studies, industry reports and experts assumptions. Standardization of many GMP manufacturing pathways, especially of the producer cell lines and continuous purification pipelines, are compulsory to reduce costs and to convert EV from premium procedures into mass-market products. Therefore, EV's market accessibility and adoption will greatly depend on industrial process innovation than on scientific validation alone [170,171,172].

Epigenetic reprogramming, while scientifically compelling due to epigenetic clock reversal and huge skin rejuvenation potential, remains at the frontier of biomedical research as a long-term and high-impact strategy, whose translation path towards mass adoption would require several decades. Partial epigenetic reprogramming is an extremely high-cost strategy, by far the most expensive (over USD 10,000–100,000 per vector batch) and technically demanding due to GMP-grade viral or non-viral vector engineering, major safety constraints and intensive off-target risk monitoring, and regulatory/ethical hurdles. Its adoption will be slow, restricted, and likely confined to specialized medical centres for the foreseeable future [25,38,84,114].

The above data sustaining the stratification of the emerging (epi)genomic-based skin rejuvenation strategies according to their widespread accessibility and market adoption are synthesized in Table 12.

The accessibility and widespread adoption of the emerging (epi)genomic-based skin rejuvenation strategies is also illustrated in Figure 4 as a heatmap in which to each strategy is assigned an estimative comparative score by integrating expert-opinions and evidence-based reports regarding their production complexity, clinical progress, regulatory status, sample stability, known and predicted costs from scalable manufacturing and administration prices.