Stem cells are undifferentiated cells with the ability to self-replicate, self-renewal, homing and plasticity potential. They can differentiate into various cell types, such as nerve cells, cardiomyocytes, liver cells and skin cells (Jin et al., 2023). They possess anti-inflammatory properties, promote epithelial cell proliferation, inhibit wound scarring, maintain homeostasis, repair tissue injuries, and accelerate healing (Jin et al., 2023; Khandpur et al., 2021; Zhang and Huang, 2023). Stem cells also secrete bioactive molecules, including growth factors, cytokines, chemokines and exosomes, which are responsible for the paracrine effects of these cells in bone tissue and nervous system regeneration, as well as in wound healing and endothelial cells (Tran et al., 2023; Wu et al., 2024).

There are different sources for using stem cells in regenerative medicine classified according to their differentiation potential (Khandpur et al., 2021), including embryonic stem cells (ESCs), umbilical cord mesenchymal stem cells (UCMSCs), induced pluripotent stem cells (iPSCs), MSCs, ADSCs and bone marrow mesenchymal stem cells (BMMSC) (Jin et al., 2023; Semsarzadeh and Khetarpal, 2022; Tran et al., 2023).

MSCs are originated from the mesoderm and ectoderm, and can differentiate into various cell types, such as osteocytes, chondrocytes, adipocytes, neurons and endothelial cells (Ma et al., 2023; Tran et al., 2023). Additionally, MSCs can be found in bone marrow, adipose tissue, synovial tissue, muscle, lung, liver, and umbilical cord blood (Khandpur et al., 2021; Huynh et al., 2022; Jin et al., 2023). MSCs play a pivotal role in wound healing and exhibit additional functions including immunomodulation, anti-inflammatory and anti-apoptotic effects, and pro-angiogenic activity (Khandpur et al., 2021; Wu et al., 2024).

Currently, there are several therapies using MSCs for the treatment of graft-versus-host disease, bone defects, ischemic heart failure, burns, autoimmune-related skin conditions, and diabetic foot (Jin et al., 2023; Wu et al., 2024; Farabi et al., 2024). Since MSCs can differentiate into skin cells such as keratinocytes, fibroblasts, and endothelial cells, they can be used to enhance the healing process. This is not only due to their cell differentiation capabilities but also because of their crosstalk with macrophages, which play a role in the wound repair process (Wu et al., 2024).

MSCs can be applied to burn injuries by inhibiting cellular inflammation through the release of anti-inflammatory cytokines. Additionally, MSCs exert paracrine effects that polarize macrophages from an inflammatory phenotype (M1) to a wound-healing phenotype (M2), promoting tissue repair and clearance (Ma et al., 2023; Wu et al., 2024; Zhang and Huang, 2023). Moreover, they stimulate the formation of new blood vessels, which results in increased blood perfusion and the delivery of nutrients to affected areas, thereby accelerating the healing process (Jin et al., 2023; Semsarzadeh and Khetarpal, 2022). On top of that, MSCs improve the wound structure by secreting proteins that make up the extracellular matrix (ECM), such as collagen, elastin and fibronectin, enhancing the reconstruction of the dermis (Mazini et al., 2020; Tran et al., 2023; Wu et al., 2024).

Beyond that, bone marrow-derived MSCs can be used for scars reduction, anti-aging, and systemic sclerosis (Tran et al., 2023). Conget et al. (2010) conducted a study in which they used these cells to treat two patients with severe generalized recessive dystrophic epidermolysis bullosa (EB). After 12 weeks of intradermal treatment, the ulcers healed completely, however this effect only lasted 4 months.

Another type of stem cells are the iPSCs. They are capable of differentiating into more cell types compared to MSCs, and they have the ability for unlimited self-renewal (Zhang and Huang, 2023). Studies show that these cells promote angiogenesis, perfusion, collagen deposition, and accelerate the natural healing process in murine models (Clayton et al., 2018; Farabi et al., 2024).

Besides MSCs and IPSCs, ADSCs are another promising cell type for regenerative therapy. ADSCs have shown potential applications in dermatology and aesthetics, including scar reduction, anti-aging, wrinkle reduction, and hair loss treatment (Khandpur et al., 2021; Suh et al., 2019; Tran et al., 2023). Anderi et al. (2018) injected ADSCs, derived from liposuction, into 20 patients with alopecia areata, and after 6 months of follow-up, they observed a statistically significant difference in hair growth rates among all treated patients. Indeed, Zhang et al. (2014) evaluated the antioxidant effects of ADSCs in a mouse model and found that, after 28 days of injection, ADSCs reversed the aging phenotype, increased dermal thickness and collagen content, and enhanced skin vascular density. In addition, Chen et al. (2020) showed that ADSCs were effective in improving the appearance of the skin, particularly in reducing wrinkles in UV-damaged skin. These results highlight the paracrine effects of ADSCs in promoting skin rejuvenation.

Interestingly, Li et al. (2016) used the conditioned medium from ADSCs (ADSC-CM) to evaluate its effect on hypertrophic scars ex vivo, injecting them into rats. Through Western blotting, they analyzed key proteins involved in healing, such as collagen I, collagen III, and α-SMA (alpha-smooth muscle actin). The results showed that the use of ADSC-CM reduced collagen deposition in hypertrophic scar tissues and improved fibrosis in these tissues.

Not only ADSC-CM, but also conditioned medium from MSCs (MSC-CM) have been investigated to be used on regenerative medicine because it contains anti-apoptotic, anti-inflammatory and anti-aging substances. Because of that, there are pre-clinical studies using MSC-CM to show whether it influence on lung progenitor cell development or its paracrine effect influences tissue regeneration (Smolinská et al., 2023).

Yet another ADSCs derivative, nano-fat, obtained through a multi-step process involving mechanical digestion and filtration of fat tissue, has demonstrated several benefits, including reduced scar size, improved skin color, and enhanced overall skin quality (Suh et al., 2019; Hajimortezayi et al., 2024).

In addition, UCMSCs can also be used in regenerative medicine, having advantages over other types of stem cells because they are abundant, easy to collect, cause no harm to donors, have low immunogenicity, and high differentiation capacity (Jin et al., 2023; Li et al., 2024). They can be used for the treatment of cardiovascular diseases, liver diseases, degenerative muscle diseases, and autoimmune diseases (Wu et al., 2024). They can also be used for treating burns and psoriasis vulgaris (Tran et al., 2023) and for treating chronic wounds, facial and body rejuvenation, even combination therapies with other biomaterials (Li et al., 2024).

The Stromal Vascular Fraction (SVF) is isolated from adipose tissue and contains various cell types, such as MSCs, endothelial cells, stromal cells, and immune cells (Surowiecka and Strużyna, 2022). For example, in a pilot study, SVF was used for the treatment of alopecia, where a significant increase in hair growth in patients was observed (Semsarzadeh and Khetarpal, 2022).

While various stem cell types exist, extensive preclinical and clinical research suggests that mesenchymal stem cells (MSCs) and MSC-based products offer the most promising balance of safety and efficacy, with a low risk of tumor formation and minimal immune rejection (Hoang et al., 2022; Farabi et al., 2024). Despite their widespread use, MSCs have limitations, such as a decline in viability and activity with age (Wu et al., 2024).

PRP was first used in 1954 to improve wound healing in dentistry (Kingsley, 1954). Since its inception, the application of PRP has grown significantly, becoming a valuable tool in tissue repair and regeneration across various medical fields. (Cecerska-Heryć et al., 2022). The treatment is based on injections of the patient’s own platelets highly concentrated in plasma and separated from other blood components by centrifugation cycles (Samadi et al., 2019; Pixley et al., 2023).

The peripheral blood contains at least five times fewer platelets than PRP. This increased platelet concentration in PRP enhances its potential for wound healing (Davies and Miron, 2024). Platelets are involved in a wide range of growth factors, proteins, cytokines and other biological agents that have effects in processes like cellular migration, proliferation, differentiation, angiogenesis, tissue regeneration and collagen synthesis (Pincelli et al., 2024). The variety of molecules derived from PRP enables efficient wound healing, as the healing process involves multiple molecules and complex pathways (Samadi et al., 2019; Davies and Miron, 2024).

Platelet-derived molecules could be secreted by α-granules, dense granules and lysosomes. Platelet derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), EGF, TGF-β, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are secreted from α-granules, in addition to adhesive proteins, coagulation factors, angiogenic regulators, cytokines and exosomes. They are the more abundant secretory granules and are responsible for releasing the greatest number of molecules with a direct effect on wound healing (Cecerska-Heryć et al., 2022; Pincelli et al., 2024). On the other hand, dense granules contribute to platelet activation and subsequent release of α-granules constituents, in which it has been shown that PRP lysosomes functions participate in antimicrobial activity and the degradation of extracellular matrices (Everts et al., 2024).

The composition of PRP can vary depending on the preparation. It can be categorized as pure (P-PRP), leukocyte-poor (LP-PRP), leukocyte-rich (LR-PRP) or platelet-rich fibrin (PRF), considering the centrifugation time, speed and the presence or absence of non-autologous anticoagulants (Karimi and Rockwall, 2019; Everts et al., 2024). PRF specifically has gained prominence in regenerative medicine in recent years due to its benefits, such as the release of platelet-related therapeutic granules over a longer period and at a slower rate than PRP (Narayanaswamy et al., 2023). In this way, PRF also offers a valuable adjunct to both surgical and non-surgical interventions, demonstrating great potential for enhancing treatment outcomes.

PRP treatment offers several advantages, including low cost, ease of preparation, versatility, and safety. However, further research is necessary to standardize preparation procedures and establish regulations regarding the composition of PRP injectables. Additionally, the efficacy of PRP and its various categories remains uncertain in relation to specific diseases and clinical conditions (Everts et al., 2020; Cecerska-Heryć et al., 2022; Pincelli et al., 2024).

Micro-vesicles, currently known as exosomes, were first identified in 1983. Initially, exosomes were considered just a cell waste, however, later research revealed that exosomes play important roles in cell communication and signal transduction. As an alternative for cell-based therapies, exosomes emerged as potential tools for treating skin’s conditions, such as improving wound healing and even skin rejuvenation. Since exosomes are small vesicles secreted by different cells, there are many possibilities for their isolation and use as an effective carrier for bioactive compounds or genetic material. Exosomes not only transport and protect molecules from degradation, but also exhibit biocompatibility, reducing the risk of immune reactions and tumorigenesis. In this way, clinical application of exosomes is a promising avenue for free-cell therapies (Hajialiasgary Najafabadi et al., 2024; Peña and Martin, 2024; Quan, 2023; Sonbhadra and Pandey, 2023; Zhou et al., 2023).

Exosomes are extracellular vesicles, approximately 30–200 nm in size, which can be obtained from cell culture and that function as membrane-bound carriers of biomolecules and metabolites, reflecting their cellular origin. These vesicles, spherical in solution, exhibit a lipid bilayer structure, which decreases the risks of immune responses and provides protection of their cargo from degradation. Furthermore, exosomes pose a low risk of uncontrolled cell proliferation and differentiation, minimizing concerns related to tumorigenicity. Their ability to carry and deliver bioactive substances to cells makes exosome-based therapies a promising possibility for therapeutic applications like various skin conditions, including psoriasis, atopic dermatitis and vitiligo, as well as for promoting skin regeneration, such as in diabetic wound healing, hypertrophic scarring and keloid formation, and even for addressing skin aging (Gurung et al., 2021; Hajialiasgary Najafabadi et al., 2024; Yu et al., 2024).

Exosomes are ubiquitous in various body fluids, including serum, saliva, milk, cerebrospinal fluid, urine and semen. Among these, stem cell-derived exosomes have been extensively investigated for their potential role in mediating the biological effects of paracrine factors, particularly in wound healing (Zhou et al., 2023).

Tissue repair is a complex process involving initial clot formation, followed by inflammatory cell signaling, cell proliferation and remodeling. These phases overlap, each with its own purpose and time before the next starts. Mesenchymal stem cells exosomes (MSC-exos) derived from different tissues, like ADSC, hold a promise for cutaneous repair due to their ability to stimulate fibroblast activity. In the dermis, fibroblasts play a pivotal role in wound healing through collagen synthesis, making MSC-exos, which enhance fibroblast function, valuable contributors to the healing microenvironment and the promotion of wound repair. ADSC-derived exosomes demonstrate significant potential for treating diabetic wounds due to their ability to induce collagen I and III production in the early stages of healing, which can help prevent scar and keloid formation (Song et al., 2023; Zhou et al., 2023; Peña and Martin, 2024).

Data from an ECM loaded with ADSC-exosomes indicate that this combination is also a possible therapeutic approach for both normal and pathological wound healing. The results demonstrated that ECM-loaded exosomes promoted increased cell growth, cell migration, collagen deposition, and decreased inflammation in vivo (Song et al., 2023).

Moreover, given their role in wound healing, stem cell-derived exosomes could potentially be used to treat skin photoaging, a condition marked by uneven pigmentation and wrinkles (Hajialiasgary Najafabadi et al., 2024).

Park et al. (2023) demonstrated that exosomes derived from human foreskin fibroblasts (BJ-5ta Exo) can mitigate oxidative stress by upregulating the expression of antioxidant genes CAT, SOD-1, SOD-2, and GPX. Additionally, BJ-5ta Exo promoted a decrease in programmed cell death and cell cycle arrest.

In addition, it was suggested that exosomes obtained from 3D culture of human dermal fibroblasts (HDFs) may be able to promote collagen synthesis and reduce skin inflammation. Data also revealed that 3D-HDF-exos, through tumor necrosis factor alpha (TNF-α) downregulation and TGF-β upregulation, promoted a procollagen type I increase while reducing matrix metalloproteinase-1 (MMP-1) expression. Given the age-related decline in HDF collagen production and repair, coupled with increased MMP-mediated ECM degradation, these findings suggest that exosomes may possess anti-aging properties (Xiong et al., 2021).

Given the abundance of exosomes in bovine milk, recent studies have explored the potential of milk-derived exosomes (MK-exo) as novel anti-aging compounds. These investigations have revealed that MK-exo can stimulate the expression of filaggrin and CD44 in keratinocytes, as well as hyaluronidase levels in fibroblasts. Furthermore, MK-exo protected collagen biosynthesis from UV-induced damage. Notably, these exosomes also stimulated increased cell migration rates in fibroblasts (Lu et al., 2024).

However, while exosomes demonstrate significant promise as therapeutic agents, several challenges must be addressed for their clinical application. These include standardizing isolation and analysis, ensuring safety and purity, and preserving exosome activity during preparation and storage (Lu et al., 2024; Rezaie et al., 2022).

Despite above stated challenges, exosomes, being versatile carriers of biomolecules, offer promise for clinical applications. Their biocompatibility, low risk of immune responses and tumorigenicity make them attractive candidates for treating diseases. In the context of skin, their potential to influence collagen synthesis and inflammation suggests their value for wound healing and skin rejuvenation. Future research should focus on exploring these possibilities and ensuring the safety of exosomes for medical use.

Dermal fillers, also known as facial fillers, are soft, gel-like substances injected beneath the skin. Over time, the loss of soft tissue volume, fat redistribution, reduced skin elasticity, and thickness contribute to the formation of wrinkles and folds characteristic of aging. In this context, injectable fillers are an option for treating wrinkles, scars, folds, and areas under the skin lacking volume (Callan et al., 2013; Maio, 2018; Colon et al., 2023; Faris, 2024).

The principle behind the use of these products is based on strengthening the ECM in the dermal layer. They are made of various low-crosslinking polymeric ingredients with different effects on the skin (Yi et al., 2024). Among the most used ingredients are hyaluronic acid, poly-L-lactic acid, polymethylmethacrylate, and calcium hydroxyapatite (CaHA) (Colon et al., 2023).

Hyaluronic acid is the most commonly used dermal filler, naturally found in the skin, being safe and effective for use (Yi et al., 2024). Poly-L-lactic acid is synthetic, acting as a collagen stimulator, recommended for softening lines and treating wrinkles (Ao et al., 2024). Similarly, according to the American Board of Cosmetic Surgery (2022), polymethylmethacrylate comprises synthetic microspheres that remain under the skin indefinitely, providing continuous support and containing collagen in their composition. In addition, CaHA is a natural substance found in bones that helps stimulate the skin’s natural collagen production and is recommended for treating deeper lines.

Focusing on hyaluronic acid fillers due to their widespread use, it is the most abundant glycosaminoglycan found in the human dermis, contributing to tissue hydration and volume, as well as providing structural support (Ballin et al., 2015; Wongprasert et al., 2022). A recent study by Chen et al. (2023) demonstrated the anti-aging ability of hyaluronic acid fillers by inhibiting the expression of MMP-1, promoting collagen accumulation, and increasing the expression of dermo-epidermal junction proteins.

One of the most significant advantages of hyaluronic acid fillers is that they can be easily removed by injecting hyaluronidase (Wongprasert et al., 2022). However, some limitations persist in their use, which can sometimes extend to other types of fillers, such as the duration of the compounds, discomfort caused by the injections, and the ability to achieve precise delivery of the fillers to the intended location and skin layer (Colon et al., 2023).

Growth factors are polypeptides or proteins that regulate physiological processes in and between cells. They are naturally found in the skin, secreted by various cell types in this tissue. Many growth factors are involved in wound healing, both acute and chronic, and are some of the most important signalers during this process (Pamela, 2018; Yamakawa and Hayashida, 2019; Vaidyanathan, 2021). Furthermore, the processes of aging and wound healing naturally stimulate the release of growth factors, which affect critical biochemical repair pathways in the dermal matrix and have inspired the use of these factors to improve skin appearance (Kremer and Burkemper, 2024).

Some of the growth factors used in skin treatment are EGF, FGF, hepatocyte growth factor (HGF), and IGF-1, which can be used in combination (Vaidyanathan, 2021; Yamakawa and Hayashida, 2019). They can come from various sources, including human and non-human cell cultures and recombinant sources (Quinlan et al., 2023).

Treatments involving the use of different growth factors have been reported to improve wrinkles, skin texture, photo-damage, and the overall appearance of facial skin (Quinlan et al., 2023). In addition to cosmetic use, human EGF (hEGF) is also applied in regenerative medicine for the treatment of alopecia, dermatitis following chemotherapy, burns, diabetic foot ulcers, and post-surgical ulcers (Kong and Hong, 2013; Kim et al., 2017; Esquirol-Caussa and Herrero-Vila, 2019; Jeon et al., 2019; Kahraman et al., 2019; Lou, 2021; Vaidyanathan, 2021).

The use of EGF for aesthetic purposes also shows broad applications, presenting a regenerative effect in the aging skin process by promoting the migration of aged fibroblasts and increasing the synthesis of hyaluronic acid and collagen in this tissue (Kim et al., 2015; Miller-Kobisher et al., 2021; Vaidyanathan, 2021). Growth factors can also be combined with other treatments for apparent skin rejuvenation, such as lasers, microneedling, radiofrequency, or chemical peels, to amplify results or improve side effects from these treatments (Quinlan et al., 2023). However, in recent years, the development of cell-penetrating peptides associated with growth factors has improved the topical application of these factors, increasing their ability to penetrate the skin and epithelial cells (Chen et al., 2017; Choi et al., 2018; Jeon et al., 2019, Lee et al., 2020, Xie et al., 2020.

Aiming to correct genetic defects and thereby prevent or cure genetic disorders, gene therapy has been applied to skin regenerative medicine. All types of skin diseases are candidates for gene therapy, from inflammatory diseases to skin cancers and genodermatoses (Ain et al., 2021), as well as more aesthetic aspects like skin regeneration and scar and keloid treatment (Hosseinkhani et al., 2023; Luo et al., 2023).

Gene therapy technologies include the use of messenger RNA, silencing RNA, antisense oligonucleotides (AON), plasmid DNA, minicircle DNA, mini-string DNA, and CRISPR/Cas9 technology (Wan et al., 2021; Guri-Lamce et al., 2024; Tenchov et al., 2024), which must have specific action on the desired area of the skin, representing one of the current challenges in this type of therapy.

Currently, there are three main methods of delivering gene therapies to the skin: viral delivery, nanoparticles and physical methods. In this perspective, viral delivery is the most used and effective method (Picanço-Castro et al., 2020), though it presents challenges such as limitations in efficacy due to pre-existing immunity, the inability to redose, and genome integration capacity, which increases the risk of unwanted insertional mutagenesis. Additionally, there are size limitations for the genetic cargo (Anzalone et al., 2019; Ain et al., 2021). Lipid-based nanoparticles, a more recent and promising method (Guri-Lamce et al., 2024); polymeric nanoparticles, capable of associating with negatively charged genetic cargoes and forming spherical complexes, but which still present high toxicity related to particle size (Blakney et al., 2020); and physical methods, such as electroporation, ultrasound application, or microneedles (Wan et al., 2021), which have limitations for clinical application, such as limited cargo capacity and challenges with whole-body administration (Ain et al., 2021).

Other substances can be incorporated into skin care products, playing an active role in tissue regeneration or inflammation by improving ECM synthesis or inhibiting its degradation, neutralizing free reactive species, or reducing proinflammatory factors (Makpol et al., 2013; Wang H. et al., 2021; Torres et al., 2023). Among these substances, zinc-based compounds stand out. This metal has been shown to reduce inflammation and the risk of infections, being involved in cell proliferation and migration and collagen synthesis, thus promoting epithelialization (Lin et al., 2017; Chen et al., 2022; Pino et al., 2023). Other substances primarily act as moisturizers, provided by ingredients that increase the synthesis of structural skin lipids, directly restoring the skin barrier, such as vitamin A, or hygroscopic substances like dexpanthenol, which bind to and retain water in the stratum corneum (Liu, 2022). Other substances are lipids, oils, and fatty acids, known as emollients, such as petrolatum derivatives, which also promote hydration by directly replacing missing fatty acids in the tissue (Elias, 2022; Torres et al., 2023).

Smart dressings were developed to enhance wound care management based on the injury type and patient conditions (Raju et al., 2022). Multifunctional wound dressings promote wound healing by encapsulating bioactive substances, sustaining the release of medicines by stimuli-responsive technology (Raju et al., 2022). Biopolymers such as alginate, chitosan, polyvinyl alcohol (PVA), and collagen are increasingly used to create innovative wound dressings due to their cost-effectiveness and eco-friendliness. Among these, alginate dressings are the most popular because they promote skin regeneration, accelerate wound closure, minimize scarring, absorb exudates effectively, and are biocompatible. Alginate’s gelling properties and stability in warm environments make it ideal for various applications, including hydrogels, nanofibers, dermal patches, films, and foams (Nqoro et al., 2022).

Other than that, hydrocolloids are effective in wound care because they can absorb significant amounts of wound fluid and are impermeable to water vapor, creating a moist healing environment. They also block oxygen, which speeds up epithelialization and collagen synthesis while lowering the pH to reduce bacterial growth (Nguyen et al., 2023). Nonetheless, new smart hydrogel wound dressings with embedded sensors have been rapidly developed to monitor wound conditions. Notable examples include flexible pH-sensing alginate-based hydrogel fibers for skin wounds and PVA/xyloglucan (PVA/XG) hydrogel membranes that absorb exudate and release biological factors (Tamayol et al., 2016; Ajovalasit et al., 2018; Tavakoli and Klar, 2020).

Although high-end wound dressings have been developed in recent years, the products face several limitations, including a complicated production process, inadequate quality assurance for biological materials and questions about the effectiveness of their components for widespread use. In addition to that, more trials and experiments are needed to assess the true effectiveness of these advanced dressings in wound healing (Nguyen et al., 2023).

Studies in the last 20 years have involved the use of different skin cell cultures (mainly keratinocytes), umbilical cord mesenchymal stem cells differentiated to keratinocytes, or co-culture of skin cells with other cell types, including immune cells and dermal fibroblasts, in a two-dimensional (2D) monolayer (Abaci et al., 2017; Santos et al., 2023), to study the signaling pathways of skin diseases such as psoriasis or melanoma, wound healing, also to test the efficacy of safety treatments (Karras and Kunz, 2024). However, 2D models do not often represent a sufficient level of complexity to assess the various cell-cell and cell-extracellular matrix interactions, as well as oxygen and nutrient gradients (Loke et al., 2021; Santos et al., 2023).

On the other hand, it is possible to better understand most complex skin diseases in the three-dimensional (3D) microenvironment by involving additional cell lineages, such as immune cells or skin appendages as innervation type, to generate more effective in vitro skin models, using spheroids or skin constructs, for example, to improve skin replacement therapy (Abaci et al., 2017; Karras and Kunz, 2024; Loke et al., 2021).

The in vivo model can be better mimicked in spheroid cultures compared to 2D models, representing a more complex tissue architecture, with increased cell-cell contacts and heterogeneous cell growth. In addition, spheroids have been used for semi-high-throughput drug screening, as well as being used in co-culture models to evaluate different tissue responses under paracrine stimulation (Raghavan et al., 2016; Loke et al., 2021; Karras and Kunz, 2024).

The application of spheroid cultures in skin models can be generated from cell aggregates of fibroblast or keratinocyte lineages, for example, under non-adherent conditions, by so-called spinner culture, hanging drop, magnetic levitation or gel incorporation (Abaci et al., 2017; Klicks et al., 2019; Schäfer et al., 2021; Ohguro et al., 2024). To improve the formation of the spheroids, it is important to choose an ECM based on biomaterials, such as hydrogels or collagens, for the architecture of the dermal matrix (Enyedi et al., 2023; Santos et al., 2023).

Some methods for evaluating the different cell configurations in spheroids still remain limited, but the main ones based on microscopy, such as phase contrast microscopy, are used to analyze the size and shape of spheroids. Other methods, such as cell surface staining and flow cytometry, are used to analyze the presence of specific molecules. Cryosectioning, after fixation in formalin and embedding in paraffin, is used for a deeper view of the sectioned spheroid (Filipiak-Duliban et al., 2022; Habanjar et al., 2021; Karras and Kunz, 2024).

Due to the cell aggregate structure, spheroid nuclei are exposed to low oxygen conditions, as well as limited access to nutrients and metabolites, which can lead to an increase in apoptotic cells (Karras and Kunz, 2024; Loke et al., 2021). On the other hand, the cells on the periphery are proliferative, due to the availability of oxygen and nutrients. Interestingly, the middle layer contains quiescent and senescent cells, and as a result, this spheroid configuration becomes a suitable model for testing pathophysiological conditions (Karras and Kunz, 2024; Loke et al., 2021; Ohguro et al., 2023).

Another model of organotypic cultures are the so-called “raft” cultures, also known as skin reconstructs. Cells are established in a manner as to allow the stratified epithelium and the dermal component of the skin, to be reconstituted in a tissue culture environment. Keratinocytes, for example, are seeded in a dermal equivalent containing fibroblast and, when raised to the air-liquid interface, reproduce the process of stratification and terminal differentiation of keratinocyte (Klicks et al., 2019; Santos et al., 2023). Histological analysis of these skin reconstructs shows the similarity and tissue organization to human skin, with a cornified epidermal-equivalent appearing on top of a dermal, containing human fibroblasts (Klicks et al., 2019; Loke et al., 2021; Santos et al., 2023).

This skin reconstruct is a useful system for testing pharmacological dynamics, efficacy tests, analysis of absorption by different forms of administration, or for preclinical screening of drugs and cosmetics (Torre et al., 2020; Portugal-Cohen et al., 2023; Suthar et al., 2024). This model takes less time to obtain results and is less expensive than performing experiments using animals (Abaci et al., 2017; Karras and Kunz, 2024). Therefore, it has been emphasized in recent years that organotypic cultures for skin reconstructions can also be obtained using the bioprinting technique, in order to construct highly reproducible dermal equivalents, with architecture similar to the in vivo (Cubo et al., 2016; Fernandes et al., 2022; Bian et al., 2024), to be widely used in regenerative medicine or in strategies for testing immunotherapy (Ao et al., 2022; Santos et al., 2023).

Tissue bioengineering has been expanding as a new strategy by employing advanced techniques of bioprinting, biopolymer engineering, stem cell research and nanomedicine (Augustine, 2018; Pasierb et al., 2022; Wei et al., 2024). Bioprinting has attracted attention as a promising technique, in which the technology aims to generate, precisely, a controlled and organized complex with similar architectures of native tissues (Loukelis et al., 2024; Bian et al., 2024). Bioprinting has been used to generate tissues and transplants, including skin and its multilayers, tracheal splints, cardiac tissue and cartilaginous textures (Kaur et al., 2019; Miguel et al., 2019; Bian et al., 2024).

In fact, bioprinting technique has the potential to revolutionize contemporary regenerative medicine, considering that by taking advantage of tissue regeneration techniques. Using approaches that facilitates the production of skin and consequently its use in cases of wound closure, it is also possible within this model to mimic characteristic inflammatory profiles, in order to study drug-related toxicity or investigate the pathological mechanism of some skin diseases, including psoriasis and atopic dermatitis (Randall et al., 2018; Lorthois et al., 2019; Derr et al., 2019; Liu et al., 2020; Deepa and Bhatt, 2024).

The simultaneous incorporation of different cells into the bioprint, including fibroblasts and melanocytes in dermal equivalents, makes it possible to study the impact of UV radiation (Pasierb et al., 2022). Compared to machining prototypes, bioprinting makes skin production cheaper and faster, as the prototype can be finished in hours, allowing the process to be efficient, even with design modifications in production. The manufacturing process can also reduce material costs, as it uses only the amount of material needed for the prototype itself, minimizing or eliminating waste (Wang H. et al., 2021; Wang Z. et al., 2021).

Other advantages of using the bioprinting technique include: 1) customization of the skin to be used. Depending on the shape and depth of the wound surface, imaging technology using computer digitization can quickly print the skin graft compatible with the wound. Indeed, this technique confers the characteristics of punctuality, high flow and high repeatability (Weng et al., 2021). 2) the use of bioinks, which can be deposited flexibly and precisely with different biological agents, including living cells, nucleic acids, growth factors, among others, is usually required to help build skin structures (Zhu et al., 2016; Wei et al., 2024; Loukelis et al., 2024). According to the different printing materials, three different techniques of bioprinting can be mentioned, such as: droplet-based bioprinting (DBB), laser-assisted bioprinting (LAB) and extrusion-based bioprinting (EBB) (Gudapati et al., 2016; Weng et al., 2021; Kang et al., 2022).

DBB technique includes the drop-by-drop mode. In this model, drops of biomaterial on a substrate are deposited when necessary. DBB-based bioprinters are suitable for deposition and patterning of materials, due to their high precision and minimal biomaterial waste. In addition, DBB mainly uses piezoelectric, thermal or electrostatic forces to generate droplets, which can precisely deposit the biomaterial to make a spatially heterogeneous tissue structure (Gudapati et al., 2016; Matai et al., 2020; Kang et al., 2022). Its non-contact printing mode is more suitable for biological printing directly onto a wound, for example,. Some studies have used the DBB technique to print human keratinocytes and fibroblasts directly onto dermal wounds on the backs of mice. Compared to the control group without any biological dressing, the skin grafts in the experimental group promoted wound healing (Gudapati et al., 2016; Matai et al., 2020; Wang Z. et al., 2021). On the other hand, DBB has some limitations. Its inkjet injector is small, measuring up to 150 μm, which can be easily blocked by biomaterials. Only low-viscosity hydrogels or other low-concentration biological agents can be used (Wang H. et al., 2021).

LAB technique consists of the emission of laser light, that is focused on the metal film on the back of the silicate glass and heated locally, so that the bioink deposited on the equipment evaporates and is sprayed onto the substrate in the form of liquid drops (Matai et al., 2020; Wang Z. et al., 2021). The LAB technique mainly uses a nanosecond laser with ultraviolet wavelengths as an energy source, and its printing resolution can reach the picogram level, performing bioprinting without direct contact with the substrate and can print cells with high resolution. (Zhang et al., 2023). However, LAB does not have a suitable rapid gelling mechanism yet, which limits its ability to produce high-performance prints (Wang H. et al., 2021; Zhang et al., 2023).

EBB technique makes controllable impressions using fluid distribution systems and automated machines. Under the control of a computer, the biomaterial passes through a catheter, using pneumatic, piston or screw approaches (Zhang et al., 2023). Hydrogels better perform in bioprinting by pneumatic extrusion, because it is kept in this material the profile of printed filaments, after extrusion. The screw-driven structure can bioprint biomaterial at high viscosity, which is conducive to producing a more stable bioprinted tissue (Zieliński et al., 2023). In addition, extrusion bioprinting can print a porous grid structure, promote the circulation of nutrients and metabolites, which allows better control over porosity, shape and distribution of cells in the printed prototype (Pasierb et al., 2022; Zhang et al., 2023). Compared to DBB and LAB models, the advantages of EBB include faster bioprinting speed, more usable bioink types (including cell clusters, high-viscosity hydrogels, microcarriers and cell matrix components) (Pasierb et al., 2022; Zhang et al., 2023), more versatility and suitability for manufacturing prosthetic implants for tissue bioengineering. However, the limitation of this technique is that it has a lower resolution of at least 100 µm (Zhang et al., 2023; Zieliński et al., 2023).

In general, different approaches in bioprinting techniques are used, in order to allow specialists to acquire more precision and high resolution in the regeneration of the skin and its appendages, including hair follicles, sebaceous and sweat glands. The same approaches might be used on selecting between the different biomaterials, which make the skin cell lineages remain highly viable and metabolically active, to keep the accuracy in replicating the tissue layers and to not compromise functionality (Weng et al., 2021).

Biomaterials can be of natural, synthetic, or a combination of both origins and are of great interest in tissue engineering due to their properties of biocompatibility, biodegradability, promoting cell adhesion and migration in scaffolds, potential resemblance the extracellular matrix, and presenting controllable properties and architecture (Chaudhari et al., 2016; Liu et al., 2023).

Natural biomaterials are primarily derived from proteins, such as collagen and spider silk, for example, (Liu et al., 2023), but can also originate from carbohydrates, such as alginate (Farshidfar et al., 2023).

Firstly, collagen is a protein that contains triple helices capable of forming strong and stable fibers through cross-linking. This stability can be used to form scaffolds that resemble the ECM of living tissues (Chattopadhyay and Raines, 2014; Amirrah et al., 2022; Zhu et al., 2022). Thus, in regenerative medicine, collagen as a biomaterial can be used as a wound dressing, promoting healing, or as a supplement for the skin, improving aspects such as elasticity and hydration (Ghomi et al., 2021).

Spider silk, as a natural biomaterial, is explored for its biocompatibility and low density. Because it is difficult to cultivate directly from arachnids, this protein has been produced recombinantly for the construction of scaffolds for cell culture. Using these supports, the regeneration of bone, cartilage, muscle, nerve, and epidermal tissues, especially in burn patients, has been studied. (Salehi et al., 2020).

It is important to highlight that some materials have limited properties, such as alginate, which has low stability due to its chemical properties, and polydopamine, which has low hydrogel-forming capacity. However, they can be used in combination with other materials, such as hydroxyapatite, chitosan, gelatin, and collagen, to achieve results and applications in tissue engineering. Thus, in combination, they can be used for bone tissue repair, corneal reconstruction, wound healing and covering, and even in drug delivery systems (Farshidfar et al., 2023; Yazdi et al., 2022).

Another way to apply the biomaterials, in general, is using as hydrogels, which provide a moist environment with the ability to retain proteins, growth factors, and nutrients within the gel structure and release these molecules into the medium (Berthiaume et al., 2011; Lei et al., 2022). Due to technological advancements in tissue engineering, it has become possible to work on an increasingly smaller scale of these gels, creating nanogels which can reach smaller and more internal wounds than hydrogels, ensuring drug release in the region, facilitating wound healing and tissue regeneration (Grimaudo et al., 2019; Brianna et al., 2024). In addition to hydrogels, there are also nanomaterials, which can be made of a single chemical element, such as silver or gold, that possess antimicrobial characteristics, can stimulate cell growth and can be delivered in systems along with nanogels (Bellu et al., 2021). The field of nanostructures in regenerative medicine and tissue engineering is still a novelty and shows extreme promise with ongoing research advancements.