Elastogenesis in Focus: Navigating Elastic Fibers Synthesis for Advanced Dermal Biomaterial Formulation

Abstract

Elastin, a fibrous extracellular matrix (ECM) protein, is the main component of elastic fibers that are involved in tissues’ elasticity and resilience, enabling them to undergo reversible extensibility and to endure repetitive mechanical stress. After wounding, it is challenging to regenerate elastic fibers and biomaterials developed thus far have struggled to induce its biosynthesis. This review provides a comprehensive summary of elastic fibers synthesis at the cellular level and its implications for biomaterial formulation, with a particular focus on dermal substitutes. The review delves into the intricate process of elastogenesis by cells and investigates potential triggers for elastogenesis encompassing elastin‐related compounds, ECM components, and other molecules for their potential role in inducing elastin formation. Understanding of the elastogenic processes is essential for developing biomaterials that trigger not only the synthesis of the elastin protein, but also the formation of a functional and branched elastic fiber network.

Elastin and elastic fibers are essential components that provide elasticity and resilience to various tissues in the human body, including the skin. The main challenge arises after severe wounding, when elastic fibers need to be regenerated. This review provides a comprehensive summary of potential triggers for elastogenesis that may be further applied in novel dermal biomaterial formulations.

1. Introduction

Elastic fibers provide elasticity and resilience to tissues and enable them to undergo reversible extensibility and endure repetitive mechanical stress. Elastin, a fibrous extracellular matrix (ECM) protein, is the main component of elastic fibers.^{[ 1 ]} Major blood vessels, elastic ligaments, lungs, and skin represent tissues with a relatively high content of elastin. Unlike other ECM proteins, elastin appeared relatively late in evolution, as it is present in almost all vertebrates, but absent in invertebrates and primitive jawless fishes.^{[ 2 ]} Moreover, elastin is one of the most durable proteins in the human body due to its very limited solubility and high metabolic stability. Its degradation rate varies from tissue to tissue. While it is more rapid for skin, it is slow for aorta and lungs with a half‐life of 74 years for elastic fibers in lung parenchymal tissue.^{[ 3 ]} Although there are no specific half‐life values provided for human skin, rat experiments showed a twofold higher turnover compared to lungs.^{[ 4 ]} In the course of our lives, degradation is getting more abundant than elastin synthesis, as evidenced by lower expression levels of tropoelastin and increased release of elastin degradation products such as (iso)desmosine.^{[ 5 ]} Next to degradation rate, the architecture of elastic fiber network in skin differs significantly from that in aorta due to their distinct functional requirements and different content. Elastin comprises less than 5% of the dry weight of the skin,^{[ 6 ]} where it is architecturally represented in a loosely organized, mesh‐like network interwoven with collagen fibers in the dermis. In contrast, the elastin content in large arteries accounts for more than half of the dry weight,^{[ 6 ]} with elastic fibers arranged in densely packed lamellar units with pores.

Elastic fibers are crucial in tissue homeostasis, but cannot be easily replaced when degraded.^{[ 7 ]} Vollenweider Roten et al. clearly demonstrated the problem using a traditional elastin staining (Figure  1 ). Non‐wounded skin showed a network of elastic fibers in the papillary dermis, but scar tissue either showed thin and separate elastic fibers, thick elastin deposits, or even a lack of elastic fibers.^{[ 8 ]} The protein elastin is not the only element of elastic fibers, as microfibrils are incorporated within and around the fibers. Microfibrillar components include, but are not restricted to, fibrillins, fibulins, latent transforming growth factor β binding proteins (LTBPs), microfibril‐associated glycoproteins (MAGPs), elastin microfibril interface–located proteins (EMILINs), and proteoglycans (e.g., decorin, biglycan, versican, perlecan).^{[ 9 ]}

Figure 1.

Elastic fiber networks in normal skin and scars. After skin repair, new extracellular matrix is synthesized, but human skin does not restore the network of elastic fibers (in blue/black) in the dermis (left). Instead, Miller's stain shows relatively isolated and thin elastic fibers (middle) or thick deposits that do not look interconnected (right). Miller's stained images were reproduced from Vollenweider Roten et al. with permission.^{[ 8 ]} Copyright 1996, John Wiley and Sons.

All full‐thickness skin wounds share one consequence: they lack the ability to regenerate and to gain the properties and architecture that were present before the injury, as observed from non‐restored elasticity, absence of appendages, and presence of scar tissue. At present, autologous split‐thickness skin grafting remains the primary and one of the most effective choice after severe skin injuries.^{[ 10 ]} Split‐thickness graft involves transplanting a thin layer of skin, including the epidermis and part of the dermis. In terms of skin appearance after healing and formation of a scar, split‐thickness graft showed better outcomes compared to a full‐thickness graft, which involves the entire skin thickness.^{[ 11 ]} However, coverage with autologous skin can be extremely challenging to perform when a huge part (e.g., >80%) of the total body surface area is damaged and there is a lack of donor site.^{[ 12 ]} One option to overcome this can be the application of a dermal substitute with a silicon layer to prevent water loss and protect the wound bed from infection until the donor site regrows.^{[ 13 ]} Further benefits of the application of dermal substitutes are that they act as scaffolds for new tissue ingrowth by facilitating cell attachment, migration, and proliferation. Therefore, they need to be porous, non‐immunogenic, and biodegradable, but they must also not only retain fluids enriched with nutrients crucial for cellular survival but also promote angiogenesis, ensuring the essential delivery of oxygen and nutrients while facilitating the efficient removal of waste products. The majority of dermal biomaterials on the market have collagen as the primary component,^{[ 14 ]} although chitosan, silk fibroin, and other (bio)polymers may be used as alternative base components.^{[ 15 ]} It is important to mention, that reports on currently available products gave varying results in literature. Some were reported to perform superior to skin autografting without any biomaterial applied in terms of clinical results of skin elasticity, diminishing wound contracture, and providing better appearance,^{[ 16 ]} although several competitive biomaterials showed similar outcomes compared to each other.^{[ 17 ]} While others published that tested dermal biomaterials were not superior to skin grafting.^{[ 14} , ^{18 ]} If we specifically focus on the pliability of healed regions and the formation of new elastic fibers with dermal biomaterials, positive flexibility outcomes in clinics were still accompanied by immature, short, and thick elastic fibers.^{[ 19 ]}

In light of this, we would like to investigate the possibilities to design dermal biomaterials that may overcome the limitations of incomplete restoration of the elastic fibers network after wound healing and are able to fully regain lost resilience and elasticity.^{[ 19 ]} Elastogenesis, the formation process of elastic fibers, can be described by three consecutive stages: i) tropoelastin synthesis; ii) coacervation; and iii) deposition and crosslinking. Tropoelastin is synthesized and subsequently secreted by elastogenic cells such as fibroblasts, endothelial cells, and smooth muscle cells (SMCs).^{[ 20 ]} Upon secretion into the ECM, tropoelastin molecules self‐assemble directed by hydrophobic forces, a process called coacervation. Tropoelastin aggregates are deposited onto a preformed microfibrillar network for further initiation of the crosslinking process by lysyl oxidase (LOX) and lysyl oxidase‐like (LOXL) enzymes.^{[ 21 ]} Elastin itself, its precursor tropoelastin, and synthetic analogs are promising candidates for biomaterials. However, some properties of elastin protein, such as its extreme insolubility, have generated challenges to the use of elastic fibers in biomaterials. At the same time, elastin is not merely a structural protein, but it contributes to various cellular activities as do elastin degradation products. That is why it is also important to understand the underlying processes inside the cell, such as the activation of receptors and signaling pathways. Although, in many cases, the corresponding information may be scarce or even uninvestigated. The increased understanding of the significance of elastin for wound healing is leading to a wider exploitation of biomaterials.

Creation of a high‐quality, outperforming biomaterial requires a complex, interdisciplinary approach involving various specialists. The primary goal of a new biomaterial is the formation of extensive dermal elastic fiber network, resulting in reversible skin extensibility with a pleasant and healthy‐like appearance, and the complete ability to perform any original physical movements. In this way, innovative dermal substitutes should enrich mechanical properties, providing a robust material for cell attachment, proliferation, and differentiation, which are crucial for effective tissue regeneration. We prioritize maximum patient safety; thus biomaterials must be biocompatible and non‐toxic, thereby mitigating the risk of severe adverse reactions. For instance, we emphasize on carefully considered use of growth factors to avoid potential side effects, such as tumor cell proliferation. Cost‐effective production techniques with utilization of readily available materials may attract both manufacturers and clinicians, since such substitutes can be scaled‐up without compromising quality and are accessible to a broader market. In other words, the simplicity and versatility of a novel approach should satisfy the needs of both industry and healthcare sectors, with a clear mode‐of‐action of the components, bridging the gap between cutting‐edge research and practical, real‐world applications.

This review provides an overview of elastin synthesis on the cellular level and its potential use for formulation in biomaterials, particularly dermal substitutes, to induce the formation of functional elastic fibers rather than merely elastin protein synthesis. We have evaluated various agents and different classes of compounds, including elastin‐based substances, ECM‐related proteins, growth factors, glycosaminoglycans, hormones, antioxidants, vitamins, metal ions, and natural products. These substances demonstrated promising results in the in vitro, and some in vivo, platforms in increasing elastin production and supporting the formation of functional elastic fibers. These studies provide valuable information for the design of innovative biomaterial formulations to promote elastogenesis and improve clinical outcomes. However, further investigation is necessary to overcome foreseen obstacles, such as the molecular stability of these components, the ability for industrial production, safety tests, reasonable price, etc. The potential applications of these findings are considerable, and they are able to advance the field of tissue engineering and regenerative medicine.

2. Elastic Fiber Formation

The ELN gene is mainly expressed during the prenatal state and the first years of postnatal life after which its expression is almost completely repressed. Elastic fiber formation comprises the process from tropoelastin mRNA expression to elastic fiber crosslinking (Figure  2 ). Upon extensive alternative splicing, mature tropoelastin mRNA is transported out of the nucleus and translated on the rough endoplasmic reticulum. After the release of the signal peptide, the protein is passed to the Golgi apparatus.^{[ 22 ]} In order to prevent aggregation and premature degradation of tropoelastin, the resulting 60–70 kDa polypeptide is chaperoned by the 67 kDa elastin‐binding protein (EBP or S‐Gal, an enzymatically‐inactive splice variant of the lysosomal β‐galactosidase with a sustained galactolectin binding site) and transferred to the plasma membrane in acidic intracellular compartments.^{[ 23 ]} At the cell membrane, EBP associates with the 61 kDa neuraminidase‐1 (Neu‐1) and the 55 kDa protective protein/cathepsin A (PPCA). All three components together, EBP, PPCA, and Neu‐1, represent the elastin receptor complex (ERC). PPCA ensures the integrity of the two other components and Neu‐1 takes the central role in elastogenesis because its subsequent activation promotes the desialylation of microfibrillar proteins.^{[ 24 ]} Exposure of galactosyl moieties on microfibrillar proteins allows binding of EBP, leading to the release of tropoelastin with subsequent self‐aggregation through coacervation, deposition on microfibrils, crosslinking under the influence of lysyl oxidase and EBP recycling into the cell via secretory vesicles.^{[ 23} , ^{24 ]} In this process, microfibrils act as a framework that drives and guides elastin deposition. Elastin undergoes several post‐translational modifications.^{[ 25 ]} These modifications involve but are not limited to processes such as controlled proteolysis (cleavage of the signal peptide when the protein enters the lumen of the rough endoplasmic reticulum^{[ 22 ]}), hydroxylation of proline residues in the endoplasmic reticulum,^{[ 26 ]} and oxidative deamination of lysine residues outside the cell.^{[ 27 ]} These modifications can tune elastin properties. For instance, hydroxyproline incorporation showed increased coacervation temperatures, changed the alignment of elastin in a microfibrillar network, assisted crosslinking, stiffer fiber formations, and eventually decreased susceptibility to enzymatic degradation.^{[ 26 ]} Experiments with fish skin revealed a higher content of proline and hydroxyproline residues in warm‐water species compared to cold‐water species.^{[ 28 ]}

Figure 2.

Elastogenesis at the cellular level. Tropoelastin mRNA is transported out of the nucleus, translated on the rough endoplasmic reticulum, and elastin protein is chaperoned by the elastin‐binding protein (EBP), passed to the Golgi apparatus, and then transferred to the plasma membrane. EBP associates with transmembrane neuraminidase‐1 (Neu‐1) and the protective protein/cathepsin A (PPCA) to form the elastin receptor complex (ERC). Neu‐1 activation promotes desialylation of microfibrillar proteins, while exposure of galactosyl moieties facilitates the interaction with EBP, followed by the release of tropoelastin. Subsequent self‐aggregation of tropoelastin occurs via coacervation. Tropoelastin aggregates that are deposited onto microfibrils are further crosslinked by lysyl oxidase and EBP is recycled back into the cell via secretory vesicles. The figure is based on reports from Pshezhetsky and Hinek,^{[ 32 ]} Bennasroune et al.,^{[ 24b ]} Schmelzer et al.,^{[ 27 ]} and Tembely et al.^{[ 23c ]}

The protein tropoelastin contains alternating hydrophilic and hydrophobic domains. Hydrophilic domains, containing lysine residues, are responsible for crosslinking, while valine, proline, alanine, and glycine in hydrophobic domains are important for elastin's elasticity.^{[ 29 ]} Many crosslinks in elastin are represented by unique amino acids characteristic for elastin, e.g., desmosine and isodesmosine (Figure  3 ). Enzymes in the ECM, lysyl oxidase and lysyl oxidase‐like 2, catalyze the reaction of oxidative deamination of lysine residues in tropoelastin.^{[ 30 ]} The resulting allysine residues can spontaneously form bonds with other allysine or lysine residues through aldol condensation or formation of a Schiff base. These bifunctional products of condensation can attach to other fragments and form tri‐ (dehydromerodesmosine and neodesmosine), tetra‐ (desmosine and isodesmosine), or even pentafunctional (pentasine and allodesmosine) crosslinks. Moreover, crosslinks can be classified by the number of participating monomers of tropoelastin (intramolecular or intermolecular) or crosslinking domains (intradomain or interdomain).^{[ 27 ]} Condensation can be interrupted at any stage through reduction reactions. For example, dehydrolysinonorleucine and dehydromerodesmosine can be transformed into lysinonorleucine and merodesmosine, respectively. Desmosine and isodesmosine are used as biomarkers due to their unique appearance in elastin.^{[ 31 ]}

Figure 3.

Molecular crosslinks found in elastin. Lysyl oxidase (like) enzymes (LOX) catalyze oxidative deamination of lysine residues. Aldol condensations and formation of Schiff bases are the major reactions for combining the molecular fragments. The formed products can form bifunctional, tri‐, tetra‐, or pentafunctional crosslinks. Desmosine and isodesmosine can be found only in elastin and are used as biomarkers. Amino acids are depicted in different colors, highlighting their input in the obtained structures.

Some pathological conditions and genetic mutations affect proper elastogenesis, and can be associated with mutations in genes encoding for elastin or elastin‐associated proteins.^{[ 33 ]} Some diseases are causing abnormalities in dermal elastic fibers and skin characteristics. For instance, cutis laxa, a condition characterized by loose, sagging, and wrinkled skin is a genetically diverse condition including mutations in genes coding for elastin, fibulin‐4, fibulin‐5, lysyl oxidase, latent TGFβ‐binding protein 1, and ATPA7, a protein involved in copper transport.^{[ 34 ]} Such skin harbors sparce and fragmented elastic fibers, and lacks elasticity, elastic recoil, and resilience. Next to skin, other organs, notably the lung, are affected. Marfan syndrome, a multi‐systemic disease caused by mutations in the gene encoding fibrillin‐1, is associated with aortic aneurysm/dissection as a life‐threatening condition.^{[ 35 ]} It is characterized by a tall and thin stature with long extremities including arms, legs, and fingers. Degradation and loss of elastic fibers lead to a weakened skin with stretch marks (striae).^{[ 36 ]} However, in stiff skin syndrome, also associated with mutations in the gene for fibrillin‐1, the skin is thick and hard, and excessive skin fibrosis is seen.^{[ 37 ]} Although the phenotypes of Marfan syndrome and stiff skin syndrome are quite different, disregulation of TGFβ seems a common denominator, as fibrillin‐1 is known for its role in sequestering TGFβ. The deletion 7q11.23 syndrome (Williams‐Beuren syndrome) is caused by a hemizygous deletion of 1.5 – 1.8 Mb on chromosome 7q11.23 spanning about 28 genes, including the elastin gene. Soft, lax skin and arterial stenosis are characteristics of the disease.^{[ 38 ]} The vocal cord abnormalities (hoarse or low‐pitched voice) have been correlated to the elastin deficiency.^{[ 39 ]} In the 7q11.23 duplication syndrome, a duplication is seen of a similar DNA segment that is deleted in the Williams‐Beuren syndrome, including the elastin gene.^{[ 38b ]} It has been suggested that the extra copy of the elastin gene may result in an increase in the amount of tropoelastin giving rise to the aortic dilatation observed in this syndrome. Taken together, pathologies caused by genetic defects in genes coding for proteins that make up the elastic fiber network underscore its relevance not only for endowing structural elasticity to organs, but its role in preserving overall body homeostasis.

While the mechanism of elastic fiber synthesis is well investigated,^{[ 21} , ^{40 ]} many questions remain about what induces or halts this process. A variety of compounds, however, have been implicated as promotors of elastogenesis, as can be appreciated in the next section.

3. Potential Inducers of Elastic Fiber Formation

In order to steer repair more toward a regenerative process, functional elastogenesis induced by biomaterials is one of the goals of dermal substitute materials to restore elastic and resilient properties of the healed skin.

To ensure a comprehensive narrative review, we performed our search in PubMed and Google Scholar using corresponding keywords, such as “elastin”, “elastogenesis”, “elastic fibers”, “elastogenic effect”, “dermal substitutes”, “biomaterials” and “wound healing” for articles published up to 2023 in English. Inclusion criteria involved peer‐reviewed studies focused on (dermal) elastogenesis, and the development and application of dermal substitutes. Exclusion criteria included non‐peer‐reviewed articles, personal websites, irrelevant studies, and those with obvious methodological flaws. Titles and abstracts were screened for relevance, followed by a detailed full‐text review and data extraction. Some articles were included based on information (reference list) of retrieved papers. A summarizing table of evidence was included in order to better compare the selected compounds and making a selection of the most promising candidates.

Different elastin derivatives have been investigated as potential ingredients in biomatrices, but their effect on cells was highly dependent on various factors such as the state of elastin (soluble or insoluble), concentration of soluble compounds, amino acid sequence, and cell type.^{[ 41 ]} For instance, low concentrations of elastin hydrolysate in 2D cell culture (10–100 µg mL⁻¹) led to cell proliferation of human lymphocytes, whereas high concentrations induced apoptosis in these cells.^{[ 42 ]} The ERC does not only play a role in elastogenesis to deposit elastin onto microfibrils and proper assembly of the fibers,^{[ 24c ]} but it can also transduce intracellular signals and modulate bioactive elastin degradation products, so‐called elastokines.^{[ 43 ]} Binding of compounds to the ERC activates internal signaling pathways which can cause cellular responses, like elastogenesis,^{[ 20} , ^{44 ]} chemotaxis,^{[ 45 ]} proliferation,^{[ 46 ]} angiogenesis,^{[ 47 ]} and apoptosis.^{[ 42b ]} While EBP protected with PPCA can form complexes with elastin peptides and galactosugars (e.g., lactose), Neu‐1 is crucial because of its ability to desialylate glycoproteins and glycolipids, and consequently modulate the signal transduction pathways of such compounds. Neu‐1 can interact with a variety of additional surface receptors, expanding the biological or pathological impact of elastin‐derived or elastin‐resembling peptides.^{[ 22 ]} Elastin‐derived peptides may interact with other proteins than the ERC, e.g. galectin‐3.^{[ 48 ]} Moreover, other receptors that are known to sense elastin‐derived peptides include a lactose‐insensitive receptor (or ribosomal protein SA^{[ 49 ]}) and cell adhesion transmembrane receptors (e.g., integrins α_Vβ₃ ^{[ 50 ]} and α_Vβ₅ ^{[ 51 ]}). Hence, depending on the type and concentration of an elastogenic compound, the underlying mechanism may differ, as could the eventual effects.

In the following sections we discuss different strategies to promote elastogenesis, i.e., by applying elastin‐based components or by other compounds that trigger elastic fiber production, (extracellular matrix components, hormones, potassium channel openers, natural products, metals, antioxidants, vitamins, etc). Table  1 summarizes the elastogenic evidence for most of the compounds discussed, indicating their level of evidence. We here focus on compounds that promote the restoration of a network of elastic fibers resembling those in healthy skin tissues. Where such effects have not been tested in dermal cells, different cell types are exemplified.

Table 1.

Proof of evidence for dermal elastogenic effect of various substances.

3.1. Elastin‐Based Components

Elastin‐based components, i.e., insoluble elastic fibers, elastin‐derived peptides and tropoelastin protein/mRNA, are particularly interesting as they mimic natural conditions and/or compositions and either contain the natural crosslinks or may be crosslinked later (Figure  4 ). Depending on their physical properties and stability these components can be used in different ways, such as by incorporation into biomaterials^{[ 52 ]} or as intradermal injections,^{[ 53 ]} and can be found naturally in decellularized extracellular matrix.^{[ 54 ]} Different species possess particular differences in amino acids composition of elastin proteins, thus porcine, bovine, and human elastin are not identical but share a lot of similarities.^{[ 55 ]} Using sequences available in the UniProt database and the Basic Local Alignment Search Tool, we have compared the alignment of rat, porcine, and bovine elastin proteins with humans and obtained 77%, 82%, and 86% amino acid alignments, respectively. When animal‐origin elastin is used for biomaterials, possible issues related to this aspect, e.g., immunogenic response and calcification, should be considered during in vivo testing of novel biomaterials.^{[ 52} , ^{56 ]} Since it is difficult to appreciate the specific effects related to elastin in decellularized biomaterials due to their complex composition with many other extracellular matrix components, they will not be discussed in this review.

Figure 4.

Major elastin‐based components and their interconnection. Translation of tropoelastin mRNA leads to the soluble tropoelastin protein, that may be mimicked by (smaller) elastin peptides and polypeptides. Mature insoluble elastic fibers may also be solubilized by chemical agents or digested by enzymes, leading to soluble fragments.

3.1.1. Insoluble Elastic Fibers

Elastic fibers provide the necessary mechanical characteristics of resilience and elasticity that are crucial for the functioning of elastic tissues. Although the approach of using elastic fibers in biomaterials formulation for enhancing elasticity is compelling, there are several complications with this idea. First of all, elastic fibers in healthy tissues form a complex network with other ECM components and it is challenging to replicate such networks.^{[ 57 ]} Naturally crosslinked elastin that is present in elastic fibers (≈90%^{[ 57 ]}) in its intact state is highly insoluble in water and rather inert to both intrinsic and external impacts. When elastic fibers are isolated and purified they differ from intact fibers due to mechanical processing (like pulverization) and chemical treatments.^{[ 9c ]}

Insoluble elastic fibers as the sole component of biomaterials are not likely to be used as a component of elastogenic biomaterials, which may be due to the above‐mentioned inertness and high insolubility of the fibers. Nevertheless, insoluble elastin demonstrated its ability to impact the physical properties of biomaterials by decreasing the rigidity of collagen scaffolds and enhancing viscoelastic characteristics.^{[ 58 ]} Biological effects of biomaterials containing isolated insoluble elastin included promotion of a contractile phenotype of SMCs in collagen‐elastin and selective proliferation of primary human keratinocytes co‐cultured with fibroblasts.^{[ 59 ]} In addition, these materials exhibited calcification of soft tissue in a young Sprague‐Dawley rat model, highly sensitive to forming calcium deposits.^{[ 52d ]} Combining obstacles with the utilization of insoluble elastin, researchers focused on solubilized forms of insoluble elastin.

3.1.2. Solubilized Elastin

Elastin is repeatedly subjected to a range of enzymatic, chemical, and biophysical stimuli during our lifespan that may (partly) solubilize elastin.^{[ 7 ]} Generally, there are two approaches to solubilize elastin from its insoluble form in the laboratory: enzymatic digestion and chemical treatment.^{[ 60 ]} Like tropoelastin during elastogenesis, digested and solubilized elastin displays lower critical solution temperature behavior that allows it to coacervate from a mixture above a certain temperature and revert to the soluble form when the temperature is lowered under the lower critical temperature.^{[ 61 ]} Self‐assembly properties are also influenced by amino acid composition, pH, and ionic strength.^{[ 62 ]} The stimulus‐responsive properties make solubilized elastins a promising material in drug delivery systems, medical devices, and tissue‐engineered constructs, including dermal substitutes. Their coacervation profile, however, is also dependent on the solubilization method, where significant prolongation of hydrolysis time can even deprive this ability of solubilized elastin preparations.^{[ 61 ]}

Enzymatic Digestion

Elastolytic enzymes can be divided into four categories: serine, cysteine (mainly cathepsins), aspartic proteases, and zinc‐dependent endopeptidases (matrix metalloproteinases). For each category, data about cleavage specificity for elastin and tropoelastin are available.^{[ 60 ]} Elastic fibers that experienced some prior damage are more readily degraded by enzymes.^{[ 63 ]} Preparation of solubilized elastin via degradation of naturally occurring elastic fibers is a promising approach for the induction of elastogenesis due to its properties to promote elastin‐enriched matrix deposition and mimicking processes that are occurring in the human body.^{[ 64 ]} For instance, bovine elastin digested with proteinase K resulted in a preparation with a molecular weight of ≤10 000 Da and showed enhanced elastogenesis with primary human dermal fibroblasts, skin explants, and pre‐treated fibroblasts implanted in the dermis of athymic nude mice.^{[ 64 ]} The in vitro experiments demonstrated the induced formation of collagen fibers and elastic fibers along with an elevated proliferation of the cells, accompanied by decreased expression of fibronectin and chondroitin sulfate proteoglycans. More elastin deposits were noticed in ex vivo experiments as well. Furthermore, abundant elastic fiber network production was seen in mice after injection of fibroblasts stimulated with the elastin digest.^{[ 64 ]} Also fish‐derived elastin treated with two enzymes—protin AC10 and protease N—resulting in a digest with a molecular weight of ≈1000 Da, increased proliferation and elastin production by primary human dermal fibroblasts.^{[ 65 ]} Although enzymatic digestion results in specific cleavage at certain amino acid residues in elastin, they are costly (especially for human elastases), may be contaminated with endotoxins, and still give a large molecular weight distribution of soluble elastin. In addition, the high affinity of enzyme to elastin makes it difficult to remove the enzyme after solubilization. These drawbacks to produce active ingredients for biomaterial formulation may cause serious issues in further clinical translation and substantially raise costs for production.

Chemical Hydrolysis

In contrast to enzymatic digestion, bond cleavages are non‐specific when solubilization is performed using oxalic acid that results in α‐elastin along with β‐elastin or potassium hydroxide that results in κ‐elastin.^{[ 61 ]} Fractionation after chemical treatment may be performed, for instance by chromatographical methods like size exclusion chromatography. We would like to stress that the terms “α‐elastin” and “κ‐elastin” still refer to mixtures of soluble compounds and not to a single component. Thus, it is important to check the characteristics of such formulations from different publications when comparing biological activities. Note that specificity of chemical hydrolysis is by definition lower than for elastases. The use of large amounts of bases and organic solvents or acids is a drawback of the methodology, as is the extremely wide range of molecular weight for obtained preparations (up to 2000 kDa). However, these methods come with straightforward removal of the used compounds and obtained molecular weights can be lowered with longer incubation or higher temperature.

Elastin‐derived peptides from acidic or alkaline hydrolysis can promote elastogenesis and replenish the elastin content in wounds when incorporated in biomaterials.^{[ 52} , ^{66 ]} In a porcine model, the elastin component in scaffolds containing type I collagen α‐elastin was responsible for reduced contraction of the wound along with decreased numbers of myofibroblasts and formation of native collagen bundles.^{[ 66} , ^{67 ]} Additionally, these scaffolds revealed fewer fibroblasts in the early wound healing phase compared to other tested constructs.^{[ 67 ]} Implantation of a scaffold containing a similar combination of elastin‐derived peptides produced by oxalic acid hydrolysis and type I collagen in young rats induced neovascularization and synthesis of thin elastic fibers containing fibrillin‐1.^{[ 52d ]} MatriDerm, a commercially available dermal substitute composed of types I, III, and V collagen and α‐elastin hydrolysate, evidenced an improved healing process in terms of function and appearance by minimizing contractures and increasing skin elasticity.^{[ 68 ]} κ‐elastin fraction with a specified molecular weight of 75 kDa enhanced adhesion and alignment of insoluble elastic fibers to human dermal fibroblasts and may serve as a reagent for oriented elastogenesis.^{[ 69 ]} It is noteworthy that KOH‐solubilized elastin can ultimately lead to insulin resistance of the cells due to desialylation of the insulin receptor by activated Neu‐1 and reduced insulin receptor signaling as seen in mice.^{[ 70 ]} In biomaterials, chemical modifications can provide additional properties to solubilized elastin preparations on demand. Conjugation of acrylate groups onto elastin solubilized with oxalic acid resulted in an injectable hydrogel that was photocrosslinked by visible light. This hydrogel accelerated dermal wound healing along with active recruitment of neutrophils and macrophages, followed by subsequent polarization of macrophages primarily toward the pro‐regenerative M2 phenotype. Recruitment of innate immune cells thereby promoted angiogenesis, collagenesis and dermal repair in a murine full‐thickness skin defect model.^{[ 52b ]}

3.1.3. Synthetic Elastin Peptides

Soluble elastin compounds can also possess much smaller molecular weights compared to solubilized elastins. Small bioactive elastin peptides from human elastin are generally generated from building blocks of Gx, Px, PGx, and GGx (where x = A, G, V, I or L) of the hydrophobic domains, VGVAPG sequence being the most investigated one.^{[ 60 ]} The biological activity of the GxxPG sequence was explained by its capacity to take a type VIII β‐turn conformation, crucial for binding to EBP.^{[ 60 ]} Different xGVxxG sequences, found in elastin, rice bran, and the human IGF‐1 binding protein 1, activated elastogenesis in cultures of human primary dermal fibroblasts.^{[ 44 ]} Moreover, the presence of such moieties in high molecular weight hydrolysates or the exposure of such sequences in damaged elastic fibers may explain sensing of these compounds by transmembrane receptors to activate different intracellular signaling cascades.

Elastin‐like peptides (ELPs) and elastin‐like recombinamers (ELRs) resemble hydrophobic sequences of natural tropoelastin and represent a vast category of synthetically made or genetically engineered polymers made up of repeated oligopeptides, generally with a specific pH‐temperature‐ionic strength coacervation profile.^{[ 71 ]} ELPs/ELRs are beyond the scope of this review, however they serve as a promising tool in wound healing.^{[ 72 ]} For further familiarization with ELPs/ELRs some publications are recommended.^{[ 73 ]}

3.1.4. Tropoelastin Protein

Another approach to stimulate elastogenesis is to skip the tropoelastin synthesis phase within the cell, and directly introduce the elastin precursor tropoelastin. Initially, tropoelastin used to be obtained from copper‐deficient animals^{[ 74 ]} (copper is an essential cofactor for crosslinking by lysyl oxidase) or by administration of lysyl oxidase inhibitors^{[ 75 ]} as part of the animals’ dietary regimen. However, nowadays tropoelastin can be obtained from fetal and neonatal tissues, but recombinant techniques provide the largest yields.^{[ 76 ]} Recombinant human tropoelastin has shown to be exploited as a substrate for the production of branched elastic fibers network in vitro.^{[ 77 ]} Meanwhile in rats, immunostaining of injected human recombinant tropoelastin monomers and host elastin did not show colocalization. However, tropoelastin has been tested crosslinked with hyaluronic acid, an essential ECM component (see section 3.2.2) that is also involved in wound healing processes among various other characteristics.^{[ 78 ]} The crosslinked combination of tropoelastin and hyaluronic acid demonstrated proper incorporation of exogenous tropoelastin in a newly synthesized elastic fiber network in rats.^{[ 77 ]} Ex vivo experiments with such a crosslinked preparation demonstrated improved moisture content and deposition of acidic glycosaminoglycans compared to untreated controls.^{[ 77 ]} Chemical modifications have also been applied to tropoelastin to enable visible light photocrosslinking of biomaterials. Conjugation of methacrylate groups onto recombinant human tropoelastin and gelatin offered a novel antimicrobial sprayable and flexible adhesive for future management of chronic wounds.^{[ 79 ]} This approach can also be used for the production of dermal biomaterials.

Despite several examples of promising biomaterials based solely on recombinant tropoelastin for healing of full‐thickness skin wounds in pigs and mice,^{[ 80 ]} tropoelastin usually serves as an additive to biomaterials.^{[ 20} , ⁵² , ^{81 ]} Elastogenic potential was not assessed after application of scaffolds made from tropoelastin alone, however it was studied upon injections of aqueous tropoelastin in a swine burn wound model. When tropoelastin was administered after wound closure, it showed substantially increased elastic fiber synthesis than when supplied earlier after wounding. Newly formed elastic fibers appeared thin, disorganized and fragmented.^{[ 53 ]} Hence, it is essential to investigate the long‐term outcomes in order to comprehensively assess the impact on triggered elastogenesis. Tropoelastin incorporation into biomaterials can lead to improved dermal wound healing with induced elastogenesis. For instance, stimulated elastogenesis by human vascular smooth muscle cells in culture was achieved in a controllable manner using tropoelastin applied onto the scaffolds fabricated from fibrin, type I collagen, and chondroitin‐6‐sulfate. It is noteworthy that only a particular drop‐loading technique of tropoelastin onto the cells incorporated in the scaffold resulted in lamellar mature elastic fibers.^{[ 20a ]} Commercial two‐layered Integra Dermal Regeneration Template (crosslinked collagen with chondroitin‐6‐sulfate glycosaminoglycan covered with a silicone sheet) can be additionally functionalized with tropoelastin. One approach was to seed patient's fibroblasts on the collagen‐glycosaminoglycan layer of Integra, culturing them in the presence of tropoelastin to prefabricate elastic fibers before implantation.^{[ 82 ]} Isolated from donors ranging from neonatal to more than 90 years old, fibroblasts originating from older participants produced fewer and less branched elastic fibers. The other method of functionalization of Integra with tropoelastin was based on covalent conjugation of tropoelastin during the production stage.^{[ 83 ]} Compared to Integra alone, tropoelastin‐Integra demonstrated early vascularization in mice and pigs with small capillaries after 2 weeks and considerably more new blood vessels in the deep neodermis. However, no specific report was made on the synthesis of elastic fibers.

Moreover, skin substitutes using human type I collagen and recombinant human tropoelastin were produced by electrospinning, a technique that yields fibers/threads using electric forces.^{[ 52} , ^{81 ]} Such scaffolds were able to promote re‐epithelization and deposition of new elastic fibers as well as a more natural extracellular matrix at the healed site in mice.^{[ 81 ]} Full‐thickness wounds in diabetic mice treated with an electrospun collagen–tropoelastin skin substitute demonstrated better resemblance of tissue mechanics with intact skin compared to commercial Oasis Wound Matrix (an acellular ECM derived from porcine small intestinal submucosa).^{[ 52a ]} Hence, scaffolds that contain tropoelastin may particularly aid healing of chronic wounds along with a lower chance of wound dehiscence.

3.1.5. Tropoelastin mRNA

Even though it is widely acknowledged that elastin biosynthesis ceases in adult tissues, it was demonstrated that transcription levels of pre‐mRNA of tropoelastin in adults are similar to neonatal lung tissue.^{[ 84 ]} It was hypothesized that posttranscriptional mechanisms rather than transcriptional changes influence elastin deposition since the change was mainly caused by decreased stability of tropoelastin mRNA.^{[ 85 ]} Supporting this hypothesis, mRNA stability is also reduced by exogenous factors such as ascorbic acid.^{[ 86 ]} It may be a promising approach to find factors that enhance elastogenesis by stabilizing tropoelastin mRNA.

One such example is that synthetically modified tropoelastin‐encoding mRNA enhanced the stability by attaching a poly‐A tail at the 3′ and a cap analog at the 5′ end, thereby increasing elastin synthesis in umbilical vein endothelial cells, fibroblasts, and mesenchymal cells.^{[ 5a ]} After intradermal administration of synthetic mRNA by microinjection, elastin protein content in porcine skin increased by about 20%.^{[ 5a ]} Although ex vivo proof of principle is given, extensive in vivo studies are needed to assess whether elastin deposits result in functional elastic fibers.

Despite the use of synthetic mRNAs as a novel therapy for the creation of desired proteins in cells has attracted increasing attention in recent years,^{[ 87 ]} limited stability of mRNA is the obstacle that should be taken into consideration. Nonetheless, mRNA vaccination strategies, obviously progressed after the SARS‐CoV‐2 outbreak, with various delivery systems and the gained knowledge can be used in the development of tropoelastin mRNA delivery methods.^{[ 88 ]} For instance, peptide‐mRNA nanoparticles in porous collagen scaffolds have shown efficient mRNA transfection of cells in vitro which resulted in local protein production.^{[ 89 ]} Thus, this approach may be utilized in dermal biomaterials in order to promote elastogenesis.

3.2. Extracellular Matrix Components

Extracellular matrix components not only ensure structural integrity but are also required for elastogenesis and have been investigated to understand how they contribute to elastogenesis based on four categories: i) elastic fiber‐associated components, ii) proteoglycans and glycosaminoglycans, iii) ECM‐associated growth factors/cytokines, and iv) collagens. It is noteworthy that growth factors and cytokines are not typically components of the extracellular matrix proper itself, but are generally stored there and are produced by other cells than they are acting on. However they play important roles in the cellular activities that occur within the extracellular matrix.

3.2.1. Elastic Fiber Associated Components

Elastic fiber‐associated proteins such as fibulins, fibrillins, fibronectin, and latent TGFβ binding protein‐4 (LTBP‐4), play a role during various elastogenesis stages, instructing and/or facilitating particular processes.^{[ 90 ]} Exogenous administration of some of these components have improved elastogenesis, e.g., in cell culture, and they may thus be of interest for incorporation in dermal templates.

Fibrillin‐1 is involved in the construction of microfibrils, and plays an important role in wound healing as was shown with a periodontal disease model, where wound healing is delayed with decreased fibrillin‐1 expression^{[ 91 ]} and fibroblasts differentiated into alpha smooth muscle actin (α‐SMA)‐positive myofibroblasts.^{[ 92 ]} In the absence of cells, recombinant fibrillin‐1 protein improved the self‐assembly of the recombinant elastin‐like polypeptide.^{[ 93 ]}

Fibronectin is also required for elastic fiber assembly.^{[ 94 ]} Fibronectin is an adhesion molecule that contributes to the formation of extracellular matrix during wound healing by providing cell–cell and cell–extracellular matrix signaling via specific binding sites in its structure.^{[ 95 ]} Exogenous fibronectin has been shown to play a role in scaffold remodeling by SMCs seeded in collagen gels toward the production of an elastic fiber‐containing ECM.^{[ 96 ]} Furthermore, it improves biomaterial stability,^{[ 97 ]} cell adhesion, migration, and proliferation,^{[ 98 ]} making it an excellent candidate for use in skin tissue materials. Human dermal fibroblast adhesion and proliferation are enhanced by fibronectin‐functionalized hydrogel in comparison to fibronectin‐free hydrogel.^{[ 99 ]}

Fibulin‐5 plays a crucial role in the formation and organization of elastic fibers by interacting with them and acting as a ligand for specific cell surface integrins, namely α₅β₁, α_vβ₃, and α_vβ₅. This interaction allows fibulin‐5 to bind to important structural components such as tropoelastin and fibrillin‐1, as well as crosslinking enzymes that aid in the formation and assembly of elastic fibers. Thus, fibulin‐5 helps to stabilize and organize elastic fibers in various structures, including the skin, lungs, and blood vessels.^{[ 100 ]} In serum‐free skin fibroblast cultures, the addition of fibulin‐5 protein enhanced the development of elastic fibers, whereas tropoelastin was not organized into elastic fibers in its absence.^{[ 101 ]}

Latent TGFβ binding protein‐4 (LTBP‐4) is another significant protein for elastogenesis and interacts with fibulin‐5 in order to cause a proper assembly of elastic fibers.^{[ 102 ]} Human dermal fibroblasts were found to assemble microfibrillar components and elastin protein into elastic fibers when the cell culture medium was supplemented with recombinant LTBP‐4S protein, a short isoform of LTBP‐4.^{[ 102 ]} Beneficial effects of recombinant LTBP‐4S protein were further exploited using crosslinked collagen scaffolds. (Immuno)histological analysis demonstrated that the presence of LTBP‐4S in both 3D cell cultures of dermal fibroblasts on collagen scaffolds and in collagen dermal substitutes implanted between the dermis and cutaneous muscle of mice improved elastogenesis.^{[ 103 ]}

As elastic fiber components are needed for proper assembly, alignment, and crosslinking of tropoelastin, they are interesting candidates for functional elastogenesis in dermal substitutes.

3.2.2. Proteoglycans and Glycosaminoglycans

Proteoglycans consist of a core protein to which one or more glycosaminoglycan chains (GAGs) are bound. They are important structural components of the extracellular matrix, and their ability to modulate elastogenesis is promising in the regulation of elastin synthesis and deposition during wound healing. As polyanions, GAGs bind to positively charged lysine residues in a temperature and concentration‐dependent manner and may facilitate tropoelastin release from EBP to promote coacervate formation and elastin deposition.^{[ 104 ]} However, not all GAGs are advantageous for elastogenesis. GAGs containing galactosamines, such as chondroitin sulfate and dermatan sulfate, bind to EBP and disrupt elastic fiber formation.^{[ 104} , ^{105 ]} Gene delivery of V3, a versican isoform that lacks chondroitin sulfate, enhanced elastic fiber assembly and reduced SMC proliferation.^{[ 106 ]} Upregulated expression of versican V3 led to enhanced tissue stiffness of engineered skin and vascular grafts, and elevated both tropoelastin expression and insoluble elastin protein deposition.^{[ 107 ]} In Hurler disease and Costello syndrome, the opposite is seen as accumulations of chondroitin sulfate and dermatan sulfate proteoglycans are linked to impaired elastogenesis.^{[ 107} , ^{108 ]} Chondroitin sulfate containing dermal matrix, Integra, did not demonstrate the elastic fiber formation when implanted in mice skin wound, unlike other tested dermal matrices without chondroitin sulfate.^{[ 109 ]} This dermal matrix also did not show elastic fibers formation after 2 years from application in humans.^{[ 110 ]} Disaccharides from marine chondroitin sulfate, however, increased elastin protein synthesis by human dermal fibroblasts.^{[ 111 ]}

Hyaluronan or hyaluronic acid (HA), a large non‐sulfated GAG, is a key component in skin tissue regeneration and wound healing. This compound is known to possess a high affinity for water and readily attract and retain moisture, provide structural support during early stages wound healing, and can demonstrate molecular weight‐dependent functions, such as anti‐inflammatory responses, angiogenesis, antioxidative properties, cells migration, and proliferation.^{[ 112 ]} In addition to abovementioned properties, hyaluronan plays a key role in elastogenesis by promoting tropoelastin self‐assembly, lysyl oxidase‐mediated crosslinking, and preserving elastic fibers from degradation.^{[ 113 ]} A mixture of low and high molecular weight HA resulted in elastin gene expression and elastin protein deposition in a co‐culture of human keratinocyte and dermal fibroblast cell lines.^{[ 114 ]} Synthesis of collagen and elastin at the mRNA and protein level was improved upon injection with a high molecular weight hyaluronan gel in Phenion, a commercial full‐thickness skin model. The elastogenic effect of hyaluronan gel was higher when combined with sulfate‐free chondroitin.^{[ 115 ]} HA‐based dermal fillers added to dermal fibroblast cultures improved the production of elastin, type I, and type III collagen together with TGF‐β1 levels in the cultures.^{[ 116 ]} Exogenous administration of hyaluronan oligosaccharides (tetra‐ to octamers) enhanced tropoelastin synthesis and elastin deposition by SMCs nearly threefold. Furthermore, surface‐immobilized tetramers improved elastic fiber organization and induced SMCs to generate significantly more elastin‐stabilizing desmosine cross‐links.^{[ 117 ]} Encapsulation of rat aortic SMCs in a hydrogel containing hyaluronan tetramers doubled their elastin‐matrix production in 21 days, which is thought to be related to HA oligomer interaction with cell surface receptors such as CD44.^{[ 118 ]} Moreover, intradermal injections of different HA‐containing dermal fillers showed elastogenic properties in vivo and in a clinical trial with 60 patients.^{[ 119 ]} There is also a commercially available Hyalomatrix dermal template, an esterified hyaluronan scaffold covered with a silicon layer.^{[ 120 ]} To the best of our knowledge, there is a lack of convincing evidence for significant elastogenic properties in vivo with Hyalomatrix,^{[ 121 ]} though a thorough assessment with appropriate competitors is recommended.

Heparan sulfate and heparin are both GAGs with a similar chemical structure, but heparin has a higher degree of sulfation. The binding sites for heparan sulfate and heparin in fibrillin‐1 play a crucial role in the binding of fibrillin‐1 molecules along heparan sulfate chains and the assembly of microfibrils.^{[ 122 ]} For instance, heparan sulfate is involved in the assembly and stability of elastin coacervates and has been shown to promote tropoelastin assembly when exogenously provided to human dermal fibroblasts.^{[ 123 ]} Heparin mediates the coacervation of positively charged tropoelastin due to its high negative charge density^{[ 124 ]} and possesses anti‐proteolytic properties that inhibited leukocyte elastase by 85–95%, which may be exploited as a protecting agent for existing elastic fibers.^{[ 125 ]} In actively growing SMCs, heparin increased elastin mRNA expression 2–3 fold, but this effect weakened when cells aged.^{[ 126 ]} Moreover, a heparin‐releasing biomaterial system based on silk enhanced elastic fiber production by SMCs.^{[ 127 ]}

HA and heparin may be the most interesting glycosaminoglycans for biomaterials as they can be commercially obtained. Hyalomatrix, a HA containing collagen template, improved wound healing in burn patients, however, further research is needed to determine its effect on elastogenesis.^{[ 128 ]} In addition to these GAGs, the effect of recombinant human versican V3 protein may be investigated, especially taking into account its elastogenic effect when overexpressed in skin fibroblasts.^{[ 107} , ^{129 ]}

3.2.3. Growth Factors and Cytokines

Growth factors are responsible for performing specific functions by binding to receptors on target cells. They are generated by producer cells when required. Their intrinsic drawbacks, instability or quick dispersion from the delivery site, may be avoided by combining them with biomaterials to enable controlled release.^{[ 130 ]}

Among these compounds, insulin‐like growth factor 1 (IGF‐1) and TGF‐β1 showed the most positive results in terms of inducing elastin synthesis.^{[ 5c ]} The involvement of IGF‐1 in elastogenesis is demonstrated by enhanced elastin gene expression and soluble elastin production in rat aortic SMCs with elevated IGF‐1 concentrations.^{[ 131 ]} Moreover, in cultures of rat aortic SMCs treated with IGF‐1, tropoelastin mRNA levels were found to be 3–5 fold higher, peaking 8 h after administration of IGF‐1.^{[ 132 ]} Next to IGF‐1, the IGF‐1 receptor has been demonstrated as an important player in elastogenesis with various compounds, including xGVxxG peptides and glucagon‐like peptide‐1.^{[ 44 ]} The xGVxxG peptide enhanced elastin gene and protein expression and elastic fiber development in human dermal fibroblasts and explants via activating the IGF‐1 receptor.^{[ 133 ]} Despite glucagon‐like peptide‐1 being an insulinotropic hormone, it did not alter insulin secretion in cardiac fibroblasts but induced elastin production in the absence of a classic glucagon‐like peptide‐1 receptor via cross‐activation of the IGF‐1 receptor.^{[ 44 ]} Furthermore, administration of a combination of IGF‐1 and fibroblast growth factor‐2 (FGF2) elevated the expression of lysyl oxidase in vivo and may assist in formation of crosslinked elastin.^{[ 134 ]} IGF‐1 treatment showed an elastogenic effect on SMCs, but had no elastogenic effect on rat pulmonary fibroblasts, indicating that the control of elastin gene expression may be tissue‐specific.^{[ 131b ]}

TGF‐β1 is an important regulator of collagen and fibronectin production in wound healing and a known pro‐fibrotic growth factor associated with scar formation. The importance of TGF‐β1 signaling in elastin formation is shown by abnormalities in elastic fiber structure induced by cells lacking functional TGF‐β1 receptors.^{[ 135 ]} TGF‐β1 effects in elastogenesis goes two ways; it can increase tropoelastin mRNA levels and elastin formation by modulating tropoelastin promoter activity, but on the other hand, diminish tropoelastin mRNA levels and increase elastin degradation.^{[ 5} , ¹⁰⁰ , ^{136 ]} In vitro systems designed based on biological compounds and biomechanical environments are promising to improve elastin synthesis. For instance, in a bioreactor system TGF‐β1 and mechanical stress treatment of primary human vascular SMCs seeded on a scaffold resulted in higher levels of deposited microfibrillar components compared to standard 2D controls.^{[ 137 ]} TGF‐β1‐coated polymeric discs were implanted layer by layer subcutaneously in Wistar rats, and in three weeks, the controlled release of TGF‐β1 from the discs enhanced elastin synthesis compared to discs without TGF‐β1.^{[ 138 ]} Elastin production, however, seems to be age‐specific, since TGF‐β1 stimulated elastin formation in neonatal rat lung fibroblasts and smooth muscle cells, but not in adult equivalents,^{[ 139 ]} even though enhanced elastogenesis is especially needed in adults. In 3D cell culture experiments with vocal fold fibroblasts, TGF‐β1 did not show elastogenic potential, but hepatocyte growth factor (HGF) did.^{[ 140 ]} As TGF‐β1 has progressively been linked with several pathologies (e.g., apoptosis, fibrosis, formation of granulation tissue), it would be controversial to select this molecule as a candidate to induce elastic fibers in biomaterials.^{[ 136} , ^{141 ]}

Pro‐inflammatory cytokines can negatively influence elastin synthesis and promote degradation. For example, TNF‐α reduced tropoelastin mRNA levels and stimulated elastin digestion by increasing the release of elastolytic enzymes such as MMP‐2, and MMP‐9.^{[ 5c ]} In contrast, interleukin (IL)−1β increased elastin gene expression in human dermal fibroblast cultures and in mice skin after subcutaneous injections.^{[ 142 ]}

More research is needed on dermal fibroblasts to fully understand the effects of various cytokines on elastogenesis, but especially IGF‐1 shows potential in directing elastogenesis when used in the formulation of biomaterials.

3.2.4. Collagen

Collagen is a structural protein in skin and an important component of biomaterials that serves as a template for cell adhesion, migration, and proliferation to remodel lost ECM in wounds.^{[ 143 ]} Type I collagen, a common component of biomaterials, may have elastogenic potential as it has been reported that human type I collagen alpha‐2 (COL1A2) peptides increase wound healing efficacy, collagen and elastin synthesis, cell proliferation, and migration by simultaneous activation of the TGF‐β signaling pathway.^{[ 144 ]} When human dermal fibroblasts were cultured in the presence of recombinant human COL1A1‐collagen effective domain (CED), it increased cell proliferation and migration, as well as collagen and elastin deposition.^{[ 145 ]} Collagen peptides isolated from fish skin alone as well as in combination with soy peptides showed mRNA upregulation of elastin, fibronectin, type I and type II collagen in dermal fibroblasts.^{[ 146 ]} Elastin expression was also elevated at both gene and protein levels when type II collagen isolated from sturgeon cartilage was used in human dermal fibroblast culture and a mouse full‐thickness lesion.^{[ 147 ]} While there are promising indications of elastogenic potential of different collagen peptides, it should be noted that type I collagen alone as a biomaterial is insufficient to fully restore the elastic properties of the skin.

3.3. Hormones

The skin has developed complex mechanisms that include, among others, steroidogenic ability in order to maintain proper homeostasis, meanwhile defending the body from external interventions and biological influences.^{[ 148 ]} Hormones and hormonal imbalances have a substantial impact on the morphology and physiology of the skin, as well as on its numerous functions, including wound healing.^{[ 149 ]} Skin cells possess the whole biochemical machinery required to produce glucocorticoids, androgens, and estrogens from systemic precursors or, alternatively, through the conversion of cholesterol to biologically active steroids, e.g., estradiol, dihydrotestosterone, cortisol, etc. Circulating hormone levels, for instance growth hormones and sex steroids, gradually decline with age. While topical application of some hormones demonstrated positive effects on wound healing in animals and humans,^{[ 150 ]} it is worthy to elucidate their potential utilization in elastogenic dermal biomaterials.

3.3.1. Steroids

Besides being one of the most important female sex hormones and one of the most potent estrogens, 17β‐estradiol (E2) may increase synthesis of elastin in the skin, enhance epidermal thickness, and repair extracellular matrix.^{[ 151 ]} In vivo experiments showed that injection of estradiol elevated elastin levels in vocal fold injuries in rabbits.^{[ 152 ]} Synergy between estradiol and progesterone may help to properly align the extracellular matrix.^{[ 153 ]} Since 17β‐estradiol possesses sex‐dependent effects on elastin synthesis (enhanced for female dermal fibroblasts), these sex distinctions may be abolished with incorporation of cyclic adenosine monophosphate in dermal substitutes.^{[ 153 ]} It will be a challenge to retain the effects of estradiol to the biomaterial site of application. As estradiol has a wide variety of effects beyond the skin,^{[ 152} , ^{154 ]} its systemic effects must be included in experimental designs.^{[ 155 ]}

Aldosterone is a mineralocorticoid steroid hormone produced by adrenal glands and responsible for water and electrolyte balance in the body, e.g., regulation of blood pressure and levels of Na⁺ and K⁺ ions in plasma. Aldosterone demonstrated induction of elastogenesis in human skin and cardiac fibroblasts as well as in skin explants derived from striae distensae, independent of mineralocorticoid receptor signaling.^{[ 156 ]} Where aldosterone enhances collagen production via the mineralocorticoid receptor, activation of elastogenesis involves the IGF‐1 receptor.^{[ 156a ]} Using aldosterone in combination with mineralocorticoid receptor inhibitors may selectively induce elastogenesis without induction of collagenesis, which may consequently prevent from the formation of hypertrophic and keloid scars.^{[ 156b ]}

Dexamethasone is a synthetic glucocorticoid steroid hormone with a very broad spectrum of clinical applications ranging from asthma and allergies to different cancer types. While this compound possesses suppressive impact on collagenesis and hyaluronic acid synthesis by human dermal fibroblast cell lines,^{[ 157 ]} its elastogenic properties are ambiguous. For example, injection of dexamethasone showed no effect on elastin levels in vocal fold injuries in rabbits,^{[ 152 ]} but demonstrated elastogenic potency (up to 3 times increase) in cell culture experiments with fibroblasts derived from bovine ligamentum nuchae,^{[ 158 ]} where especially in older bovine ligament fibroblasts elastin synthesis was induced.^{[ 159 ]} In keloid explants, dexamethasone alone did not increase elastin synthesis.^{[ 156b ]} Dexamethasone has also been reported to suppress elastin mRNA expression in normal human fibroblasts.^{[ 85} , ^{160 ]} Additional experiments conducted with primary human fibroblasts focusing on protein deposition and architecture are needed to provide conclusive evidence regarding the impact of dexamethasone on dermal elastogenesis.

Compared to dexamethasone, other glucocorticoids, especially cortisol, its medication form hydrocortisone, and methylated derivative, methylprednisolone, demonstrated much lower potency in stimulation of elastin production by fetal bovine fibroblasts.^{[ 159 ]} Cortisol inhibited elastin and elastin mRNA production in normal human dermal fibroblasts but not in keloid cells.^{[ 160 ]} The elastogenic effects of hydrocortisone may be dependent on age, as in chickens the effect was quite prominent in embryos and 1‐day‐old organisms, but lost in 1‐ or 2‐weeks‐old chickens.^{[ 161 ]}

3.3.2. Peptide Hormones

Insulin is a peptide hormone and growth factor released by pancreatic beta cells and plays a crucial role in regulating blood glucose levels in the body.^{[ 162 ]} It is also able to improve wound healing as was seen when insulin was topically administered in vivo, resulting in a decreased healing time, reduced wound contraction, and enhanced angiogenesis and re‐epithelization.^{[ 150a ]} Its potential may be expanded to trigger elastin synthesis.

In human aortic SMCs, low insulin doses induced an elastogenic effect mediated by the insulin receptor,^{[ 163 ]} without promoting deposition of type I collagen and fibronectin. Insulin results in elastic fiber deposition by activating elastin gene expression and enhancing tropoelastin secretion. In human skin fibroblast cultures, supplementation of insulin alone and especially in combination with epidermal growth factor (EGF) promoted the synthesis of dermis‐like sheets, and production of elastin, type I collagen and fibrillin‐1.^{[ 164 ]} Interestingly, two fibrillin‐derived peptide hormones influence insulin levels. These hormones, asprosin and placensin (140 and 133 amino acids residues, respectively), are derived from the C‐terminal region of profibrillin 1 and 2 respectively, by furin‐mediated cleavage.^{[ 165 ]} Both hormones are glucogenic and act by cAMP‐PKA signaling, likely through a G protein‐coupled receptor. It remains to be investigated what role they play in skin regeneration, but the direct coupling of hormone production and elastic fiber formation is challenging.

3.4. Potassium Channel Openers

Potassium channels in cell membranes play a crucial role in the regulation of membrane potential and cellular excitability. Potassium channel openers are a class of drugs that influence the movement of potassium ions across the cell membrane, leading to hyperpolarization and reduced cellular excitability. Some studies suggest that potassium channel openers may have beneficial effects on wound healing, including the skin,^{[ 166 ]} and it may even lead to elastic fiber formation.^{[ 167 ]}

The effect of minoxidil, an ATP‐dependent potassium channel opener that causes vasodilation and is applied topically to promote hair growth, has mostly been studied on SMCs.^{[ 167 ]} There minoxidil‐induced tropoelastin mRNA levels and elastin synthesis independently from cell proliferation.^{[ 168 ]} Ad libitum administration of minoxidil in tap water to adult^{[ 169 ]} and aged mice^{[ 169} , ^{170 ]} as well as in young^{[ 167 ]} and adult rats^{[ 171 ]} preserved integrity of existing elastic fibers, improved elastic fiber synthesis, lowered blood pressure, and decreased aortic stiffness, with gender‐specific differences. Adverse effects included cardiac hypertrophy, an effect that may be avoided when an angiotensin II–receptor antagonist (e.g., irbesartan) is simultaneously administered.^{[ 167 ]} Another potassium channel opener, diazoxide, had a three times lower elastogenic effect compared to minoxidil in young rats, but could reduce side effects.^{[ 167 ]} Chemical derivatives of diazoxide and cromakalim also enhanced elastogenesis in vascular SMCs at a ten times lower dose than their unmodified counterparts.^{[ 172 ]} The potassium channel opener nicorandil demonstrated a very selective increase in aortic elastin protein content when orally administered to young rats.^{[ 173 ]}

Regarding dermal elastogenesis, the evidence is limited to minoxidil in cell culture experiments in chicken skin fibroblasts, where medium supplementation stimulated elastin synthesis and overexpression of elastin mRNA.^{[ 174 ]} Local application of potassium channel openers may stimulate elastogenesis while avoiding systemic side effects.

3.5. Metals

Metal ions such as copper and iron directly or indirectly affect wound repair, and their effects on elastogenesis are being studied.

3.5.1. Copper

Copper is a necessary element for skin formation and wound healing, as it is involved in the synthesis of elastin by fibroblasts and the cross‐linking of collagen and elastin as a cofactor for the lysyl oxidase enzymes.^{[ 175 ]}

Several studies used copper sulfate, CuSO_4, to administer Cu²⁺. In rat aortic SMCs, CuSO₄ boosted elastin crosslinking, promoted elastin synthesis fourfold, and increased LOX protein production 2.5 times.^{[ 176 ]} Copper nanoparticles, used to overcome the mild cytotoxicity of copper ions, with hyaluronan led to mature elastic fiber formation rather than amorphous accumulations.^{[ 177 ]} In a human fibroblast cell line, CuSO₄ administration showed increased elastin and type I collagen alpha 2 chain gene expression and increased deposition of elastin and type I collagen protein.^{[ 178 ]} When combining copper sulfate with a mixture of amino acids (Gly, Pro, Ala, Val, Leu, Lys), these cells demonstrated a synergetic effect of improved elastogenesis.^{[ 178 ]} It was also shown that valine and alanine were essential for elastin expression in primary human skin fibroblasts, while proline and glycine were responsible for collagenesis.^{[ 179 ]} In a small clinical trial with five volunteers, a commercial product containing Gly, Pro, Ala, Val, Leu, Lys, copper ions and hyaluronic acid showed reduced wrinkles, improved moisture, and enhanced elasticity.^{[ 180 ]} A combination of low‐dose CuSO₄ together with heparin was proposed to promote elastogenesis in lungs as inhalation therapy in patients with emphysema.^{[ 181 ]}

Other forms of copper also showed elastogenic potential. Copper oxide particles stimulated elastin deposition in dermal fibroblasts along with other ECM components.^{[ 182 ]} A Gly‐His‐Lys‐copper complex (GHK‐Cu) stimulated collagen and elastin production in primary human dermal fibroblasts even at low concentrations (0.01 nm), increased mRNA expression levels and the ratio of tissue inhibitors of metalloproteinases (TIMPs) to MMPs.^{[ 183 ]} In a clinical study with 40 female middle‐aged volunteers, topical application of GHK‐Cu reduced wrinkle volume and depth.^{[ 183 ]}

3.5.2. Iron

Iron has also been implicated in elastogenesis, although its mechanism is not clear. Low dosage of iron supplementation (2–20 µm Fe(III) citrate) to cultured human dermal fibroblasts induced elastogenesis with a threefold increase of tropoelastin mRNA and a significant enhancement of elastic fiber formation.^{[ 184 ]} Additionally, iron citrate protected elastic fibers in rat vascular SMCs and prevented fragmentation of elastic fibers of calcified aortic rings.^{[ 185 ]} Iron(II,III) oxide nanoparticles revealed a concentration‐dependent and shape‐dependent stimulatory effect on elastin deposition in spheroids of primary rat aortic SMCs.^{[ 186 ]} The relationship between iron ions and elastogenesis is complex, and an imbalance in iron homeostasis can have negative effects. For example, excessive iron or iron overload can lead to oxidative stress, which may contribute to the degradation of elastin and other structural proteins.^{[ 187 ]} Iron deficiency on the other hand has detrimental impact on the development of elastic fibers.^{[ 188 ]}

3.6. Antioxidants and Vitamins

Various antioxidants serve as promising candidates for ingredients of biomaterials due to their capacity to eliminate free radicals and protect cells.^{[ 189 ]} Natural extracts that possess antioxidant properties due to the presence of complex phytochemicals will be reviewed in the next section.

Retinol, or vitamin A₁, is a fat‐soluble vitamin that enhances both elastogenesis and collagenesis in monolayered and 3D‐cultured human skin fibroblasts.^{[ 190 ]} Topical retinol treatment of skin explants increased elastin, fibrillin‐1, and type I collagen gene expression together with elastic fiber network formation ex vivo. In in vivo experiments, full‐thickness punch biopsies were collected from sun‐protected buttock skin of elderly individuals who followed topical retinol application demonstrated that retinol activated ECM deposition (elastin, type I collagen, and fibronectin) by fibroblasts with epidermal thickening along with increased endothelial cell proliferation.^{[ 191 ]} The vitamin A derivative, all‐trans retinoic acid, decreased proliferation of chicken embryonic dermal fibroblasts, but almost doubled elastin protein production.^{[ 192 ]} A similar elastogenic effect was observed in neonatal rat lung fibroblasts.^{[ 193 ]} In UVA‐treated human foreskin fibroblast cell line, retinoic acid‐loaded nanoparticles lowered MMP‐1 production and enhanced the synthesis of type I collagen and elastin.^{[ 194 ]} Retinoic acid nanoencapsulation decreased cytotoxicity in human dermal fibroblasts and reduced skin damage in a nude mouse model after topical administration of nanoparticles compared to free retinoic acid.^{[ 138 ]}

Polyphenols are antioxidants with multiple phenol structures in their chemical makeup. They represent a large and diverse group of naturally occurring compounds found in plants. The polyphenols pentagalloyl glucose (PGG) and epigallocatechin gallate (ECGC) promote production, alignment, crosslinking, and preservation of elastin by binding to monomeric tropoelastin, improving coacervation and enhancing lysyl oxidase production.^{[ 195 ]} Upon short‐term UVA exposure, they promote significant elastin (30‐45x) and collagen extracellular matrix deposition,^{[ 196 ]} without changing intracellular tropoelastin production.^{[ 197 ]} Upon addition of another polyphenolic compound, ellagic acid, to human dermal fibroblasts, elastin mRNA synthesis was threefold higher than retinol‐supplemented culture. Moreover, elastin deposition improved significantly in the retinol‐coupled ellagic acid group, demonstrating that combining polyphenols with retinol could be promising to improve elastin synthesis and deposition.^{[ 198 ]}

Ascorbic acid or vitamin C has a well‐known stimulatory effect on collagen synthesis. However, initially it was believed to have a negative impact on elastogenesis as seen in murine SMCs.^{[ 199 ]} Also, a direct correlation between ascorbic acid concentration and incorporation of hydroxyproline in elastin protein was seen in those experiments. In vivo study with vitamin C‐deficient guinea pigs revealed that lack of ascorbic acid prevented hydroxylation of already existing proline residues in aortic elastin but did not affect the synthesis of elastin protein in scurvy.^{[ 200 ]} Later it was discovered that ascorbic acid was capable of increasing elastin synthesis by chicken aortic SMCs and in a rat model, as did its stable analogue 2‐O‐D‐glucopyranosyl L‐ascorbic acid in rat pulmonary fibroblasts.^{[ 201 ]} Moreover, sodium l‐ascorbate improved the production and preservation of elastic fibers by human skin fibroblasts.^{[ 202 ]}

Nicotinamide, a form of vitamin B₃, and its derivatives (e.g., 2,6‐dihydroxynicotinamide) may also have elastogenic potential as shown in skin fibroblasts^{[ 203 ]} and in clinical trials with topical cream application in 50 women.^{[ 204 ]} Nicotinamide mixed with polydeoxyribonucleotide and vitamin C was tested by microneedle injections in UVB‐irradiated mouse skin, where especially the combination of the three components led to prominent elastin synthesis.^{[ 205 ]}

Coenzyme Q₁₀ is a crucial component of the electron transport chain and a widely used antioxidant in cosmetic formulations. The cofactor exists as an equilibrium between its oxidized (ubiquinone) and reduced (ubiquinol) form. Compared to ubiquinone, ubiquinol was more efficient in reverting the expression of the senescent phenotype of fibroblasts.^{[ 206 ]} In human primary foreskin fibroblasts, it induced elastin and type IV collagen gene expression, while suppressing MMP‐1 levels.^{[ 207 ]} At specific doses, coenzyme Q₁₀ promoted elastin mRNA expression in human dermal fibroblasts.^{[ 208 ]}

3.7. Natural Products

Screening of natural products for induction of elastic fiber production seems to become more popular in recent years. It could be economically advantageous to use available natural products rather than synthetic compounds, especially if their synthesis and purification are laborious and as some natural compounds cannot be synthesized yet because of their steric complexity. Natural extracts represent a complex mixture of components whose properties may not be directly related to those of a single component. It may be difficult to compare studies from different research groups as variations in extraction process likely result in varying final products.

3.7.1. Defined Chemicals from Natural Sources

The beneficial dermal effect of notoginseng or Chinese ginseng is mainly attributed to steroid glycosides, protopanaxadiol and protopanaxatriol, present in extracts. In vitro experiments with protopanaxadiol and protopanaxatriol resulted in enhanced mRNA expression of elastin, type I collagen, and fibrillin‐1 in human skin fibroblasts isolated from both young (20‐years‐old) and old (60‐years‐old) donors.^{[ 209 ]} A crude extract of nanosized ginseng stem increased the cell proliferation and synthesis of ECM proteins (collagen, fibronectin, elastin) of human keratinocyte and skin fibroblast cell lines and accelerated the wound closure in mice by decreasing inflammation.^{[ 210 ]}

Gamma‐aminobutyric acid, commonly known as GABA, is the primary inhibitory neurotransmitter in the central nervous system. GABA promoted production of elastin intracellularly in human dermal fibroblasts, but no extracellular elastic fibers were observed.^{[ 211 ]} In another study, skin elasticity was found to be improved after topical application, but a detailed ECM analysis was not conducted.^{[ 212 ]} This compound also triggered type I collagen transcription, diminished MMP‐1 expression,^{[ 213 ]} and abated atopic dermatitis skin lesions via suppression of IgE and IL‐4 production.^{[ 214 ]} Additional experiments are required to investigate the exact impact of GABA on the ECM during wound healing and elucidate its potential applications in biomaterials.

Tetrahydrocurcumin is a metabolite of curcumin with stronger antioxidative properties^{[ 215 ]} and was able to elevate elastin, collagen, and HA content in a human dermal fibroblast cell line.^{[ 216 ]} Camphor, a compound found in essential oils of various herbs, induced gene expression of elastin in primary human skin fibroblasts and UV‐exposed murine skin.^{[ 217 ]} In addition, its safety and anti‐aging properties have recently been assessed in reconstructed skin substitutes.^{[ 218 ]} Two main compounds from sheep laurel extract, avicularin and epicatechin‐(2β→O→7,4β→6)‐ent‐epicatechin, were identified for their bioactivity to upregulate elastin and type I collagen expression and increasing skin thickness.^{[ 218 ]}

Linoleic acid, an essential polyunsaturated omega‐6 fatty acid, is a structural component of cell membranes and a key contributor to the lipid barrier of the skin, helping to prevent excessive water loss and maintaining skin hydration.^{[ 219 ]} Linoleic acid‘s analog 8‐[2‐(2‐pentyl‐cyclopropylmethyl)‐cyclopropyl]‐octanoic acid (DCP‐LA) upregulated elastic fiber deposition and fibulin‐5 expression in human skin fibroblasts, without changing their mRNA expression.^{[ 220 ]}

The phytoestrogen tanshinone IIA is a lipophilic diterpene quinone that possesses anti‐inflammatory and antioxidant properties.^{[ 221 ]} In human cardiac fibroblasts, tanshinone IIA enhanced elastin production while hindering collagenesis.^{[ 222 ]} Subsequently, it would be worthwhile to investigate the consistency of this effect on dermal fibroblasts as this could aid in preventing hypertrophic scars characterized by excessive collagen deposition.

3.7.2. Extracts

Several herbs have been investigated for their potential for elastogenesis and include low‐cost products like dill. A study in mice with dill extract in drinking water showed elastic fiber neosynthesis and protection of existing fibers from degradation in blood vessels together with increased tropoelastin and LOXL‐1 mRNA expressions.^{[ 223 ]} Also in skin, dill extract protected the elastic network from digestion by elastase in injured mice.^{[ 224 ]} In human skin fibroblasts, dill extract induced elastin production together with upregulated gene expression of LOXL‐1.^{[ 225 ]} Recently, combined use of dill and blackberry extracts enhanced the effects both in vitro (human dermal fibroblasts) and in vivo (clinical trials with a topical lotion) by inducing elastin and collagen production, inhibiting elastases and forming a functional ECM.^{[ 226 ]}

As mentioned in Section 3.2.4, soy peptides with collagen peptides induced elastin expression in dermal fibroblasts.^{[ 146 ]} In rat cardiac myoblasts, soybean extract showed elastic fiber stimulation with enhanced elastin promoter activity and suppressed elastase activity, while topical application induced elastic fiber formation in murine and porcine skin.^{[ 227 ]}

Some evidence of natural extracts is more incidental and require further investigation. We will give some examples thereof. An aqueous wheat extract increased expression of type I collagen and elastin in a human keratinocyte cell line.^{[ 228 ]} Blackcurrant extract promoted protein deposition of elastin and type I and III collagen by a dermal fibroblast cell line and oral intake by ovariectomized rats raised elastin, collagen, and HA levels in skin.^{[ 229 ]} An extract of the Orpheus flower upregulated elastin and type VI and XVI collagen mRNA expression in H₂O₂‐treated normal human skin fibroblasts and topical application demonstrated better skin elasticity in a small clinical study with 20 middle‐aged women.^{[ 230 ]} Sponge gourd stem sap inhibited MMP‐1, MMP‐2, and MMP‐9 expression,^{[ 231 ]} and stimulated elastin and type I procollagen synthesis in human dermal cells in a dose‐dependent manner by controlling the epidermal growth factor receptor and proliferator‐activated receptor gamma (PPARγ) signaling.^{[ 232 ]} Tasmanian pepper berry extract enhanced synthesis of elastin and type I collagen in five human skin explants and induced skin smoothness, restored dermal thickness, and reduced scar appearance of stabilized stretch marks upon topical application in 30 women.^{[ 233 ]}

New trends include the use of fermented extracts and probiotics. Fermented adlay bran extract added to human primary dermal fibroblasts increased elastin mRNA levels and decreased MMP‐12 and MMP‐9 gene expression.^{[ 234 ]} Rhodiola rosea fermented by Lactobacillus plantarum shielded fibroblasts from UVA‐induced oxidative stress by lowering levels of reactive oxygen species and raising the antioxidant level. Moreover, it diminished MMP activity, and enhanced collagen and elastin production in comparison to non‐fermented Rhodiola rosea.^{[ 235 ]} Upon topical application of a living Lactobacillus plantarum Lp90, a probiotic, to Epiderm, elastin expression was elevated, while other studied ECM components, collagen, and hyaluronan, did not change.^{[ 236 ]} In conclusion, investigation of elastogenic properties of various natural extracts and fermentation methods can shift the focus to a completely different field—probiotics and microbiome. However, detailed studies on the active ingredients in such an extract would reduce the impact of cultivation, extraction methods, and raw materials and allow for more consistent and robust results.

4. Discussion and Future Outlook

In this review, we collected available information about compounds that may be capable of inducing elastogenesis in biomaterial formulations. Our particular focus lied in the realm of skin regeneration after severe excisional and burn injuries as there is ample room for improvement in terms of reducing scar formation, and improving healing pace and skin elasticity.^{[ 237 ]} The distinguishment between induction of the elastin protein only, elastic fiber deposition, and functional elastogenesis leading to enhanced biomechanical properties is quite challenging. Thus, our findings are consolidated in Table 1, where abovementioned candidates are assessed based on the published evidence for the creation of novel elastogenic biomaterials. A compound receiving three green ticks is considered as a possible elastogenic candidate to perform further investigation (highlighted in bold), while a compound with more than three ticks is deemed a promising elastogenic substance (highlighted with bold green font style). Our primary emphasis was directed towards dermal cells (e.g., skin fibroblasts), although encouraging results for other cells sometimes were also included (e.g., SMCs, cardiac fibroblasts), as these compounds may be worthy to study in a dermal setting as well. To date, there is no universal manual for the investigation of functional elastic fibers, which is why we pointed out the major aspects to look at: firstly whether the effect was observed in cell culture, explants, and/or living organisms, and secondly on what level the effect was found, i.e., gene expression, protein deposition (with immunoblotting, mass spectrometry, etc.), histological & morphological findings (e.g., histological assays and immunofluorescent detection) and/or biomechanical tests (e.g., elastic modulus, stiffness, tensile strength, strain to failure, etc.). Note that an increased gene expression does not automatically result in an increase of protein expression. At the same time, protein synthesis cannot guarantee the formation of elastic fibers (that have microfibrillar components, see Figure 2). Even if elastic fibers are formed, they should be tested for biomechanical properties, in order to show whether they really improve elasticity and resilience on the tissue level.

When we discuss advanced elastogenic skin biomaterials, we should consider multiple aspects of the supplementary compounds, i.e., their properties for restoration of the complex network of elastic fibers, biocompatibility, integration in the wound, potential sustained or controlled release and ultimate elimination from the implantation site. Additionally, angiogenesis and innervation in tissue‐engineered constructs is crucial to ensure clinical translation.^{[ 243 ]} The goal is to propose new options for biomaterials that not only provide structural support but also actively contribute to the formation of new elastic fibers and skin regeneration. Advances in nanotechnology allow the creation of smart materials with unique properties that can respond to specific environmental conditions and provide release mechanisms for bioactive compounds.^{[ 244 ]} From Table 1, the most encouraging substantiation was for the following type of compounds: recombinant human tropoelastin and digested elastin peptides preparations, hyaluronan, dill extract, derivatives of vitamin A, minoxidil and 17β‐estradiol. A direct comparison of these bioactive substances in experimental setups will provide deeper insights to this topic, as will the testing of combinations of compounds. Tropoelastin, as the building block for crosslinked elastin primarily obtained through recombinant techniques, can serve both as a biomaterial main component and as an additive. Its main advantage is that it bypasses the elastin secretion by cells. While exogenous tropoelastin can serve as a substrate to produce more elastic fibers, there is the evidence that it (in the form of the crosslinked tropoelastin‐derivatized HA matrix) can also be exploited in the formation of new elastic fiber networks in skin tissue in rats.^{[ 77 ]} Enzymatically digested elastin, generally with a lower molecular weight than tropoelastin, is less likely to be directly incorporated in newly formed elastic fibers but shows proficiency to induce elastogenesis by cells. Enzymatic digestion results in degradation products that can be released in the human body, thus forming peptides that can be sensed by cells, who respond by launching corresponding internal signaling pathways, similar to matrikines or growth factors. Stable pyridinium core structures may play the key role in the recognition of elastin‐derived compounds by the cells and leading to activation of particular effects. Such digests can stimulate cell migration, proliferation, and differentiation, promoting skin repair and regeneration of damaged tissues. The complete signaling picture by elastokines is unclear, but the phosphoinositide 3‐kinase (PI3K)/protein kinase B (PKB/Akt) pathway can be triggered by soluble elastins.^{[ 133} , ^{245 ]} At the same time, this pathway is targeted in cancer treatment as its aberrant activation leads to tumorigenesis, cancer progression, and drug resistance.^{[ 246 ]} Processes in cancer biology, like cell proliferation and migration, are also essential for the repair of damaged tissues. However, effects of elastokines could be beneficial for biomaterials if they can be spatiotemporally controlled.^{[ 247 ]} Hyaluronan is pivotal in wound healing with its affinity to water, structural support, anti‐inflammatory responses, angiogenesis and may particularly stimulate elastogenesis, especially considering its effects on promoting elastin gene expression and protein deposition.^{[ 119a,b ]} At the same time, experiments with crosslinking of tropoelastin with hyaluronan resulted not only in induced elastogenesis, but also in a proper incorporation of the applied biomaterial.^{[ 77 ]} Despite this compound shows molecular weight‐ and crosslinking state‐dependent properties, it is an important substance to consider in formulation of novel elastogenic biomaterials. While minoxidil is primarily known for its vasodilatory effects and use in the treatment of hair loss, its dermal elastogenic potential is of interest. This potassium channel opener showed improved wound healing, as well angiogenesis, when applied topically in rats.^{[ 248 ]} Although this compound has been extensively tested for aortic elastogenesis in vivo, it is worthwhile to check the dermal elastogenic effect upon topical or local application, which will also prevent its off‐target effects, such as weight gain and cardiac hypertrophy.^{[ 170 ]} Dill extract, derived from a low‐cost and easily accessible herb, shows promise in elastic fiber neosynthesis and in protection of existing elastic fibers. Natural extracts constitute complex mixtures of various compounds usually not easily synthesized in the lab and the driving force of this extract remains unknown. It could be a specific component of the extract or multiple components in the mixture. Vitamin A and its derivatives demonstrated elastogenic potency, although collagen synthesis was also enhanced. Excessive collagenesis goes hand in hand with scar formation.^{[ 249 ]} Thus, vitamin A compounds should be used in combination with collagen‐suppressive molecules, such as human cathelicidin peptide LL‐37^{[ 250 ]} or Lipo‐PGE₁ (a lipid‐encapsulated preparation of prostaglandin E₁).^{[ 251 ]} It is crucial to highlight that the application of additional compounds can have other consequences in wound healing (e.g., increased cyclic AMP signaling by prostaglandin E₁ ^{[ 252 ]}) and may increase the complexity of biomaterial production. 17β‐estradiol (E2), the major female sex hormone and the most potent estrogen found in humans, possesses broad biological activity, including elastogenic potential. Despite some methods to eliminate its sex‐specificity are available,^{[ 153 ]} systemic effects should be studied when applied to dermal biomaterials. Estradiol and hyaluronan were reported to act on elastogenesis through the TGF‐β/Smad signaling pathway.^{[ 119} , ^{253 ]} Therefore, investigating their combination is recommended, as it may lead to the amplification of each other's effects. At the same time, crosslinking of tropoelastin with hyaluronan resulted not only in induced elastogenesis but also in the proper incorporation of the applied biomaterial. Hence, a comprehensive assessment of the individual and synergistic effects of candidates could contribute significantly to improving our understanding of their roles in promoting functional elastogenesis, thereby enhancing the development of skin biomaterials for wound healing and tissue regeneration.

We here propose multiple components that may act as stimulators of elastic fiber formation. While some researchers investigated both elastogenic potentials and activated corresponding internal signaling pathways, others have yet to publish information about the possible underlying mechanisms. It was seen that certain compounds can either exert the effect via elastin receptor complex,^{[ 133 ]} show cross‐activation of multiple receptors,^{[ 44} , ^{133 ]} possess the mechanism described for particular cell types^{[ 169 ]}—not directly applicable for dermal cells—or demonstrate an opposing effect when used together with another elastogenic compound.^{[ 70 ]} Undoubtedly, when a compound is reported to trigger production of elastic fibers, a detailed and comprehensive analysis of its effects is needed. Such unveiled cellular mechanisms not only explain the nature of the effect but offers insight into alternative approaches or potentially more efficacious candidates. As a future outlook, it is important that scientists more systematically study the cell biological pathways responsible for elastogenic effects, not only looking at elastin gene expression or elastin protein, but also effective elastic fiber formation including its microfibrillar components. When proper mode‐of‐actions are established, it can be studied whether the elastogenic effect is maintained in a biomaterial formulation and the compound itself is compatible with the production process. Biomaterials offer an effective spatial administration route, where a sustained release of active compound can reduce systemic off‐target or toxic effects. The key principle that needs to be adopted in this respect is to keep it as simple as possible, while maintaining the desired effect. At the same time, ensuring patient safety is paramount, necessitating biocompatibility and an appropriate degradation rate, while excluding inflammation, toxicity, and other adverse effects. Aligning to this reasoning, single (pure) biological or chemical compounds are favored over complex mixtures or extracts as batch‐to‐batch variation may give rise to variations in effects. The whole process of biomaterial production must be reproducible in order to deliver a new product to the market. In addition, compounds that can act extracellularly are favored over intracellular effector molecules, as these do not need to be taken up by the cell and route to the desired cell organelle.

In summary, it is highly important to continue research on elastogenesis. In the process of identifying novel substances with the potential to augment the synthesis of new elastic fibers, elucidation of either the comprehensive mechanisms, previously undisclosed facets of functional elastic fiber production, and/or the revelation of a novel class of elastogenic compounds may be uncovered. At the same time, biomaterial formulation is a complex topic where we should think about its components, mechanical and physical properties, and state.^{[ 254 ]} Even though the construction of elastic fibers in vivo must be significantly more challenging than in in vitro 2D culture, this review gives ample suggestions for components of dermal substitutes that can be further investigated.

Conflict of Interest

The authors declare that Gizem Coşar Kutluoğlu, Dominique Manikowski, and Claudia Doberenz are employed by MedSkin Solutions Dr. Suwelack AG, a company that produces and markets MatriDerm, a dermal substitute. However, the opinions expressed in this review were not influenced by the company.

Biographies

Roman Krymchenko obtained his B.Sc. and M.Sc. in organic chemistry from Taras Shevchenko National University of Kyiv, Ukraine. Later, he received a scholarship for the Erasmus Mundus Joint Master Degree in Translational Cosmetic and Dermatological Sciences at the University of Eastern Piedmont, Italy, and the University of Namur, Belgium. Currently, he is a Ph.D. candidate at Radboud University Medical Center, the Netherlands, and a Marie Skłodowska‐Curie fellow. His research interests focus on the preparation and analysis of elastin hydrolysates, and the construction and assessment of corresponding regenerative biomaterials for skin healing, both in vitro and in vivo.

Gizem Coşar Kutluoğlu obtained her B.Sc. degree in bioengineering from Ege University, Izmir and her M.Sc. degree in bioengineering from Yildiz Technical University, Istanbul, Türkiye both with a focus on biomaterials. She is currently employed as a Development Specialist at MedSkin Solutions Dr. Suwelack AG, Germany, and is a Ph.D. candidate at Radboud University Medical Center, the Netherlands, as a Marie Skłodowska‐Curie fellow. Her doctoral research is focused on the development of dermal substitutes to manage the wound healing process and tip the scales in the direction of regeneration.

Krymchenko R., Coşar Kutluoğlu G., van Hout N., Manikowski D., Doberenz C., van Kuppevelt T. H., Daamen W. F., Elastogenesis in Focus: Navigating Elastic Fibers Synthesis for Advanced Dermal Biomaterial Formulation. Adv. Healthcare Mater. 2024, 13, 2400484. 10.1002/adhm.202400484