Abstract
In vitro engineered skin models are emerging as an alternative platform to reduce and replace animal testing in dermatological research. Despite the progress made in recent years, considerable challenges still exist for the inclusion of diverse cell types within skin models. Blood vessels, in particular, are essential in maintaining tissue homeostasis and are one of many primary contributors to skin disease inception and progression. Substantial efforts in the past have allowed the successful fabrication of vascularized skin models that are currently utilized for disease modeling and drugs/cosmetics testing. This review first discusses the need for vascularization within tissue‐engineered skin models, highlighting their role in skin grafting and disease pathophysiology. Second, the review spotlights the milestones and recent progress in the fabrication and utilization of vascularized skin models. Additionally, advances including the use of bioreactors, organ‐on‐a‐chip devices, and organoid systems are briefly explored. Finally, the challenges and future outlook for vascularized skin models are addressed.
Keywords: 3D skin models, alternate to animals, dermatology, in vitro, skin tissue engineering, vascularization
Vascularized human skin models are an emerging alternate platform for drug testing. The need for incorporation of blood vessels within in vitro models emerges from the fact that skin diseases and the ensuing therapy influence vessel morphology, functionality, and growth. Use of bioreactors, biofabrication techniques, and synthetic cell‐mimics are predicted to pave a pathway for more advanced vascularized skin models.
1. Introduction
Skin‐related ailments are globally prevalent and have a detrimental impact on patient well‐being.[ 1 , 2 , 3 ] To combat skin disorders, patients rely on a limited number of drugs accessible on the market. Some of the challenges associated with the availability of novel drugs are their slow advancement from pre‐clinical laboratory settings to clinical trials, failed clinical trial outcomes, and unexpected side effects. Advancements in pre‐clinical testing techniques are crucial to resolve these issues and facilitate the translation of novel therapeutics into clinical products. Beyond the extensive use of conventional 2D skin cell monolayers and animals as tools for pre‐clinical drug testing in dermatology, a rapidly emerging alternative involves the utilization of in vitro‐engineered 3D human skin models. Existing tissue‐engineered skin models recapitulate the elementary anatomy and physiology of both native healthy and diseased skin tissue and can provide valuable readings related to drug penetration, absorption, and toxicity. Yet, there is an increasing demand for more complex skin models that can more precisely and realistically mimic skin tissue architecture and functionality and, therefore, have the potential to fully replace conventional pre‐clinical models.
To establish in vitro skin models as a mainstream platform for pre‐clinical studies, researchers have attempted to develop novel skin models with the following properties: a) enhanced barrier properties, b) encompassing a variety of cell types comparable to the cellular diversity of native skin tissue, and c) optimal media composition to facilitate model survival. Moreover, several technologies such as microfluidics and bioreactors have been adapted to enable the long‐term maintenance of skin models. It is recognized that skin models require a certain level of complexity to recapitulate the physiology of human tissues. For instance, the reconstructed human epidermis (RHE) models that comprise only the epidermal compartment of the skin are the simplest representation of a skin tissue. However, the need for an underlying fibroblast‐consisting dermis compartment to facilitate optimal epidermal differentiation is widely accepted.[ 4 , 5 ] Similarly, the inclusion of other cell types such as endothelial cells (ECs), immune cells, neuronal cells, lymphatic cells, and skin appendages could be highly relevant in the context of novel drugs and cosmetics applications.
The inclusion of ECs in skin models holds a key role in creating realistic models, due to their significant impact on skin homeostasis through the formation of a network of blood vessels (BVs). Tissues thicker than 100–200 µm require sufficient vascularization for optimal transport of essential nutrients, growth factors, and oxygen to the cells as well as the elimination of metabolic waste products.[ 6 ] Apart from their primary role as biological pipelines, ECs are also strongly involved in cellular cross‐talk with many diverse cell types, the foundation for maintaining tissue homeostasis. BVs also have a vast implication in several skin‐related ailments. For instance, in inflammatory skin diseases such as psoriasis and atopic dermatitis (AD), BVs spearhead disease progression. Additionally, in skin grafts for burn injuries, pre‐vascularization has been shown to improve integration with the host tissue, enabling long‐term survival of the implanted grafts. Therefore, there is a need for the development of fully‐vascularized in vitro human skin models to mirror disease progression and to be utilized as skin grafts and platforms for pre‐clinical assays.
Numerous techniques have been developed in the past to fabricate vascularized human skin models (VHSMs). Major advancements include 1) the development of an optimal 3D environment for the ECs to form vascular networks, 2) the development of vascular‐permissive hydrogels, 3) progress in understanding co‐culture systems, 4) the use of sacrificial channel micro‐fabrication to create a framework for the attachment of ECs, 5) the ability to integrate angiogenic growth factors either in soluble form or attached to the scaffold for prolonged release, 6) the use of decellularized tissues with an intricate framework for vascularization as scaffolds, 7) the development of scaffold‐free techniques that use cell‐derived extra‐cellular matrix (ECM) to provide an EC‐permissive environment, 8) the utilization of genetically modified cells to express angiogenic factors and prevent cell apoptosis, 9) the utilization of patient‐derived induced pluripotent stem cells (iPSC), which allows the development of patient‐specific VHSMs, and 10) progress in microfluidic approaches with multiple cellular compartments. These advancements have directed the progress of VHSMs as a promising alternative platform for pre‐clinical drug testing. The future of vascularized skin models entails the development of personalized skin models and body‐on‐a‐chip devices as well as the integration of synthetic cell‐like materials within VHSMs to assist optimal vascularization. This review summarizes past and current techniques used for the fabrication of VHSMs, emphasizes the necessity of vascularization, and highlights some of the current challenges in the fabrication and utilization of VHSMs.
1.1. The Need for Alternatives to 2D and Animal Models
Conventional drug and cosmetic screenings are usually carried out using 2D cultures and animals. 2D cultures comprise cells seeded as a monolayer and offer a representation of human skin tissues that is both minimalist and premature. Simplicity and an amenability for high‐throughput screening are benefits of these cultures, yet, cellular monolayers are inept in mirroring the complexity and intricacy of native tissue architecture.[ 7 ] Cellular viability, morphology, motility, and differentiation are directly associated with the culture environment.[ 7 , 8 ] The homogenous distribution of culture medium on a 2D monolayer supplies essential growth factors, nutrients, and soluble oxygen to individual cells uniformly, which, in fact, does not mirror the reality of nutrient distribution within a thick native tissue. Monolayer culture conditions do not favor optimal cell–cell and cell‐ECM interactions, because cells on a flat surface physically communicate only with their adjacent cells. Conversely, animal models possess the complexity of human tissues and can provide better predictions on the efficacy of drugs and cosmetics. As animal tissues have been continually scrutinized for their anatomical, physiological, and molecular resemblance to human tissues and their ability to predict therapeutic outcomes for humans,[ 9 ] the consensus is that porcine skin anatomically resembles human skin best. There are, however, differences between porcine and human skin pertaining to the elastic modulus, possibly caused by differences in elastic fiber and lipid contents.[ 10 , 11 ] Gallagher et al. recently studied the mechanical properties of burned skin tissues from porcine and human sources and reported significant differences in modulus values.[ 10 ] This was attributed to differences in elastic fiber and lipid content as well as the ability to retain moisture at higher temperatures.[ 10 ] In addition, most drug testing assays are performed on thawed porcine skin, which has been shown to exhibit an abnormal increase in permeability. This, in turn, can result in a substantial unpredictability with regard to drug penetration as well as discrepancies in the generated data.[ 12 ]
The most common animal models used to mimic human skin diseases and wounds are murine models. This is despite the fact that there are obvious anatomical and functional differences between mouse and human skin tissue. First, there is a difference in skin thickness (≈100 µm for human skin compared to <25 µm for murine skin).[ 13 ] Human skin also comprises more cellular layers in the epidermis than murine skin. Furthermore, the renewal of the epidermis in murine skin takes <10 days, whereas human skin takes much longer (≈28 days) to renew completely.[ 14 ] These differences in skin architecture can have major implications for the accuracy of skin disease modeling and the testing of novel therapeutics, particularly with regard to drug absorption test outcomes. Murine and human skin also differ in their cutaneous wound healing mechanisms. Murine skin heals wounds primarily via a contraction mechanism, whereas human skin wounds develop granulation tissue and undergoes re‐epithelialization.[ 15 ] Moreover, murine skin comprises the panniculus carnosus, a layer of thin striated muscle responsible for wound contraction, which is only present as remnants in distinct anatomical sites of the human skin and are considered as vestigial.
In addition to morphological differences, gene‐level differences between animal and human skin cells also exist. Global transcriptomic profiling of six common murine models has revealed that murine models were unable to accurately recapitulate two main clinical features of atopic dermatitis (AD), namely epidermal barrier disruption and immune response.[ 16 ] The transcriptomic analysis showed murine profiles to substantially differ from human AD samples.[ 16 ] None of the six mouse models were able to fully recapitulate AD features. In another study, gene expression in five selected mouse models of psoriasis was found to exhibit similarities with that of clinical psoriasis.[ 17 ] However, the study also highlighted that there were significant differences in immune‐associated gene expression between the mouse and human models of psoriasis. In conclusion, a consensus on the pre‐selection of murine models for skin disease modeling has yet to be reached. The selection of incorrect murine models could result in an inaccurate interpretation of disease pathophysiology and possibly even failed clinical trial outcomes. Thus, beyond ethical concerns, the dissimilarity of animal skin to human skin and the limitations of animal models highlight the need to replace animal models in dermatological research.
1.2. Anatomy of the Vascularized Human Skin
Skin is a 3D hierarchical tissue that comprises three distinct layers: epidermis, dermis, and hypodermis (Figure 1A,B).[ 18 ] The outermost layer of the skin, the epidermis, endures physical, chemical, and microbial traumas in order to guard the body against infections. Also, the epidermis functions to prevent dehydration and to eradicate toxins.[ 19 , 20 ] The epidermis houses several cell populations, namely keratinocytes (90%), pigment‐producing melanocytes, antigen‐presenting Langerhans cells, and Merkel cells.[ 18 , 21 ] It is composed of several sub‐layers with distinct functionalities (Figure 1B). Keratinocytes interact with each other via desmosomes and tight junctions to create an intact barrier (Figure 1C). The stratum basal, the deepest cell layer of the epidermis, firmly adheres to the underlying dermal compartment via dermal‐epidermal junctions (DEJs). During the differentiation process, a few cells from the stratum basal detach, proliferate, and migrate upward, forming the other upper layers, namely stratum spinosum, stratum granulosum, stratum lucidum, and finally the stratum corneum (the exterior layer of the epidermis). The granular layer contains squamous keratinocytes that expresses filaggrin and loricrin, which are essential for maintaining skin barrier properties. The differentiated cells that form the stratum cornea are anucleated, flattened, and are continuously replaced by inner keratinocytes that travel toward the skin surface.[ 19 , 22 , 23 ] Both migration and the upward directed proliferation of keratinocytes are of special importance during the wound healing process.[ 19 ]
Figure 1.
Anatomy of vascularized skin. A) The distribution of BVs in the skin shows two primary horizontal arterial and venous plexuses that ascend to the superficial plexuses. Capillary loops from the superficial plexuses distribute and diffuse nutrients and oxygen to the epidermal compartment. B,C) A magnified view of the dermo‐epidermal junction and the epidermal compartment shows interaction between basal keratinocytes and the underlying basement membrane via anchoring filaments. Keratinocytes interact with each other via tight junctions and desmosomes.
The dermis encompasses the bulk of the skin and is known to provide structure and strength. It is divided into two sub‐layers, the superficial papillary and the deeper reticular dermal layers. The papillary dermal layer mainly consists of fibroblasts that play a key role in maintaining the skin's elasticity and tensile strength by depositing collagen and elastin.[ 24 ] The reticular dermal layer mainly consists of dense connective tissue containing bundles of collagen fibers and elastic fibers, which give the skin its flexibility.[ 25 ] The deepest layer of the skin is the hypodermis layer, which mainly contains adipose cells. The hypodermis offers cushioning between the skin layers, muscles, and bones. Moreover, the presence of proteoglycans and glycosaminoglycans serves as a nutrient reservoir by absorbing fluid into the tissue and giving it mucous‐like properties.[ 26 , 27 ] In addition to fibroblasts and keratinocytes, the skin contains large numbers of hair follicles, immune cells, melanocytes, Merkel cells, nerve fibers and BVs.
The dermal and hypodermal compartments comprise an intricate network of BVs that supply nutrients, growth factors and oxygen to the avascular epidermal layers. Figure 1A depicts the schematic of vessel distribution in skin.There are two primary horizontal plexuses composed of arteries and veins in the dermal compartment, namely the superficial sub‐papillary plexus (SSP) and the deeper cutaneous plexus (DCP). Arterioles that supply blood to the muscles and the hypodermal layer form the DCP, which reside between the subcutaneous and the cutaneous compartment. The arteries and veins arrange in a vertical vascular pattern to connect to the SSP, which is located between the epidermis and the papillary dermal compartment. The vessels in the DCP differ in morphology to those in the SSP. DCP vessels have a wider diameter (10–35 µm in the SSP and 40–50 µm in the DCP) and thicker walls.[ 28 ] Individual arterioles in the dermal papilla form separate capillary vessel loops that have an ascending limb, an intra‐papillary loop, and a descending limb which fuses with post‐capillary venules.[ 29 , 30 ] The capillary loop supplies blood to a skin area that is between 0.04 and 0.27 mm2 in size.[ 28 , 31 ] There are numerous anastomoses in the skin vasculature through which blood flows that play an important role in temperature regulation.[ 32 ]
1.3. Historical Development of In Vitro Skin Models
The fact that basal keratinocytes in the human epidermis divide once every 14 days has been known for some time. Nevertheless, their expansion in vitro have presented a considerable challenge.[ 33 ] Rheinwald and Green in 1975 serially cultivated epithelial cells (a mouse teratoma cell line).[ 34 ] However, the cells were unable to proliferate in normal culture media after low density plating. Researchers then discovered that these epithelial cells were able to propagate into epidermal colonies and grow indefinitely in co‐culture with lethally irradiated 3T3 fibroblast cells.[ 35 ] This indicated the importance of fibroblast‐keratinocyte interactions for epidermal formation and differentiation. Achieving in vitro serial cultivation of human keratinocytes into stratified squamous epithelial layers from single human keratinocytes was an important milestone in the development of modern skin models.[ 36 , 37 ] Following this, Green et al. fabricated keratinocyte sheets that could be harvested as skin grafts and used in clinical applications. Sheets were produced by seeding human keratinocytes on top of a supporting 3T3 feeder cell layer.[ 38 ] Such epidermal sheets have since been used for various applications such as allografts for skin ulcers,[ 39 ] vitiligo,[ 40 ] epidermodysplasia verruciformis,[ 41 ] and burn injuries.[ 42 ]
In 1981, Bell et al. fabricated a bi‐layered human skin model (HSM) by seeding keratinocytes on top of collagen gel comprised of fibroblasts.[ 43 ] The contraction of the collagen matrix caused by fibroblasts facilitated the formation of a dermal framework. The keratinocytes cultured on the collagenous dermis differentiated to generate the epidermal layers. The presence of fibroblast in the HSMs significantly enhanced keratinocyte outgrowth, which was attributed to the release of fibroblast diffusible factors.[ 43 ] Bell's HSMs were the first commercialized organ‐mimicking model systems. These models were vigorously tested for their morphological, biochemical and functional properties. Various substances, such as toluene, formaldehyde and resorcinol, were tested on these bi‐layered HSMs and native human skin to compare their responses to chemical irritants.[ 44 ] As time progressed, several derivations of the collagen‐based models were established and various novel scaffold‐based and scaffold‐free techniques were developed, many of which (with a focus on vascularized models) will be discussed later in this review.
Since the development of HSMs in the 1970s, there has been a trend to commercialize novel HSMs for clinical applications. These commercialized models have ranged from RHE to collagen‐based bi‐layered to immune‐competent models. A list of some of selected commercially available skin models and their applications are shown in Table 1 . However, it should be noted that the commercially‐available skin models lack the inclusion of functional BVs.
Table 1.
Selection of commercially available skin models and their applications.
| Supplier | Model | Description | Applications | Availability |
|---|---|---|---|---|
| Episkin | SkinEthic RHE | Human epidermis model from normal human keratinocytes cultured on an inert polycarbonate filter at air‐liquid interface | Skin irritation and corrosion, UV exposure, DNA damage, bacterial adhesion, permeability test | Commercial |
| T‐Skin | Human full‐thickness model consisting of human fibroblasts and keratinocytes on a tissue culture insert | UV exposure, DNA damage, bacterial adhesion, omics analysis, permeability | Commercial | |
| SkinEthic RHE‐LC | Standard epidermal model including Langerhans cell progenitors | Skin immune response, UV exposure, bacterial adhesion, omics analysis, permeability | Commercial | |
| MatTek Life Sciences | EpiDerm FT | A full‐thickness skin model cultured from fibroblast and keratinocytes at the air‐liquid interface in tissue culture inserts | Anti‐aging, wound healing, skin hydration, UV protection efficacy of formulations |
Commercial |
| EPIDerm | RHE model with highly differentiated normal human keratinocytes | Skin corrosion test, skin irritation test, Phototoxicity |
Commercial |
|
| Melanoma | The model consists of human malignant melanoma cells, keratinocytes, and fibrobasts | Tumor invasion, anti‐melanoma drug screening | Commercial | |
| Psoriasis | The diseased tissue model consists of healthy human keratinocytes and psoriatic fibroblasts harvested from psoriatic lesions | Anti‐psoriasis drug screening | Commercial | |
| Phenion | Phenion FT long life | A full‐thickness human skin model with a 50‐day lifespan | Monitoring of physiological, biochemical and genetic processes, provides an in vitro platform for development and treatment of skin tumors. | Commercial |
| Phenion FT AGED Skin Model | A full‐thickness human skin model characterized by connective tissue with senescent fibroblasts, reduced synthesis of ECM proteins, and elevated MMP secretion | Study of basic biology of the skin ageing mechanism, supports developing formulations that can delay/counteract ageing | Commercial | |
| Straticell | RHE models | Epidermis models with melanocytes, or skin microbiota | Modeling of inflammatory diseases such as AD and psoriasis, cosmetic and chemical testing | Service provider |
| LabSkin Creations | 3D vasculoskin | Bilayer skin model featuring microvascular‐like networks | Basic skin research, Physiologically‐relevant, efficacy and safety evaluations | Service provider |
| Biosolution | KeraSkin‐VM | RHE models, multilayered, highly differentiated | Corrosion/irritation tests, phototoxicity, genotoxicity, cosmetic testing | Commercial |
| KeraSkin‐FT | Collagen‐based full‐thickness model with dermis and epidermis | Skin permeation tests, wound healing models | Commercial | |
| Creative Bioarray | Neuro‐dermatology cell model | This model is generated by co‐culturing a) sensory neurons and skin cells or b) co‐culturing melanocytes and skin cells | Skin ageing, inflammatory skin disorder, skin pigmentation, hair growth | Commercial |
| Psoriasis model | 3D highly differentiated psoriatic human tissue phenotype | Drug screening, testing of new pharmacological approaches, study of the disease | Commercial | |
| GenoSkin | InflammaSkin | A fully human T‐cell driven skin inflammation model | Efficiency of anti‐psoriasis topical drugs and subcutaneously injected biologics | Commercial |
| HypoSkin | Excised donor skin for subcutaneous injections | Drug and vaccine development, injection site reactions, immunotoxicity and profiling | Commercial |
2. The Need for Vasculature in Skin Models
2.1. The Role of Vasculature in Inflammatory Skin Diseases
Vasculature plays a critical role in skin homeostasis and pathogenesis. Vascular remodeling and activation are often associated with the progression of numerous skin diseases.[ 45 ] In a normal healthy skin tissue, angiogenesis is a conserved process where a delicate balance between the production of pro‐angiogenic and anti‐angiogenic factors leads to stable vasculature. However, in diseases, there is an imbalance of angiogenic factors, thereby leading to excessive angiogenesis or BVs regression. Since one of the end goals of fabricating physiological HSMs is to recapitulate skin diseases, the presence of BVs within the model is a prerequisite.
Inflammation is one of the immune system's biological responses that is triggered by various stimuli. A cascade of events induces inflammation, including a) recognition of harmful stimuli by cell receptors, b) activation of inflammatory pathways, c) release of inflammatory cytokines and d) attraction of immune cells to the tissue.[ 46 ] Traditionally, the response of the body to inflammation is characterized by rubor, tumor, calor, and dolor referring to redness, swelling, heat, and pain, respectively. These responses are related to an increased blood flow to the local vasculature of the affected area. Furthermore, as metabolic activity increases, vessels become more permeable (leaky), resulting in swelling accompanied by pain.[ 47 ] There is evidence suggesting that BVs act as a primary contributor to the inception and progression of inflammatory skin diseases. Table 2 describes the involvement of BVs in the progression of several skin diseases.
Table 2.
Involvement of blood vessels in several skin diseases.
| Condition | Angiogenic factors | Observation | Reference |
|---|---|---|---|
| Psoriasis | VEGF, HIF, TNF‐α, IL‐8, IL‐17, E‐selectin |
|
[49, 52, 53, 54, 55, 56] |
| Atopic Dermatitis | VEGF‐A, VEGF‐B, VCAM‐1, Increased serum level of soluble ICAM‐1, ICAM‐3, E‐selectin |
|
[57, 58, 59] |
| Rosacea | VEGF‐A, mTORC1, LL‐37, TNF‐α, IL‐8, D2‐40 (lymphatic endothelium marker) |
|
[60, 61, 62, 63] |
|
Bullous Pemphigoid |
VEGF, TGF‐β |
|
[64, 65] |
| Melanoma | VEGF, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), TGF‐α and β, IL‐8 |
|
[66, 67, 68] |
Psoriasis is an immune‐mediated inflammatory skin ailment characterized by the hyper‐proliferation of epidermal keratinocytes. It is also related to the onset of other serious diseases such as psoriatic arthritis and cardiovascular complications.[ 48 ] It can be observed that the vessels in psoriatic lesions are dilated and leaky, leading to an enhanced accumulation of immune cells at the lesion site.[ 49 ] In 1961, Telner et al. observed microvascular abnormalities in patient biopsies even prior to the appearance of histological features that are suggestive of psoriatic lesions.[ 50 ] Structural abnormalities of the vasculature in early stages of psoriasis result from angiogenic inflammatory growth factors derived from infiltrating immune cells.[ 51 ]
Most psoriasis‐related alterations in vascular homeostasis occur due to the increased presence of angiogenic factors and decreased anti‐angiogenic factors. For instance, Nickoloff et al. showed that psoriatic keratinocytes promote angiogenesis through a significant reduction in the angiogenesis inhibitor thrombospondin‐1 and increased production of pro‐angiogenic interleukin IL‐8.[ 69 ] Similarly, Bhushan et al. showed that the tissue levels of endothelial stimulating angiogenesis factor (ESAF) and vascular endothelial growth factor (VEGF) were significantly upregulated in psoriatic plaques and correlate with disease severity.[ 70 ] Previous observations have indicated the enhanced migration and proliferation of BVs in psoriasis via an increase in αVβ3 integrin levels.[ 49 , 71 ] These studies indicate that vasculature plays a major role in psoriasis disease progression, highlighting the importance of BVs as a part of in vitro skin models of psoriasis. Figure 2 summarizes key changes in the psoriatic vasculature compared to healthy skin.
Figure 2.
The role of BVs in psoriasis inflammatory skin diseases has a direct impact on vascularization. The increased presence of VEGF and other angiogenic growth factors from psoriatic keratinocytes and infiltrating immune cells leads to enhanced angiogenesis. In turn, vascular enlargement and fenestrations lead to an accumulation of immune cells in the inflamed area of the skin, thus leading to a vicious circle of disease progression.
Similar trends have been observed in other inflammatory skin diseases such as AD, the most prevalent chronic inflammatory disease of the skin (Table 2). A distinguishing characteristic of AD is chronic cutaneous inflammation accompanied by dry skin and dysfunction of the epidermal barrier. AD previously has been linked to the presence of increased numbers of BVs compared to normal skin tissue.[ 72 ] Angiogenesis in AD has been correlated to elevated levels of angiogenic growth factors in AD lesions that exhibit a significant upregulation of VEGF expression.[ 57 ] Similarly, enhanced vascular permeability has been observed in Bullous pemphigoid, an autoimmune skin disease, where an increased expression of VEGF has been reported.[ 65 ] Rosacea is a chronic inflammatory skin condition that primarily affects the patient´s face and is visually characterized by persistent redness and rashes. Although the pathophysiology of rosacea is yet to be fully understood, modulations in the innate immune response and cutaneous vasculature are presumed to be the primary triggers. Increase in dermal expression of VEGF and CD31 (BVs marker) has been reported in rosacea patients.[ 62 ] Moreover, immunohistochemistry and gene‐level studies have highlighted vasodilation as an early indicator of rosacea.[ 73 ] Abnormal BVs is a hallmark of cancer. In skin cancer such as melanoma, there is a shift in the balance of angiogenic and angiostatic growth factors, leading to unrestrained angiogenesis. Several growth factors such as VEGF, FGF, PDGF, angiopoietin (ANG), and interleukins (ILs) are involved in angiogenesis during melanoma progression.[ 74 ]
Vascular abnormalities are not only evident in skin‐related ailments. Skin is one of the primary organs that exhibit signs of other internal diseases present in the human body. For instance, diabetes can lead to manifestations of diabetic dermopathy, diabetic blisters, and complications with skin wound healing, which are accompanied with the modulations in skin BVs. Several studies have therefore utilized VHSMs to deliberate the influence of diabetes on skin and the BVs.[ 75 , 76 , 77 ] Recently, BS Kim et al. developed 3D printed VHSMs to mimic the diabetic skin.[ 78 ] Bioinks were prepared from decellularized porcine‐derived ECM. The decellularized tissues were digested, lyophilized, and neutralized to form printable bioinks. The prepared bioinks were dermis‐derived ECM (SdECM), hypodermis‐derived ECM (AdECM), and vascular tissues‐derived ECM (VdECM). The models incorporated either human diabetic dermal fibroblasts (dHDFs) or normal human dermal fibroblasts (NHDFs).[ 78 ] The epidermis formed on top of dHDFs‐incorporated SdECM gels were thinner as compared to NHDFs‐incorporated SdECM gels. Using wound models, they showed that the dHDFs‐based models healed slower compared to NHDFs‐based skin model, thereby replicating clinical diabetic pathophysiology. Additionally, to understand the influence of diabetic adipose tissue on BVs, the models incorporated perfusable vasculature within the diabetic hypodermal compartment. The compartment included AdECM bioink either with human subcutaneous preadipocyte cells or diseased human subcutaneous preadipocyte cells isolated from diabetic type II donors. In these models, endothelial dysfunction (leakiness) was observed under hyperglycemic conditions, where high glucose containing media was perfused through the formed perfusable BVs.[ 78 ] As a proof‐of‐concept, the diabetic VHSMs were perfused with common diabetic drugs (metformin and eicosapentaenoic acid). Such 3D printed, multi‐compartmental, multi‐cellular VHSMs could in the future be used as a testing platform for diabetes therapy.
In addition to the influence of vasculature on skin disease pathophysiology, therapeutics applied to alleviate skin diseases can have both a direct and an indirect impact on BVs. For instance, Markham et al. tested infliximab, a tumor necrosis factor (TNF)‐α blocker, on psoriatic patients.[ 79 ] The anti‐TNF‐α therapy was shown to significantly downregulate angiogenic factors such as VEGF, ANG1, ANG2, and Tie2 receptors. Furthermore, a decrease in endothelial adhesion protein and vascularity was observed. The inhibition of inflammation and EC deactivation by low‐dose infliximab treatment led to a promising clinical response.[ 79 ] In a more direct approach, the angiogenesis inhibitor Neovastat (Æ‐941), was used in a randomized phase I/II clinical trial. Treatment with Neovastat lead to an improvement in the Psoriasis Area and Severity Index (PASI) after treatment of psoriatic plaques.[ 80 ] These examples imply that the requirement of vasculature is a prerequisite not only in models mimicking psoriatic disease progression but also in models that serve as a platform for anti‐angiogenic drug testing.
2.2. Vasculature in Skin Grafts
Deep wounds and large burns require the use of skin grafts. However, there are numerous challenges associated with successful grafting. These include 1) a lack of integration of the graft with host tissue, 2) a lack of adherence leading to a loosening of the graft over time, 3) immunological reactions leading to graft rejection, 4) the possibility of infections, and 5) improper vascular anastomosis with the host vasculature, leading to persistent hypoxia and eventual necrosis of the grafted tissue.[ 81 ]
Skin allografts (transplanted from an external donor) with biodegradable synthetic materials are susceptible to graft rejection in the first 1–2 weeks, due to the activation of the immune cells by both the biomaterial as well as the donor cells. Similarly, scaffold‐free cellular allografts are prone to rejection due to donor‐recipient human leukocyte antigen (HLA) incompatibility.[ 82 ] Conversely, autografts (a patient's own tissue transplanted from a different part of the body) elicit minimal graft rejection. However, utilizing a patient´s own tissue for covering large burns and wounds remains a challenge. Alternatively, acellular grafts composed of naturally‐derived ECM components such as collagen and hyaluronic acid also elicit low immune rejection. Acellular grafts, however, display poor adhesion to host tissue, often leading to incomplete wound healing and slow recovery from the burn.[ 83 ] Tissue‐engineered skin grafts containing fibroblasts and keratinocytes represent a promising alternative, despite the fact that they are susceptible to graft necrosis due to a lack of vascularization and the inability to repopulate skin appendages.[ 83 ]
One of the limiting factors in the in vitro fabrication of viable thick tissue is the transport of oxygen and nutrients to individual cells. Thin tissues can be nourished via diffusion but thicker (>1 cm) tissues demand vascular networks to prevent cell apoptosis and tissue necrosis.[ 84 ] Figure 3 shows the differences between skin graft integration (vascularized and non‐vascularized skin grafts) into a recipient wound bed. Young et al. studied the significance of vascularization for artificial skin graft survival.[ 85 ] They found that skin grafts survive in the host by 1) imbibition, in other words, diffusion of essential nutrients from the host wound bed to the graft, 2) vascularization due to inosculation on day 2–5, followed by 3) neovascularization. Inosculation is the process where the host vasculature anastomoses with the pre‐existing graft vasculature (Figure 3). Neovascularization, on the other hand is the formation of new vascular sprouts between the host vasculature and the graft. Neovascularization was observed in the grafted skin replacements only after two weeks, a clear indicator of the lengthy amount of time it would take to vascularize avascular tissue. This also means imbibition would be insufficient for keeping an avascular skin substitute alive long enough for neovascularization to take place. Creating a pre‐existing vasculature within the skin graft, on the other hand, would favor inosculation and prevent graft failure.[ 85 , 86 ]
Figure 3.
The schematic shows the importance of pre‐vascularization in skin grafting. Initially, the nutrients are diffused to the graft via plasma imbibition. Due to the presence of BVs in the grafts, inosculation and neovascularization processes are accelerated in vascularized compared to non‐vascularized grafts. A lack of BVs causes delayed inosculation and vascularization, leading to graft damage and necrosis.
Recent studies have further stressed the importance of vascularization in both scaffold‐based and scaffold‐free skin grafts.[ 87 , 88 , 89 , 90 ] Miyazaki et al. fabricated miniature scaffold‐free grafts with or without BVs and implanted these onto excised wounds of immuno‐deficient mice.[ 91 ] It was observed that non‐vascularized grafts had limited perfusion 7 days post‐grafting, with host BVs only observed in the periphery of the graft 14 days after grafting. Pre‐vascularized grafts, on the other hand, displayed complete perfusion, followed by anastomosis with the host vasculature 7 days post‐grafting. In conclusion, the study showed that pre‐vascularization of scaffold‐free grafts aided in wound healing and resulted in high graft survival.
3. Strategies to Fabricate Vascularized Skin Models
The preliminary step in the formation of VHSMs is the appropriate choice of a 3D environment. There are two main categories of VHSM environments: 1) scaffold‐based and 2) scaffold‐free. Scaffold‐based VHSMs include both natural or synthetic ECM components, where cells adhere and proliferate. Hydrogels, electro‐spun nanofibers, decellularized tissue scaffolds, and porous substrates are examples of scaffold‐based models. The biochemical and mechanical properties of these models can be fine‐tuned, allowing epidermal differentiation and vascularization. Moreover, angiogenic growth factors can be included within the scaffold to facilitate optimal vascularization. However, scaffold‐based models are susceptible to tissue contraction due to cellular traction forces, and are susceptible to enzymatic degradation leading to vascular regression.[ 92 ] This challenge both the longevity and stability of the formed VHSMs.
In contrast, scaffold‐free models rely on a cell‐derived ECM and cell‐cell interactions to form the tissue. Cells release ECM proteins such as collagen, laminin, elastin, and fibronectin, which are deposited around the cells. With time, the assembly of deposited proteins and cells result in the formation of intact tissue. Examples of scaffold‐free systems are organoids, spheroids, cell sheets obtained by self‐assembly/stacking (CS), and 3D cellular multilayers created using the ECM‐coating/accumulation technique. These models closely mimic skin tissue as they are comprised of cell‐secreted ECM. Additionally, compared to scaffold‐based skin tissues, they stimulate the development of a thicker epidermal compartment and proliferative keratinocytes.[ 93 ] However, the requirement of a large quantity of cells as well as the time required to assemble the cells and deposit the ECM hinder the large‐scale utilization of scaffold‐free models for VHSMs. Several notable scaffold‐free and scaffold‐based VHSMs will be summarized in the following sections.
3.1. Scaffold‐Based Skin Models
3.1.1. Collagen‐Based Skin Models
Collagen is the most abundant ECM protein of the skin and historically has been used for skin tissue engineering. Collagen in human tissue is highly organized and provides structural support and flexibility to the tissue. Pure collagen‐based models, however, have certain limitations as a biomaterial for VHSMs. These include 1) the constriction of gels due to cellular traction forces, 2) the time‐dependent degradation of the hydrogel due to enhanced enzymatic degradation, 3) inadequate or immature vascularization, 4) the time‐dependent regression of vessels, and 4) poor mechanical properties. To overcome these limitations, researchers now utilize a hybrid mixture of collagen with supplementary biomaterials to construct the VHSMs. In 1998, Black et al. reported capillary‐like tubular structures in chitosan‐linked collagen‐glycosaminoglycan sponge skin models.[ 94 ] A combination of HUVECs and dermal fibroblasts were seeded and cultured under media submersion. Keratinocytes were added on top of the formed dermis to create the epidermal compartment. The models were then shifted to an air–liquid interface (ALI). It was observed that no additional factors were necessary for angiogenesis, due to secreted ECM and growth factors from fibroblasts and keratinocytes.[ 94 ] Hudon et al. went on to further analyze these VHSMs, examining the influence of angiogenic and angiostatic molecules on them.[ 95 ] They reported that the capillary‐like tubes were modulated by the addition of angiogenic factors, demonstrated by the increased number of capillary tubes and a reduction in vessels after the addition of an angiostatin factor.[ 95 ] These observations made it evident that collagen‐glycosaminoglycan models could be utilized as an in vitro platform for the screening of angiogenic compounds and drugs.[ 95 ]
In order to facilitate vessel formation in the VHSMs, researchers have utilized genetically modified cells that overexpress angiogenic factors and assembled these with collagen‐based hydrogels. For instance, Supp et al. employed genetically modified keratinocytes that overexpress VEGF to fabricate a full‐thickness VHSM.[ 96 ] The models with the VEGF‐expressing keratinocytes showed better vascularization and attachment to the wound bed after grafting than models with normal keratinocytes.[ 96 ] Subsequently, Supp et al. fabricated an endothelialized collagen‐glycosaminoglycan skin substitute by mixing human dermal endothelial cells (HDMECs), fibroblasts and keratinocytes derived from a single autologous biopsy sample. At day 3, the model was exposed to an ALI and cultured for 15 days. On the upside, after implantation in mice the skin models with HDMECs showed the ability to deposit basement membrane proteins and form vascular analogs. But on the downside, only a low number of HDMECs could be detected at the end of the culture period.[ 97 ] Tremblay et al. used a collagen/glycosaminoglycan/chitosan sponge along with fibroblasts and HUVECs as the dermal compartment to fabricate vascularized skin models and observe capillary‐like tubes within their model before grafting it onto mice.[ 98 ] Less than 4 days post‐transplantation red blood cells (RBCs) were observed in the capillary‐like structures of the fabricated skin model. The presence of RBCs in graft vasculature demonstrates the anastomosis of vessels from the host tissue to the graft. It was observed that a non‐epithelialized skin model required at least 14 days in order to achieve similar results. Auxefans et al. constructed VHSMs using the common method of seeding ECs and fibroblasts together on a scaffold and later adding keratinocytes on top.[ 99 ] Collagen‐glycosaminoglycan‐chitosan was used as the base scaffolding. The novelty of their approach came from the use of adipose‐derived stem cells. Both progenitor and pre‐differentiated cells were tested, and only the pre‐differentiated ECs appeared successful. When ECs and fibroblasts were seeded together, the skin equivalent displayed capillary‐like structures.[ 99 ]
Owing to the successful use of collagen for the fabrication of VHSMs, recent studies have expanded the use of collagen‐based VHSMs by integrating novel techniques such as 3D bioprinting. In a recent article, Baltazar et al. fabricated a 3D‐printed collagen‐based HSM comprising of ECs and pericytes.[ 89 ] This 3D‐printed skin had a morphology reminiscent of native skin. The long‐term presence of BVs was observed within the VHSMs and the addition of pericytes allowed for stable vascularization. The printed models were then implanted in an immune‐deficient mouse. Vessel perfusion and anastomosis with the host vasculature was observed in the implanted VHSMs. Non‐vascularized grafts severely contracted while VHSMs with and without pericytes remained unaffected.
A vascularized collagen‐based skin‐on‐a‐chip model was developed by Sun et al. to study Herpes simplex virus (HSV) infections.[ 100 ] The chip contained pre‐organized perfusable BVs to allow the circulation of immune cells and antiviral agents (Figure 4A). The epidermis of the collagen‐based HSMs was infected with HSV, followed by the perfusion of neutrophils through the input channel of the skin chip (Figure 4B). It was observed that the perfused neutrophils migrated up toward the infected epidermis and interacted with the HSV‐infected cells (Figure 4C). This study highlights the possibility of using collagen‐based and perfusable bio‐fabricated skin‐on‐a‐chip systems to mimic skin infections and subsequent immune responses. Recently, an edgeless collagen‐based skin tissue was fabricated without any open edges.[ 101 ] Most skin models are characterized by open boundaries, which stands in contrast with the enclosed arrangement of native skin. The process of edgeless skin formation starts by generating a computer‐aided model of a body part. Figure 4D shows the design of a human hand. A hollow scaffold with permeable porous walls is generated. Next, using carbon digital light sensitive technology, collagen type I and fibroblast mixture is cast onto the exterior of the scaffold utilizing a polydimethylsiloxane (PDMS) negative mold. The structure is then kept in submerged culture conditions for 14 days. Primary keratinocytes are added and the construct exposed to ALI culture. Next, the construct is continually exposed to perfusion culture. The fabricated skin models are vascularized via perfusion with human dermal blood ECs (HDBECs), which attach to the inner walls of the dermis. The ECs aggregate together with the underlying fibroblasts, forming vascular islands (Figure 4E1–3). The enclosed geometry provided by the edgeless skin model resulted in enhanced deposition of the ECM, improved mechanical properties, and site‐specific differences in cellular and ECM organization. In the future, such collagen‐based edgeless skin can be used as personalized wearable skin grafts.
Figure 4.
Recent advancements in skin models utilizing collagen as a biomaterial for skin tissue engineering. A) Schematic representation of a collagen‐based skin‐on‐a‐chip device. The device incorporates keratinocytes, fibroblasts, perfusable vasculature, and immune cells. The fabricated BVs were perfusable and allowed the infiltration of immune cells and antiviral agents. B) HSV infection was mimicked by seeding the virus on top of the skin models. Subsequently, the inlet tubes of the chip were perfused with Neutrophils. C) depicts the spatial distribution of the cells. The neutrophils (red, CD15 stained) migrated against gravity toward the infected keratinocytes (HSV infected epidermis, green). Images reproduced from,[ 100 ] Copyright 2022, Distributed under Creative Commons Attribution License 4.0 (CC‐BY). D) Overview of edgeless collagen‐based vascularized skin formation. A computer‐aided drawing of the anatomical site is prepared and this template utilized to 3D print a porous scaffold. A PDMS negative mold is prepared, after which collagen ink containing fibroblasts is injected. After dermal contraction, keratinocytes are injected to form a full‐thickness skin (Scale bar = 1 cm), E1) Human dermal blood ECs are introduced for skin grafting applications. EGFP‐tagged BVs are stained with CD31 (red). E3) The magnified image of E1 shows the orientation of the BVs and vascular protrusions. E2) Interconnections between BVs (green, CD31) and fibroblasts (red, vimentin) were observed within the vascular islands. Images are reproduced with permission from,[ 101 ] Copyright 2023, Distributed under a Creative Commons Attribution License 4.0 (CC‐BY).
3.1.2. Hyaluronic Acid‐Based Skin Models
Hyaluronic acid (HA) is an abundant ECM protein present in skin tissue. Hyaluronan in the skin is associated with collagen and elastin fiber spacing and the structural integrity of the skin.[ 102 ] The ester derivative of hyaluronan, commercially known as HYAFF11, is a resorbable non‐woven scaffold mesh that has been used in the past as skin grafts.[ 103 ] The advantages of such scaffolds are its biocompatibility, long shelf life, biodegradability, and limited immunogenicity. Tonello et al. fabricated a dermal equivalent with BVs using a HYAFF11 scaffold, which incorporated HDMECs and fibroblasts. They reported that the production of the ECM (collagen type I, III, and IV) by the fibroblasts within the HA‐scaffold assisted in the proliferation and organization of the ECs into micro‐capillary‐like structures. An open lumen was observed after 21 days of culture.[ 104 ] Cerqueira et al. combined gellan gum‐HA spongy‐like hydrogels with human adipose stem cells (hASCs) and human adipose microvascular endothelial cells (hAMECs) to fabricate VHSMs. The presence of hAMECs facilitated neo‐vessel formation.[ 105 ] Tonello et al. improved upon their previously reported epithelialized dermal equivalent through the addition of keratinocytes to create an epidermal compartment. Using the HA‐based non‐woven scaffold, they seeded dermal fibroblasts followed by HDMECs, and cultured the scaffold for 10 days. Keratinocytes were seeded and after one week of culture exposed to ALI for 21 days. Histology showed evidence of dermal and epidermal compartments as well as ECs organized into rings similar to capillary structures with a lumen. They reported the production of a two‐layered skin construct with a micro‐capillary network.[ 106 ]
HA has been used either in the form of non‐woven scaffolds or as a combinatory ECM blended together with collagen type I or fibrin in order to improve their mechanical properties.[ 107 ] Most of the studies conducted with HA‐based HSMs are as skin grafts and the potential use of such HSMs in disease modeling and drug testing has not yet been thoroughly evaluated. Although the limitations of HA‐based non‐woven scaffolds and hydrogels has not been deliberated extensively in the existing literature, some of the challenges may include 1) difficulty in penetration of fibroblasts and ECs throughout the scaffold leading to poor vascular distribution, 2) the need to optimize scaffold degradation rate to align with tissue regeneration, 3) inability to recapitulate the ECM diversity and the concentration of HA comparable to native skin tissue, and 4) large scale production of pure HA, and 5)limited data pertaining to skin disease modeling and drug testing.
3.1.3. Fibrin‐Based Skin Models
Fibrin is a natural and biodegradable scaffold that holds a significant role in wound healing. Fibrin has several advantages over other hydrogels, namely: 1) the option to tune gel properties such as porosity and stiffness, 2) superior wound healing properties, 3) its applicability in providing an angiogenic 3D environment for vascular modification, and 4) limited foreign body reaction due to its autologous nature.[ 108 , 109 ] Chen et al. fabricated fibrin‐based tissues that were pre‐vascularized. Fibrin was mixed with HUVECs and fibroblasts to construct the dermal compartment. After 7 days, the vascularized tissues were implanted in immune‐deficient mice and collected at different time points. As early as day 5, RBCs were found in HUVECs‐lined vessels. Post‐implantation, tissues containing ECs showed a greater number of perfused lumens than controls. The pre‐vascularization of the tissue was shown to strongly and positively influence the necessary tissue inoculation time with host vasculature and further promoted cellular activity indicative of tissue remodeling.[ 110 ]
Chan et al. fabricated a skin model consisting of adipose stem cells that were isolated from discarded burn skin.[ 111 ] The epithelial and hypodermal compartments were composed of collagen hydrogel, whereas the vascularized dermal construct was a collagen PEGylated fibrin‐based bi‐layered hydrogel. The advantage of this hydrogel was that it does not require pre‐differentiation of the ASCs before seeding.[ 111 ] A PEGylated‐fibrin environment allowed the differentiation of ASCs into tubular networks that resembled the vascular phenotype, whereas the collagen layer allowed the ASCs to maintain their fibroblast‐like phenotype. The collagenous hypodermal compartment was nourished by adipogenic media triggering the differentiation of ASCs into adipocytes. Recently, Liu et al. fabricated 3D printed, fibrin‐based VHSMs to mimic AD.[ 112 ] The dermal compartment was composed of fibroblasts, iPSC‐derived ECs and pericytes. To stimulate the AD‐like diseased phenotype, IL‐4 was added to the medium during ALI culture. A decrease in trans‐epithelial electrical resistance (TEER) values in AD‐models compared to non‐diseased models was observed and found to correlate with decreased epidermal barrier differentiation protein expression in the vascularized disease models. The addition of IL‐4 also upregulated the production of endothelial adhesion molecules VCAM‐1 and ICAM‐1.[ 112 ]
3.1.4. Decellularized Scaffolds
Researchers have also focused on using decellularized tissues for skin tissue engineering. In this technique, tissues (human cadaver tissues, human donated tissue, porcine, and bovine tissues) are decellularized. This results in the elimination of the donor cells and cellular components (DNA and RNA), and the preservation of the deposited ECM scaffold. The scaffolds are then subsequently recellularized with human cells. The advantage of decellularized tissue is the presence of an intact scaffolding with intricate architecture, which can be beneficial for vascularization.
Schechner et al. utilized cadaveric acellular pieces of the dermis to fabricate skin models.[ 113 ] Keratinocytes were added on top of the decellularized dermis to create the epidermal compartment. A day before implantation into mice, HUVECs that were modified to express Bcl‐2 endothelial survival gene were seeded on the reticular dermis or the bottom side of the skin equivalent. The VHSMs were sutured superficially on the excised skin of mice and demonstrated graft perfusion. The Bcl2 modification also appeared to promote perfusion and maturation of the vasculature.[ 113 ] To fabricate vascularized skin grafts, Sahota et al. used acellular donor dermis and seeded dermal fibroblasts and HDMECs on the reticular surface toward the top. After 24 h the dermis was flipped over, thus leaving the reticular surface facing down and the papillary dermis facing up. Thus positioned, the exposed surface could be seeded with primary epidermal keratinocytes.[ 114 ] The fibroblasts, HDMECs, and keratinocytes were extracted from a single donor biopsies. Penetration of HDMECs BVs within the graft was observed. However, challenges ensue such as time required to culture HDMECs from the patient and the slow penetration of HDMECs within the decellularized dermis. Decellularized tissues can also be transformed into dermal hydrogels. In this technique, tissues are decellularized and the residual tissue‐derived ECM (dECM) is homogenized, lyophilized, enzymatically digested, and gelated to form hydrogels.[ 115 ] Blending dECM together with fibrinogen, collagen type‐ I, and gelatin has shown to improve mechanical and biological properties of the formed hydrogel.[ 116 , 117 ] Such dECM have been used in the past as bioinks for 3D printing of VHSMs.[ 78 , 118 , 119 ] Limited supply of dECM, use of animal‐derived dECM, and reproducibility (due to varying dECM sources) are the current challenges.
3.2. Scaffold‐Free Skin Models
3.2.1. Self‐Assembled Cell Sheets
Scaffold‐free approaches exploit the cell´s ability to produce its own ECM. The advantages of scaffold‐free methods include: 1) the physiological similarity to native skin tissue, 2) improved epidermal formation, and 3) improved vascularization and decrease in regression.[ 93 , 120 ] The techniques utilized to fabricate scaffold‐free skin models involve the stacking of self‐assembled cell‐sheets (CS), a layer‐by‐layer ECM cell‐coating and accumulation technique, and skin organoids.[ 121 , 122 ] Figure 5 gives a schematic overview of scaffold‐free VHSM fabrication methods.
Figure 5.
Different techniques for fabricating scaffold‐free VHSMs. A) The self‐assembly and cell‐sheet technology involves the long‐term cultivation of highly confluent sheets of fibroblasts and ECs with ascorbic acid supplementation. The formed sheets are stacked to form a thick vascularized tissue. B) The ECM cell‐coating and assembly method involves layer‐by‐layer deposition of ECM moieties (for instance, fibronectin and gelatin). The coating of ECM proteins on single cells and the assembly of these cells together with ECs in a confined space lead to the rapid formation of a thick vascularized dermal compartment. Schematics adapted from Rimal et al.[ 123 ] Copyright 2021, Published by Elsevier Ltd. Distributed under Creative Commons CC‐BY‐NC‐ND License.
The CS technique involves the stacking of dermal sheets to form a thick tissue. Fibroblasts and ECs are cultivated with media supplemented with ascorbic acid, which triggers the cells to rapidly produce ECM. The formed sheets are then extracted and stacked to form thick tissues. Next, keratinocytes are added on top of the stacked sheets. They differentiate to form the epidermal compartment. In contrast, the cell‐coating and accumulation technique involves rapid construction of thick tissues through the accumulation of ECM‐coated cells. The coated ECM nano‐layers allow cells to interact with one another and rapidly form a 3D tissue. The dermis forms within 24‐h, followed by the addition of keratinocytes and their subsequent differentiation.[ 121 ]
Cerqueira et al. utilized CS technology to fabricate VHSMs for wounds[ 124 ] employing dermal sheets composed of fibroblasts, keratinocytes and HDMECs. The wound healing capability of cell sheets composed of different cell types was analyzed. 21 days after transplantation it was observed that sheets with either 1) fibroblasts, HDMECs, and keratinocytes or 2) fibroblasts and keratinocytes showed the highest percentage of wound closure compared to the control. Furthermore, the inclusion of ECs resulted in neovascularization.[ 124 ] Recently, Bourland et al. fabricated a vascularized 3D melanoma model utilizing CS technology.[ 125 ] Melanoma tumor spheroids were integrated with a vascularized dermal sheet and keratinocytes subsequently added. The integrated melanoma spheroids proliferated and migrated in the 3D model. There was an increased secretion of ANG‐2 and CCL21 in these VHSMs, suggesting metabolically active ECs.[ 125 ]
3.2.2. ECM‐Cell Coating Technique
The ECM‐cell coating technique relies on a layer‐by‐layer coating of ECM moieties onto the cell surface and the assembly of the coated cells to form 3D tissues. This technique is relatively faster than the self‐assembly technique and has been used to fabricate various in vitro tissues in the past.[ 120 , 121 ] Matsusaki et al. fabricated skin models with blood and lymph capillaries by using the cell‐coating technique.[ 122 ] Fibronectin and gelatin‐coated dermal fibroblast along with HUVECs and hLECs were utilized to fabricate the VHSMs. Miyazaki et al. utilized the same model to use as a functional engraftment.[ 91 ] In contrast to hydrogel‐based skin models, these VHSMs are insusceptible to graft contraction and shrinkage after transplantation. Furthermore, 7 days after transplantation, non‐vascularized models showed epidermolysis which was not observed in VHSMs.[ 91 ] Table 3 elaborates the advantages and limitations of different scaffold‐based and scaffold‐free VHSMs.
Table 3.
Advantages and challenges of scaffold‐based and scaffold‐free models.
| Scaffold | Advantages | Challenges | Reference |
|---|---|---|---|
| Collagen |
|
|
[27] |
| Fibrin |
|
|
[108] |
| HA |
|
|
[126, 127] |
| Organoids |
|
|
[128, 129] |
| Scaffold‐free self‐assembly and CS models |
|
|
[130] |
| Scaffold‐free cell coating and assembly models |
|
|
[121, 123] |
| Decellularized skin Models |
|
|
[131] |
| Ex‐vivo human skin from discarded human skin tissue |
|
|
[132, 133] |
4. Current Trends in VHSMs Fabrication
4.1. Cultivation of VHSMs in a Dynamic Environment
Emerging technologies such as 3D bioprinting allow the fabrication of durable and thick viable tissues.[ 134 ] In recent years, 3D printing technologies, sacrificial scaffolds, and microfluidic methods have enabled the fabrication of perfusable in vitro VHSMs. VHSMs featuring vessel perfusion offer many advantages, including 1) the transport of essential growth factors and nutrients into the tissue, 2) the removal of cell‐derived by‐products, 3) the ability to study circulating cancer cells or immune cells even as they infiltrate skin tissue, 4) the possibility to observe drug absorption, 5) the conditions to gain greater insight into the transport of intra‐venously injected drugs/nanoparticles in skin tissue, and 6) the opportunity to construct thick skin models with an enhanced deposition of cell‐derived ECM components. Some notable perfusable VHSMs are highlighted below.
Abaci et al. utilized iPSC‐derived ECs to fabricate a perfusable skin model.[ 88 ] A micro‐pattern of sacrificial alginate gel was prepared on 3D printed molds. Collagen gel containing dermal fibroblasts was then added on top of the patterned vasculature. After polymerization around the channels, keratinocytes were seeded on top of the matrix, cultured under submerged conditions, and exposed to ALI culture conditions for 7 days. The sacrificial alginate was treated with sodium citrate to dissolve the alginate, followed by the addition of iPSC‐derived ECs. In another study, Mori et al. fabricated collagen‐based skin models with perfusable vasculature.[ 135 ] Here, a 3D‐printed bioreactor with anchoring connectors was manufactured and nylon wires were fixed across the printed device. Next, collagen with human dermal fibroblasts was assembled in the reactor. After solidification of the collagen gel, the nylon wires were extracted giving rise to hollow openings in the dermal compartment, which were then filled with ECs (Figure 6A,B). The perfused skin equivalent showed a higher cell density than non‐perfused models as well as showing promising results in predicting vascular absorption.[ 135 ]
Figure 6.
Skin models cultured in a dynamic flow environment. A) The schematic shows a method for the formation of large vessels within the dermal compartment by creating microchannels, B) H&E images show perfused and non‐perfused skin models. The outer surface of the perfused channel is stained with CD31 vascular marker (green). Images reproduced from,[ 135 ] Copyright 2016, Elsevier Ltd. C) Schematic of the long‐term culture of vascularized scaffold‐free models in a 3D‐printed bioreactor. D) On day 21, static VHSMs show abnormal vascularization, perfusion flow culture of the VHSMs positioned within the 3D‐printed bioreactor stabilizes the BVs. Top image: static culture‐treated, bottom image: flow culture‐treated, CD31 is stained red, scale bar = 100 µm. E) Flow culture leads to the formation of vascular openings on the basal‐side of the scaffold‐free skin models due to the media flow. The openings were connected to the vessel spanning the dermis, shown by the presence of fluorescent beads in the dermal vasculature. Images reproduced from,[ 123 ] Copyright 2021, Published by Elsevier Ltd. Distributed under Creative Commons CC‐BY‐NC‐ND License. F) Schematic of the experimental procedure for the fabrication of VHSMs: a) human keratinocytes, fibroblasts and ECs are isolated from juvenile foreskin biopsies, b) cells are re‐seeded on a vascularized scaffold (BioVasc) produced by decellularization of porcine jejunum, c) the vessels are inoculated with arterial inflow (AI) and venous outflow (VO). G) H&E staining of the formed VHSMs shows epidermal and dermal layers as well as intact vasculature (black arrow). The enlarged vascular structure shows the lumen and the seeded ECs. Scale bar = 50 µm. Images reproduced from,[ 139 ] Distributed under Creative Commons Attribution License 4.0 (CC‐BY).
In a recent study, Kim et al. fabricated a fully perfusable skin model comprising a hypodermal, a dermal, and an epidermal compartment.[ 136 ] The bio‐inks of the hypodermis and dermis were composed of adipose‐derived and skin‐derived dermal‐ECM with bovine fibrinogen, respectively. A gelatin hydrogel was used for the fabrication of vascular channels in the skin model.[ 136 ] The study showed that HSMs comprising an epidermal, a dermal, and a hypodermal compartment were better at recapitulating native skin complexity. Helmedag et al. used a fibrin‐based scaffold to fabricate VHSMs.[ 137 ] The fibrin gel and the fibroblast allowed for rapid vascularization of the tissue. The fibrin‐based VHSMs were cultivated on a 3D‐printed bioreactor and under flow perfusion culture. The study showed that increases in the flow rate were associated with decreased vascular branching and vessel length. Jusoh et al. described a microfluidic device where a fibrin‐based vascularized skin model was constructed.[ 138 ] The device comprised of several compartments separated by micro‐posts. Compartments included a fibrin compartment containing human dermal fibroblasts, a 2D keratinocyte compartment, and an EC compartment. Stimulated growth factors from the keratinocytes led to the migration of vessels toward the keratinocytes and the micro‐posts aided in the formation of endothelial lumen. The VHSMs were used for skin irritation testing. Chemical irritants such as sodium lauryl sulfate (SLS) and steartrimonium chloride (SC) were tested on keratinocytes and their effect on the vascularization was assessed. The increase in sprout length, vessel proliferation, and lumen formation after the addition of SLS and SC showed the potential of such models for application in platforms for skin irritation testing.[ 138 ]
In our recent study, scaffold‐free models and 3D‐printed bioreactors were utilized to cultivate scaffold‐free VHSMs. We fabricated VHSMs using the ECM‐coating and assembly method. We showed that employing flow culture prevented the degradation of the vascularized dermal compartment and allowed for the maintenance of vascular and dermal integrity during a 28‐day culture period (Figure 6C,D).[ 123 ] The angiogenesis of the VHSMs in static culture (Day 21) was uncontrolled, leading to abnormal vascular coverage within the VHSMs while the VHSMs in flow culture showed an optimal vascular architecture and coverage (Figure 6D). Perfusable sites were observed in the basal compartment of the skin due to the flow culture (Figure 6E). Furthermore, it was demonstrated that flow culture led to faster wound healing in laser‐irradiated VHSMs compared to static models, thereby emphasizing the need for dynamic perfusion of tissue models in disease modeling.[ 123 ]
Decellularized scaffolds can also be employed as a perfusable platform. For instance, Groeber et al. utilized a biological vascularized scaffold taken from a segment of the jejunum as a vascularized bed (Figure 6F). After de‐cellularization, the scaffold was placed in a bioreactor.[ 139 ] Human microvascular ECs lined the walls of the formed vessels which could be perfused with a physiological volume flow (Figure 6G). Dermal fibroblasts were seeded on the surface of the vessels and the vessels connected to a fluidic system that provided medium, thus creating a perfused vascular system.[ 139 ] Another study from the same group fabricated perfusable VHSMs to study skin fibrosis.[ 140 ] Transforming growth factor‐beta (TGF‐β) was added to induce the fibrosis process. Furthermore, the anti‐fibrosis drug Nintedanib was tested on the VHSMs. Nintedanib reduced TGF‐β‐induced ECM production as well as fibroblast to myofibroblast transition.[ 140 ]
4.2. Multi‐Cellular VHSMs and Multi‐Organ Systems
Skin consists of numerous cell types that continually cooperate with one another to maintain homeostasis. The future of physiologically‐relevant skin models would be one that includes diverse cell types such as neurons, lymphatic vessels, immune cells, and skin appendages. Adding additional cell types influences the vasculature via cell‐cell interactions and/or by the release of essential angiogenic growth factors. As the inclusion of these cells into skin models becomes more widespread, the requirement for vascularization becomes more apparent. It can be predicted that there will be a demand for VHSMs (both healthy and diseased) as biological templates to which other ancillary cell types can be supplemented.
Several studies have incorporated immune cells, neuronal cells, and lymphatic cells together with ECs. Marino et al. fabricated both collagen‐based and fibrin‐based skin models containing vascular and lymphatic capillaries.[ 141 ] Both HDMECs and hLECs formed intricate vascular and lymphatic networks spanning the entire tissue construct. Transplantation studies showed the anastomosis of lymphatic vessels between the skin models and the host. Kreimendahl et al. fabricated a fibrin‐based VHSMs with macrophages.[ 142 ] Wound healing experiments have elucidated the interplay between vasculature and macrophages. Laser‐treated samples in the presence of macrophages showed significantly higher levels of vascularization than laser‐treated skin models without the addition of macrophages. The presence of macrophages also led to faster wound healing after laser treatment. Furthermore, the detection of macrophage‐specific markers CD14 and CD163 after wound infliction points to the transformation of the quiescent macrophage phenotype to an activated phenotype (M2‐macrophages), which has implications in angiogenesis and wound healing.[ 142 ]
Kwak et al. engineered a skin model in a microfluidic chip comprising BVs and immune cells to study the immunological response.[ 143 ] HUVECs were cultured on the bottom of a porous PDMS membrane and exposed to the fluidic compartment. The upper compartment comprised the collagen‐based dermis and the epidermis. Toxicity tests with doxorubicin were conducted, revealing that the presence of HUVECs in the model resulted in an increased viability of dermal fibroblasts in the presence of the drug. To recapitulate the immune response in the skin, the models were exposed to UV irradiation followed by the addition of leukocytes to the fluidic compartment. UV irradiation resulted in a higher recruitment of leukocytes that migrated from the fluidic compartment through the HUVECs monolayer and into the dermal compartment.[ 143 ] Recently, Attiogbe et al. fabricated an autologous, immune‐competent and scaffold‐free vascularized skin model using self‐assembly and the CS protocol.[ 144 ] A high number of viable cells (fibroblasts, EC, immune cells) were isolated from the same human biopsy. The immune cells in the VHSMs displayed functionality, more specifically, the CD45+ immune cells secreted TNF‐α and GM‐CSF cytokines in response to LPS stimulation. Such immune‐competent VHSMs, in future, could improve the understanding of cell interactions and serve as a platform for drug testing.
Skin, as the largest organ in the human body, has continual interactions with all the other organs of the body via a network of BVs. A multiorgan‐on‐a‐chip platform interconnecting multiple organ‐like functional in vitro tissues would enable an in‐depth understanding of disease progression and drug interactions. Numerous skin diseases have shown to correlate with multi‐organ disease progression. Such correlations can be substantiated in vitro using multi‐organ‐on‐a‐chip systems. For instance, such systems can help elucidate the correlations between psoriasis and psoriatic arthritis, AD and asthma, AD and cardiovascular risk as well as melanoma metastasis and other secondary organs. In order to fabricate such multi‐organ chip systems, vascularization of individual in vitro tissues would be an important step.
Preliminary studies on multiorgan‐on‐a‐chip systems have been performed by several research groups. Wagner et al. constructed a multi‐organ chip system that combined a 3D skin model and a human liver model.[ 145 ] Skin biopsies and liver spheroids were integrated into the chip and a continuous medium flow established. The cross‐talk between the liver spheroids and the skin was demonstrated by the consumption of liver‐released albumin by the skin tissue. Troglitazone was tested on the system, revealing a dose‐dependent sensitivity to the drug.[ 145 ] Similarly, Maschmeyer et al. fabricated an organ‐on‐a‐chip platform comprising of interconnected human kidney, skin, liver and intestine models.[ 146 ] The skin, however, was acquired from prepuce samples, from which biopsies of the required diameter were excised and integrated into the chip. The individual models showed high tissue viability and an intact architecture during the 28‐day culture period.[ 146 ] The future could entail multiple vascularized organs including VHSMs integrated into a single organ‐on‐a‐chip system that provides a platform for fast and efficient drug testing.
4.3. Personalized Skin Models
The concept of treating patients as an individual rather than a part of a large patient population could be the next step to treat specific skin diseases more effectively. Personalized skin models could be fabricated 1) through the extraction of cells (immune cells, keratinocytes, fibroblasts, ECs) from the patient and subsequent formation of HSMs or 2) from iPSCs that would be differentiated into different cell lineages and subsequently used to fabricate HSMs.[ 147 ] In a recent article, Girardeau‐Hubert et al. demonstrated that skin models created from cells derived from different ethnic sources lead to divergent epidermal fates.[ 148 ] It was observed that collagen‐based HSMs from African skin consist of an increased number of proliferating basal keratinocytes and lower filaggrin (FLG) levels in terminally differentiated epidermal layers than HSMs from Caucasian sources. Furthermore, several genes extracted from the HSMs pertaining to lipid/ceramide metabolism, FLG processing, and epidermal differentiation varied significantly between the two groups.[ 148 ] Genetic characteristics could, therefore, be one of many criteria to define the efficacy of drugs and cosmetics both in the treatment of ailments as well as concerning the safety/side effects of the drug. The fabrication of patient‐derived personalized VHSMs could, in future, be essential in eradicating skin diseases.
4.4. Skin Organoids
Skin organoids are in vitro 3D tissue constructs that act as morphologically and functionally competent skin imitations due to their inherent complex architecture consisting of various cell types. Sophisticated skin organoids with a complex structure—comprising a fat‐rich dermis, a multi‐layered epidermis, neurons, hair follicles with sebaceous glands—can be generated from human iPSCs (hiPSCs). The structural and functional similarities of these organoids to native skin hold promise for disease model and skin regenerative therapy applications.
Lee et al. reported a method to fabricate human hair‐bearing skin entirely from hiPSCs, starting out with the manipulation of the TGF‐β and FGF signaling pathways.[ 129 ] The protocol was continued for 16–20 weeks until stratified epidermis had maturated. Dermis with hair follicles and sebaceous glands was also observed in the skin organoid. The maturation of neuroglial cells in skin organoids was observed through immunostaining (day 140 of the differentiation process) for NEFH (sensory neurons) and S100β+ (Schwann cells), which verified nervous tissue organization.[ 129 ] Major issues associated with hiPSC‐derived skin organoids are the presence of cell populations originating from cartilage and the appearance of inward growing skin (cell populations growing in a closed and spherical cyst morphology). Jung et al. addressed these issues by dissecting the hiPSC‐derived skin organoids and subsequently culturing organoids in trans‐well inserts where the skin is exposed to air.[ 149 ] This ALI culture condition enhanced planar growth of skin appendages resembling native human skin architecture and physiology. By further modulating the culture conditions for skin organoids, Lee et al. were able to produce a complex skin imitation containing stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells, and sensory neurons after 130 days of differentiation.[ 150 ]
Despite the obvious challenges in fabricating vascularized and immunocompetent iPSC‐derived skin organoids, these organoids can still be beneficial for the study of early human skin development and the modeling of skin diseases. The future entails the fabrication and utilization of vascularized skin organoids as a platform to test novel therapies.
5. Challenges and Outlook
The primary challenges in the utilization of VHSMs as skin imitations can be broadly classified into four main categories: 1) their resemblance to native human skin, 2) their lab‐to‐lab variability, 3) the challenge to scale‐up production, and 4) the longevity and maintenance of the formed BVs. These four major challenges will be discussed further in this section and an overview of the existing challenges is listed in Table 4 .
Table 4.
Prevailing questions and challenges in utilizing VHSMs as skin imitations.
| Questions | Challenges |
|---|---|
| Do in vitro VHSMs precisely mimic in vivo skin anatomy and physiology? |
|
| Why is there lab‐to‐lab variability in scaffold‐based VHSMs? |
|
| Why is there lab‐to‐lab variability in scaffold‐free VHSMs? |
|
| Why do vessels regress in vitro? |
|
| Why are there reproducibility issues with post‐fabrication image analysis and validation studies? |
|
| What are the challenges regarding VHSM longevity? |
|
| Why are vascularized skin grafts rejected post‐transplantation? |
|
| What are the challenges associated with skin organoids? |
|
5.1. Resemblance with Native Human Skin
In the previous sections, the dissimilarity between murine and porcine models with native skin was briefly discussed. However, it has to be noted that there are also differences between in vitro tissue‐engineered skin models and native skin. These include: 1) a lack of rete ridges, 2) a lack of diverse cell types, skin appendages, and a hypodermis layer, 3) mechanical properties that are unlike native human skin, 4) an organization of BVs that is different to native human skin, 5) inadequate skin barrier properties, and 6) differences in cell population ratios.
VHSMs are essential for parametric studies to understand the role of distinct cell types. However, it is still challenging to recapitulate the complexity and entirety of the responses observed in the native human skin. For instance, in inflammatory skin diseases such as AD and psoriasis, there is an intricate interaction between keratinocytes, BVs, T‐cells, dendritic cells, mast cells, and neurons. Moreover, the repercussions of the inclusion or exclusion of these cell types for disease progression and therapeutical efficacy are unknown. Although there are novel skin models that are vascularized, immune‐competent and innervated, the lack of a complete functional model hinders the utilization of HSMs as replacements for animal models, thereby limiting their usage as a complimentary rather than an alternate platform. Due to this reason, there is also a rise in the demand of commercially‐available, donor‐acquired ex vivo skin models that include several cell types. The absence of rete ridges and skin appendages in HSMs and VHSMs is another limiting factor for accurate therapeutic testing. In human skin the penetration of drugs takes place via a combination of intercellular, intracellular and follicular pathways.[ 151 ] The amount of diffused substance has been shown to be 2–4 fold higher in skin with hair follicles than skin without follicles.[ 152 ] Rete ridges, on the other hand, are essential in providing mechanical interaction between the epidermis and the dermis and are present in normal skin. It has been shown that alterations and a flattening of rete ridges occurs as a consequence of aging and several skin diseases.[ 153 ] The absence of rete ridges in HSMs should, therefore, be considered a limitation.
The formed BVs within VHSMs are either 1) randomly distributed, 2) arranged in a monolayer (e.g., in organ‐on‐a‐chip devices), 3) aligned (after removal of nylon wire), or 4) structured (using bioprinting). However, it has not yet been possible to mimic the precise architecture and diameter of the skin vasculature in vitro. The distance between the vascular capillaries and the epidermis as well as the presence of horizontal arteries and veins in the papillary dermis, the reticular dermis, and the hypodermis have to be considered in the future development of VHSMs. This is particularly important for vascularized skin grafts, as tortuous geometries of the formed BVs could result in thrombosis post‐implantation.[ 154 , 155 ] Moreover, blood extravasation has been observed once the pre‐vascularized skin graft anastomoses with the host vasculature.[ 155 ] This could be attributed to the implantation of grafts with immature and tortuous BVs with less or no pericyte coverage. One potential solution for this is to cultivate the VHSMs with pericytes and perfusion culture over a longer period of time, establishing BV stability prior to skin grafting. In addition to differences in vascular architecture, it has also been shown that both commercially available as well as in‐house HSMs are more permeable to tested substances than human cadaver skin. This could be attributed to an impairment in the formation of a cornified envelope and differences in the composition, properties and organization of lipids. A detailed review on the subject has been published by Wietecha et al.[ 156 ] In conclusion, it should be emphasized that conventional commercial and in‐house HSMs are comparatively more permeable to substances (even without the presence of hair follicles). Fabrication of in vitro models with barrier properties that are comparable to human skin, therefore, represent a challenge.
5.2. Reproducibility of VHSMs
Lab‐to‐lab variability in VHSMs is a consequence of the rapidly developing field of biomaterials and a lack of standardization. Labs employ different scaffold and scaffold‐free methods to generate VHSMs. Moreover, biomaterial concentrations, cell amounts, cell sources, media composition, culture conditions, drug treatment protocols, and even analysis methods may vary.
Several past studies utilize diverse sources of ECs in the VHSMs. HUVECs are the most common ECs used in VHSMs due to the availability and data accessibility (due to wide‐spread past usage). Alternative ECs sources include the use of ECs derived from the recipient or from circulating endothelial progenitor cells. Shepherd et al. compared Bcl2‐transduced and non‐transduced HUVECs with ECs derived from umbilical cord blood (CB‐EC) and from peripheral adult blood (AB‐EC).[ 157 ] The skin models with CB‐EC, AB‐EC and Bcl2‐transduced HUVECs showed robust vessel formation within the decellularized scaffold and were also able to promote host vascularization within the implanted graft. In contrast, non‐transduced HUVECs stimulated slow host vascularization within the graft. This study highlights the employment of alternative ECs sources for the vascularization of skin grafts.[ 157 ]
Other sources of ECs are the human dermal blood ECs (HDBECs) and the iPSC‐derived ECs (iECs). A side‐to‐side comparative study was conducted using HUVECs, HDBECS, and iECs as ECs source within collagen type I‐based VHSMs.[ 158 ] HDBECs and HUVECs showed comparable vascular network formation within the VHSMs whereas iECs were unable to form optimal vascular networks. Past studies have showed that iECs require pre‐formed bio‐fabricated lumen‐like structures to attach and form stable BVs, therefore highlighting the inability of iECs to spontaneously assemble into vascular networks within collagen type I‐based dermis.[ 88 ] It was also observed post‐engraftment that the VHSMs with HDBECs had the optimal epidermal viability and proliferative capacity followed by VHSMs with HUVECs and iECs, respectively. Moreover, HDBECs enhanced the expression of several skin‐specific ECM genes compared to models with HUVECs and iECs. A thorough examination in the consequence of using diverse ECs source within VHSMs for drug testing, skin graft survival, and skin disease modeling should be conducted. Therefore, a consensus on the cell source, choice of material, and analysis techniques needs to be reached in order to maintain reproducibility and clinical relevancy.
In the case of analyzing vascular distribution within the VHSMs, researchers generally utilize imaging techniques. This includes visualizing and analyzing immuno‐stained cryo‐cut sections. However, vascular distribution within a tissue is heterogenous, with certain sections (especially the periphery) containing more BVs than the center. Therefore, histological sections of both the periphery and the center should be analyzed. Additionally, all histological analyses should be complemented with confocal imaging (Z‐stacked images) in order to quantify vascular area, junction points, and branching. Other reasons for lab‐to‐lab variabilities are provided in Table 4. One challenge in the utilization of VHSMs to mimic skin diseases is the process of validating the models. The key question is whether the disease‐stimulated VHSMs closely resemble the actual skin disease scenario. Moreover, a consensus regarding the stimulants to be added has to be reached. For instance, in the case of AD, IL‐4 and 13 are commonly added as stimulants. However, the importance of other cytokines such as IL‐33, TNF‐α, and IL‐25 in AD progression is not entirely known. The existence of immuno‐competent skin models, where immune cells release the necessary cytokines in response to stimulants, could lead to more clinically‐relevant VHSMs.
5.3. Challenge of Vessel Perfusion and Stability
Another challenge in vascularized skin tissue engineering is to establish perfusion through the newly formed BVs. A lack of media flow through the BVs eventually leads to vessel pruning and regression. In human tissues, vascular angiogenesis and regression is a conserved process and is activated under disease conditions.[ 159 ] During development, vascular regression is a mechanism used for eliminating immature BVs with improper blood flow and stabilizing the remaining BVs. Stabilized or quiescent BVs, to an extent, are resistant to angiogenic and angiostatic stimuli, in order to prevent random vascular sprouting.[ 160 ] Within in vitro models, there is an accelerated period of angiogenesis followed by BVs regression. This could be attributed to an uncontrolled release of proteinases and angiogenic factors, a lack of pericytes for BV stabilization, low mechanical properties of the scaffold, and a lack of media perfusion. Moreover, the presence of keratinocytes and immune cells in VHSMs may lead to excessive BVs formation and subsequent enzymatic dermal degradation.[ 123 ] Therefore, the focus should also shift toward maintaining the long‐term functionality of BVs. This can be achieved by utilizing novel scaffolds with human skin‐like mechanical properties and the utilization of dynamic perfusion cultures.
5.4. Upscaling the Production of Scaffold‐Free VHSMs
The presence of a cell‐secreted ECM makes the scaffold‐free VHSMs an essential prospect in future skin‐related research and treatments. However, the construction of scaffold‐free models necessitates large quantities of cells, especially dermal fibroblasts. This is a limiting factor in the fabrication of sufficient quantities of scaffold‐free VHSMs for drug/cosmetic testing and large‐scale clinical applications such as wound coverage. In order to combat the challenge of upscaling, future scaffold‐free VHSMs could be assembled together with either synthetic ECM micro‐materials or synthetic imitations of fibroblasts, thus forming a hybrid cell‐material co‐culture system. Primarily, the synthetic ECM or cell‐mimics could provide structural integrity to the VHSMs, improving their mechanical properties. Second, they could act as a depot for angiogenic growth factors, thereby allowing optimal cell‐material interactions, enhancing BVs formation, and supporting the long‐term maintenance of vessel architecture.[ 161 ] Although the addition of synthetic cells or ECM‐like materials together with the cells have the above mentioned advantages, several challenges could ensue such as 1) tissue integrity: the challenge of providing strong cell‐material adhesion within the hybrid VHSMs, 2) reproducibility: combination of both scaffold‐free and scaffold‐based systems into a single hybrid system could lead to additional experimental and lab‐to‐lab variability, 3) choice of materials: challenge with the optimization of the material degradation kinetics, biocompatibility, and influence on vascularization, 4) biological functionality: reducing the cell number could lead to modulations in the biological outcomes such as BVs formation and morphogenesis as the amount of secreted growth factors as well as direct cell‐cell interactions diminishes in a hybrid model. Despite the above‐mentioned challenges, such cell‐material hybrid models hold the potential to alleviate the inherent limitations of scaffold‐free and scaffold‐based VHSMs.
6. Conclusion
The recent ban on cosmetic animal testing has sparked the interest of the tissue engineering community to fabricate more clinically‐relevant in vitro skin models. Animal models are still an indispensable tool in preclinical drug testing. This is due to the lack of complex, high‐throughput, multi‐organ human‐based models. Vascularization is an essential step to fabricate organ‐like tissues that can, in future, drastically reduce animal experimentation, and completely replace 2D models. The future entails the testing of drugs and cosmetics on personalized models comprising an assembly of multiple patient‐derived, vascularized tissues. VHSMs—owing to the progress of novel angiogenic biomaterials as well as advancements in scaffold‐free techniques, perfusion culture bioreactors, 3D printing, and organ‐on‐a‐chip systems—are a promising platform for future dermatological research.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
The authors acknowledge Nina Grunze for proof‐reading the manuscript. All schematics were created using Biorender
Open access funding enabled and organized by Projekt DEAL.
Rimal R., Muduli S., Desai P., Marquez A. B., Möller M., Platzman I., Spatz J., Singh S., Vascularized 3D Human Skin Models in the Forefront of Dermatological Research. Adv. Healthcare Mater. 2024, 13, 2303351. 10.1002/adhm.202303351
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