SkThin is a large and complex organ that serves protective and regulatory functions and is responsible for communication between the external environment and the inner organism. To fulfill these functions, skin has evolved as an organ with a complex anatomy derived from both the ectoderm (epidermis) and mesoderm (dermis). The skin includes not only these two major compartments but also important appendages, including hair follicles, sweat and sebaceous glands, nerve endings, and blood vessels, all of which have intricate spatial arrangements that render s is an entry focused on extrusion bioprinting for skin applications. Bioprinting technologies have the ability to combine various human cell phenotypes, signaling proteins, extracellular matrix (ECM) components, and other scaffold-like biomaterials and are currently being exploited for the fabrication of human skin, broadly aiming to achieve two main goals. The first goal is to meet the urgent clinical demand for skin equivalents, which can range in complexity from advanced dressings for chronic wounds to biomimetic skin grafts to help restore the barrier function in complex ulcers, burns, or traumatic postsurgical wounds. The second important motivation for skin biofabrication of the full skin organ challengingis to create disease models for in vitro research and drug development.
21. Skin Biology and Relevant Aspects for Bioprinting
In this context, bioprinting can also benefit from platelet-rich plasma (PRP) biotechnology, as it provides a unique pool of growth factors and cytokines that can enhance healing mechanisms . In physiology, upon skin injury and vessel disruption, extravasated blood forms a clot filling the injured area. Activated platelets and leukocytes within this clot release growth factors and cytokines, establishing a cascade of molecular signals that drives tissue repair. Taking advantage of this mechanism, PRP-based therapies have been used to treat nonhealing wounds, with different degrees of success . In fact, the platelet secretome contains more than 300 proteins, and among the crucial effectors of the repair function of PRP are platelet-derived growth factor (PDGF), Transforming growth factor (TGF), fibroblast growth factors (FGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Connective tissue growth factor (CTGF), Vascular Endothelial Growth Factor (VEGF) . Accordingly, the inclusion of PRP in bioink formulations can improve the efficacy of biofabricated skin equivalents.
32. Overview of Current Research on Extrusion-Based Skin Bioprinting
We first addressed printable cell phenotypes in hydrogel scaffolds. Next, we synthesized current research in extrusion bioprinting and classified research studies according to the two main extrusion trends: first, studies involving a biomimetic approach with natural hydrogels, i.e., fibrinogen, decellularized extracellular matrix (dECM), and collagen; second, studies focused on polymer bioink modification aiming to enhance the fabrication process.
In addition, we roughly estimated the maturity of extrusion bioprinting for skin conditions by applying TRL concepts to the retrieved studies.
32.1. Cells Applied in Skin Bioprinting
The vast majority (61%) of constructs manufactured through extrusion lack complexity and include a single cell phenotype, which is mainly dermal fibroblasts . Although these studies of a single cell phenotype play a role in furthering bioink research, they can only be considered the foundation for creating 3D-bioprinted skin constructs. Even though these methods can be applied to manufacture dermal constructs in an automated way, the resulting constructs differ little from hand-poured hydrogels seeded with fibroblasts. Unfortunately, these models fail to represent the entirety of the functions of the skin, which requires more complex systems integrating multiple cell phenotypes with complex molecular crosstalk.
Fibroblasts are crucial for dermal formation and wound repair, as in the presence of appropriate stimuli, including but not limited to PDGF, IGF-I, and TGF-β1, they synthesize ECM-forming proteins and additional signaling factors. The latter are involved in both autocrine (i.e., TGF-b, connective tissue growth factor (CTGF), VEGF, PDGF-BB) and paracrine (i.e., ICAM-1, VCAM-1, IL-6, IL-8, IL-15, MMPs, CCL2, CCL7, TIMP-1) signaling; thus, they not only participate in fibroblast communication but also coordinate their activities with surrounding cells, i.e., immune cells, endothelial cells, and stem cells in niches .
However, poor advances in bioprinting blood and lymphatic vessels have limited the translational application of skin constructs. The vascular and lymphatic systems located in the dermis are essential for the proper distribution of oxygen and nutrients and removal of waste, respectively. In addition, they are involved in inflammatory skin conditions and wound healing. Despite their importance, only 7 of the 47 articles reviewed reported the blending of fibroblasts with endothelial cells or pericytes .
The primary function of the skin, i.e., serving as a barrier to pathogen invasion, requires a healthy epidermal layer made mainly of keratinocytes. Altered barrier function is involved in inflammatory skin conditions . However, only two works employed melanocytes , which fabricate the photoprotective pigment melanin. The interplay between the two main cell phenotypes of the epidermis, i.e., keratinocytes and melanocytes, is crucial to form the EMU and distribute melanin to keratinocytes, supporting the protective function of the skin against light and heat.
Moreover, the interaction between fibroblasts and keratinocytes is required for the recovery of skin homeostasis. Indeed, keratinocytes instruct fibroblasts to produce several tissue-forming factors, i.e., keratinocyte growth factor (KGF), fibroblast growth factor (FGF), IL-6, GM-CSF, hepatocyte growth factor (HGF), IL-6, IL-19, and PDGF-BB . Nonetheless, merely eleven of the reviewed articles introduced fibroblasts and keratinocytes together in their models , and more importantly, only five of these works have successfully created vascularized, full-thickness skin substitutes .
32.2. Stem Cell Sources
The most commonly used stem cells are bone marrow-derived mesenchymal stem cells (BM-MSCs), as they were the first to be isolated . They have a multilineage differentiation capacity and can differentiate into several cell types, including skin-like cells, i.e., fibroblasts , keratinocytes, endothelial cells, and pericytes . Moreover, during physiological wound healing, circulating MSCs are recruited to the wound site and differentiate into skin cell phenotypes .
Adipose tissue-derived stem cells (ASCs) are a more advantageous type of MSC. Unlike BM-MSCs, ASCs can be easily isolated in large quantities from abundantly available human adipose tissue through a minimally invasive procedure. ASCs have also shown potential in wound healing. They can differentiate into keratinocytes, fibroblasts, and endothelial cells, as well as release a healing milieu of cytokines and growth factors that support angiogenesis, fibroblast migration, and fibronectin and collagen production . Therefore, their use is considered promising in skin regeneration, but as they were discovered later, only five of the reviewed studies used ASCs , and these studies mainly assessed cell viability. Only Kim BS et al.  proved the in vivo wound-healing properties of the fabricated scaffolds, reinforcing the benefits of including stem cells in bioprinted grafts.
On the other hand, pluripotent stem cells have further advantages, as they can differentiate into any somatic cell type of the body. Among these, embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts , and induced pluripotent stem cells (iPSCs)  are derived from somatic cells that have been reprogrammed to induce pluripotency. However, safety concerns linger because of their teratogenic potential. In addition, in the case of ESCs, very few cells are obtained from each extraction, and there are ethical concerns due to their embryonic origin. Therefore, there are critical issues regarding the application of these cells for clinical purposes, and their implementation in human therapy is challenging.
Very recently, human amniotic epithelial cells (AECs) have emerged as a safer source of pluripotent stem cells. They can be easily isolated from the inner amniotic membrane of the placenta, without invasive procedures or associated ethical issues. AECs have shown promising results in wound healing  and become great candidates for skin tissue engineering. In this way, Liu P et al.  developed a scaffold containing AECs and a special type of MSCs derived from the umbilical cord, Wharton’s jelly-derived MSCs (WJMSCs). While AECs are more likely to differentiate into keratinocytes, WJMSCs differentiate into fibroblasts and endothelial cells. Thus, in this work, they explored the development of a meaningful multi-layered skin construct with epidermal (AECs) and dermal (WJMSCs) compartments.
43. Skin Bioprinting, from Bench to Society, Technology Readiness Pathway
As depicted in Figure 2, most of the reviewed works focus on the first steps of the development. In fact, proof of concept was only achieved in eight of the works, which were the biomimetic bioinks from studies shown in Table 1. This demonstrates the immaturity of the technology, seldom validated in experimental research in vivo; no developments have reached sufficient maturity to be applied in a clinically relevant environment.
FigureFigure 2. 2. (A) Display of the reviewed publications according to the stage of development; (B) Distribution of analyzed bioinks for extrusion bioprinting according to technological development
While several companies focused on the bioprinter market business have reached TRL9 , the biofabrication of tissue equivalents for skin conditions is still in the early phase of laboratory research. In particular, research studies dealing with extrusion bioinks are at TRL3, while a few studies have progressed to TRL4 with testing of the bioprinted product in animal models (Figure 2). Indeed, no publications/clinical trials concerning the use of bioprinting for skin conditions in humans have been reported.Based on the original works identified in this review, technology transition to commercial products could be anticipated in the next future in the field of wound management. In order to meet the market and clinical demand, these bioprinted constructs should enable tissue repair and reconstruction of skin architecture in clinically relevant contexts, such as diabetic or vascular ulcers or burn wounds. To shorten the time to market, experimental research (TRL4-5) should generate data that are ready to be used in the certification of the bioprinted construct. A co-development interdisciplinary methodology should achieve constructs with high performance -cost ratio and, generate clinical data that meet regulatory issues associated with marketing authorization of the living constructs (i.e. Advanced Therapy Medicinal Products, ATMP).
54. Limitations and Future Directions
Extrusion bioprinting is highly interdisciplinary, as it involves the development of complex platforms requiring interdisciplinary knowledge, including knowledge of medical imaging, hardware and software to control multiple extruders (the printer), and advanced biomaterial development together with cell production, including a deep understanding of physiology and cellular biology. Moderate progress in extrusion bioprinting has led to a novel technology, which involves bioink extrusion in a yield stress fluid capable of supporting the extruded bioink (reviewed in ), joining competing requirements from the perspective of manufacturability (engineering) and biomimetics (life sciences).
In addition, commonly bioprinted constructs are not ready for in vivo applications and have to follow a maturation process, where architectural changes and remodeling are recognized as the fourth dimension of bioprinting . Alternatively, remodeling can take place in the host tissue. In this context, the merger of robotics with bioprinting has evolved toward intraoperative bioprinting, spanning from engineering, cellular biology and biomaterials to medical sciences and surgery . However, such far-reaching frontiers have created hype-type expectations because of the promising benefits achieved thus far.
Many intricate challenges need to be overcome before bioprinting technology achieves its full potential and transcends the accomplishments of tissue engineering. First, the so-called bioinks, i.e., cell-laden advanced biomaterials or natural polymers, have to be optimized to meet the requirements for printability, reproducibility and spatial organization of the construct; second, the living skin equivalent should be doped with a molecular pool of signaling proteins for the activation of healing mechanisms in a manner that can address the specific requirements of the skin as an organ and various medical conditions. The inclusion of cell signaling molecules in bioinks is often neglected, broadening the disparity between the in vitro and in vivo microenvironments. Thus, the confluence of the two perspectives, representing interdisciplinary inputs as reflected in bioink development, i.e., biomimicry and manufacturability, are required for further advancement toward the future translation of biofabrication.
Based on the original works identified in this review, technology transition to commercial products could be anticipated in the next future in the field of wound management. In order to meet the market and clinical demand, these bioprinted constructs should enable tissue repair and reconstruction of skin architecture in clinically relevant contexts, such as diabetic or vascular ulcers or burn wounds. To shorten the time to market, experimental research (TRL4-5) should generate data that are ready to be used in the certification of the bioprinted construct. A co-development interdisciplinary methodology should achieve constructs with high performance -cost ratio and, generate clinical data that meet regulatory issues associated with marketing authorization of the living constructs (i.e. Advanced Therapy Medicinal Products, ATMP).
Although we are still far from skin fabrication for regenerative medicine, the applications of bioprinted constructs also expand to the generation of in vitro models for drug discovery, which is technically easier with less regulatory constraints. These features help to speed TRL development and get earlier the market demand, while leveraging our accomplishments in biofabrication.
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