Skin tissue engineering has attained several clinical milestones making remarkable progress over the past decades. Skin is inhabited by a plethora of cells spatiotemporally arranged in a 3-dimensional (3D) matrix, creating a complex microenvironment of cell-matrix interactions. This complexity makes it difficult to mimic the native skin structure using conventional tissue engineering approaches. With the advent of newer fabrication strategies, the field is evolving rapidly. However, there is still a long way before an artificial skin substitute can fully mimic the functions and anatomical hierarchy of native human skin. The current focus of skin tissue engineers is primarily to develop a 3D construct that maintains the functionality of cultured cells in a guided manner over a period of time. While several natural and synthetic biopolymers have been translated, only partial clinical success is attained so far. Key challenges include the hierarchical complexity of skin anatomy; compositional mismatch in terms of material properties (stiffness, roughness, wettability) and degradation rate; biological complications like varied cell numbers, cell types, matrix gradients in each layer, varied immune responses, and varied methods of fabrication. In addition, with newer biomaterials being adopted for fabricating patient-specific skin substitutes, issues related to escalating processing costs, scalability, and stability of the constructs under in vivo conditions have raised some concerns. This review provides an overview of the field of skin regenerative medicine, existing clinical therapies, and limitations of the current techniques. We have further elaborated on the upcoming tissue engineering strategies that may serve as promising alternatives for generating functional skin substitutes, the pros and cons associated with each technique, and scope of their translational potential in the treatment of chronic skin ailments.

If controllable, stem cell activation following injury has the therapeutic potential for supporting regeneration in acute or chronic wounds. Human dermally-derived stem cells (FmSCs) were exposed to the cytokines interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) in the presence of erythropoietin (EPO). Cells were cultured under ischemic conditions and phenotypically characterized using flow cytometry. Topical EPO application was performed in three independent clinical wound healing attempts. The FmSCs expressed the receptor for EPO. EPO had a strong inhibitory effect on FmSC growth in the absence of IL-6 and TNF-α. With IL-6, the EPO effects were reversed to that of growth stimulation. TNF-α had the strongest stimulatory effect. In contrast, IL-1β had an inhibitory effect. Topically applied EPO considerably enhanced wound healing and improved wound conditions of acute and chronic wounds. Site specificity of stem cell activation is mediated by IL-6 and TNF-α. In trauma, EPO ceases its inhibitory role and reverts to a clinically relevant boosting function. EPO may be an important therapeutic tool for the topical treatment of acute and chronic wounds.

Skin has the natural ability to heal and replace dead cells regulated by a network of complex immune processes. This ability is conferred by the population of resident immune cells that act in coordination with other players to provide a homeostatic environment under constant challenge. Other than providing structure and integrity, the epidermis and dermis also house distinct immune properties. The dermal part is represented by fibroblasts and endothelial cells followed by an array of immune cells which includes dendritic cells (DCs), macrophages, mast cells, NK-cells, neutrophils, basophils, eosinophils, αβ T lymphocytes, B-cells and platelets. On the other hand, the functionally active immune cells in the epidermis comprise keratinocytes, DCs, NKT-cells, γδ T cells and αβ T cells (CD4+ and CD8+). Keratinocytes create a unique microenvironment for the cells of the immune system by promoting immune recognition and cellular differentiation. T lymphocytes exhibit tissue-specific tropism toward the epidermis and the lymphatic drainage system important for their function in immune regulation. This diversity in immune regulators makes the skin a unique organ to overcome pathogenic or foreign invasion. In addition, the highly coordinated molecular events make the skin an attractive model to understand and explore its regenerative potential.

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Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a “gold standard” in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.

Till date, no bioartificial liver (BAL) procedure has obtained FDA approval or widespread clinical acceptance, mainly because of multifactorial limitations such as the use of microscale or undefined biomaterials, indirect and lower oxygenation levels in liver cells, short-term undesirable functions, and a lack of 3D interaction of growth factor/cytokine signaling in liver cells. To overcome preclinical limitations, primary rat liver cells were cultured on a naturally self-assembling peptide nanoscaffold (SAPN) in a clinically relevant bioreactor for up to 35 days, under 3D interaction with suitable growth factors and cytokine signaling agents, alone or combination (e.g., Group I: EPO, Group II: Activin A, Group III: IL-6, Group IV: BMP-4, Group V: BMP4 + EPO, Group VI: EPO + IL-6, Group VII: BMP4 + IL-6, Group VIII: Activin A + EPO, Group IX: IL-6 + Activin A, Group X: Activin A + BMP4, Group XI: EPO + Activin A + BMP-4 + IL-6 + HGF, and Group XII: Control). Major liver specific functions such as albumin secretion, urea metabolism, ammonia detoxification, phase contrast microscopy, immunofluorescence of liver specific markers (Albumin and CYP3A1), mitochondrial status, glutamic oxaloacetic transaminase (GOT) activity, glutamic pyruvic transaminase (GPT) activity, and cell membrane stability by the lactate dehydrogenase (LDH) test were also examined and compared with the control over time. In addition, we examined the drug biotransformation potential of a diazepam drug in a two-compartment model (cell matrix phase and supernatant), which is clinically important. This present study demonstrates an optimized 3D signaling/scaffolding in a preclinical BAL model, as well as preclinical drug screening for better drug development.

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