Three-dimensional (3D) bioprinting, a flexible automated on-demand platform for the free-form fabrication of complex living architectures, is a novel approach for the design and engineering of human organs and tissues. Here, we demonstrate the potential of 3D bioprinting for tissue engineering using human skin as a prototypical example. Keratinocytes and fibroblasts were used as constituent cells to represent the epidermis and dermis, and collagen was used to represent the dermal matrix of the skin. Preliminary studies were conducted to optimize printing parameters for maximum cell viability as well as for the optimization of cell densities in the epidermis and dermis to mimic physiologically relevant attributes of human skin. Printed 3D constructs were cultured in submerged media conditions followed by exposure of the epidermal layer to the air-liquid interface to promote maturation and stratification. Histology and immunofluorescence characterization demonstrated that 3D printed skin tissue was morphologically and biologically representative of in vivo human skin tissue. In comparison with traditional methods for skin engineering, 3D bioprinting offers several advantages in terms of shape- and form retention, flexibility, reproducibility, and high culture throughput. It has a broad range of applications in transdermal and topical formulation discovery, dermal toxicity studies, and in designing autologous grafts for wound healing. The proof-of-concept studies presented here can be further extended for enhancing the complexity of the skin model via the incorporation of secondary and adnexal structures or the inclusion of diseased cells to serve as a model for studying the pathophysiology of skin diseases.

Chemical penetration enhancers (CPEs) are present in a large number of transdermal, dermatological, and cosmetic products to aid dermal absorption of curatives and aesthetics. This wide spectrum of use is based on only a handful of molecules, the majority of which belong to three to four typical chemical functionalities, sporadically introduced as CPEs in the last 50 years. Using >100 CPEs representing several chemical functionalities, we report on the fundamental mechanisms that determine the barrier disruption potential of CPEs and skin safety in their presence. Fourier transform infrared spectroscopy studies revealed that regardless of their chemical make-up, CPEs perturb the skin barrier via extraction or fluidization of lipid bilayers. Irritation response of CPEs, on the other hand, correlated with the denaturation of stratum corneum proteins, making it feasible to use protein conformation changes to map CPE safety in vitro. Most interestingly, the understanding of underlying molecular forces responsible for CPE safety and potency reveals inherent constraints that limit CPE performance. Reengineering this knowledge back into molecular structure, we designed >300 potential CPEs. These molecules were screened in silico and subsequently tested in vitro for molecular delivery. These molecules significantly broaden the repertoire of CPEs that can aid the design of optimized transdermal, dermatological, and cosmetic formulations in the future. stratum corneum ͉ spectroscopy ͉ skin irritation ͉ lipid C urrently, hypodermic needles are the only available mode for systemic delivery of macromolecular drugs into humans. Transdermal delivery offers an attractive alternative to needlebased drug administration. However, evolved to impede the flux of exogenous molecules, stratum corneum (SC), the topmost layer of the skin, provides a strong barrier to molecular delivery. This is especially problematic for relatively large drugs (molecular mass Ͼ 500 Da), which represent a large majority of active agents for therapeutic applications (1). Over 350 molecules, termed chemical penetration enhancers (CPEs), have been identified to perturb the SC barrier to facilitate molecular delivery. However, incorporation of CPEs into products has been mitigated by safety concerns related to the health of the skin membrane (2-4). Accordingly, overcoming the skin barrier in a safe and effective way still remains the bottleneck of transdermal and topical therapies.Identification of chemicals to increase skin permeability has been an area of high activity in the last three decades (5-7). After an initial rise in the number of CPEs in the 1980s, the active pool of CPEs has reached a plateau in the last decade. In an era where new chemical entities are being discovered at an exponential rate (as indicated by the entries in the Chemical Abstract Service), the plateau in the number of CPE molecules is rather surprising (only 1 in 100,000 known molecules represents a CPE). This anomaly originates from the slow rates of syntheses of CPEs when compar...

Transdermal drug delivery is an attractive alternative to conventional techniques for administration of systemic therapeutics. One challenge in designing transdermal drug delivery systems is to overcome the natural transport barrier of the skin. Chemicals offer tremendous potential in overcoming the skin barrier to enhance transport of drug molecules. Individual chemicals are however limited in their efficacy in disrupting the skin barrier at low concentrations and usually cause skin irritation at high concentrations. Multicomponent mixtures of chemicals, however, have been shown to provide high skin permeabilization potency as compared to individual chemicals without necessarily causing irritation. Here we review systems employing synergistic mixtures of chemicals that offer superior skin permeation enhancement. These synergistic systems include solvent mixtures, microemulsions, eutectic mixtures, complex self-assembled vesicles and inclusion complexes. Methods for design and discovery of such synergistic systems are also discussed.

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete.

Although transdermal drug delivery is more attractive than injection, it has not been applied to macromolecules because of low skin permeability. Here we describe particular mixtures of penetration enhancers that increase skin permeability to macromolecules (approximately 1-10 kDa) by up to approximately 100-fold without inducing skin irritation. The discovery of these mixtures was enabled by an experimental tool, in vitro skin impedance guided high-throughput (INSIGHT) screening, which is >100-fold more efficient than current tools. In vitro experiments demonstrated that the mixtures delivered macromolecular drugs, including heparin, leutinizing hormone releasing hormone (LHRH) and oligonucleotides, across the skin. In vivo experiments on hairless rats with leuprolide acetate confirmed the potency and safety of one such mixture, sodium laureth sulfate (SLA) and phenyl piperazine (PP). These studies show the feasibility of using penetration enhancers for systemic delivery of macromolecules from a transdermal patch.