Tri-layered skin implants developed using suspended layer additive manufacturing

C wounds, resulting from an imbalance in the healing equilibrium of skin, are a major health problem with limited available treatments. The fabrication of skin replacements that can fulfill the complex chemical, mechanical, and biological roles played by human skin remains a tough challenge. Researchers from the University of Birmingham and the University of Huddersfield in the United Kingdom have developed tri-layered skin implants that closely resemble human skin, using suspended layer additive manufacturing. Suspended layer additive manufacture is a method that takes advantage of a suspension reservoir, containing a suspension medium known as a fluid gel, to provide support for the printed layer until the construct can be cured. The method has a high degree of customization. “It is possible to use this method of suspended manufacture to produce a singular structure with a gradient of mechanical properties that can support the growth of different cell types. This is important for a tissue like skin, where the properties of the bottom-most layer (hypodermis) are very distinct [from] those of the upper layer (epidermis) and the requirements of the resident cells are similarly different,” Liam Grover, one of the lead researchers of this study, told MRS Bulletin. As presented in a recent issue of APL Bioengineering (https:// aip. scita tion. org/ doi/ 10. 1063/5. 00613 61), the threelayered construct was computationally designed and printed using different bio-inks to create the three main layers: epidermal, dermal, and hypodermal. To closely mimic the composition of skin, pectin and collagen were blended in different ratios to simulate the properties of each layer. The hypodermal layer was composed of collagen and pectin in a 1:1 ratio, which allowed the confinement of adipose-derived mesenchymal stem cells and the structural freedom for other cells to migrate. The dermal construct had a 2:1 collagen to pectin content with a gradient of fibroblasts cell density across the layer. Keratinocytes were seeded atop the dermal compartment to form an epidermis. Achieving high cell viability postextrusion along with high print fidelity has always been a limiting factor in three-dimensional printing. Notwithstanding, the ability of suspended layer additive manufacturing to print low viscosity fluids at reasonably slow flow rates made it an attainable goal by minimizing the stress exerted on the cells throughout the process. Gelation kinetics and mechanical properties of the constructs were studied using small deformation rheology. Gelation kinetics were characterized by an initial rapid increase in storage modulus, a value that indicates the ability of a material to store energy elastically, followed by a much smaller gradient. Pectin was responsible for the control over gelation profile, allowing the formation of a gelled network within just 1 min, thus preventing agglomeration or sedimentation of the cells. Likewise, the bulk properties of each layer were also dominated by pectin as evidenced by linear rheology, which showed responses typical of hydrogels for both dynamic frequency and strain. The addition of collagen resulted in lower values of storage moduli due to a less dense pectin network. The integrity of this construct is principally maintained by the pectin while the collagen located within the interstitial area creates a dual network that allowed various layers to form with mechanical properties comparable to those of actual skin. “Besides the pectin-collagen blend, many other material blends could be explored to create these supportive beds,” Grover says. The tri-layered construct was implanted ex vivo using a simulated porcine wound. When comparing the mechanical properties of a 14-day incubated implant to a post-curing one, aging of the material was demonstrated to increase the Young’s modulus. This is likely due to the collagen fibrillogenesis and rearrangement, which results in a stiffer material. Histological staining was used to evaluate the resemblance of the implant to human skin after 21 days of culture. A clear distinction between the different layers, especially the dermis and hypodermis, was observed, showing that the resemblance between them was not only mechanical but also structural, compositional, and on a cellular level. Visualization of the tissue-implant interface after seven days showed a seamless continuity between the tri-layered construct and the tissue indicative of full integration. Through histological staining, it was possible to identify cell structures within the dermis. Furthermore, fluorescence keratinocyte HDF ADSC