A novel approach for the formulation of human skin equivalents (hSKE) has been developed, combining droplet-based 3D bioprinting and nebulization technology.1
This technique differs from traditional manual or extrusion-based methods as it facilitates high reproducibility, uniformity, and enhanced cell viability in skin constructs.
Methodology
- Dermis was created by printing collagen bioink with human dermal fibroblasts. These were stabilized by using nebulized sodium bicarbonate as a crosslinking agent.
- Epidermis formation was achieved by printing keratinocyte bioink, followed by the development of an air-liquid interface culture to induce stratification.
- Constructs were compared with native mouse and human skin in order to confirm morphological similarity.
- A skin grafting model was used to evaluate efficacy.
Outcome
1.Bioprinted hSKE mimics native skin
hSKE fabricated via droplet-based bioprinting and nebulization techniques has the potential to effectively replicate native skin’s dermal and epidermal architecture, including keratinocytes’ keratinization process within the epidermis.
2. Cell viability
Droplet-based printing at low pressure has been shown to preserve fibroblasts and keratinocytes, offering survival rates of >84 %.
3. Therapeutic efficacy
The use of hSKE transplantation in wound healing models has been shown to:
- Accelerate wound closure
- Promote vascularization, offering two times more blood vessels versus collagen-only controls
- Support organized dermal regeneration and mature collagen deposition
4. Advantages of nebulization
Nebulization was found to prevent nozzle clogging by spraying crosslinking agents as a fine mist. This also enabled the printing of low-viscosity collagen bioinks with no cell loss.
Figure 1. Fabrication of hSKE using droplet-based bioprinting with nebulization. A: Droplet nozzle, B: Nebulizer spray, C: Printing design, D: Printed collagen, E: Fibroblasts in collagen (day zero and day one), F: Schematic of hSKE fabrication. Scale bar: 200 μm. Image Credit: Molecular & Cellular Toxicology (2023)
Figure 2. Comparison of morphological similarities between manufactured hSKE and native skin through H&E staining. A: Human native skin isolated from circum tissue. B: Mouse native skin sampled from the skin of nude mice. C: hSKE produced by printing each major cell. Scale bar: 100 μm. Image Credit: Molecular & Cellular Toxicology (2023)
Conclusion
Nebulization-enabled droplet bioprinting is a powerful fabrication method, enabling the production of scalable, reproducible, and functional human skin equivalents.
This approach also offers significant promise as a New Approach Methodology (NAM) in cosmetics, dermatology, and drug development.
Future work in this area is expected to focus on hiPSC-derived skin equivalents, facilitating the delivery of patient-specific and personalized skin therapies.
References and further reading
- Lee, S.-G., et al. (2023). Evaluation of the therapeutic efficacy of human skin equivalents manufactured through droplet-based bioprinting/nebulization technology. Molecular & Cellular Toxicology. (online) DOI: 10.1007/s13273-023-00330-9. https://link.springer.com/article/10.1007/s13273-023-00330-9.
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