中国P站

Bioprinting; Skin equivalents; Wound healing; Multicellular constructs; Layer-by-layer; Tissue regeneration; Biomaterials; Keratinocytes; Fibroblasts; Epidermis

Introduction

Skin injuries, including burns, chronic wounds, and surgical defects, represent a major clinical challenge due to the skin's complex structure and functions. Traditional wound healing therapies often fail to fully restore the skin's function and architecture, particularly in large or deep wounds. One promising approach to address this issue is the development of multicellular skin equivalents through layer-by-layer bioprinting. This technique allows for the precise deposition of cells and biomaterials, creating tissue-like structures that mimic the native skin's architecture. Skin is composed of multiple layers, including the epidermis and dermis, which serve distinct roles in protecting the body and promoting healing. To recreate this functionality, it is crucial to use a combination of keratinocytes, fibroblasts, and extracellular matrix (ECM) proteins, as these components collectively contribute to skin regeneration [1-5].

Layer-by-layer bioprinting enables the construction of these complex structures by printing successive layers of cells, hydrogel-based bioinks, and growth factors. By controlling the deposition order, cell types, and biomaterial composition, this method can closely replicate the skin's organization, promoting better integration and functionality. This study explores the application of layer-by-layer bioprinting to create multicellular skin equivalents for advanced wound healing applications, focusing on optimizing the fabrication process, cell viability, and the functionality of the engineered tissues in wound repair [6-10].

Discussion

The use of layer-by-layer bioprinting to fabricate multicellular skin equivalents provides several advantages over traditional tissue engineering methods. This technique offers precise control over cell positioning, which is crucial for creating the layered structure of skin, where keratinocytes form the outer protective epidermis, and fibroblasts within the dermis provide structural support and ECM production. The ability to print these cells in a controlled manner ensures the correct spatial arrangement and interaction between cell types, enhancing the tissue's ability to mimic native skin.

In our study, we investigated various bioinks composed of collagen, gelatin, and alginate, which provide the necessary support for cell growth and tissue formation. The bioinks were optimized for printability and crosslinking properties to ensure structural stability after printing. The inclusion of growth factors such as epidermal growth factor (EGF) and transforming growth factor beta (TGF-β) was essential to promote cell proliferation, migration, and differentiation, particularly for keratinocytes and fibroblasts. These factors enhanced the formation of a functional epidermal barrier and stimulated dermal fibroblasts to produce ECM proteins, vital for wound healing.

One of the challenges we encountered was maintaining the viability of the cells during the printing process. The shear stress applied during extrusion can damage cells, but by optimizing the printing parameters, such as nozzle diameter and printing speed, we were able to minimize cell loss. Additionally, the biomaterial choice played a critical role in promoting cell attachment and growth. The printed multicellular constructs displayed the expected keratinocyte differentiation in the epidermal layer and collagen deposition in the dermal layer. These constructs were able to form a stable tissue matrix and supported initial wound healing in vitro when tested in wound closure assays.

Further studies are needed to assess the long-term performance of these bioprinted constructs, particularly in vivo. For example, the vascularization of the dermal layer is essential for nutrient and oxygen delivery in thicker skin equivalents. Without adequate blood supply, larger constructs may fail to fully mature or integrate into host tissues. Bioprinting vascular networks alongside the skin equivalents could be a key area for future improvement.

Conclusion

The development of multicellular skin equivalents using layer-by-layer bioprinting represents a significant advancement in the field of wound healing and tissue engineering. Our study demonstrates that this technique can successfully recreate the complex architecture of skin, including both the epidermis and dermis, by carefully layering cells and biomaterials. By optimizing the bioink composition, printing parameters, and growth factor supplementation, we were able to enhance cell viability, differentiation, and tissue formation.

These bioprinted skin equivalents hold great promise for clinical applications, particularly in advanced wound healing, where they can provide an effective alternative to traditional skin grafts and substitutes. The ability to fabricate skin with patient-specific cells could also reduce the risk of immune rejection and improve the speed and quality of healing. However, challenges remain in improving the vascularization and long-term functionality of these constructs. With continued advancements in bioprinting technology and biomaterial development, the goal of creating fully functional, tissue-engineered skin for clinical applications is becoming increasingly feasible.

References

1. Guha TK, Wai A, Hausner G (2017) Computational and structural biotechnology journal 15: 146-160.

   , ,

2. Tafazoli A, Behjati F, Farhud DD, Abbaszadegan MR (2019) Iranian Journal of Public Health 48: 371.

   ,

3. Lippow SM, Aha PM, Parker MH, Blake WJ, Baynes BM (2009) Nucleic acids research 37: 3061-3073.

   , ,

4. Adli M (2018) Nature communications. 9: 1-13.

   , ,

5. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, et al. (2007) Science 315: 1709-1712.

   , ,

6. Mojica FJ, Díez-Villaseñor C, Soria E, Juez G (2000) . Molecular microbiology 36: 244-246.

   , ,

7. Sternberg SH, Doudna JA (2015) . Molecular cell 58: 568-574.

   , ,

8. Bhattacharjee G, Gohil N, Khambhati K, Mani I, Maurya R (2022) . Journal of Controlled Release 343: 703-723.

   , ,

9. Li D, Zhou H, Zeng X (2019) . Cell Biology and Toxicology 35: 403-405.

   , ,

10. Li Y, Li S, Wang J, Liu G (2019) Trends in Biotechnology 37: 730-743.

    , ,