Pilot Study of the Biological Properties and Vascularization of 3D Printed Bilayer Skin Grafts
The skin is the largest human organ, and defects in the skin with a diameter greater than 4 cm do not heal without treatment. Allogeneic skin transplantation has been used to allow wound healing, but many grafts do not survive after implantation, due to multiple complications in the procedure. In the present study, the vascularization of threedimensional (3D) printed full-thickness skin grafts was investigated. Dermal-epithelial grafts were transplanted into a nude mouse model to evaluate integration with the host tissue and the extent of wound healing. To create microvessels in the skin grafts, a bilayer structure consisting of human dermal fibroblasts, keratinocytes, and microvascular endothelial cells was designed and fabricated using an extruded 3D printer. Human dermal fibroblasts and human microvascular endothelial cells were mixed with gelatin-sodium alginate composite hydrogel as the dermis, and human keratinocytes were mixed with gel as the epithelium. Confocal imaging allowed visualization of the location of the cells in the doublelayer skin grafts. A full-thickness wound was created on the backs of nude mice and then covered with a double-layer skin graft. Various groups of mice were tested. Animals were euthanized and tissue samples collected after specified time points. Compared with the control group, wound contraction improved by approximately 10%. Histological analysis demonstrated that the new skin had an appearance similar to that of normal skin and with a significant degree of angiogenesis. The results of the immunohistochemical analysis demonstrated that the transplanted cells survived and participated in the healing process.
1. Rognoni E, Walko G, 2019, The Roles of YAP/TAZ and the Hippo Pathway in Healthy and Diseased Skin. Cells, 8(5):E411. DOI: 10.3390/cells8050411.
2. Prost-Squarcioni C, 2006, Histology of Skin and Hair Follicle. Med Sci (Paris), 22(2):131–7. DOI: 10.1051/medsci/2006222131.
3. Bernerd F, 2005, Human Skin Reconstructed in vitro as a Model to Study the Keratinocyte, the Fibroblast and Their Interactions: Photodamage and Repair Processes. J Soc Biol, 199(4):313–20.
4. Kim SW, Choi SH, Kim JT, et al., 2015, An Additional Option for Split-Thickness Skin Graft Donors: The Previous Free Flap Sites. Ann Plast Surg, 75(6):634–6. DOI: 10.1097/SAP.0000000000000143.
5. Wei LG, Chang HI, Wang Y, et al., 2019, A Gelatin/Collagen/Polycaprolactone Scaffold for Skin Regeneration. PeerJ, 7:e6358. DOI: 10.7717/peerj.6358.
6. Han XX, Courseaus J, Khamassi J, et al., 2018, Optimized Vascular Network by Stereolithography for Tissue Engineered Skin. Int J Bioprinting, 4(2):17. DOI: 10.18063/IJB.v4i2.134.
7. Ng WL, Chua CK, Shen YF, 2019, Print Me An Organ! Why We Are Not There Yet. Prog. Polym Sci, 97:45. DOI: 10.1016/j.progpolymsci.2019.101145.
8. Liu F, Liu C, Chen QH, et al., 2018, Progress in Organ 3D Bioprinting. Int J Bioprinting, 4(1):15. DOI: 10.18063/IJB.v4i1.128.
9. An J, Teoh JEM, Suntornnond R, et al., 2015, Design and 3D Printing of Scaffolds and Tissues. Engineering, 1(2):261–8. DOI: 10.15302/j-eng-2015061.
10. Choudhury D, Anand S, Naing MW, 2018, The Arrival of Commercial Bioprinters Towards 3D Bioprinting Revolution! Int J Bioprinting, 4(2):20. DOI: 10.18063/IJB.v4i2.139.
11. Zhuang P, Sun AX, An J, et al., 2018, 3D Neural Tissue Models: From Spheroids to Bioprinting. Biomaterials, 154:113–33. DOI: 10.1016/j.biomaterials.2017.10.002.
12. Chong C, Wang Y, Fathi A, et al., 2019, Skin Wound Repair: Results of a Pre-clinical Study to Evaluate Electropsun Collagen-Elastin-PCL Scaffolds as Dermal Substitutes. Burns, 45(7):1639–48. DOI: 10.1016/j.burns.2019.04.014.
13. Kandhasamy S, Arthi N, Arun RP, et al., 2019, Synthesis and Fabrication of Novel Quinone-Based Chromenopyrazole Antioxidant-Laden Silk Fibroin Nanofibers Scaffold for Tissue Engineering Applications. Mater Sci Eng C Mater Biol Appl, 102:773–87. DOI: 10.1016/j.msec.2019.04.076.
14. Baltazar T, Merola J, Catarino C, et al., 2019, Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells. Tissue Eng Part A, 12. DOI: 10.1089/ten.tea.2019.0201.
15. Ng WL, Yeong WY, 2019, The Future of Skin Toxicology Testing Three-Dimensional Bioprinting Meets Microfluidics. Int J Bioprinting, 5(2.1):44–54. DOI: 10.18063/ijb.v5i2.1.237.
16. Wang JH, Chen J, Kuo SM, et al., 2019, Methods for Assessing Scaffold Vascularization In Vivo. Methods Mol Biol, 1993:217–26. DOI: 10.1007/978-1-4939-9473-1_17.
17. Li Y, Lu H, Gu Y, et al., 2017, Enhancement of NK Cells Proliferation and Function by Shikonin. Immunopharmacol Immunotoxicol, 39(3):124–30. DOI: 10.1080/08923973.2017.1299174.
18. Zhou S, Cui Z, Urban J, 2011, Dead Cell Counts During Serum Cultivation are Underestimated by the Fluorescent Live/Dead Assay. Biotechnol J, 6(5):513–8. DOI: 10.1002/biot.201000254.
19. Kuppan P, Sethuraman S, Krishnan UM, 2017, In Vitro Co- Culture of Epithelial Cells and Smooth Muscle Cells on Aligned Nanofibrous Scaffolds. Mater Sci Eng C Mater Biol Appl, 81:191–205. DOI: 10.1016/j.msec.2017.07.050.
20. Yanez M, Rincon J, Dones A, et al., 2015, In Vivo Assessment of Printed Microvasculature in a Bilayer Skin Graft to Treat Full-Thickness Wounds. Tissue Eng Part A, 21(1-2):224–33. DOI: 10.1089/ten.TEA.2013.0561.
21. Nye R, Robinia K, Peterson P, et al., 2018, Efficacy of a Nitric Oxide Dressing in Decreasing Bacterial Counts on Human Skin. J Wound Care, 27(7):S19–25. DOI: 10.12968/ jowc.2018.27.Sup7.S19.
22. Garcia M, Escamez MJ, Carretero M, et al., 2007, Modeling Normal and Pathological Processes Through Skin Tissue Engineering. Mol Carcinog, 46(8):741–5. DOI: 10.1002/mc.20327.
23. Yannas IV, Tzeranis DS, So PTC, 2017, Regeneration of Injured Skin and Peripheral Nerves Requires Control of Wound Contraction, not Scar Formation. Wound Repair Regen, 25(2):177–91. DOI: 10.1111/wrr.12516.
24. Feldman AT, Wolfe D, 2014, Tissue Processing and Hematoxylin and Eosin Staining. Methods Mol Biol, 1180:31–43. DOI: 10.1007/978-1-4939-1050-2_3.
25. Ferringer T, 2015, Immunohistochemistry in Dermatopathology. Arch Pathol Lab Med, 139(1):83–105. DOI: 10.5858/arpa.2014-0075-RA.
26. Ng WL, Qi JTZ, Yeong WY, et al., 2018, Proof-of-Concept: 3D Bioprinting of Pigmented Human Skin Constructs. Biofabrication, 10(2):025005. DOI: 10.1088/1758-5090/aa9e1e.
27. Huang S, Yao B, Xie J, et al., 2016, 3D Bioprinted Extracellular Matrix Mimics Facilitate Directed Differentiation of Epithelial Progenitors for Sweat Gland Regeneration. Acta Biomater, 32: 170–7. DOI: 10.1016/j.actbio.2015.12.039.
28. Kim BS, Kwon YW, Kong JS, et al., 2018, 3D Cell Printing of In Vitro Stabilized Skin Model and In Vivo Pre-Vascularized Skin Patch Using Tissue-Specific Extracellular Matrix Bioink: A Step Towards Advanced Skin Tissue Engineering. Biomaterials, 168:38–53. DOI: 10.1016/j. biomaterials.2018.03.040.
29. Chen H, Xing X, Tan H, et al., 2017, Covalently Antibacterial Alginate-Chitosan Hydrogel Dressing Integrated Gelatin Microspheres Containing Tetracycline Hydrochloride for Wound Healing. Mater Sci Eng C Mater Biol Appl, 70:287–95. DOI: 10.1016/j.msec.2016.08.086.
30. Nezhad ZM, Poncelet A, Fervaille C, et al., 2019, Comparing the Host Reaction to CorMatrix and Different Cardiac Patch Materials Implanted Subcutaneously in Growing Pigs. Thorac Cardiovasc Surg, 67(1):44–9. DOI: 10.1055/s-0037-1607332.
31. Limandjaja GC, van den Broek LJ, Breetveld M, et al., 2018, Characterization of In Vitro Reconstructed Human Normotrophic, Hypertrophic, and Keloid Scar Models. Tissue Eng Part C Methods, 24(4):242–53. DOI: 10.1089/ten.TEC.2017.0464.
32. Walmsley GG, Hu MS, Hong WX, et al., 2015, A Mouse Fetal Skin Model of Scarless Wound Repair. J Vis Exp, 95:52297. DOI: 10.3791/52297.
33. Tranquillo RT, Murray JD, 1993, Mechanistic Model of Wound Contraction. J Surg Res, 55(2):233–47. DOI: 10.1006/jsre.1993.1135.
34. Oosterwijk AM, Mouton LJ, Schouten H, et al., 2017, Prevalence of Scar Contractures After Burn: A Systematic Review. Burns, 43(1):41–9. DOI: 10.1016/j.burns.2016.08.002.
35. Lee V, Singh G, Trasatti J P, et al., 2014, Design and Fabrication of Human Skin by Three-Dimensional Bioprinting. Tissue Eng Part C Methods, 20(6):473–84. Doi: 10.1089/ten.TEC.2013.0335.
36. Lee MS, Ahmad T, Lee J, et al., 2017, Dual Delivery of Growth Factors with Coacervate-Coated Poly(Lactic-Co-Glycolic Acid) Nanofiber Improves Neovascularization in a Mouse Skin Flap Model. Biomaterials, 124:65–77. Doi: 10.1016/j.biomaterials.2017.01.036.
37. Lalley AL, Boyce ST, 2019, Fabrication of Chimeric Hair Follicles for Skin Tissue Engineering. Methods Mol Biol, 1993:159–79. DOI: 10.1007/978-1-4939-9473-1_13.
38. Brandenburger M, Kruse C, 2019, Fabrication of a Co- Culture System with Human Sweat Gland-Derived Cells and Peripheral Nerve Cells. Methods Mol Biol, 1993:139–48. DOI: 10.1007/978-1-4939-9473-1_11.