The cellular responses of human macrophages seeded on 3D printed thermoplastic polyurethane scaffold

Year 2021, , 40 - 45, 31.12.2021

Mehtap Yuksel Egrılmez , Ufkay Karabay , Selma Aydemir , Başak Baykara , R. Bugra Husemoglu

https://doi.org/10.51934/jomit.1042774

Cited By: 1

Abstract

Tissue engineering is an interdisciplinary field for the design of functional constructs that aid to repair damaged or diseased tissue. Three-dimensional (3D) printing is a growing technology that offers new opportunities for tissue engineering. Thermoplastic polyurethane (TPU) is a member of the polyurethane class. TPUs are commonly used in medical applications with their biocompatible, superior mechanical properties and shape memory behavior. Macrophages are key regulators of tissue homeostasis, inflammation, and regeneration. They play crucial roles in initial immune response to implants. In this study, we aimed to investigate the viability, adhesion, and distribution properties of human THP-1 macrophages seeded on 3D printed TPU scaffolds in vitro. The expression of CD68 and CD10 was also analyzed in human THP-1 macrophages on 3D TPU scaffolds. THP-1 macrophages treated with phorbol-12-myristate-13-acetate (PMA) were seeded on 3D TPU scaffolds or tissue culture plastic plates as control and cultured for 1, 3, 7, and 14 days. 3D TPU scaffolds were prepared using a custom made fused deposition modeling printer. The cell viability was measured by WST-1 assay on days 1 and 3. The cell adhesion was evaluated by scanning electron microscopy (SEM). The cell distribution was analyzed by hematoxylin and eosin (H&E) staining. Expression of CD10 and CD68 was analyzed by immunohistochemical (IHC) staining. The viability of THP-1 macrophages on 3D TPU scaffolds was lower than their control groups on days 1 and 3. SEM images showed THP-1 macrophage attachment on the 3D TPU scaffold surface with round and elongated morphologies. H&E staining demonstrated that THP-1 macrophages showed eosinophilic cytoplasm and large nuclei. CD68 staining was more intense in THP-1 macrophages on 3D TPU scaffolds on day 3 compared to days 1, 7 and 14. CD10 staining was more intense on day 1 compared to days 3, 7, and 14. Our results show that 3D TPU scaffolds are biocompatible with macrophages and might be a potential biomaterial for medical applications.

Keywords

macrophages, 3d printed, TPU, cell culture

3B Baskılı termoplastik poliüretan iskeleye ekilen insan makrofajlarının hücresel yanıtları

Abstract

Doku mühendisliği, hasarlı veya hastalıklı dokuyu onarmak için destekleyici fonksiyonel yapıların tasarımı için disiplinler arası bir alandır. Üç boyutlu (3B) baskı, doku mühendisliği için yeni fırsatlar sunan ve büyümekte olan bir teknolojidir. Termoplastik poliüretan (TPU), poliüretan sınıfının bir üyesidir. TPU'lar, biyouyumlulukları, üstün mekanik özellikleri ve şekil hafızalı davranışları ile tıbbi uygulamalarda yaygın olarak kullanılmaktadır. Makrofajlar, doku homeostazı, inflamasyon ve rejenerasyonun anahtar düzenleyicileridir. İmplantlara karşı ilk bağışıklık yanıtında çok önemli rol oynarlar. Bu çalışmada, 3B baskılı TPU doku iskelelerine ekilen insan THP-1 makrofajlarının canlılık, adezyon ve dağılım özelliklerini in vitro olarak araştırmayı amaçladık. Ayrıca, 3B TPU doku iskelelerine ekilen insan THP-1 makrofajlarında CD68 ve CD10'un ekspresyonu da analiz edildi. Forbol-12-miristat-13-asetat (PMA) ile uyarılan THP-1 makrofajları, 3B TPU doku iskeleleri ve kontrol olarak doku kültürü plastik plakaları üzerine ekildi ve 1, 3, 7 ve 14 gün boyunca kültüre edildi. 3B TPU doku iskeleleri, özel yapılmış bir eriyik yığma modelleme (FDM) yazıcısı kullanılarak hazırlandı. Hücre canlılığı, 1. ve 3. günlerde WST-1 kiti ile ölçüldü. Hücre adezyonu, taramalı elektron mikroskobu (SEM) ile değerlendirildi. Hücre dağılımı, hematoksilen ve eozin (H&E) boyaması ile analiz edildi. CD68 ve CD10 ekspresyonu, immünohistokimyasal (IHC) boyama ile analiz edildi. THP-1 makrofajlarının 3B TPU doku iskeleleri üzerindeki canlılığı, 1. ve 3. günlerde kontrol gruplarından daha düşük tespit edildi. SEM görüntüleri, THP-1 makrofajların 3B TPU doku iskeleleri yüzeyinde yuvarlak ve uzun morfolojilere sahip olarak tutunduğunu gösterdi. H&E boyaması ile THP-1 makrofajlarının eozinofilik sitoplazma ve büyük çekirdekli morfolojide olduğu gösterildi. 3B TPU doku iskelelerindeki THP-1 makrofajların 3. günde CD68 boyaması 1, 7 ve 14. günlere kıyasla daha yoğundu. CD10 boyaması 1. günde 3, 7 ve 14. günlere kıyasla daha yoğundu. Sonuçlarımız, 3B TPU doku iskelelerinin makrofajlarla biyolojik olarak uyumlu olduğunu ve tıbbi uygulamalar için potansiyel bir biyomateryal olabileceğini göstermektedir.

Keywords

makrofaj, 3b baskı, TPU, hücre kültürü

References

• Langer R, Vacanti JP. Tissue engineering. Science. 1993;260:920-6.
• Richards DJ, Tan Y, Jia J, Yao H, Mei Y. 3D Printing for tissue engineering. Isr J Chem. 2013;53:805-14.
• Harynska A, Kucinska-Lipka J, Sulowska A, Gubanska I, Kostrzewa M, Janik H. Medical-Grade PCL based polyurethane system for FDM 3D Printing-Characterization and Fabrication. Materials (Basel). 2019 Mar 16;12:887.
• Joseph J, Patel RM, Wenham A, Smith JR. Biomedical applications of polyurethane materials and coatings. Trans Inst Met Finish. 2018;96:121-29.
• Xiao J, Gao Y. The manufacture of 3D printing of medical grade TPU. Prog Addit Manuf 2017; 2: 117-123.
• Anderson JM. Biological responses to materials. Annu Rev Mater Res 2001; 3: 81-110.
• Martin KE, García AJ. Macrophage phenotypes in tissue repair and the foreign body response: Implications for biomaterial-based regenerative medicine strategies. Acta Biomaterialia 2021;133:4-16.
• Farrugia BL, Brown TD, Upton Z, Hutmacher DW, Dalton PD, Dargaville TR. Dermal fibroblast infiltration of poly(ε-caprolactone) scaffolds fabricated by melt electrospinning in a direct writing mode. Biofabrication. 2013;5: 025001.
• Huerta RR, Silva E, Ekaette I, El-Bialy T, Saldaña MDA. High-Intensity ultrasound-assisted formation of cellulose nanofiber scaffold with low and high lignin content and their cytocompatibility with gingival fibroblast cells. Ultrason Sonochem 2020;64:104759.
• Chen WC, Wei YH, Chu IM, Yao CL. Effect of chondroitin sulphate C on the in vitro and in vivo chondrogenesis of mesenchymal stem cells in crosslinked type II collagen scaffolds. J Tissue Eng Regen Med 2013;7:665-72.
• Griffin MF, Naderi N, Kalaskar DM, Seifalian AM, Butler PE. Argon plasma surface modification promotes the therapeutic angiogenesis and tissue formation of tissue-engineered scaffolds in vivo by adipose-derived stem cells. Stem Cell Res Ther 2019;10:1-14.
• Tatai L, Moore TG, Adhikari R, Malherbe F, Jayasekara R, Griffiths I, Gunatillake PA. Thermoplastic biodegradable polyurethanes: The effect of chain extender structure on properties and in-vitro degradation. Biomaterials 2007;28:5407-17.
• Mi HY, Jing X, Salick MR, Cordie TM, Peng XF, Turng LSh. Properties and fibroblast cellular response of soft and hard thermoplastic polyurethane electrospun nanofibrous scaffolds. J Biomed Mater Res B Appl Biomater . 2015;103:960-70.
• Woitschach F, Kloss M, Schlodder K, Borck A, Grabow N, Reisinger EC, Sombetzki M. In vitro study of the interaction of innate immune cells with liquid silicone rubber coated with zwitterionic methyl methacrylate and thermoplastic polyurethanes. Materials 2021;14:5972.
• Gordon S, Plüddemann A, Martinez Estrada F. Macrophage heterogeneity in tissues: phenotypic diversity and functions. Immunol Rev 2014;262:36–55. Erdos EG, Skidgel RA. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB Journal 1989;3:145-151.
• Huang X, He C, Lin G, Lu L, Xing K, Hua X, Sun S, Mao Y, Song Y, Wang J, Li S. Induced CD10 expression during monocyte-to-macrophage differentiation identifies a unique subset of macrophages in pancreatic ductal adenocarcinoma, Biochem Biophys 2020;524:1064-71.