Comparison of the Biological Behaviour of Human Dermal Fibroblasts seeded on 3D Printed Polylactic acid, Polycaprolactone and Polyethylene Terephthalate Scaffolds in vitro

Abstract

Regenerative medicine is a scientific field that improves and repairs diseased and injured tissues. Three-dimensional (3D) printing is an innovative technology that provides a new application field for regenerative medicine. 3D printed scaffolds by programming pore sizes and shapes serve as a temporary basis for cells until the natural extracellular matrix (ECM) is reconstructed. Dermal fibroblasts are mesenchymal cells located in the dermal skin layer that produce and organize ECM components. They play an essential role in skin wound healing and fibrosis. The aim of this study is to analyze the viability, adhesion, distribution, and collagen IV expression of human dermal fibroblasts (HDFs) seeded on 3D printed polylactic acid (PLA), polyethylene terephthalate (PET), and poly-ε-caprolactone (PCL) scaffolds in vitro. HDFs were seeded on scaffolds or tissue culture plastic plates as control and were cultured for 1 and 3 days. 3D PLA, PCL, and PET 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 distribution was analyzed by hematoxylin and eosin (H&E) staining. Collagen IV expression was analyzed by immunohistochemical (IHC) staining. On day 1, the viability of HDFs on the 3D PLA scaffolds was significantly higher than PCL scaffolds. On day 3, the viability of HDFs on 3D PLA and PET scaffolds was significantly higher than PCL scaffolds. SEM images showed that HDFs on 3D PLA scaffolds attached the surfaces, filled the interfiber gaps and maintained their tissue specific morphology on day 3 compared to PCL and PET scaffolds. Histological images stained with H&E demonstrated that the distribution of HDFs on 3D PLA scaffolds was uniform on days 1 and 3. Collagen IV staining was more intense in HDFs on 3D PLA scaffolds on days 1 and 3. This study shows that 3D PLA scaffolds create a appropriate environment for cell viability, adhesion, distribution and may provide a high advantage in skin tissue regeneration.

Keywords

• Human dermal fibroblasts
• 3D printed scaffolds
• Skin tissue regeneration

Öz

Rejeneratif tıp, hastalıklı ve yaralı dokuları iyileştiren ve onaran bir bilimsel alandır. 3 boyutlu (3B) baskı, rejeneratif tıbba yeni bir uygulama alanı sağlayan yenilikçi bir teknolojidir. Gözenek boyutları ve şekilleri programlanarak 3B baskı ile üretilen doku iskeleleri, doğal hücre dışı matriks (ECM) yeniden yapılandırılıncaya kadar hücreler için geçici bir destek görevi görmektedir. Dermal fibroblastlar, ECM bileşenlerinin üretimi ve düzenlenmesinde rol oynayan dermal deri tabakasında bulunan mezenkimal hücrelerdir. Dermal fibroblastlar yara iyileşmesi ve deri fibrozisinde temel rol oynayan hücrelerdir. Bu çalışmanın amacı, 3B baskılı polilaktik asit (PLA), polietilen tereftalat (PET) ve poli-ε-kaprolakton (PCL) doku iskelelerine ekilen insan dermal fibroblastlarının (HDF) canlılığı, adezyonu, dağılımı ve kollajen IV ekspresyonlarının in vitro analiz edilmesidir. Doku iskelelerine ve kontrol grubu olarak doku kültür plastik plakalara ekilen HDF’ler 1 ve 3 gün boyunca kültüre edilmiştir. Doku iskeleleri özel tasarım birleştirmeli yığma modellemesi (FDM) ile 3B yazıcı kullanılarak hazırlanmıştır. Hücre canlılığı WST1 ile, hücre adezyonu taramalı elektron mikroskobisi (SEM) ile, hücre dağılımı hematoksilen & eozin (H&E) ve kollajen IV ekspresyonu immünohistokimyasal (IHC) boyamalar ile analiz edilmiştir. 1. günde 3B PLA iskelelerindeki HDF’lerin canlılığı, PCL iskelelerindeki HDF’lerden istatiksel olarak anlamlı yüksek bulunmuştur. 3. günde ise, 3B PLA ve PET iskelelerdeki HDF'lerin canlılığı PCL iskelelerindeki HDF’lerden istatiksel olarak anlamlı yüksek tespit edilmiştir. SEM görüntüleri, 3B PLA iskelelerdeki HDF'lerin yüzeylere bağlandığını, fiberler arası boşlukları doldurduğunu, PCL ve PET iskelelerine kıyasla özellikle 3. günde dokuya özgü morfolojilerini koruduğunu göstermiştir. HDF'lerin 3B PLA iskelelerindeki dağılımı 1. ve 3. günlerde geniş yayılım göstermiştir. 3B PLA iskelelerindeki HDF'lerde kollajen IV boyamasının 1. ve 3. günlerde daha şiddetli olduğu gözlenmiştir. Sonuçlarımız, 3B PLA iskelelerinin hücre canlılığı, adhezyonu, dağılımı için uygun bir ortam oluşturduğunu ve deri rejenerasyonunda avantaj sağlayabileceğini göstermektedir.

Anahtar Kelimeler

• Dermal fibroblast
• 3B Baskılı iskeleler
• Deri rejenerasyonu

Kaynakça

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