Doğal protein katkılı biyobozunur polimer doku matrikslerinin geliştirilmesi ve adipoz kök hücreler ile etkileşiminin araştırılması
Evaluation of biodegradable polymeric scaffolds with natural protein and interaction with adipose derived stem cells
Yazar: NERGİS ZEYNEP RENKLER DEĞİRMENCİ
Danışman: PROF. KADRİYE TUZLAKOĞLU
Yalova Üniversitesi / Fen Bilimleri Enstitüsü / Polimer Mühendisliği Ana Bilim Dalı / Polimer Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2019
Konu: Biyomühendislik = Bioengineering ; Polimer Bilim ve Teknolojisi = Polymer Science and Technology

Özet:
Bu tez çalışması, doku mühendisliği uygulamalarında kullanılmak üzere, mevcut sentetik ve doğal kompozit polimerik doku iskelelerine alternatif olarak literatürde ilk kez yumurta beyazı proteininin (HEW) kullanımını ve bu proteinin yüzey kimyası özelliklerine etkisini ve adipoz kaynaklı mezenkimal kök hücreler (ASCs) ile etkileşimini incelemektedir. Bu amaçla, HEW ve poli(-kaprolakton) (PCL) polimerinden elektroeğirme ile nanofiber doku iskeleleri üretildikten sonra, bu yapıların çeşitli yöntemler ile karakterizayonu gerçekleştirilmiştir. Bir sonraki aşamada, doku iskelelerinin üzerine ASC'ler ekilerek hücre canlılık analizleri yapılmıştır.
İlk aşamada elde edilen liyofilize HEW proteinine SDS-page analizi yapılmıştır ve HEW'in içeriğindeki ana proteinlere ait bantlar gözlenmiştir. Taramalı elektron mikroskobu (SEM) ile yapılan morfolojik analizler sonucunda düzgün yüzeylere sahip fiberlerden oluşan membranların başarıyla elde edildiği görülmüştür. Ayrıca, HEW içeren fiberlerin (50-254nm) yalnızca PCL'den oluşan fiberlere (0.90-1.60μm) göre daha ince olduğu tespit edilmiştir. Nanofiberler üzerindeki HEW dağılımı, anti-ovalbumin antikoru kullanılarak immünohistokimyasal olarak gösterilmiştir. Fourier Dönüşümlü Kızılötesi Spektroskopisi (FTIR-ATR) analizi ile de membran üzerindeki protein varlığı doğrulanmıştır. PCL/HEW membranının termal özelliklerinin belirlenmesi ve ayrıca membran içeriğinde bulunan PCL ve HEW miktarının tayini amacıyla termogravimetrik diferansiyel termal analiz (TGA) yapılmıştır. TGA sonucuna göre her iki bileşenin dekomposizyonu görülmüş ve kompozit nanofiberlerdeki HEW protein oranının %36 civarında olduğu saptanmıştır. Fiberlerin ıslanabilirliğini analiz etmek amacıyla su ile yapmış olduğu temas açıları ölçülmüştür. Hidrofobik özellikte olduğu bilinen PCL membranların temas açısının 90o'de sabit kaldığı gözlemlenirken, PCL/HEW membran yüzeylerinde başlangıçta ölçülen 70o'lik temas açısının zamanla azaldığı ve dolayısıyla proteinin membranlara su absorplama özelliği katarak hidrofilik karakterde bir matris oluşumuna sebep olduğu bulunmuştur.
Hücre kültürü çalışmalarında, doku iskeleleri üzerine ekilen ASC'lere hücre canlılığının belirlenmesi amacıyla 2, 7 ve 14. günlerde canlı/ölü boyaması ve alamar blue çalışması yapılmıştır. Ayrıca, hücresel iskeletteki aktin filamentleri ile çekirdek görüntülemesi için sırasıyla phalloidin ve draq5 boyaması yapılmıştır. Yapılan analiz ve görüntülemeler sonucunda HEW içeren örnekler üzerinde ekili hücrelerin 14 gün boyunca canlılıklarını koruyarak çoğaldıkları; kontrol grubu olan sadece PCL'den oluşan örnekler üzerinde ekili hücre sayısında ise 7. günden sonra azalma olduğu gözlenmiştir.
Hücre tutunması ve çoğalması için doğru biyofiziksel özelliklere sahip doku iskelesi üretilmesi amacıyla sentetik polimerlerin doğal bileşenlerle karıştırılması, doku iskelelerinde istenen özelliklere ulaşmak için basit ve etkili bir yoldur. Bu amaç doğrultusunda yapılan bu tez çalışmasında sonuç olarak basit, hızlı ve düşük maliyetli bir imalat yöntemi ile PCL/HEW doku iskeleleri oluşturulmuş; biyobozunurluğu, yüzey yapısı ve mekanik özellikleri açısından yeterli olduğu, hücre tutunmasını ve çoğalmasını büyük ölçüde desteklediği anlaşılmıştır ve böylelikle çeşitli doku mühendisliği uygulamaları için uygulanabilirliği gösterilmiştir.

Abstract:
This thesis examines the use of hen egg white protein (HEW) and its effect on surface chemistry and its interaction with adipose-derived mesenchymal stem cells (ASCs) for the first time in the literature as an alternative to existing synthetic and natural composite polymeric scaffolds for use in tissue engineering applications.
For effective applications in tissue engineering requires a scaffold with superior properties in terms of biological and physical performances. For this reason, various manufacturing technologies and engineering methods are used together to obtain the desired properties in tissue scaffolds. Tissue scaffolds formed from synthetic polymers may exhibit good structural and mechanical properties, but may not be suitable for cell adhesion and tissue formation. In contrast, natural polymers can provide very good biological properties. However, natural polymers also do not exhibit good mechanical strength and stability in physiological environments. For this reason, the use of composite scaffolds made up of synthetic and natural components is becoming increasingly common.
In this thesis, composite nanofiber scaffold was formed by electrospinning from lyophilized HEW protein and synthetic and biodegradable polymer poly (-caprolactone) (PCL). The effect of bioactive natural component HEW protein on the surface properties of the nanofiber membrane was investigated and its cellular response to the ASCs was tested. These nanofiber tissue scaffolds were formed by electrospinning method because of the properties of electrospinning being one of the production methods of polymeric tissue scaffolds and the ability to form micro and nanoscale fibers that can mimic extracellular matrix fibers. Furthermore, the nanofiber membranes obtained have a high surface area and porosity and thus have great potential for use in tissue engineering.
PCL is a biodegradable and biocompatible synthetic polymer that is widely used in tissue engineering. However, its hydrophobicity presents a disadvantage in terms of cell attachment to the surface. To overcome these disadvantages, it has advantages to use PCL tissue scaffold together with bioactive materials to promote cell adhesion and proliferation. As for HEW is an inexpensive protein source, containing more than 100 types of soluble proteins with different molecular weights, isoelectric points, and concentrations, including ovalbumin, ovotransferrin, ovomuciod, ovamucin and lysosome at high concentrations. HEW proteins provide further benefits for human health, with their antihypertensive, antioxidant, antidiabetic and anticancer features. Therefore, HEW protein was added to the nanofiber PCL membrane structure. It was thought that the hydrophobicity of PCL, which has a disadvantage in terms of cell adhesion, could be reduced by changing the surface chemistry by adding protein. ASCs were seeded on the characterized scaffolds and cell viability analyzes were performed. The cells were isolated from fresh human subcutaneous adipose lipoaspirates.
The SDS-page analysis was performed on the lyophilized HEW protein obtained in the first step and bands of the main proteins in HEW (ovalbumin, ovotransferrin, ovomucoid, avidin, and lysozyme) content were observed. The total protein concentration was calculated to be about 5 mg/ml. Fiber morphology of the nanofiber scaffold was examined by scanning electron microscopy (SEM) and it was observed that smooth surfaces and without any bead formation were obtained. Also, HEW-containing fibers were found to be thinner (50-254nm) than neat PCL fibers (0.90-1.60μm). The distribution of HEW on nanofibers was demonstrated with a confocal laser scanning microscope after staining with antibody against ovalbumin. For this, the scaffolds were incubated with the primary rabbit anti-ovalbumin antibody and then incubated with secondary antibody, anti-mouse Alexa Fluor 488 so, homogeneous distribution of HEW was achieved throughout the nanofibrous mats. Fourier Transform Infrared Spectroscopy (FTIR-ATR) analysis of the nanofiber membrane confirmed the presence of both components in the produced scaffolds with bands associated with proteins as expected. Thermogravimetric differential thermal analysis (TGA) was performed to determine the thermal properties of the PCL/HEW membrane and also to determine the amount of PCL and HEW contained in the membrane. According to TGA results, decomposition of both components was observed and HEW protein content in composite nanofibers was found to be around 36%. To analyze the wettability of the fibers, their contact angles with water were measured. While the contact angle of PCL membranes known to be hydrophobic was observed to be constant at 90°, it was found that the contact angle of 70° initially measured on PCL/HEW membrane surfaces decreases with time and thus the protein causes water-absorbing matrix formation to membranes.
Human ASCs were used to determine the cell adhesion and proliferation behavior on the nanofibrous mats. The cells were isolated from fresh human subcutaneous adipose lipoaspirates. ASCs at passage 3 were suspended in culture medium and seeded onto scaffolds using a density of 25×103 cells/sample and then allowed to grow for 2 weeks at 37°C in a humidified atmosphere containing 5% CO2 with medium changes every 2–3 days. To determine the cell viability of ASCs that were seeded on tissue scaffolds during the cell culture stage, live/dead staining and Alamar blue staining were performed on days 2nd, 7th, and 14th. Live/dead staining were observed with confocal laser scanning microscopy. The morphology and cytoskeleton organization of the cells on the mats were analyzed under a confocal microscope at day 14 after staining of F-actin and nuclei with phalloidin and draq5, respectively. Neat PCL nanofiber mats were used as a control for all experiments. Following the observations of live/dead staining, more uniform cell spreading and distribution was observed on PCL/HEW mats throughout the culture. Cellular morphology was further examined by SEM at each time point. As a result of the analysis and imaging, it was observed that the cultivated cells proliferated for 14 days on HEW containing samples; the number of cultivated cells was decreased after 7th days on the control group PCL-only samples.
Mixing synthetic polymers with natural components to produce tissue scaffolds with the correct biophysical properties for cell attachment and proliferation is a simple and effective way to achieve the desired properties in tissue scaffolds. In this thesis, PCL/HEW scaffolds were formed with a simple, fast and low-cost manufacturing method. Moreover, having sufficient properties in terms of biodegradability, surface structure, mechanical properties and greatly supports cell adhesion and proliferation thus is applicable for various tissue engineering applications. The
addition of HEW to the structure has led to a decrease in fiber diameter, as well as significant changes in surface chemistry and wettability, which are beneficial for cells. Thus, the cell culture studies indicated that the PCL/HEW electrospun mats could support robust ASC attachment and viability more than neat PCL counterparts. Overall, results obtained from the present work indicate that PCL/HEW electrospun mats possess favorable morphological and biochemical properties to serve as an artificial matrix for stem cell growth and can be used as a scaffold for the regeneration of different tissues.