Dondurarak kurutma ve çözücü döküm-parçacık uzaklaştırma tekniği ile PCL bazlı doku iskelesinin karakterizasyonu
Characterization of PCL-based tissue scaffold using freeze drying and solvent casting-particulate leaching
Yazar: CANAN DURUKAN GÜL
Danışman: PROF. DR. MEDİHA İPEK
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyomedikal Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Biyomühendislik = Bioengineering ; Mühendislik Bilimleri = Engineering Sciences ; Polimer Bilim ve Teknolojisi = Polymer Science and Technology

Özet:
Doku hasarı ve doku kaybı milyonlarca insanın yaşadığı sorunlardan biridir. Yaşanılan doku hasarını ve doku kaybını onarma ve yenileme potansiyeline sahip iki farklı yöntem kullanılarak bir doku iskelesinde olması gereken optimum özelliklere sahip iki farklı doku iskelesi geliştirilmesi öngörülmüştür. İlk yöntem olan dondurarak kurutma ile üretilen doku iskelelerinin istenilen iskele özellikleri sağlaması ve çift katmanlı olması amaçlanmıştır. İkinci yöntem olan çözücü döküm ve parçacık uzaklaştırma yöntemi ile üretilen doku iskelelerinin ise istenilen iskele özellikleri sağlaması ve ilaç salınımı yapılması amaçlanmıştır. Doku hasarını ve doku kaybını iyileştirmek için biyolojik olarak parçalanabilen doku iskeleleri üretilmesi amaçlanmıştır. İskelelerin üretiminde ana malzeme olarak polikaprolakton (PCL) tercih edilmiştir. PCL doku oluşumu için biyouyumlu ve biyobozunur bir polimerdir. Dondurarak kurutma yönteminde PCL'nin yapısına destek olabilmesi için Kitosan (CHI) yardımcı polimer olarak tercih edilmiştir. Oluşan bu yapıya destek sağlamak ve çift katmanlı bir yapı oluşturması için farklı oranlarda karbon nanotüp (CNT) eklenmiştir. Çözücü olarak asetik asit (AA) tercih edilmiştir. Bu biyomalzemeler kullanılarak dört farklı çözelti hazırlanmıştır. Çözeltilere dondurarak kurutma (liyofilizasyon) yöntemi uygulanmıştır. Hazırlanan çözeltiler -20°C'de dondurulmuştur. Donmuş çözeltiler daha sonra liyofilizasyon işlemi için basıncın vakum pompasıyla düşürüldüğü liyofilizatöre yerleştirilmiştir ve -60°C'de on gün bekletilmiştir. Liyofilizatörden çıkan numuneler iki gün kurumaya bırakılmış ve kuruduktan sonra çift katmanlı doku iskelelerinin üretimi tamamlanmıştır. Üretilen çift katmanlı doku iskelelerinin gözenek yapılarını incelemek için SEM, organik ve inorganik bileşikleri karakterize etmek için FTIR, iskelelerin kimyasal yapılarını doğrulamak için XRD, iskele yüzeylerinin ıslanabilirliğinin kontrolü için temas açısı ve serbest yüzey enerjisi analizleri incelenmiştir. SEM ile yüzeylerin gözenekliliği belirlenmiştir. FTIR analizi ile malzemeler için karakteristik pikler görülmüştür. XRD ile PCL karakterize edilmiştir, temas açısı ve serbest yüzey enerjisi analizi ile iskelenin ıslanabilirliği ölçülmüştür. Temas açısı ölçümü ile iskelenin hidrofilik olduğu tespit edilmiştir. Literatür taramalarında doku iskeleleri dokuya yapışması için en az 40 mN/m yüzey enerjisine sahip olması gerektiği tespit edilmiştir. Serbest yüzey enerjileri 40 mN/m'nin üzerindedir. Analiz sonucunda başarılı bir şekilde çift katmanlı bir doku üretildiği tespit edilmiştir. İkinci çalışmada ise PCL'ye ek olarak Polietilen Glikol (PEG) kullanılmıştır ve çözeltiye farklı oranlarda Polilaktik Asit (PLA) eklenmiştir. Çözücü olarak bu polimerlerin ortak çözücüsü olan kloroform tercih edilmiştir. Farklı oranlarda polimerler ile dört farklı çözelti hazırlanmıştır. Hazırlanan bu çözeltilere çözücü döküm ve partikül uzaklaştırma yöntemi uygulanmıştır. Çözeltilere toplam 24  
g tuz (NaCl) eklenmiş ve manyetik karıştırıcıda 15 dakika karıştırılmıştır. Çözeltiler, 24 saat boyunca bir çeker ocak içinde serbest bırakılmıştır. Daha sonra çözeltilere distile su ilave edilerek 60 saat suda bekletilmiştir. Çözeltilerin suda bekletilmesi NaCl ve PEG'i çözmek için tasarlanmıştır. Daha sonra sudan alınan numuneler iki gün boyunca havada kurutulmuştur ve dört farklı doku iskelesi üretimi tamamlanmıştır. Üretilen doku iskelelerinin gözenek yapılarını incelemek için SEM, organik ve inorganik bileşikleri karakterize etmek için FTIR, su tutma kapasiteleri için şişme analizi, iskele yüzeylerinin ıslanabilirliğinin kontrolü için temas açısı ve serbest yüzey enerjisi ve ilaç taşınmasını gözlemlemek için ilaç salınım analizi yapılmıştır. SEM analizinde iskelenin gözenekleri nanometre ve mikrometre boyutlarındadır. İskelelerdeki gözeneklilik NaCl porojenleri ve PEG ile sağlanmıştır ancak SEM görüntüleri incelendiğinde porojenlerin tamamının iskelelerden uzaklaştırılamadığı görülmüştür. FTIR spektrumunda bileşenlerin tüm karakteristik pikler görüldüğü gözlemlenmiştir. PCL oranı en yüksek doku iskelesinde en az su emme gözlenmiştir. Yapılan ölçümlerde 60. dakikadan sonra şişme oranında azalma meydana geldiği tespit edilmiştir. En iyi temas açısına sahip doku iskelesi ise %14PCL/PEG içeren doku iskelesidir. Literatür taramalarında doku iskeleleri dokuya yapışması için en az 40 mN/m yüzey enerjisine sahip olması gerektiği tespit edilmiştir. Dört doku iskelesi içinde ölçülen değer 40 mN/m'ye yakın değerler elde edilmiştir. Yapılan ilaç salınımı analizi sonucu çizilen doku iskelelerinin %DOX salınımı grafiğine bakıldığı zaman ilacın 1. saatten itibaren dört numune içinde salınım yaptığını 720. saatte %100'e ulaştığı gözlemlenmiştir. Doku iskelesinde kullanılan iki farklı yönteme bakıldığında dondurarak kurutma yöntemi ile üretilen iskele yapısının istenilen iskele yapısına daha yakın olduğu tespit edilmiştir. Çözücü döküm ve parçacık uzaklaştırma yönteminde PCL'nin yanında PLA'nın kullanılması üretilecek iskelenin hidrofobik özelliğini arttırmıştır. Bu tez çalışmasında hem malzeme açısından hem de yöntem açısından dondurarak kurutma yöntemi ile istenilen amaca en uygun doku iskelesi üretimi sağlanmıştır.

Abstract:
Tissue damage and tissue loss are problems experienced by millions of people. Damaged tissues, it can be regenerated using donor graft tissues such as autografts, allografts, and xenografts. However, clinical applications for this renewal process are limited because the number of donors is insufficient. Tissue engineering is an interdisciplinary field that applies engineering principles aimed at repairing and remodeling lost or damaged tissues. Intensive research is being carried out to develop tissue scaffolds that can perform the functions of tissues and organs with the tissue engineering approach. The desired properties of the tissue scaffold are that it is biocompatible, has a biodegradable and porous structure, and has mechanical strength. The scaffold must have appropriate surface chemistry to ensure biocompatibility. Biodegradable scaffolds replace the implanted scaffold with the body's own cells over time. For this reason, the scaffold must be biodegradable, allowing cells to propagate their own extracellular matrix. The porous structure is It helps in cell formation, tissue renewal and tissue growth, and helps in maintaining mechanical stability over a period. The tissue scaffold must have sufficient mechanical strength during implantation and must not lose this property after implantation. In tissue scaffolds, it is very difficult to create two different features in a single scaffold at the same time. For this reason, in tissue engineering, they can create scaffolds with two or more tissue layers to recruit different cell types and restore their functions. Multilayer tissue scaffold helps repair multiple damages occurring in the scaffold. A tunable drug release mechanism in the scaffold may further facilitate new tissue formation and repair of damaged tissue. Drug carriers release a therapeutic drug onto the scaffold over a controlled and prolonged period by targeting infected tissue and cells, adjusting the dose required to cure the patient. The materials used for tissue scaffold production are called biomaterials. Biomaterials; metals, ceramics, polymers, and composites. Processing and shaping ceramic and metal materials is difficult. Tissue scaffolds of specific geometries can be produced by processing at high temperatures using ceramic and metal materials. Polymer materials are easier to process than metal and ceramic materials. Polymer materials are mostly used in tissue scaffold production. Polymer materials are divided into two: natural and synthetic. The processability feature of synthetic polymers allows the porosity and degradation properties of the tissue scaffold to be produced to be controlled. In the human body, three-dimensional structures are organized by cells and tissues. Tissue scaffolds are produced using different methods to design tissues and aid cell distribution. The first of these methods is the freeze-drying method. In this method, the polymer is dissolved in a suitable solvent and then the solution is frozen by bringing it to a temperature below the freezing point of the entire solution. It forms a network structure with polymer molecules among the ice crystals formed in the solution. The frozen solutions are then placed in the lyophilizer for the lyophilization process, where the pressure is reduced by a vacuum pump. In this way, porous scaffolds are obtained. Thus, many interconnected porous structures are formed. The second method is solvent casting and particle leaching method.Tissue scaffolds whose porosity properties and pore size can be controlled are produced by solvent casting and particle leaching method. This method does not require high temperatures and special equipment. The polymers to be used in this method are dissolved in the appropriate solvent. Then, particles that dissolve in water and have a pore-forming effect, such as salt and sugar, are added to the solution and placed in a container. In this solution taken into the container, the solvent is removed by evaporation. After the solvent is removed, the particles that have a pore-forming effect are removed from the structure with the help of water. In this way, a porous tissue scaffold is formed by solvent casting and particle leaching. Based on this, two different tissue scaffolds with the optimum properties that a tissue scaffold should have should be developed by using two different methods that have the potential to repair and regenerate the tissue damage and tissue loss experienced in this study. The tissue scaffolds produced by freeze drying, which is the first method, are intended to provide the desired scaffolding properties and be double layered. The tissue scaffolds produced by the second method, solvent casting, and particle leaching, are intended to provide the desired scaffolding properties and release drugs. In both methods, polycaprolactone (PCL) was preferred as the main material in scaffold production. PCL is a biocompatible and biodegradable polymer for tissue formation. Chitosan (CHI) was preferred as the copolymer to support the structure of PCL in the freeze-drying method. Different amounts of carbon nanotubes (CNT) were added to this composite structure to provide support and create a double-layer structure. Acetic acid (AA) was preferred as the solvent. 5% (w/v) PCL and 1% (w/v) CHI were used in all four different mixtures. CNTs were added as 0% (w/v), 0.005% (w/v), 0.01% (w/v) and 0.02% (w/v), respectively. Four different 20 mL solutions were prepared by pouring PCL, CHI into four different AA solvents. Freeze drying (lyophilization) method was applied to the solutions. The prepared solutions were frozen at -20°C. The frozen solutions were then placed in the lyophilizer, where the pressure was reduced by a vacuum pump for the lyophilization process. The solutions were kept in the lyophilizer at -60 °C for ten days. The samples coming out of the lyophilizer were left to dry for two days, and after drying, double-layered tissue scaffolds were produced. SEM was examined to examine the pore structures of these produced double-layer tissue scaffolds, FTIR to characterize organic and inorganic compounds, XRD to verify the chemical structures of the scaffolds, and contact angle and free surface energy analyzes to control the wettability of the scaffold surfaces. The porosity of the surfaces was determined by SEM. Characteristic peaks were observed for the materials with FTIR analysis. PCL was characterized by XRD, and the wettability of the scaffold was measured by contact angle and free surface energy analysis. By contact angle measurement, it was determined that the scaffold was hydrophilic. In literature reviews, it has been determined that tissue scaffolds must have a surface energy of at least 40 mN/m to adhere to the tissue. Their free surface energy is over 40 mN/m. As a result of the analysis, it was determined that a double-layer tissue was successfully produced. In the second study, the solvent casting particle leaching method, Polyethylene Glycol (PEG) was used in addition to PCL and Polylactic Acid (PLA) was added to the solution at different rates. PLA was preferred because it has a biocompatible hydrophobic polyester structure. PEG is a hydrophilic, uncharged, non-volatile, and colorless polymer. Since PCL-PEG performed better than PCL in studies, PEG polymer was added to the structure of PCL polymer. Chloroform, the common solvent of these polymers, was preferred as the solvent. Four different solutions were created using PCL, PLA, and PEG. The PEG ratio was kept constant, and the amount of PLA and PCL was changed proportionally. The first solution was prepared using 14% (w/v) PCL, 14% (w/v) PEG, and 72% (w/v) CHCL3. This mixture was allowed to dissolve in the magnetic stirrer. After half an hour, a homogeneous mixture was obtained and completely dissolved. The second mixture was prepared using 13.3% (w/v) PCL, 0.7% (w/v) PLA, 14% (w/v) PEG, and 72% (w/v) CHCL3. The mixture was stirred in a magnetic stirrer for 2-3 hours and a homogeneous mixture was obtained. The third mixture was prepared using 12.6% (w/v) PCL, 1.4% (w/v) PLA, 14% (w/v) PEG, and 72% (w/v) CHCL3. The mixture was stirred in a magnetic stirrer for 2-3 hours and a homogeneous mixture was obtained. The fourth sample was prepared using 11.9% (w/v) PCL, 2.1% (w/v) PLA, 14% (w/v) PEG, and 72% (w/v) CHCL3. The mixture was stirred in a magnetic stirrer for 2-3 hours and a homogeneous mixture was obtained. Casting and particle leaching methods were applied to these prepared solutions. A total of 24 g of salt was added to the solutions and mixed for 15 minutes in a magnetic stirrer. The solutions were released in a fume hood for 24 hours. Then, distilled water was added to the solutions, and they were kept in water for 60 hours. The water of the solutions was changed every 12 hours. Soaking the solutions in water is designed to dissolve NaCl and PEG. Then, the samples taken from the water were dried in air for 2 days and four different tissue scaffolds were produced. SEM was performed to examine the pore structures of the produced tissue scaffolds, FTIR to characterize organic and inorganic compounds, swelling analysis for water retention capacities, contact angle and free surface energy to control the wettability of the scaffold surfaces, and drug release analysis to observe drug transport. Porosity in the scaffolds was provided by sodium chloride porogens and PEG, but when SEM images were examined, it was seen that not all porogens could be removed from the scaffolds. It was observed that all characteristic peaks of the components were seen in the FTIR spectrum. In the swelling test, the scaffold with the highest swelling ratio was the scaffold containing 11.9% PCL/2.1%PLA/PEG, and the swelling ratio increased with the increase in polylactic acid ratio. The scaffold with the best contact angle is the scaffold containing 14% PCL/PEG. Studies have determined that tissue scaffolds must have a surface energy of at least 40 mN/m to adhere to the tissue. The calculated free surface energy value of the produced tissue scaffolds is close to this value, with 35.1-38.8 mN/m. When the %DOX release graph of the tissue scaffolds drawn because of the drug release analysis was looked at, it was observed that the drug was released in four samples starting from the 1st hour and reached 100% at the 720th hour. When we look at the two different methods used in tissue scaffolding, it has been determined that the scaffold structure produced by the freeze-drying method is closer to the desired scaffold structure. The use of PLA alongside PCL in the solvent casting and particle removal method increased the hydrophobic properties of the scaffold to be produced. In this thesis study, the most suitable tissue scaffold production for the desired purpose was achieved by the freeze-drying method, both in terms of material and method.