İpek fibroin kriyojellerinin sentezi ve mekanik özelliklerinin incelenmesi
Preparation of silk fibroin cryogels and investigation of their mechanical properties
Yazar: FATİH AK
Danışman: PROF. DR. OĞUZ OKAY
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Kimya = Chemistry ; Polimer Bilim ve Teknolojisi = Polymer Science and Technology
Dizin: Hidrojeller = Hydrogels ; Çapraz bağlama = Cross linking ; İpek fibroini = Silk fibroin

Özet:
Ġpek genellikle Bombyx mori adı verilen ipek böcekleri tarafından üretilen birbiyopolimerdir. Ġpek; fibroin ve serisin olarak isimlendirilen iki proteindenoluĢmakta olup ipek liflerinin yapısal proteini olan fibroini bir arada tutan serisinyüksek alkali çözeltilerde çözünür. Ġpek fibroini, baĢlıca glisin ve alanin amino asitünitelerinden oluĢan büyük hidrofobik bloklar ile bunların aralarında ve zinciruçlarında daha küçük hidrofilik bloklardan (arjinin ve rizin üniteleri) ibaret çokbloklubir kopolimer mimarisine sahiptir. Hidrofilik bloklar suda çözünürlüğüsağlarken, hidrofobik bloklar arası asosiyasyonlar, fibroinin rastgele yumakyapısından β-tabaka yapısına bir konformasyon geçiĢine neden olur. Ġpek fibroininyapısındaki β-tabakaları malzemeye dayanıklılık ve sertlik kazandırırken, dahadüzensiz olan hidrofilik bloklar tokluğu ve elastisiteyi arttırır. Ġpek fibroindejelleĢmenin fibroin proteininin ikincil yapısındaki konformasyonel geçiĢlerlemeydana geldiği bilinmektedir. Çapraz bağlayıcı ile ipek fibroindekikonformasyonel geçiĢler kısa sürede gerçekleĢerek jel elde edilebileceği son yıllardagrubumuzda yapılan çalıĢmalar sonucunda ortaya konmuĢtur. Fibroin molekülleri,birbirlerine 1,4 bütandioldiglisidileter (BDDE) çapraz bağlacısıyla bağlandıkça,moleküllerin hareketliliği azalır. Bunun bir sonucu olarak, moleküller arasıhidrofobik etkileĢmeler kuvvetlenerek -tabaka yapısının çekirdeklenme ve büyümesüreci kolaylaĢır. Böylece kısa süre içerisinde jelleĢme meydana gelir.Bu çalıĢmanın amacı mekanik olarak dayanıklı, dıĢarıdan gelen uyarılara hızlı cevapverebilen kemik hücre mühendisliğinde kullanıma uygun makrogözenekli ipekfibroin iskeletlerinin sentezlenmesidir. Bunun için literatürde ilk defa olarak fibroinmoleküllerinin kriyojelleĢme yani düĢük sıcaklık jelleĢme yöntemi uygulanarak-18oC sıcaklıkta donmuĢ sulu çözeltilerinde çapraz bağlanması sağlanmıĢtır.KriyojelleĢme tekniğinde; jelleĢme reaksiyonları, reaksiyon sisteminin donmanoktasının altında ilerlediği için ortamdaki çözücü kristalleri kalıp etkisi yaparakgözenekli bir yapının oluĢmasını sağlamıĢ ve böylece makrogözenekli fibroiniskeletleri elde edilebilmiĢtir. KarĢılaĢtırma amacıyla +50oC' de fibroin hidrojelleride sentezlenmiĢtir. Sentezler suda çözünmüĢ fibroin molekülerinin 1,4bütandioldiglisidileter çapraz bağlayıcısı ile N,N,N',N' tetrametiletilendiamin(TEMED) katalizörlüğünde -18oC ve +50oC'de gerçekleĢtirilmiĢitir. JelleĢmereaksiyonları sırasında fibroin molekülleri üzerindeki fonksiyonel grupların BDDEile çapraz bağlanma reaksiyonu fotometrik yöntemlerle izlenmiĢtir. Elde edilenjellerin ĢiĢme davranıĢları, mekanik ve morfolojik özellikleri incelenmiĢtir.

Abstract:
Silk fibroin derived from Bombyx mori is a fibrous protein exhibiting extraordinarymaterial properties such as good biocompatibility, biodegradability, high strengthand toughness, and ease of processability. Silk fibroin has been used for cell culture,wound dressing, drug delivery, enzyme immobilization, and as a scaffold for bonetissue engineering. Silk fibroin has a blocky structure consisting of less orderedhydrophilic and crystallizable hydrophobic blocks. Hydrophilic blocks providesolubility in water and, are responsible for fibroin elasticity and toughness, whilehydrophobic blocks form intermolecular -sheet structures leading to the insolubilityand high strength of fibroin. Several techniques have been developed to produceporous fibroin scaffolds such as freeze-thawing, porogen leaching, gas foaming,electrospinning, and freeze-drying. It was shown that porogen leaching and gasfoaming techniques produce scaffolds having larger pores (100-200 m) ascompared to the scaffolds formed by freeze-drying (10-50 m). The compressivemoduli of the scaffolds vary depending on the preparation conditions between 10 kPaand 3 MPa.An alternative simple route to produce 3D highly porous fibroin networks is the lowtemperature gelation technique, known as cryotropic gelation or cryogelation. Sincethe pioneering works of Lozinsky and coworkers, cryogelation technique has beenwidely used to produce macroporous gels (cryogels) of high toughness and superfastresponsivity. By this technique, polymerization and/or cross-linking reactions areconducted in apparently frozen reaction solutions. During freezing of an aqueouspolymer solution containing a chemical cross-linker, the polymer chains and thecross-linker molecules expelled from the ice concentrate within the liquid channelsbetween the ice crystals, so that the cross-linking reactions only proceed in theseunfrozen domains. After cross-linking and, after thawing of ice, a macroporousmaterial is produced whose microstructure is a negative replica of the ice formed. Incontrast to the mechanically weak macroporous gels prepared by phase separationtechnique, cryogels are very tough and withstand very large strains withoutpermanent deformation or fracture.The aim of the present study is the preparation of silk fibroin cryogels with tunableproperties starting from frozen fibroin solutions in the presence of 1,4-butanedioldiglycidyl ether (BDDE) as a crosslinker. BDDE contains epoxide groups on bothends that can react with nucleophiles, including amino groups, sulfhydryls, andhydroxyls. Recently, our research group has shown that the introduction of ethyleneglycol diglycidyl ether (EGDE) cross-links between the fibroin molecules decreasesthe mobility of the chains, which triggers the conformational transition from randomcoilto -sheet structure and hence fibroin gelation. Gelation reactions conducted at+50oC showed formation of strong to weak fibroin hydrogels depending on the pH of the solutions, which was adjusted by the addition of N,N,N',N'-tetramethylethylenediamine (TEMED).Here, we conducted the gelation reactions of fibroin in frozen solutions -18oC. Thereactions were first carried out at a fibroin concentration of 4.2 w/v % in the presenceof 20 mmol/g epoxide while the amount of TEMED was varied. Gelation at -18oCresults in cryogels with a gel fraction Wg = 1 over the whole range of TEMEDinvestigated. Both the swelling ratio and the modulus of the cryogels do not changemuch with the TEMED content and they remain at 16 and 53 kPa, respectively. Thisis in contrast to the fibroin hydrogels where the modulus decreases while theswelling ratio increases with rising amount of TEMED and, no gel forms at or above0.25 % TEMED.The reaction between BDDE and fibroin was assessed by the ATR-FTIR spectra offreeze-dried fibroin samples. In addition to a shift of the Amide I absorption band tolower wavenumbers, new bands at 1040 - 1100 cm-1appear upon gelation, whichwere assigned to the ether stretching bands of BDDE cross-linkages. Amide I bandregion of the spectra presenting the carbonyl stretching vibration of amide groups onsilk fibroin is characterized by a peak at 1640 cm-1 indicating the presence ofprimarily random coil and/or -helix conformations. After cryogelation, all samplesdisplay a main peak at 1620 cm-1 which was assigned to -sheet conformation. Inaddition to the main peak, shoulders at 1660 and 1698 cm-1 appear after gelation,which can be assigned to -helix and -turn conformations, respectively. Thisindicates the occurrence of a conformational transition from random coil to -sheetstructure in frozen fibroin solutions. A further evidence for the -sheet formationcomes from the X-ray profiles of freeze-dried cryogels. Silk fibroin before gelationexhibits a broad peak at around 22o indicating an amorphous structure. Aftergelation, all the cryogels show a distinct peak at 20.9o and two minor peaks at 9.8and 24.5o. These are the characteristic peaks of the -sheet crystalline structure ofsilk fibroin corresponding to -crystalline spacing distances of 4.3, 9.0, and 3.6 Aorespectively. To estimate the conformation of the fibroin network chains, peakseparation of Amide I band was carried out after base line correction by selecting aGaussian model for curve fitting. The peak positions were fixed at 1620, 1640,1660, and 1698 cm-1 representing -sheet, random coil, -helix, and -turnconformations, respectively. The results of -sheet contents show that fibroin chainsbefore gelation have 12 ± 2 % -sheet structures, while their contribution increasesto 33 ± 2 %, independent on the amount of TEMED.Mechanical tests showed that the fibroin hydrogels formed at 50oC fractured underlow deformation indicating that the mechanical stress applied is localized withouteffective dissipation. However, fibroin cryogel remained mechanically stable up tocomplete compression. Important point is that, as the cryogel is squeezed under thepiston or, via manual hand compression, the gel releases all its water so that it cancompletely be compressed. After the release of the load, the gel sample immediatelyrecovers its original shape by absorbing the released water. Successive compressiontests conducted on the same gel sample between 0 and 99.8 % strain showedreversibility of the stress-strain curves of the cryogels revealing that no crack occursduring the experiments. The results indicate that fibroin cryogels prepared as beadsor high flow-path monolith columns can be used in separation processes in which theseparated compounds such as particulate matter or proteins deposited in the internalpore surface can easily be recovered by compression of the gels under a piston. xxiiiMacroporous fibroin scaffolds were obtained by freeze-drying of the cryogels andhydrogels under identical conditions. The hydrogel samples were transparent (> 0.10% TEMED) or translucent, indicating the existence of scattering centers for light,while all the cryogels had an opaque white color. After freeze-drying, scaffoldsderived from the cryogels retained their original shape while a lateral distortion in thecylindrical shape of the hydrogel scaffolds was observed. SEM images of thesesamples after freeze-drying also show that the cylindrical shape of the hydrogelscaffold is partially destroyed due to the weak pore structure. In contrast, cryogelscaffolds were mechanically stable and consisted of regular pores of sizes 101which are typical for macroporous networks created by the cryogelation technique.The pore size of the cryogel scaffold could be regulated depending on thecryogelation conditions. Increasing fibroin concentration CSF led to scaffolds withthicker pore walls but a smaller pore size. For instance, the pore diameter decreasedfrom 33 to 10 m as CSF increased from 4.2 % to 12.6 %. The inverse relationbetween CSF and the pore size is possibly related to the higher amount of unfrozenmicrodomains during gelation as the amount of fibroin is increased.Mechanical properties of the scaffolds were investigated by uniaxial compressiontests. Three different regimes were observed in the stress-strain curve of the cryogelscaffold. First, the curve is quite linear indicating that the porous structure remainsmechanically stable in this range of strain. This linear elastic regime is followed by anear-plateau regime indicating that the network easily deforms due to the collapse ofits pores under the pressure. The critical stress corresponding to the plateau regime,denoted by p, is thus a measure of the mechanical stability of the porous structure.Finally, the steep increase of the curve in the third regime corresponds to thecompression of the nearly non-porous fibroin network. Although freeze-driedhydrogels are also porous, no distinct plateau was observed in stress-strain curves,which is attributed to the weak pore structure and irregularity of the porous structure.Inspection of stress-strain curves of cryogel scaffolds revealed that the critical stressp increases with decreasing size of the pores, that is, with increasing fibroin contentCSF . The results thus suggest increasing mechanical stability of the porous structureof fibroin scaffolds with decreasing size of the pores. Indeed, the compressivemodulus and the compressive stress measurements of the scaffolds support thisfinding. The cryogel scaffolds formed at CSF = 4.2 % exhibit a compressive modulusE of 8 MPa over the whole range of TEMED with a compressive nominal stresscomp of 0.224 MPa. These values are about 1 order of magnitude higher than thoseof the hydrogel scaffolds formed below 0.25 % TEMED (E = 1.0 MPa, comp = 0.030MPa). The most significant variations in the modulus and strength of the cryogelnetworks were observed by changing fibroin concentration CSF at the gel preparation.E increases from 7 to 48 MPa while comp increases from 0.2 to 1 MPa as CSF isincreased from 4.2 to 12.6 %. The extraordinary strength of the cryogel scaffoldsoriginates from the high fibroin concentration of the pore walls; gelation in frozensolutions confines fibroin in a small region of the reaction volume forming the porewalls of the final material. This also provides a high degree of toughness to cryogelsin their swollen states.