The purpose of this study is to understand the thermally induced phase in poly(lactic acid) (PLA)/dialkyl phthalate system, control the final morphology, and subsequently fabricate the PLA scaffold suitable for cell culture. Both poly(DL-lactic acid) (PDLLA) and poly(L-lactic acid) (PLLA), which are stereoisomers of PLA, were used and a series of dialkyl phthalate, in which the length of alkyl chain was changed from the methyl to hexyl, was employed to control the interaction between the polymer and solvent. Thermally induced phase separation was adopted as fabrication method because the morphology could be tailored by adjustment of the process parameter such as polymer concentration, quenching temperature and quenching method.
To control the final morphology, it is essential to understand the phase separation behavior. For this reason, we investigated the relationship between the liquid-liquid phase separation and crystallization in PLA/dialkyl phthalate system through phase diagram in chapter 2. Experimental phase diagrams, which were composed of the liquid-liquid phase separation temperature and crystallization temperature, for poly(lactic acid)/dialkyl phthalate systems were constructed. From the phase diagram, it is confirmed that when the molecular weight of PLLA is increased, the liquid-liquid phase separation occurs at a higher temperature and the crystallization temperature remains almost constant. And as the number of carbon atoms of alkyl chain in the phthalate was decreased, liquid-liquid phase separation temperature decreased considerably while crystallization temperature was not significantly influenced. The final morphology of PLLA solutions was investigated by changing the variables of the TIPS process. The pore size increased when quenching temperature and solvent quality were decreased, which allow liquid-liquid phase separation to progress further before crystallization. When the polymer concentration was low and the PLLA molecular weight was decreased, the rate of liquid-liquid phase separation is increased because of low viscosity and ease of diffusion, so large pores were obtained. The domain size increased with longer coarsening time and higher coarsening temperature.
Some of thermodynamic parameters for PLA/dialkyl phthalate systems have been investigated in chapter 3. Theta temperatures of PLA/dialkyl phthalate system were determined and subsequently the second virial coefficient and the interaction parameter were evaluated. In a given solvent, theta temperature of PDLLA solution was slightly higher than that of PLLA solution. As the number of carbon atoms of alkyl chain in the phthalate was increased, the theta temperature increased systematically about 40~50℃ for one additional carbon atom. Light scattering measurements showed the temperature and solvent dependence of interaction parameter and radius of gyration. Interaction parameter decreased with shorter alkyl chain in the phthalate and at higher temperature. It was confirmed that the solvent quality in PLA solution is improved when the dialkyl phthalate has a shorter alkyl chain. Radius of gyration expanded in the better solvent and shrank at higher temperature. Interaction parameter of each PDLLA/dialkyl phthalate system was changed systematically according to the length of alkyl chain of phthalate. On the basis of the temperature-dependent interaction parameter, phase diagrams were calculated using Flory-Huggins theory and agreed well with the observed values.
In chapter 4, we focused on the intrinsic viscosities of PDLLA solution in dialkyl phthalate. The unperturbed dimensions, end-to-end distances of the PDLLA in a series of dialkyl phthalate could be deduced from the temperature-dependent intrinsic viscosity data by the extrapolation methods such as Kurata-Stockmayer-Fixman, Berry and Inagaki-Suzuki-Kurata equations. The intrinsic viscosity of PDLLA solution increased with the solvent having shorter alkyl chain. The higher molecular weight of the polymer and lower temperature induced the increment in intrinsic viscosity. Temperature increase caused the shrinkage of the unperturbed chain dimension while the solvent effect on unperturbed chain dimension was negligible. In contrast, the actual chain dimension increased when the temperature and length of alkyl chain in solvent was decreased. When considering favorable polymer-solvent interaction at higher temperature, these results demonstrated that the effect of the polymer conformation was dominant over the effect of interaction between the polymer and solvent.
When PLLA is employed to the scaffold material, it has some disadvantages. Its hydrophobic property inhibits the cells from thoroughly penetrating into the scaffold. Moreover it has no functional sites that could induce specific interaction with the cells and subsequent cell attachment. To overcome these weak points, the surface of PLLA was modified by coating poly-[N-p-vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide] (PVLA), which was employed to improve the hepatocyte adhesion because of its amphiphilic property and the presence of hepatocyte recognition part. We characterized the surface properties through water contact angle, electron spectroscopy for chemical analysis (ESCA) and scanning probe microscopy (SPM). The effect of PVLA coating on the efficiency of hepatocyte adhesion was evaluated by protein assay and optical microscopy. The surface morphology was influenced by the concentration of PVLA coating solution and it played a critical role in hepatocyte adhesion. It was confirmed that enhanced hepatocyte adhesion on the PVLA-coated surface was attributed to both of the improved hydrophilicity and the galactose moieties in PVLA, which could bind to the asiologlycoprotein receptor (ASGPR) on the hepatocyte.
본 연구의 목적은 폴리락틱산/디알킬프탈레이트 계에서의 열유도상분리 현상을 이해함으로써 몰폴로지를 조절하여 세포 배양에 적합한 지지체를 제조하는 것이다. 폴리락틱산의 광학이성질체인 DL-폴리락틱산 (PDLLA)과 L-폴리락틱산 (PLLA)를 모두 사용하였으며, 용매로는 디알킬프탈레이트를 사용하였다. 디알틸프탈레이트의 알킬 사슬의 길이에 따라 디메틸프탈레이트에서 디헥실프탈레이트까지 총 여섯 가지의 용매를 사용하였다. 열유도상분리 방법은 조작 변수를 제어함으로써 다양한 몰폴로지를 얻을 수 있다는 장점이 있기 때문에 본 연구에서 세포지지체의 제조 방법으로 채택하였다.
우선 주어진 계에서의 열유도상분리를 이해하기 위하여 상도를 작성하였다. 디알킬프탈레이트 내의 알킬 사슬이 짧아지고 고분자의 분자량이 작을수록, 액체-액체 상분리 (liquid-liquid phase separation) 온도는 급격히 감소하는 반면 결정화 (crystallization) 온도는 거의 영향을 받지 않았다. 상도를 통해 디알킬프탈레이트의 알킬 사슬의 길이가 짧아질수록 용매 세기가 증가함을 정성적으로 확인할 수 있었다. 몰포로지를 관찰한 결과 용매 세기가 감소하거나 급냉온도가 낮을수록 기공의 크기가 커지고, 고분자의 분자량이 감소하거나 용액의 농도가 감소함에 따라 기공의 크기가 증가함을 확인하였다. PDLLA 용액에 대해 세타 온도 (theta temperature) 와 상호작용인자 (interaction parameter) 를 측정하였다. PDLLA의 세타 온도가 PLLA의 세타 온도보다 약간 높은 것으로 나타났다. 상호작용인자는 용매 내 알킬 사슬의 길이가 짧을수록, 온도가 높을수록 높은 값을 나타냈다. 고유점도 (intrinsic viscosity )를 측정하여 계산한 사슬의 무동요 상태의 길이 (unperturbed dimension) 는 용매 세기와는 무관하고 온도가 올라감에 따라 감소하였다. 본 연구에서는 PLLA 표면에 양친성 물질인 poly-[N-p-vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide] (PVLA)를 코팅하여 간세포의 점착을 살펴보았다. 표면 분석을 통해 PVLA가 코팅한 PLLA 표면의 몰폴로지가 간세포 점착에 큰 영향을 미치는 것을 확인할 수 있었으며, PVLA 내의 galactose 부분이 간세포와 상호작용하여 간세포 점착이 향상됨을 확인하였다.
