聚麩胺酸及幾丁聚醣複合生醫基材之製程探討、性質改良及制放應用
γ-poly(glutamic acid)/chitosan composite tissue engineering scaffolds – investigation of fabrication process, enhancement of cytocompatibility and application in controlled release
Date Issued
2005
Date
2005
Author(s)
Hsieh, Chien-Yang
DOI
en-US
Abstract
Tissue engineering provides a new way to recover physiological function by seeding cells onto scaffolds, together with the use of signaling molecules, to modulate cell growth. Hence, the cooperation between scaffolds, cells, and signaling molecules is becoming the major theme in tissue engineering. The scaffolds themselves must be biocompatible. Further, in many cases in which signaling molecules play an important role, a satisfactory release profile for signaling molecules is required. Thus, the biocompatibility of scaffolds and the release profile of signaling molecules are two important factors which should be considered.
To establish a tissue-engineered prosthesis, the first task is to fabricate suitable scaffolds. In this research, we attempted to fabricate various chitosan-based porous scaffolds using a newly developed “freeze-gelation” method. This method saves time and energy, and there is less residual solvent. For porous scaffolds, the mechanical properties are very important characteristics. The fabrication processes and cross-linking treatments significantly affect these characteristics. In Chapter 3, we investigate the influence of three process variables (freezing temperature, concentration of acetic acid, and ethanol concentration in the rinse buffer) on the freeze-gelation method and also the effect of adding the cross-linking reagents of glutaraldehyde (GTA), N-(3-dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride (EDC), and tripolyphosphate (TPP). For the three process variables, both the tensile strength and the elongation at tensile strength of the porous chitosan scaffolds increased with the freezing temperature and the concentration of acetic acid, whereas increasing the ethanol concentration in the rinse buffer only slightly increased the tensile strength but decreased the elongation. For cross-linking reagents, the data showed that both the tensile strength and elongation increased with the addition of GTA, while the effects of EDC and TPP were not obvious. Further, we successfully established a correlation to describe and predict the mechanical properties of scaffolds within our operating ranges of these three process variables.
After obtaining porous chitosan scaffolds, we tried to enhance their cytocompatibility. Although chitosan is non-toxic and biodegradable, it is not compatible with certain types of cells. In Chapter 4, we chose γ-poly(glutamic acid) (γ-PGA), a hydrophilic and biodegradable polymer, and blended it with the chitosan to prepare composite scaffolds. We developed a new method to obtain a homogeneous solution of chitosan and γ-PGA without the formation of polyion complexes. Both dense and porous γ-PGA/chitosan composite scaffolds were fabricated using the freeze-gelation method. The photographs of fluorescence staining confirmed the homogeneity of the γ-PGA/chitosan composite porous scaffolds. SEM micrographs showed that an interconnected porous microstructure was present in the porous scaffolds. The data of swelling ratio and contact angles both indicated that the hydrophilicity was obviously improved by γ-PGA. Additionally, the tensile strength of the porous γ-PGA/chitosan scaffolds was higher than that of the unmodified chitosan scaffolds. In the cytocompatibility test, the density of rat osteosarcoma (ROS) cells] on the 20% γ-PGA-modified surfaces was almost triple that on the unmodified chitosan surfaces on day 5. Therefore, the γ-PGA/chitosan composite scaffolds, due to their better hydrophilicity, cytocompatibility, and mechanical properties, are very promising biomaterials for tissue engineering applications.
Combining scaffolds, cells, and signaling molecules to induce the regeneration of tissues has become the central theme of tissue engineering. In Chapter 5, we demonstrate the use of signaling molecules in tissue-engineered scaffolds. Bone morphogenetic proteins (BMPs) can promote the formation of bone and cartilage tissues, and are utilized to cure bone defects. In order to provide for the stable and sustained release of BMPs, we used freeze-gelled γ-PGA/chitosan composite porous scaffolds as carriers for delivering BMP-2. For comparison, scaffolds made of freeze-dried chitosan, freeze-dried PLLA, and freeze-gelled chitosan were also prepared. From the controlled release data of BMP-2, the freeze-gelled γ-PGA/chitosan composite scaffolds provided the most-satisfactory release curve, followed by the freeze-gelled chitosan, freeze-dried chitosan, and freeze-dried PLLA scaffolds. In the stability test of BMP-2 in various solvents, p-dioxane (the solvent for PLLA) seriously deteriorated BMP-2, whereas acetic acid (the solvent for chitosan) did not. In brief, our results indicated that the freeze-gelled γ-PGA/chitosan composite scaffold is very promising as a carrier system for BMP-2. This novel biomaterial can be further developed and potentially applied to the therapy of bone defects and other related diseases.
This research investigated in detail the effects of process variables on the mechanical properties of the scaffolds. The chitosan scaffolds fabricated by the freeze-gelation method were further modified by γ-PGA, and the cytocompatibility of γ-PGA/chitosan scaffolds was examined using ROS cells. Finally, the scaffolds were used as carriers for a signaling molecule, BMP-2. Our results clearly indicated that the γ-PGA/chitosan scaffold is a very promising tissue engineering material. It has good biodegradability, cytocompatibility, and hydrophilicity. The microstructure and mechanical properties of this scaffold can easily be adjusted and enhanced by changing the conditions of the freeze-gelation and cross-linking processes. It can also be a good carrier for the delivery of BMP-2. In summary, the freeze-gelled γ-PGA/chitosan scaffold developed in this research is a very promising tissue engineering biomaterial, and can be applied to the therapy of bone defects and other diseases.
Subjects
幾丁聚醣
聚麩胺酸
chitosan
γ-poly(glutamic acid)
Type
thesis
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