Chitosan/Chondroitin Sulfate/Glutamic Acid Composite Scaffolds – Investigation of Fabrication Process, Enhancement of Properties and Applications
Date Issued
2009
Date
2009
Author(s)
Yuan, Nai-Yi
Abstract
In the tissue engineering research, scaffolds usually have to fulfill basic requirements such as high porosity, favorable pore size distribution, satisfactory mechanical strength and cytompatibility, and so on. Furthermore, the developments of novel biological materials are focusing on the design and fabrication of materials for the particular function and need. The trend of new biomaterial development includes the use of several materials to fabricate composite scaffolds for improving the performance of the scaffolds. Therefore, this research focused on the improvement in the fabrication method of porous scaffold, and the development of a novel composite material.n the first part of research, using fast-cooling (FC) and slow-cooling (SC) modes in freeze-gelation method, polysaccharide scaffolds made of chitosan (CH), alginate (AL), and carboxymethyl cellulose (CMC) were fabricated. The temperature profiles with respect to time in cooling process were measured, and mechanical properties of the scaffolds were determined. In addition, the pore structure of the scaffolds was observed by scanning electron microscope (SEM) and optical microscope. The results showed that SC mode (0.83 ºC/min) induced simultaneous ice nucleation and formed the isotropic pore distribution of the scaffolds, with the mean pore size about 60-100 μm. In contrast, the use of FC mode for preparing the scaffolds would induce non-simultaneous ice nucleation and formed stratiform pore structure due to greater temperature gradient, and the mean pore size of the scaffolds was larger than that of SC mode. Moreover, for all cooling mode, the supercooling phenomenon occurred during the cooling processes. Regarding the effect on mechanical properties of the scaffolds, the normalized tensile strength of the scaffolds prepared by using SC mode (60 N/g) was two to three-folds higher than that of using FC mode (20 N/g). It demonstrates that cooling mode was able to affect the pore structure, and then induce the difference in mechanical properties of the scaffolds.n the second part of research, the preparation of the chitosan/chondroitin sulfate (CH/CS) and CH/CS/glutamic acid (Glu) composite scaffolds were carried out by freeze-gelation method. During the preparation of scaffold solution,DC/NHS was used as the crossing-linking reagent, and the viscosities, pH value of solution, CS binding efficiency were determined. After the fabrication of scaffolds, water contact angle, internal porosity, water uptake, and mechanical properties of the scaffolds were measured. The microstructures of scaffolds were observed by SEM. In the CH/CS solution, strong ionic interactions lead to the precipitation especially under the highly CH/CS concentrations. Therefore, the third component-glutamic acid (Glu) was added to the solution to decrease the amount of protonated amines and reduce the ionic interactions between CH and CS because carboxyl groups of Glu could interact with amino groups of CH, then acetic acid was added slowly to the solution to reduce precipitation. The results of tensile strength and compressive test of scaffolds showed that the addition of a small amount of CS to the scaffold improved mechanical strength of CH scaffolds. However, a large amount of CS in the scaffold was unfavorable to the mechanical strength of CH scaffolds. For the addition of Glu to the CH/CS scaffolds, the tensile strength increased with increasing the content of Glu in CH/CS/Glu scaffold (molar ratio of CH/CS/Glu increasing from 96/4/0 to 96/4/16, and the tensile strength increase in tensile strength: from 150 N/g to 250 N/g), the compressive strength may also increased from 143.39 N (96/4 scaffold) to 160.50 N (96/4/16 scaffold). In hydrophilicity test and water uptake measurement, the addition of CS and Glu caused the contact angle to decrease from 95º to 50º (92/8/8 scaffold surface), and the water uptake increased from 15.32 to 21.34, demonstrating that hydrophilicity of scaffolds increases.n addition, we used Electron Spectroscopy for Chemical Analysis (ESCA) to determine the elements C, N, O and S of the scaffolds. The result showed that the chitosan had an average degree of deacetylation of 92%. For the N1S spectrum of CH/CS scaffolds showed that after reacted with EDC/NHS coupling agent, new covalent bonds of C=N was formed. Moreover, the C=N proportion of 92/8 group (29.06%) was higher than 96/4 group (20.92%). That is to say, the addition of small amount of CS to the scaffold increased tensile strength of the scaffold due to the covalent crosslinkages was increased. The S2P spectrum of CH/CS showed that the sulfonate groups (having affinity to water) increased in the scaffolds that led to the peak of bound water in the DSC graph shifted to the highIemperature region as the CS content increased. From N1S spectrum analyses, the C=N (398.9 eV) proportion of the three component scaffolds (CH/CS/Glu) were higher than two component scaffolds (CH/CS), and the S2P spectrum of the scaffolds showed that the sulfate groups of the scaffolds was decreased. It demonstrated that the addition of Glu increased covalent crosslinkage again and reduced the ionic interaction between CH and CS, thus improving the tensile strength of the scaffolds. Besides, the amount of sulfonate groups increased in CH/CS/Glu to enhance the hydrophilicity of the scaffolds. As the result of the cell compatibility test using murine osteoblast-like cells (7F2 cells) revealed that the addition of a small amount of CS to CH is beneficial to the proliferation of 7F2 cells. And the addition of Glu, according to osteocalcin and the type I collagen gene expression analysis and the calcium ion measurement, CH/CS/Glu composite material was not only suitable for the 7F2 cell proliferation, but also beneficial to osteogenic the differentiation and the matrix mineralization.n conclusion, this research used different cooling modes in the freeze-gelation method for fabricating polysaccharide scaffolds with different pore structure and thus different mechanical strength. And then a novel composite scaffold made of CH/CS/Glu was successfully fabricated. This scaffold possessed higher hydrophilicity and mechanical strength than other CH-based scaffolds. Besides, the proliferation, maintenance of osteogenic phenotype, and mineralization behavior of the osteoblast-like (7F2) cells were also improved. Therefore, we suggest that this novel composite biomaterial consisting of CH, CS, and Glu has great application potential in the field of bone-related tissue engineering.eywords: chitosan, chondroitin sulfate, glutamic acid, freeze-gelation method, composite, tissue engineering, osteoblast-like cells.
Subjects
chitosan
chondroitin sulfate
glutamic acid
freeze-gelation method
composite
tissue engineering
osteoblast-like cells
Type
thesis
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