Preparation of Cell-Specific Scaffolds by Peptide-Grafting for Bone Regeneration
Date Issued
2005
Date
2005
Author(s)
Ho, Ming-Hua
DOI
zh-TW
Abstract
Freeze-extraction and freeze-gelation methods are presented in this thesis which can be used to prepare highly porous scaffolds. The porous structure was generated after freeze of a polymer solution, following that either the solvent was extracted by a non-solvent or the polymer was gelled under the freezing condition; thus, the porous structure would not be destructed during the subsequent drying stage. Compared with the traditional freeze-drying method, the presented methods are time and energy saving, with less residual solvent, and easier to scale-up. Besides, by the methods presented, the limitation is lifted so only solvent with low boiling point can be used for scaffold preparation. With the freeze-extraction and freeze-gelation methods, porous PLLA, PLGA, chitosan and alginate scaffolds were successfully fabricated.
Chitosan scaffolds were modified with peptides, RGDS (Arg-Gly-Asp-Ser), KRSR (Lysine-Arginine-Serine-Arginine) and FHRRIKA (Phenylalanine- Histidine-Arginine-Arginine-Isoleucine-Lysine-Alanine), via an amide-bond forming reaction between amino groups in chitosan and carboxyl groups in peptides. Successful immobilization was verified with FTIR spectroscopy, and the immobilized amount was determined with an amino acid analyzer to be in the order. The RGDS immobilization can enhance the attachment of cells onto the chitosan, resulting in cells with higher density attached to the RGDS-modified scaffold than to the unmodified scaffold. Consequently, when being applied to culture of ROS (rat osteosarcoma cells), more cells were on the RGDS-modified scaffold than on the unmodified scaffold, which tended to form bone-like tissues. The immobilizations of KRSR and FHRRIKA made the chitosan scaffolds specific to osteoblastic cells, promoting attachment of ROS cells but ineffective on human fetal skin fibroblasts. For PF (periodontal fibroblast) cells, the graft of KRSR and FHRRIKA also increased the initial density of attached cells, although less effectively than the graft of RGDS did. However, the PF cells cultured on the KRSR and FHRRIKA immobilized chitosan expressed more significant markers in osteoconduction. The grafted KRSR and FHRRIKA might induce the attachment of the osteoblastic subgroup in the PF cells or make the non-osteoblastic subgroup in PF cells transform to osteoblastic cells. The results suggested that the immobilization of KRSR and FHRRIKA could make chitosan scaffolds osteoblastic cell specific and more suitable for regeneration of bones.
The peptides were also grafted on the PLLA scaffolds with the plasma grafting technique. The successful graft was confirmed with FTIR spectroscopy and ESCA, and the graft amount was determined with an amino acid analyzer. The grafted RGDS enhanced cell attachment and the grafted KRSR and FHRRIKA had specific effects on osteoblastic cells, just like what was observed for chitosan scaffolds.
In the last part, a mathematical model was proposed to describe the attachment and growth of ROS cells in scaffolds. For chitosan scaffolds, it revealed that the enhancement on the initial cell attachment by the grafted RGDS should be the major reason for the observed effect and the cell doubling time remains changeless with different modification. Besides, the model can well describe the occurrence of a plateau in cell density at long culture time, which might be caused by the space limitation in the scaffold. For PLLA scaffolds, the graft of RGDS can enhance not only the initial attached cell number but also the cell growth rate.
Subjects
骨再生
接枝
細胞專一性
生物基材
胜肽
peptide
graft
tissue engineering
bone regeneration
cell-specific
scaffold
Type
thesis