Yang, Yang
(2018)
Porous scaffolds and soft hydrogel scaffolds for soft tissue engineering.
PhD thesis, University of Nottingham.
Abstract
Three dimensional (3-D) porous scaffolds are valuable in tissue engineering as they can provide the micro-environment for cell adhesion, proliferation, migration and induce tissue regeneration. Collagen is regarded as the most valuable biomaterial in tissue engineering as it is the most important and abundant structural protein in the human body.
This thesis consists of three parts. Part one describes investigation of porous scaffolds fabricated from gelatin-chitosan; part two concerns a study of porous scaffolds developed based on recombinant human collagen-polypeptide (RHC); part three investigates soft hydrogels scaffolds based on recombinant collagen-polypeptide.
Firstly, porous gelatin-chitosan scaffolds were developed in order to refine and understand fabrication methods, as well as to improve characterization techniques for highly porous scaffolds. Gelatin/chitosan (Gel/Chi) porous scaffolds were fabricated using a freeze-drying method and cross-linked by proanthocyanidin (PA) or glutaraldehyde (GA). Porous micro-structures, swelling in aqueous media, in vitro degradation and mechanical strength were characterized in this study. Cytocompatibility of the fabricated porous scaffolds was investigated by seeding 3T3 fibroblasts into the porous structures, and the cellular metabolic activity, proliferation, distribution, and morphology were investigated.
Secondly, Recombinant Human Collagen-polypeptide (RHC) and RHC-chitosan (RHC-CHI) porous scaffolds were fabricated by a freeze-drying method and cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Porous structures, cross-linking mechanisms, cross-linking degree, swelling ratio, in vitro degradation and mechanical properties were investigated. Cytocompatibility of the porous scaffolds was investigated using 3T3 fibroblasts as before.
Finally, a series of RHC based soft hydrogel scaffolds were fabricated through cross-linker induced polymerization, and these hydrogels were studied to determine their feasibility as potential biomaterials. Changes in cell morphology and proliferation in response to hydrogel composition were also investigated.
Scanning electron microscopy and micro computed tomography (micro-CT) indicated that highly porous structures had been obtained in freeze-dried gelatin and collagen based porous scaffolds. Fourier transform infrared spectroscopy determined that cross-linking had occurred through covalent bonding between the biopolymer molecules. The degree of cross-linking was determined using high performance liquid chromatography, and the results confirmed that the biopolymers in the porous scaffolds were efficiently cross-linked. In vitro degradation tests indicated that the porous scaffolds showed acceptable biostability. The mechanical tests showed that mechanical stiffness of the porous scaffolds could be tailored to their end-use application by either adjusting the biopolymer or cross-linker concentration and their mechanical strengths were found to be comparable to biological soft tissues. Cytocompatibility tests using Alamar Blue and DNA assays confirmed that gelatin and RHC based porous scaffolds had no toxicity to fibroblasts and could support cell proliferation, while fluorescence microscopy and cell morphology showed the adhesion, migration and proliferation of seeded cells in porous scaffolds. Quantitative reverse transcription-polymerase chain reaction further showed a high expression of extracellular matrix associated protein (β-integrin, collagen I and collagen III) genes as tissue regeneration progressed.
Results from soft hydrogel scaffold characterization found that the gelation time could be optimized by adjusting the RHC fraction, biopolymer concentration, or reaction temperature. Acceptable mechanical properties and biostability were verified in mechanical and in vitro degradation tests, and as with the porous scaffolds, mechanical strengths could be tuned by modifying either RHC fraction or total polymer concentration. Cytotoxicity tests showed that the fabricated soft hydrogels had no toxicity to fibroblasts and cytocompatibility tests indicated that they promoted adhesion and proliferation. The DNA assay and cell morphology study also confirmed that cellular activities were affected by both mechanical properties and polymer composition, however RHC fraction in these hydrogel scaffolds was the major factor influencing cellular activity.
In conclusion, the results of this study show that gelatin and RHC based porous scaffolds as well as homogeneous soft hydrogel scaffolds will be highly applicable in current and future applications in tissue engineering.
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