Date

2017

Author

Sayın, Esen

Metadata

Show full item record

Item Usage Stats

197
views

215
downloads

Cite This

Cues of microenvironment that guide both mature and stem cells determine the success of tissue engineered constructs. To prove and emphasize this expectation, various parameters such as surface topography, scaffold (cell carrier, scaffold) chemistry, 2D vs 3D microenvironments and mechanical stimulation were included into the microenvironment. Surfaces with two distinct physical cues pillar and groove-ridge type micropatterns were transferred to the surfaces of the films by casting the collagen type I and silk fibroin biopolymers on poly(dimethylsiloxane) (PDMS) templates which were replicated from photolithographically produced micropatterns on silicon wafers. Bombyx mori silk fibroin a structural biopolymer, was blended with collagen type I protein, to obtain high mechanical properties and biodegradability. Adipose derived stem cells (ADSCs) were cultured on collagen–silk fibroin films with microchannel and micropillar patterns to investigate the effects of cell morphology changes on osteogenic differentiation. While higher ADSC proliferation profiles were obtained on micropillar patterned film, microchannel patterned films, however, caused twice higher aspect ratio and effective orientation of cells. Alkaline phosphatase activity of ADSCs was several times higher on microchannel surface when the measured activities were normalized to cell number. Effective deposition of collagen type I and mineral after cell culture was observed for patterned and unpatterned films and these extracellular matrix (ECM) components were oriented along the axis of the microchannels. The use of collagen–fibroin blend film with microchannel topography increased the aspect ratio and alignment of cells significantly, and it was also effective in the differentiation of ADSCs into osteogenic lineage. As an additional biochemical cue of microenvironment defining element, elastin-like recombinamer (ELR) with a hydroxyapatite depositing amino acid sequence was incorporated into films of collagen-silk fibroin blend carrying microchannel patterns to stimulate anisotropic cell growth and osteogenesis. The Young's modulus and the ultimate tensile strength (UTS) of unseeded films were 0.58 ± 0.13 MPa and 0.18 ± 0.05 MPa, respectively. After 28 days of cell culture, ADSC seeded films had a Young's modulus of 1.21 ± 0.42 MPa and UTS of 0.32 ± 0.15 MPa which were about 3 fold higher than human osteoblast (HOB) seeded films. The difference in Young's modulus was statistically significant (p = 0.02). ADSCs attached, proliferated and produced calcium phosphate mineral on films better than the HOBs. In the light of these results, ADSCs served as a better cell source than HOBs for bone tissue engineering of collagen-fibroin-ELR based constructs used in this study. In vitro systems generally rely on 2D test media whereas in vivo can best be mimicked if a 3D test medium is used. This is important because the maturation of a tissue engineered product should be different than on a 2D surface due to the kind of interactions and the accumulation of molecular signals in 2D and 3D systems are expected to be different. 3D scaffolds were created by folding long strips of engineered films on a rod to investigate the contributions of 3D microenvironment over mesenchymal stem / multipotent stromal cells (MSCs) by comparing with 2D films. Additionally, the contribution of hypoxia and arterial oxygen pressure was studied further to look into the concept of oxygen limited microenvironments of the native MSCs and osteoblast niche. Hypoxia maintained the stemness of the MSCs on TCPS and 2D scaffold. Interestingly, MSCs had elevated VEGFA level and osteogenic differentiation on 3D construct while preserving their stemness at normoxic conditions. Strikingly, osteogenic and angiogenic marker expressions were 13200 and 266 fold higher, respectively on 3D construct than 2D scaffold at 21% oxygen. At all of the oxygen tensions that were tested, UTS was found to be similar with the unseeded scaffolds at less preferable 2D scaffolds by MSCs. Distinctively, during the transition from hypoxia to normoxia, Young’s modulus and UTS of the MSC seeded 3D scaffolds were enhanced from 1.13 ± 0.33 MPa to 2.16 ± 0.81 MPa and 0.51 ± 0.12 MPa to 1.82 ± 0.27 MPa, respectively, owing to the contribution of cells by secreting their ECM onto the surface of 3D scaffold surface. MSC proliferation was dependent both on oxygen tension and cyclic strain exposure (10%) on collagen hydrogel embedded scaffolds. On the other hand, strain was able to alter MSC shape by elongating them on the force direction and inhibit angiogenic activity both at hypoxia and normoxia. In summary, this study showed that mimicking the bone ECM was possible through the use of microchannel patterns especially in 3D microenvironments that overcome the limitations of 2D substrates. The transition from 2D to 3D microenvironment enhanced the osteogenic and angiogenic activity of MSCs and along with the tensile properties both at normoxia and oxygen limited physiological environments.

Subject Keywords

Tissue engineering., Stem cells.

URI

http://etd.lib.metu.edu.tr/upload/12621554/index.pdf
https://hdl.handle.net/11511/26975

Collections

Graduate School of Natural and Applied Sciences, Thesis