- Title
- Exploring novel application of tissue engineering strategies to human myometrium
- Creator
- Heidari Kani, Minoo
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Understanding human uterine smooth muscle cell interactions within tissue and their cellular mechanisms are important to completely understand how the human uterus generates the contractions of labour. Research insights into uterine function and the mechanisms of labour have been hindered by the lack of suitable animal and cellular models. The use of traditional culturing methods limits the exploration of complex uterine functions, such as cell interactions, connectivity and contractile behaviour, as it fails to mimic the three-dimensional (3D) nature of uterine cell interactions in vivo. Animal models are an option; however, use of these models is constrained by ethical considerations as well as translational limitations to humans. Evidence indicates that these limitations can be overcome by using 3D culture systems, or 3D Bioprinters, to model the in vivo cytological architecture of the tissue in an in vivo environment. Artificially created tissue can not only be used as an appropriate model in which to study cellular function and organisation but could also be used for regenerative medicine purposes including organ or tissue transplantation, organ donation and obstetric care. In model systems, the tissue-level mechanisms are most dependent upon which cell type and whether 2D or 3D culture techniques are used. In order to model uterine systems, it is essential to have an in-depth knowledge of gene expression, protein profiles, and proliferation characteristics of term myometrial tissue and that of the cell types that might be used in culture. However, there are only a few studies in these areas. In this thesis, these major limitations are addressed by introducing novel studies and methods to human myometrial tissue engineering. Firstly, with a focus on providing a base line in 2D culture as a comparison for 3D culture studies, three commonly used cellular models: primary myometrial, hTERT, and PHM cells were used. Expression of contraction-associated proteins (CAPs) and genes in cell models were identified by performing RT-PCR and confocal immunofluorescence. Furthermore, principal component analysis was performed to compare gene profiles of these cells to term, non-labouring myometrium. The analysis indicated that PHM and primary cells have 2D profiles closer to term non-labouring myometrium, however, protein expression profiles of primary and hTERT cells are more similar to myometrium. Gene and protein profiles of the three 2D cell culture models became of the baseline for interpretation of the 3D culturing studies. Ultimately, these cells will provide the ability to investigate mechanisms underlying human labour and mimic the in vivo 3D environment of myometrial cells. 3D culture of cells and 3D printing of cell structures requires large numbers of cells to provide an appropriate environment for cell adhesion, proliferation, migration and differentiation within the culture. Primary myometrial smooth muscle cells obtained from uterine biopsies have limited proliferative capacities, which slow the process of culturing these cells. Preparing and developing 3D models of myometrial tissue may therefore necessitate the use of alternative sources of cells in order to provide sufficient numbers of functional SMCs that can be cultured in a short time. To this end, TGF-β1 and vitamin C were introduced to the culturing of primary myometrial, hTERT and PHM cells in order to move the CAPs expression profile in these cells toward those of in vivo myometrium. Results suggested that alterations of the gene expression profile occurred when primary myometrial, hTERT and PHM cells were stimulated by TGF-β1, whereas vitamin C had minimal impact on cellular differentiation. Secondly, the survivability of commonly used uterine smooth muscle cells in 3D culturing systems was assessed. Each scaffolding method has its own properties. Thus, the feasibility of culturing cells on a variety of scaffolds (4 different types) to form 3D structure was tested. Cells were cultured on Nanofiber inserts, glass wool and AlgiMatrix and microscopic observations showed cellular proliferation and migration in 3D. Although, their functionality remained unanswered, as they did not respond to contraction agonists or tension stimuli in contraction bioassay. Changing the strategy by reconstructing myometrial tissue strips from primary myometrial cells or explants resulted in contractile strips, but no phasic contractions was recorded. Thirdly, the innovative approach of decellularising human myometrial tissue was applies to facilitate the development of a normal tissue 3D architecture. The decellularised scaffolds were repopulated with primary myometrial, hTERT and PHM cells, and the scaffold suitability for cell culture experimentation and cellular adhesion, proliferation and migration was confirmed with immunohistochemistry. However, non-transparency of human myometrial tissue is an obstacle in depicting a detailed picture of tissue structure for tissue engineering purposes. Fourthly and finally, for the first time, human myometrium was optically cleared by removing lipids, while preserving cellular structure and network. This was to provide better insight into myometrial interactions with the structural elements of the uterine tissue; that might influence uterine function and contraction during pregnancy and labour. The study describes initial steps toward creating human uterine tissue. Merging biological principles with engineering in an interdisciplinary area is an appropriate platform to develop myometrial tissue engineering.
- Subject
- tissue engineering; regenerative medicine; 3D cell culture; pregnancy; uterus
- Identifier
- http://hdl.handle.net/1959.13/1385416
- Identifier
- uon:32223
- Rights
- Copyright 2018 Minoo Heidari Kani
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT01 | Thesis | 7 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 439 KB | Adobe Acrobat PDF | View Details Download |