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Differentiation of Human Dermal Fibroblasts and Applications in Tissue Engineering
Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns . Linköping University, Faculty of Health Sciences. (Laboratory for Reconstructive Plastic Surgery)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors.

One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin.

The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications.

Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV).

In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2010. , 62 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1202
Keyword [en]
Tissue Engineering, Human Dermal Fibroblasts, Differentiation, Adipogenesis, Chondrogenesis, Endotheliogenesis, Osteogenesis
National Category
Surgery
Identifiers
URN: urn:nbn:se:liu:diva-60879ISBN: 978-91-7393-326-1 (print)OAI: oai:DiVA.org:liu-60879DiVA: diva2:359630
Public defence
2010-11-19, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2010-10-29 Created: 2010-10-28 Last updated: 2010-10-29Bibliographically approved
List of papers
1.
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2.
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3. In Vivo Implantation of Osteogenic Induced Human Dermal Fibroblasts in a Fracture Model
Open this publication in new window or tab >>In Vivo Implantation of Osteogenic Induced Human Dermal Fibroblasts in a Fracture Model
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Fracture healing is a complex event involving cells and growth factors. When healing is impaired it substantially affects quality of life and increases medical costs. To overcome difficulties with impaired bone healing several methods using biomaterials have been tested. Osteogenic biomaterials, which are scaffolds loaded with osteocompetent cells, have been proposed when the defect is large. In this study we wanted to investigate the potential of osteogenic induced human dermal fibroblasts grown on gelatin microcarriers combined with platelet rich plasma (PRP) in a femoral gap surgical model in athymic rats. The gaps were transplanted with one of the following six combinations: 1; NaCl, 2; PRP, 3; microcarriers + PRP, 4; human dermal fibroblasts on microcarriers + PRP, 5; human osteoblasts on microcarriers + PRP, 6; osteogenic induced human dermal fibroblasts on microcarriers + PRP. The gaps were analysed 4 weeks postoperatively with computer tomography, routine histological staining, fluorescence in situ hybridization (FISH) and polyclonal antibodies directed towards osteocalcin and osteonectin. Radiographs taken 4 weeks post surgery did not reveal callus in any of the groups. Gaps transplanted with osteogenic induced human dermal fibroblasts on microcarriers (group 6) contained dense cell clusters with large amounts of extracellular matrix. These cell clusters were not found in the other groups and stained highly positive for osteocalcin and osteonectin. FISH analysis revealed viable human cells in gaps filled with cell-seeded microcarriers confirming survival of transplanted cells. In conclusion osteogenic induced human dermal fibroblasts survive in this new niche and display bonelike structures in the gaps.

Keyword
Tissue engineering, Stem cells, Human dermal fibroblasts, Differentiation, Osteogenic induction, Bone formation
National Category
Surgery
Identifiers
urn:nbn:se:liu:diva-60876 (URN)
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2010-10-29
4. Evaluating Multi-Lineage Induction of Human Dermal FibroblastsUsing Gene Expression Analysis
Open this publication in new window or tab >>Evaluating Multi-Lineage Induction of Human Dermal FibroblastsUsing Gene Expression Analysis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

During the past decades, several adult stem cell populations from a range of tissues have been characterized. Since principally all human cells contain the same genetic material, the specific gene expression profile determines the cell phenotype. The notion of terminally differentiated somatic cells being necessarily restricted to one phenotype has been challenged, and instead an inherent range of plasticity for any given cell type has been suggested. We have in previous work shown that normal human dermal fibroblasts have an inherent plasticity and can be induced to differentiate towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages when subjected to defined induction media. The aim of the present study was to further study the differentiation of human dermal fibroblasts on a gene expression level. This was achieved by employing genome wide expression analysis using microarray technology. Selected gene expression was also evaluated over time using real-time PCR. Several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes, were found regulated in the respective induced cultures. The results obtained in this study strengthen previously published results showing an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that adipogenic, chondrogenic, endotheliogenic and osteogenic induction results in novel phenotypes that show a genetic readiness for lineage-specific biological functionality.

Keyword
Adipogenesis, Chondrogenesis, Endotheliogenesis, Osteogenesis, Fibroblasts, Microarray
National Category
Surgery Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-60877 (URN)
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2010-10-29

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