A major challenge facing the tissue engineering discipline is cartilage tissue repair
and engineering, because of the highly specialized structure and limited repair
capacity that cartilage possesses. Dental pulp stem cells (DPSCs) were identified
about a decade ago as a potential candidate for cell based therapy and tissue
engineering applications. The present study aimed to utilize gene therapy with
isolated DPSCs to induce chondrogenic transgene expression and chondrogenic
lineage differentiation, with the ultimate goal of engineering cartilage tissue-like
constructs.
We isolated DPSCs from human teeth extracted for orthodontic treatment. We
further enriched the isolated population using immunomagnetic bead selection,
which increased stem cell markers: Stro-1 and CD146, compared to unselected
population.
The DPSCs showed the ability to differentiate into the chondrogenic lineage when
induced with recombinant hTGFβ-3 and when transduced with hTGFβ-3
transgene. We successfully constructed the recombinant adeno-associated viral
vector encoding the human TGFβ-3, and determined the best multiplicity of
infection for DPSCs. The transduced DPSCs highly expressed hTGFβ-3 for up to
60 days. Expression of chondrogenic markers; Collagen IIa1, Sox9, and aggrecan
was verified by immunohistochemistry and mRNA.
We successfully fabricated an electrospun nano-fiber scaffold upon which
morphology, proliferation and viability of the DPSCs were examined. DPSCs
attached and proliferated on nano-fiber scaffolds demonstrating better viability
compared to micro-fiber scaffolds.
Transduced cells expressed hTGFβ-3 protein up to 48 days. Cells seeded on nanofiber
scaffolds showed higher expression levels compared to micro-fiber scaffolds
or culture plate.
Scaffolds seeded with DPSCs were implanted in nude mice.
Immunohistochemistry for TGFβ-3 DPSCs constructs (n=5/group) showed
cartilage-like matrix formation with glucoseaminoglycans as shown by Alcian
blue. Immunostaining showed positivity for Collagen IIa1, Sox9 and aggrecan.
Semi-thin sections of the transduced DPSCs constructs examined by transmission
electron microscopy (TEM) showed chondrocytic cellular and intra-cellular
features, as well as extracellular matrix formation (n=2/group).
In vivo constructs with the TGFβ-3 DPSCs showed higher collagen type II and
Sox9 mRNA expression relative to non-transduced DPSCs constructs
(n=5/group). Western blot analysis confirmed this expression pattern on the
protein level (n=3/group).
Engineered constructs mechanical properties were examined and compared to
patellar bovine cartilage to assess functionality (n=5/group). TGFβ-3 transduced
DPSCs constructs showed a higher equilibrium elastic modulus compared to nontransduced
constructs. Micro-fiber scaffolds constructs showed a higher elastic
modulus (0.11 MPa, 18% of bovine cartilage), compared to nano-fiber constructs
modulus (0.032 MPa, 6% of bovine cartilage). Nano-fiber based constructs
showed a similar Poisson‘s ration to bovine cartilage, while that of micro-fiber
scaffolds was lower.
As an alternative gene delivery method, electroporation parameters for DPSCs
transfection were optimized, and compared to commonly used chemical
transfection methods. TGFβ-3 transfected DPSCs showed a significantly higher
relative TGFβ-3 mRNA and protein expression compared to non transfected
control and to eGFP transfected DPSCs. Transfected DPSCs showed increased
relative expression of chondrogenic markers; Collagen II, Sox9 and aggrecan,
compared to non transfected DPSCs.
Successful chondrogenic differentiation of DPSCs gene therapy with TGFβ-3
transgene, and seeding them on PLLA/PGA scaffolds makes it a potential
candidate for cartilage tissue engineering and cell based therapy. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174469 |
| Date | January 2011 |
| Creators | Rizk, Ahmed El Sayed Mahmoud. |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47752890 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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