Osteoporotic fractures are still the major health concerns in many developed
societies especially when the incidence of that tremendously increased with the aging
population. However, the outcomes of osteoporotic fracture treatment have not been
entirely satisfactory due to the poor quality of bone substance. Inspiringly, bone
morphogenetic protein 2 (BMP2) with the ability to accelerate bone formation
showed advantages over the conventional treatment. The only problem needed to
overcome is its short half-life which resulted in the requirement of readministration
and extremely high cost. As a solution to that, gene therapy provides a promising way
to sustainably release this protein at the regeneration site. Since viral vectors have
been hampered by genetic toxicity and immunogenicity, nanoscaled non-viral vectors
offer an attractive means for gene delivery.
Chitosan as non-viral vector has been widely investigated for its excellent
biocompatibility. Most efforts have been given to improve its low transfection
efficiency. In this study, chitosan was first modified with octaarginine, one of cell
membrane penetrating peptides, and showed enhanced transfection activity, but which
was not significant as expected. Following that, low molecular weight
polyethyleminine (PEI) was introduced to modify chitosan through bioreducible
disulfide linkage, denoted as Chitosan-ss-PEI. PEI is an efficient non-viral vector but
hampered by molecular-weight dependent toxicity. The developed Chitosan-ss-PEI
showed good biocompatibility in MTT assay in three different cell lines, during which
cells were maintained 80% of viability when the concentration of this vector was up
to 100 μg/mL. The optimal transfection efficiency of Chitosan-ss-PEI was higher than
that of PEI 25k and comparable to Lipofectamine in delivering luciferase reporter
gene. GFP expression mediated by Chitosan-ss-PEI also showed similar results.
Chitosan-ss-PEI was then applied to deliver BMP2 gene to skeletal system cells
and exhibited the osteogenic ability. For C2C12 myoblast cells, this system inhibited
their myoblast differentiation and induced the osteogenic differentiation. It also
showed stronger effect in promoting the differentiation of immature osteblast-like
MG63 cells and in inducing C3H10T1/2 mesenchymal stem cells osteogenic
differentiation in term of ALP activity and mineralization ability compared with other
commercial available non-viral vectors. Primary MSCs such as bone marrow stromal
cells (BMSC) and human umbilical cord blood mesenchymal stem cells
(hUCB-MSC), are usually more difficult to transfect, but they showed stronger
osteogenic differentiation ability induced by this system comparing with the cell lines.
BMP2 usually requires extremely high concentration to realize its function.
Through the investigation of BMP2 signaling regulation in this study, it was found
that parathyroid hormone (PTH) could increase the access of BMP2 ligands to their
receptors by negatively influencing BMPs antagonist network, resulted in enhanced
BMP2 activity in bone remodeling and in promoting the commitment of MSC to
osteoblast lineage both in vitro and in vivo. This course involved the endocytosis of
PTHR with a complex of LRP6, which organized antagonist network on the cell
surface to shield the BMPs receptors. Novel approaches are expected to be developed
based on this mechanism with the purpose of intensifying the therapeutic effect of
BMPs. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/146135 |
| Date | January 2011 |
| Creators | Zhao, Xiaoli, 赵晓丽 |
| Contributors | Lu, WW, Luk, KDK |
| 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/B47246078 |
| 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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