臺灣博碩士論文加值系統

ABSTRACT
Bony injury or fracture lead to compromised functional ability and reversing skeletal function
remains a challenge, which further increase in advancing aging population due to reduced
bone mineral density. Therefore, we aimed to investigate the therapeutic potential of plateletderived
biomaterials (PDB) against bone injury. Specifically, we assessed the impact of PDB
on migration and osteo-inductive characteristics of mouse embryonic fibroblasts (MEFs) in
vitro as well as in-vivo mice model. The osteogenic lineage and degree of mineralization was
determined by gene markers (RUNX2, OPN and OCN), and alizarin Red S staining,
respectively. Cell migration was determined by FAK, pFAK and Src (migration markers) as
well as E-cadherin, vimentin and snail (EMT markers). The key component of PDB, TGF-β1,
mediated therapeutic impact was confirmed by employing inhibitor of TGF-β receptor I
(TBRI). In vivo cellular migration in mice was also examined by molecular imaging through
establishing bone injury at right femurs. Our results showed that PDB markedly upregulate
expression of osteogenic markers and matrix mineralization. Increased migration and EMT
markers revealed higher migratory potential of PDB-treated MEFs. The in vivo cell migration
was manifested by expression of migratory factors, SDF-1 and CXR4, and compared to
control, PDB-treated mice exhibited an increased bone density and volume. The TBRI
treatment inhibited migration as well osteogenic potential of MEFs, affirming impact of
TGF-β1. Taken together, the outcomes of our study clearly indicated that PDB rescued from
bone injury by enhancing migratory potential of MEFs and osteogenesis.

ABSTRACT
Bony injury or fracture lead to compromised functional ability and reversing skeletal function
remains a challenge, which further increase in advancing aging population due to reduced
bone mineral density. Therefore, we aimed to investigate the therapeutic potential of plateletderived
biomaterials (PDB) against bone injury. Specifically, we assessed the impact of PDB
on migration and osteo-inductive characteristics of mouse embryonic fibroblasts (MEFs) in
vitro as well as in-vivo mice model. The osteogenic lineage and degree of mineralization was
determined by gene markers (RUNX2, OPN and OCN), and alizarin Red S staining,
respectively. Cell migration was determined by FAK, pFAK and Src (migration markers) as
well as E-cadherin, vimentin and snail (EMT markers). The key component of PDB, TGF-β1,
mediated therapeutic impact was confirmed by employing inhibitor of TGF-β receptor I
(TBRI). In vivo cellular migration in mice was also examined by molecular imaging through
establishing bone injury at right femurs. Our results showed that PDB markedly upregulate
expression of osteogenic markers and matrix mineralization. Increased migration and EMT
markers revealed higher migratory potential of PDB-treated MEFs. The in vivo cell migration
was manifested by expression of migratory factors, SDF-1 and CXR4, and compared to
control, PDB-treated mice exhibited an increased bone density and volume. The TBRI
treatment inhibited migration as well osteogenic potential of MEFs, affirming impact of
TGF-β1. Taken together, the outcomes of our study clearly indicated that PDB rescued from
bone injury by enhancing migratory potential of MEFs and osteogenesis.

TABLE OF CONTENT
ABSTRACT ............................................................................................................................................ i
CHAPTER 1. Introduction ................................................................................................................ 1
I. Overview ............................................................................................................................ 2
II. Bone composition ............................................................................................................... 2
1. Bone cells .................................................................................................................. 3
1.1. Osteoblast ........................................................................................................... 3
1.2. Osteoclast ........................................................................................................... 3
1.3. Osteocytes ........................................................................................................... 3
2. ECM .......................................................................................................................... 4
2.1 Organic ECM ..................................................................................................... 4
2.2 Inorganic ECM or Bone Minerals ......................................................................... 5
III. Bone Injuries/Fractures ................................................................................................ 5
IV. Mechanism of bone healing .......................................................................................... 6
1. Indirect bone healing ................................................................................................... 6
2. Direct Bone Healing ............................................................................................................ 7
V. Cell therapy for bone injury ........................................................................................ 7
1. Mesenchymal Stem cells ..................................................................................................... 8
1.1 Adipose-derived Stem Cell (ADSCs) ................................................................... 8
1.2 Bone Marrow Stem Cell (BMSCs) ....................................................................... 8
1.3 Dental Pulp Stem Cell (DPSCs) ........................................................................... 9
2. Embryonic Stem Cell (hESCs) .................................................................................... 9
2.1. Induced Pluripotent Stem Cell (iPSCs) ............................................................... 10
2.2. Mouse Embryonic Fibroblast Cell (MEF) ........................................................... 10
VI. Role of Growth Factors in bone healing .................................................................. 10
1. Transforming Growth Factor-β1 (TFG-β1) ....................................................................... 11
2. Bone Morphogenetic Proteins (BMPs) .............................................................................. 11
3. Platelet-Derived Growth Factor (PDGF) .......................................................................... 11
4. Fibroblast Growth Factors (FGF) ..................................................................................... 11
VII. Platelet-derived Biomaterial (PDB) .......................................................................... 12
1. Role of PDB in Bone Repair ......................................................................................... 13
2. PDB connection with Growth Factors ......................................................................... 13
2.1 PDB regulate TGFs ................................................................................................. 14
2.2 PDB regulate Insulin-Like Growth Factor 1 ....................................................... 14
2.3 The Effect of PDB in Angiogenesis Factors Promoting Bone Reparation ...... 15
VIII. Aim of Study .................................................................................................................. 16
CHAPTER 2. Materials and Methods .............................................................................................. 17
I. Cell Line and their Culture .................................................................................................. 18
II. Preparation of Platelet-derived biomaterials (PDB) ........................................................... 18
III. RNA extraction and semi-quantitative reverse-transcription PCR ...................................... 18
IV. Cell migration assay ............................................................................................................ 19
V. Immunohistochemistry and Western Blot Analysis ............................................................ 19
VI. Animal studies and ethics .................................................................................................... 20
VII. Assessment of Bone Mineral Density (BMD) .................................................................... 20
VIII. Statistical Analysis .............................................................................................................. 20
CHAPTER 3. RESULT AND DISCUSSION ................................................................................... 21
Study-1 Investigating impact of PDB on osteogenesis and migration of mouse embryonic
fibroblasts (MEFs) ............................................................................................................................. 22
I. Results............................................................................................................................................ 23
1. Effect of PDB on MEFs migratory potential to bone injury site ................................ 23
2. Impact of PDB on Migration ability of MEFs: In-vitro ............................................... 23
3. Corroborating proliferative and migratory ability of PDB-contained TGF-β through TGF-β
receptor I inhibitor (TBRI) activity ...................................................................................... 24
4. PDB-mediated regulation of cell migratory migration through TBRI activity .................... 24
5. PDB-induced regulation of cell migratory through TBRI activity ....................................... 25
II. Discussion .................................................................................................................................... 26
STUDY-2 To evaluate in-vivo Platelet-derived biomaterials-mediated Improvement of Bone
Injury through Migratory Ability of Embryonic Fibroblasts ......................................................... 29
I. Results............................................................................................................................................ 30
1. Impact of PBDB on expression of GFP in MEFs-TGL .......................................................... 30
2. Establishment of bone injury mice and transplantation of PDB-treated MEFs-tagged Cell for
in-vivo tracking ....................................................................................................................... 30
3. Bone Mineral Density analysis through μ-CT and DEXA ..................................................... 30
4. Gene expression analysis of osteogenic and migration markers ............................................ 30
II. Discussion ...................................................................................................................................... 32
CHAPTER 4. CONCLUSION ........................................................................................................... 33
CHAPTER 5. FIGURES AND TABLES .......................................................................................... 35
CHAPTER 6. REFERENCES ........................................................................................................... 47
LIST OF FIGURES
Figure 1: Schematic of in vitro & in vivo experiment protocol representing role of TGF-β-1
containing PDB in migration and osteogenic potential of embryonic fibroblast to improve bone injury
.............................................................................................................................................................. 36
Figure 2: Analysis of in vitro osteoblastic differentiation treated with PDB. ..................................... 37
Figure 3: Analysis of in vitro cell migration of osteoblasts treated with PDB. .................................. 38
Figure 4: Optimization of TBRI concentration for proliferative through TBRI activity .................... 39
Figure 5: Optimization of TBRI concentration for migratory regulation of PDB through TBRI
activity and its quantification. .............................................................................................................. 40
Figure 6: Analysis of in vitro osteoblastic differentiation of PDB induced osteoblasts treated with
PDB/T25 ............................................................................................................................................... 41
Figure 7: Analysis of in vitro cell migration of PDB induced osteoblasts treated with PDB/T25 ..... 42
Figure 8: In vitro optical imaging of GFP transfected NIH3T3-TGL cell line .................................... 43
Figure 9: In vitro bone morphologic characterizations of PDB/NIH3T3-TGL transplanted in mice .. 44
Figure 10: Analysis of bone quantity with photomicrographs ............................................................. 45
Figure 11: Diagrammatic summary of PDB and TBRI mechanism for osteoblast differentiation in
bone injury site ......................................................................................................................... 46

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