Regeneration of injured bone and articular cartilage using mouse muscle derived stem cells ; Pažeisto kaulinio ir kremzlinio audinio regeneracijos tyrimai panaudojant pelės raumeninės kilmės kamienines ląsteles

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES
MEDICAL ACADEMY








Arvydas Ūsas


REGENERATION OF INJURED BONE
AND ARTICULAR CARTILAGE USING
MOUSE MUSCLE DERIVED
STEM CELLS



Doctoral Dissertation
Biomedical Sciences, Medicine (07 B)












Kaunas, 2011
   
1The investigations were performed at the Children’s Hospital of Pittsburgh
and the University of Pittsburgh, Pittsburgh, Pennsylvania, USA during the
period of 2000–2010, and the dissertation has been prepared at Kaunas
University of Medicine during the period of 2009–2010.

Dissertation is defended extramurally.

Scientific Consultant:
Prof. Dr. Romaldas Ma čiulaitis (Lithuanian University of Health
Sciences, Medical Academy, Biomedical Sciences, Medicine – 07 B)
 
2LIETUVOS SVEIKATOS MOKSL Ų UNIVERSITETAS
MEDICINOS AKADEMIJA








Arvydas Ūsas


PAŽEISTO KAULINIO IR KREMZLINIO
AUDINIO REGENERACIJOS TYRIMAI
PANAUDOJANT PELĖS RAUMENIN ĖS
KILMĖS KAMIENINES LĄSTELES



Daktaro disertacija
Biomedicinos mokslai, medicina (07 B)












Kaunas, 2011
   
3Disertacija ginama eksternu.

Mokslinis konsultantas:
Prof. dr. Romaldas Mačiulaitis (Lietuvos sveikatos mokslų universitetas,
Medicinos akademija, biomedicinos mokslai, medicina – 07 B)

 
4CONTENT
CONTENT .................................................................................................... 5
ABBREVIATIONS ...................................................................................... 8
1. INTRODUCTION ................................................................................. 9
1.1. Study objective and specific aims ................................................. 9
1.2. Research significance and novelty ................................................ 9
1.3. Review of the literature ............................................................... 10
2. MATERIALS AND METHODS ....................................................... 19
2.1. Cell isolation and characterization .............................................. 19
2.1.1. Isolation of muscle-derived cell populations and MDSC clones ....... 19
2.1.2. Immunohistochemistry ...................................................................... 20
2.1.3. RT-PCR analysis ................................................................................ 20
2.1.4. Flow cytometry .................................................................................. 21
2.1.5. Cytogenetic and tumorigenicity assay ............................................... 21
2.1.6. In vitro cell stimulation with rhBMP-2 or rhBMP-4 .......................... 22
2.1.7.vivo myogenic differentiation of the mc13 cells ............................ 23
2.2. Retroviral vectors and transduction of MDSCs ........................... 23
2.2.1. Construction of retroviral vectors ...................................................... 23
2.2.2. Transduction of MDSCs .................................................................... 24
2.2.3. BMP4 bioassay and detection of protein secretion ............................ 24
2.3. Evaluation of osteogenic and chondrogenic differentiation
of transduced MDSCs in vitro ..................................................... 25
2.3.1. Detection of alkaline phosphatase activity after transduction ............ 25
2.3.2. Chondrogenic stimulation in monolayer and type II collagen
immunohistochemistry ....................................................................... 25
2.3.3. Cell pellet culture and staining for glycosaminoglycans .................... 26
2.4. Evaluation of osteogenic and chondrogenic differentiation
of MDSCs in vivo ........................................................................ 26
2.4.1. Intramuscular transplantation of MDSCs ........................................... 26
2.4.2. Evaluation of β-galactosidase and osteocalcin expression ................. 27
2.4.3. Immune response to allogeneic cell implantation .............................. 27
2.4.4. Alcian blue staining for cartilage formation ...................................... 27
2.4.5. Skull defect model ............................................................................. 28
2.4.6. Von-Kossa staining ............................................................................ 28
2.4.7. Repair of osteochondral defects ......................................................... 28
2.4.8. Macroscopic evaluation and detection of LacZ transgene expression ..... 29
2.4.9. Evaluation of β-galactosidase and type II collagen expression .......... 29
2.4.10. Histological evaluation and grading of cartilage repair ..................... 29
   
52.5. Evaluation of osteogenic capacity of BMP4- and VEGF-
expressing MDSCs in vivo........................................................... 30
2.5.1. Ectopic bone formation ...................................................................... 30
2.5.2. Skull defect healing............................................................................ 30
2.5.3. Immunostaining for vascular structures ............................................. 30
2.5.4. Apoptosis assay .................................................................................. 31
2.6. Evaluation of chondrogenic capacity of BMP4- and
VEGF-expressing MDSCs........................................................... 31
2.6.1. Pellet culture for in vitro chondrogenesis .......................................... 31
2.6.2. Alcian blue staining ........................................................................... 32
2.6.3. RT-PCR analysis ................................................................................ 32
2.6.4. Repair of osteochondral defects ......................................................... 32
2.6.5. Histological evaluation and grading of cartilage repair ..................... 33
3. RESULTS............................................................................................. 34
3.1. Characterization of muscle-derived cell populations .................. 34
3.1.1. Marker analysis of the pp6 cells ........................................................ 34
3.1.2. Marker analysis of the mc13 cell clone .............................................. 35
3.1.3. Osteogenic differentiation after in vitro stimulation with rhBMP-2 .. 36
3.1.4. Osteogenic differentiation after stimulation with rhBMP4 ................ 40
3.1.5. Standard cytogenetic and tumorigenicity assay ................................. 40
3.1.6. In vivo myogenic differentiation of the mc13 cells ........................... 41
3.2. Osteogenic potential of MDSCs .................................................. 43
3.2.1. In vitro osteogenic differentiation of transduced MDSCs .................. 43
3.2.2. Protein expression from transduced MDSCs ..................................... 44
3.2.3. Ectopic bone formation induced by BMP4-expressing MDSCs ........ 45
3.2.4. Immune reaction at the site of cell implantation ................................ 46
3.2.5. In vivo osteogenic differentiation of transduced MDSCs .................. 47
3.2.6. Enhancement of bone healing in skull defect model .......................... 48
3.3. Chondrogenic potential of MDSCs ............................................. 49
3.3.1. In vitro chondrogenic differentiation of MDSCs ............................... 49
3.3.2. Cell viability detected by LacZ transgene expression ........................ 51
3.3.3. In vivo chondrogenic differentiation of MDSCs ................................ 52
3.3.4. Macroscopic findings at the cartilage repair site53
3.3.5. Histological evaluation of the cartilage repair ................................... 54
3.3.6. Histological grading of the cartilage repair ........................................ 56
3.4. The effect of VEGF supply on BMP4 induced bone formation ... 56
3.4.1. Enhancement of endochondral bone formation ................................. 56
3.4.2. Enent of angiogenesis ............................................................ 59
3.4.3. Enhancement of skull defect healing ................................................. 60
3.4.4. Healing mechanism of calvarial defects ............................................ 63
 
63.4.5. Importance of proper ratio of VEGF to BMP4 in bone regeneration . 64
3.4.5. Inhibition of bone formation by VEGF-specific antagonist sFlt1 ...... 66
3.5. The effect of BMP4, VEGF and sFlt1 on chondrogenic potential
of MDSCs .................................................................................... 68
3.5.1. Expression of BMP4, VEGF, and sFlt1 by transduced MDSCs ........ 68
3.5.2. Chondrogenesis in pellet culture ........................................................ 68
3.5.3. Macroscopic and histological evaluation of cartilage repair .............. 71
3.5.4. Histological grading of the cartilage repair ........................................ 74
4. DISCUSSION ...........