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Revitalization of bone allografts using induced pluripotent stem cell-derived mesenchymal stem cells and human umbilical vein endothelial cells

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Title:
Revitalization of bone allografts using induced pluripotent stem cell-derived mesenchymal stem cells and human umbilical vein endothelial cells
Creator:
Summers, Megan Elizabeth ( author )
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
Physical Description:
1 electronic file (16 pages) : ;

Thesis/Dissertation Information

Degree:
Master's ( Master of science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Bioengineering, CU Denver
Degree Disciplines:
Bioengineering
Committee Chair:
Park, Dae Won
Committee Members:
Payne, Karin
Patel, Vikas

Subjects

Subjects / Keywords:
Bones -- Growth ( lcsh )
Regeneration (Biology) ( lcsh )
Bones -- Growth ( fast )
Regeneration (Biology) ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Tissue engineering provides the opportunity to repair damaged and diseased tissues using biocompatible scaffolds, cells, and biochemical factors. Within the field of bone tissue engineering (BTE), the use of stem cells to regenerate bone is of great interest because bone allografts lack living cells, resulting in limited angiogenic and osteogenic potential and slow or incomplete bone healing. Revitalization of bone allografts using mesenchymal stem cells (MSCs) has shown promising results, but the limited proliferative potential of MSCs in culture limits their use clinically. The discovery that induced pluripotent stem cells (iPSCs) can be directed to the mesenchymal and endothelial lineages demonstrates that iPSCs may offer a source of rejuvenated, patient-specific osteogenic and angiogenic cells. ( ,, )
Abstract:
The effect of pre-stimulating iPSC-derived mesenchymal stem cells (iPSC-MSCs) in vitro before implantation in vivo has not been widely studied. By culturing iPSC-MSCs in osteogenic differentiation medium (ODM) on bone scaffolds for varying time periods with or without the addition of BMP-2, we tested the hypothesis that pre-stimulation of iPSC-MSCs to the osteogenic lineage would lead to greater osteogenic activity in vitro, and enhance bone formation when implanted in vivo. Results were compared to unseeded scaffolds. Cells that were cultured for 1 week in ODM with the addition of BMP-2 showed high levels of alkaline phosphatase (ALP) activity, and higher bone formation after 12 weeks of implantation.
Abstract:
In terms of vascularizing a bone allograft, it is well understood that a vascular network is essential for bone healing to occur and that osteoblasts and endothelial cells closely regulate one another. However, the mechanism by which this happens is largely unknown, resulting in poor vasculature formation. By culturing iPSC-MSCs and human umbilical vein endothelial cells (HUVECs) together in fibrin gel on a bone scaffold, the hypothesis that angiogenic cues must be applied prior to osteogenic cues to enhance angiogenesis and osteogenesis was tested. Results showed the greatest ALP activity and enhanced tissue formation in the group that received endothelial medium prior to receiving osteogenic medium. In conjunction, these studies show that osteogenesis can be enhanced on bone scaffolds in vitro to enhance in vivo bone formation.
Bibliography:
Includes bibliographical references.
System Details:
System requirements: Adobe Reader.
Statement of Responsibility:
by Megan Elizabeth Summers.

Record Information

Source Institution:
University of Colorado Denver
Holding Location:
Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
on10131 ( NOTIS )
1013197400 ( OCLC )
on1013197400
Classification:
LD1193.E56 2017m S96 ( lcc )

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Full Text
REVITALIZATION OF BONE ALLOGRAFTS USING INDUCED PLURIPOTENT STEM CELL-
DERIVED MESENCHYMAL STEM CELLS AND HUMAN UMBILICAL VEIN ENDOTHELIAL
CELLS
by
MEGAN ELIZABETH SUMMERS B.S., University of Colorado Denver, 2015
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Master of Science Bioengineering Program
2017


The thesis for the Master of Science degree by Megan Elizabeth Summers has been approved for the
Bioengineering Program by
Dae Won Park, Chair Karin Payne, Advisor Vikas Patel
May 13, 2017
ii


Summers, Megan Elizabeth (M.S., Bioengineering)
Revitalization of Bone Allografts Using Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells
Thesis directed by Assistant Professor Karin Payne.
ABSTRACT
Tissue engineering provides the opportunity to repair damaged and diseased tissues using biocompatible scaffolds, cells, and biochemical factors. Within the field of bone tissue engineering (BTE), the use of stem cells to regenerate bone is of great interest because bone allografts lack living cells, resulting in limited angiogenic and osteogenic potential and slow or incomplete bone healing. Revitalization of bone allografts using mesenchymal stem cells (MSCs) has shown promising results, but the limited proliferative potential of MSCs in culture limits their use clinically. The discovery that induced pluripotent stem cells (iPSCs) can be directed to the mesenchymal and endothelial lineages demonstrates that iPSCs may offer a source of rejuvenated, patient-specific osteogenic and angiogenic cells.
The effect of pre-stimulating iPSC-derived mesenchymal stem cells (iPSC-MSCs) in vitro before implantation in vivo has not been widely studied. By culturing iPSC-MSCs in osteogenic differentiation medium (ODM) on bone scaffolds for varying time periods with or without the addition of BMP-2, we tested the hypothesis that pre-stimulation of iPSC-MSCs to the osteogenic lineage would lead to greater osteogenic activity in vitro,
and enhance bone formation when implanted in vivo. Results were compared to


unseeded scaffolds. Cells that were cultured for 1 week in ODM with the addition of BMP-2 showed high levels of alkaline phosphatase (ALP) activity, and higher bone formation after 12 weeks of implantation.
In terms of vascularizing a bone allograft, it is well understood that a vascular network is essential for bone healing to occur and that osteoblasts and endothelial cells closely regulate one another. However, the mechanism by which this happens is largely unknown, resulting in poor vasculature formation. By culturing iPSC-MSCs and human umbilical vein endothelial cells (HUVECs) together in fibrin gel on a bone scaffold, the hypothesis that angiogenic cues must be applied prior to osteogenic cues to enhance angiogenesis and osteogenesis was tested. Results showed the greatest ALP activity and enhanced tissue formation in the group that received endothelial medium prior to receiving osteogenic medium. In conjunction, these studies show that osteogenesis can be enhanced on bone scaffolds in vitro to enhance in vivo bone formation.
The form and content of this abstract are approved. I recommend its publication.
Approved: Karin Payne
IV


TABLE OF CONTENTS
I. INTRODUCTION AND GOALS.............................................................1
Bone..............................................................................1
Bone Grafts.......................................................................1
Synthetic Bone Grafts...........................................................1
Allografts......................................................................1
Stem Cells........................................................................1
Totipotent Stem Cells...........................................................1
Pluripotent Stem Cells..........................................................1
Multipotent Stem Cells..........................................................1
Endothelial Progenitor Cells....................................................1
Mesenchymal Stem Cells..........................................................1
Induced Pluripotent Stem Cells..................................................1
Approaches to Improve Bone Scaffolds..............................................1
Vascularization.................................................................1
Polymers........................................................................1
Growth Factors..................................................................1
Perfusion.......................................................................1
Goals and Hypotheses..............................................................1
II. MATERIALS AND METHODS..............................................................1
iPSC Methods......................................................................1
Generation of iPSCs.............................................................1
Culture of iPSCs................................................................1
v


Differentiation of iPSCs to MSCs....................................................1
Flow Cytometry......................................................................1
Decellularized Bone Scaffolds.........................................................1
Osteogenesis Methods..................................................................1
Differentiation of iPSC-MSCs to the Osteogenic Lineage..............................1
Cell Encapsulation and Seeding of Osteogenesis Cores................................1
In vitro Culture of Osteogenesis Cores..............................................1
Live/Dead Assay.....................................................................1
Immunohistochemistry................................................................1
In vivo Testing of Osteogenesis Cores...............................................1
Micro CT............................................................................1
Vascularization Methods...............................................................1
HUVEC Culture.......................................................................1
Cell Encapsulation and Seeding of Vascularization Cores.............................1
In vitro Culture of Vascularization Cores...........................................1
Live/Dead Assay.....................................................................1
Immunohistochemistry................................................................1
DNA Assay.............................................................................1
ALP Activity Assay....................................................................1
III. RESULTS...............................................................................1
iPSC Results..........................................................................1
Mesenchymal Differentiation of iPSCs................................................1
Osteogenic Differentiation of iPSC-MSCs.............................................1
VI


In Vitro Osteogenesis Results.......................................................1
Live/Dead.........................................................................1
ALP Activity......................................................................1
Histology.........................................................................1
Immunohistochemistry..............................................................1
In vivo Osteogenesis Results........................................................1
Micro-CT..........................................................................1
Vascularization Results.............................................................1
Live/Dead.........................................................................1
ALP Activity......................................................................1
PicoGreen.........................................................................1
Histology.........................................................................1
Immunohistochemistry..............................................................1
IV. DISCUSSION...........................................................................1
iPSC Differentiation................................................................1
Vascularization ALP Activity........................................................1
Vascularization Histology and Immunohistochemistry..................................1
Future Directions...................................................................1
REFERENCES..........................................................................2
vii


CHAPTER I
INTRODUCTION AND GOALS
CHAPTER II
MATERIALS AND METHODS
CHAPTER III RESULTS
CHAPTER IV DISCUSSION
1


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Full Text

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REVITALIZATION OF BONE ALLOGRAFTS USING INDUCED PLURIPOTENT STEM CELL DERIVED MESENCHYMAL STEM CELLS AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS by MEGAN ELIZABETH SUMMERS B.S. , University of Colorado Denver, 2015 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Master of Science Bioengineering Program 2017

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ii The thesis for the Master of Science degree by Megan Elizabeth Summers has been approved fo r the Bioengineering Program by Dae Won Park, Chair Karin Payne, Advisor Vikas Patel May 13 , 2017

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iii Summers, Megan Elizabeth (M.S., Bioengineering) Revitalization of Bone Allografts Using Induced Pluripotent Stem Cell Derived Mesenchymal Stem Cell s and Human Umbilical Vein Endothelial Cells Thesis directed by Assistant Professor Karin Payne. ABSTRACT Tissue engineering provides the opportunity to repair damaged and diseased tissues using biocompatible scaffolds, cells, and bioc hemical factor s. W ithin the field of bone tissue engineering (BTE), the use of stem cells to regenerate bone is of great interest because bone allografts lack living cells , resulting in limited angiogenic and osteogenic potential and slow or incomplete bone healing. R evitalization of bone allografts using mesenchymal stem cells (MSCs) has shown promising results, but the limited proliferative potential of MSCs in culture limits their use clinically. The discovery that induced pluripotent stem cells (iPSCs) can be dire cted to the mesenchymal and endothelial lineages demonstrates that iPSCs may offer a source of rejuvenated, patient specific osteogenic and angiogenic cells. The effect of pre stimulating iPSC derived mesenchymal stem cells (iPSC MSCs) in vitro before im plantation in vivo has not been widely studied. By culturing iPSC MSCs in osteogenic differentiation medium (ODM) on bone scaffolds for varying time periods with or without the addition of BMP 2, we tested the hypothesis that pre stimulation of iPSC MSCs to the osteogenic lineage would lead to greater osteogenic activity in vitro , and enhance bone formation when implanted in vivo . Results were compared to

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iv unseeded scaffolds. Cells that were cultured for 1 week in ODM with the addition of BMP 2 showed hig h levels of alkaline phosphatase ( AL P) activity, and higher bone formation after 12 weeks of implantation . In terms of vascularizing a bone allograft, it is well understood that a vascular network is essential for bone healing to occu r and that osteoblasts and endothelial cells closely regulate one another. However, the mechanism by which this happens is largely unknown, resulting in poor vasculature formation. By culturing iPSC MSCs and human umbilical vein endothelial cells (HUVECs) together in fibrin g el on a bone scaffold, the hypothesis that angiogenic cues must be applied prior to osteogenic cues to enhance angiogenesis and osteogenesis was tested. Results showed the greatest ALP activity and enhanced tissue formation in the group that received endo thelial medium prior to receiving osteogenic medium. In conjunction, these studies show that osteogenesis can be enhanced on bone scaffolds in vitro to enhance in vivo bone formation. The form and content of this abstract are approved. I recommend it s publication. Approved: Karin Payne

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v TABLE OF CONTENTS I. INTRODUCTION AND GOALS ................................ ................................ ............................ 1 Bone ................................ ................................ ................................ ................................ ......... 1 Bone Grafts ................................ ................................ ................................ .............................. 1 S ynthetic Bone Grafts ................................ ................................ ................................ .......... 1 Allografts ................................ ................................ ................................ .............................. 1 Stem Cells ................................ ................................ ................................ ................................ . 1 Totipotent Stem Cells ................................ ................................ ................................ ........... 1 Pluripotent Stem Cells ................................ ................................ ................................ ......... 1 Multipot ent Stem Cells ................................ ................................ ................................ ........ 1 Endothelial Progenitor Cells ................................ ................................ ................................ . 1 Mesenchymal Stem Cells ................................ ................................ ................................ ..... 1 Induced Pluripotent Stem Cells ................................ ................................ ............................ 1 Approaches to Improve Bone Sca ffolds ................................ ................................ ................... 1 Vascularization ................................ ................................ ................................ ..................... 1 Polymers ................................ ................................ ................................ .............................. 1 Growth Factors ................................ ................................ ................................ .................... 1 Perfusion ................................ ................................ ................................ .............................. 1 Goals and Hypotheses ................................ ................................ ................................ .............. 1 II. MATERIALS AND METHODS ................................ ................................ ............................ 1 iPSC Methods ................................ ................................ ................................ ........................... 1 Generation of iPSCs ................................ ................................ ................................ .............. 1 Culture of iPSCs ................................ ................................ ................................ .................... 1

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v i Differentiation of iPSCs to MSCs ................................ ................................ .......................... 1 Flow Cytometry ................................ ................................ ................................ .................... 1 Decellularized Bone Scaffolds ................................ ................................ ................................ .. 1 Osteogenesis Methods ................................ ................................ ................................ ............ 1 Differentiation of iPSC MSCs to the Osteogenic Lineage ................................ .................... 1 Cell Encapsulation and Seeding of Osteogenesis Cores ................................ ....................... 1 In vitro Culture of Osteogenesis Cores ................................ ................................ ................ 1 Live/Dead Assay ................................ ................................ ................................ ................... 1 Immunohistochemist ry ................................ ................................ ................................ ........ 1 In vivo Testing of Osteogenesis Cores ................................ ................................ .................. 1 Micro CT ................................ ................................ ................................ ............................... 1 Vascularization Methods ................................ ................................ ................................ ......... 1 HUVEC Culture ................................ ................................ ................................ ..................... 1 Cel l Encapsulation and Seeding of Vascularization Cores ................................ .................... 1 In vitro Culture of Vascularization Cores ................................ ................................ ............. 1 Live/Dead Assay ................................ ................................ ................................ ................... 1 Immunohistochemistry ................................ ................................ ................................ ........ 1 DNA Assay ................................ ................................ ................................ ................................ 1 ALP Activity Assay ................................ ................................ ................................ .................... 1 III. RESULTS ................................ ................................ ................................ .......................... 1 iPSC Results ................................ ................................ ................................ .............................. 1 Mesenchymal Differentiation of iPSCs ................................ ................................ ................ 1 Osteogenic Differentiation of iPSC MSCs ................................ ................................ ............ 1

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vii In Vitro Osteogenesis Results ................................ ................................ ................................ ... 1 Live/Dead ................................ ................................ ................................ ............................. 1 ALP Activity ................................ ................................ ................................ .......................... 1 Histology ................................ ................................ ................................ .............................. 1 Immunohistochemistry ................................ ................................ ................................ ........ 1 In vivo Osteogenesis Results ................................ ................................ ................................ .... 1 Micro CT ................................ ................................ ................................ ............................... 1 Vascularization Results ................................ ................................ ................................ ............ 1 Live/Dead ................................ ................................ ................................ ............................. 1 ALP Activity ................................ ................................ ................................ .......................... 1 PicoGreen ................................ ................................ ................................ ............................. 1 Histology ................................ ................................ ................................ .............................. 1 Immunohistochemistry ................................ ................................ ................................ ........ 1 IV. DISCUSSION ................................ ................................ ................................ ................... 1 iPSC Dif ferentiation ................................ ................................ ................................ .................. 1 Vascularization ALP Activity ................................ ................................ ................................ ..... 1 Vascularization Histology and Immunohistochemistry ................................ ........................... 1 Future Directions ................................ ................................ ................................ ..................... 1 REFERENCES ................................ ................................ ................................ ............................. 2

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1 CHAPTER I INTRODUCTION AND GOALS CHAPTER II MATERIALS AND METHOD S CHAPTER III RESULTS CHAPTER IV DISCUSSION

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