Advances in Regenerative Medicine: Role of Nanotechnology, and Engineering Principles

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Edited by

Venkatram Prasad ShastriUniversität FreiburgInstitute of Makromolekulare ChemieGermany

George AltankovICREA & Institute of Bioengineering CatalunyaBarcelonaSpain

Andreas LendleinInstitute of Polymer Research and Berlin-Brandenburg Center for Regenerative TherapiesGKSS Research Center Geesthacht GmbHTeltowGermany

Published in Cooperation with NATO Public Diplomacy Division

Advances in Regenerative Medicine: Role of Nanotechnology, and Engineering Principles

Proceedings of the NATO Advanced Research Workshop on Nanoengineered Systems for Regenerative Medicine Varna, Bulgaria 21–24 September 2007

Library of Congress Control Number: 2010929479

ISBN 978-90-481-8789-8 (PB)ISBN 978-90-481-8788-1 (HB)ISBN 978-90-481-8790-4 (e-book)

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Preface

This book summarizes the NATO Advanced Research Workshop (ARW) on “Nanoengineered Systems for Regenerative Medicine” that was organized under the auspices of the NATO Security through Science Program. I would like to thank NATO for supporting this workshop via a grant to the co-directors.

The objective of ARW was to explore the various facets of regenerative medi-cine and to highlight role of the “the nano-length scale” and “nano-scale systems” in defining and controlling cell and tissue environments. The development of novel tissue regenerative strategies require the integration of new insights emerging from studies of cell-matrix interactions, cellular signalling processes, developmental and systems biology, into biomaterials design, via a systems approach. The chapters in the book, written by the leading experts in their respective disciplines, cover a wide spectrum of topics ranging from stem cell biology, developmental biology, cell-matrix interactions, and matrix biology to surface science, materials processing and drug delivery. We hope the contents of the book will provoke the readership into developing regenerative medicine paradigms that combine these facets into clini-cally translatable solutions.

This NATO meeting would not have been successful without the timely help of Dr. Ulrike Shastri, Sanjeet Rangarajan and Ms. Sabine Benner, who assisted in the organization and implementation of various elements of this meeting. Thanks are also due Dr. Fausto Pedrazzini and Ms. Alison Trapp at NATO HQ (Brussels, Belgium). The commitment and persistence of Ms. Wil Bruins at Springer in get-ting this book published is very much appreciated. Last but not least, I would like to thank Prof. George Altankov my co-Director and Prof. Andreas Lendlein for their supportive role.

Co-Director, Freiburg V. Prasad Shastri

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Contents

1 Cell Adhesions and Signaling: A Tool for Biocompatibility Assessment ................................................................................................. 11.1 Introduction ........................................................................................ 21.2 Cell-Matrix Adhesions ....................................................................... 3

1.2.1 Integrin Receptors and Integrin Cytoplasmic Complexes ........................................................ 3

1.2.2 Focal Adhesions ..................................................................... 51.2.3 Fibrillar Adhesions ................................................................. 91.2.4 Three-Dimensional Matrix Adhesions ................................... 101.2.5 Dynamics of Cell Adhesions ................................................. 12

1.3 Concluding Remarks .......................................................................... 14References ................................................................................................... 15

2 Development of Provisional Extracellular Matrix on Biomaterials Interface: Lessons from In Vitro Cell Culture ............................................................................................... 192.1 Introduction ........................................................................................ 202.2 Initial Cell–Materials Interaction ....................................................... 20

2.2.1 Development of Focal Adhesion Complex ............................ 212.2.2 Substratum Properties Affect Focal

Adhesions Formation ............................................................. 222.3 Development of Provisional Extracellular Matrix

at the Biomaterials Interface .............................................................. 232.3.1 Development of Early Matrix ................................................ 232.3.2 Development of Late Fibronectin Matrix .............................. 25

2.4 Integrin Receptors Dynamics and Provisional Extracellular Matrix Formation ......................................................... 262.4.1 Studies on Integrin Dynamics ................................................ 262.4.2 Integrin Dynamics Depend on Substratum Properties ........... 29

2.5 Development of Extracellular Matrix at “Real” Biomaterials Interface ........................................................................ 302.5.1 Effects of Substratum Chemistry on Matrix Formation:

A View of Biosensors Application ......................................... 30

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2.5.2 Development of Extracellular Matrix on Biomimetic Hydroxyaptite Cements Surfaces ........................................... 33

2.5.3 Development of Extracellular Matrix on Different Rough Titanium Surfaces .................................. 36

References ................................................................................................... 41

3 Endothelial Progenitor Cells for Tissue Engineering and Tissue Regeneration .......................................................................... 453.1 Introduction ........................................................................................ 463.2 Vascular Networks for TE Constructs ................................................ 463.3 Sources of Human Endothelial Cells for TE

Vascular Networks ............................................................................. 473.3.1 Mature Vessel-Derived Endothelial Cells .............................. 473.3.2 Human Embryonic Stem Cells ............................................... 473.3.3 Blood-Derived EPCs .............................................................. 483.3.4 Bone Marrow-Derived Cells

for Tissue Vascularization ...................................................... 493.4 Blood-Derived EPCs for Creating Vascular Networks ...................... 50

3.4.1 Isolation and Culture of Human EPCs and HSVSMCs ....................................................................... 50

3.4.2 In Vivo Assay for Vascularization .......................................... 513.4.3 Summary: Vasculogenic Potential of Human EPCs .............. 51

References ................................................................................................... 52

4 Dermal Precursors and the Origins of the Wound Fibroblast .............. 554.1 Introduction ........................................................................................ 554.2 Mesenchymal Stem Cells ................................................................... 56

4.2.1 Marrow Derived Fibroblast Populations ................................ 574.2.2 Fibrocytes ............................................................................... 584.2.3 Factors for Mobilization and Recruitment

of Marrow Populations ........................................................... 614.3 Defining the Dermal Fibroblast ......................................................... 62

4.3.1 Dermal Progenitors ................................................................ 624.3.2 Evidence for Progenitor Populations ..................................... 63

4.4 Clinical Applications ......................................................................... 654.5 Novel Healing Properties of MSC ..................................................... 664.6 Summary ............................................................................................ 66References ................................................................................................... 67

5 Cell Based Therapies: What Do We Learn from Periosteal Osteochondrogenesis? ................................................... 715.1 General Introduction .......................................................................... 72

5.1.1 Cell Based Therapies ............................................................. 725.2 Periosteum .......................................................................................... 73

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5.2.1 Embryological Development of Bone, Cartilage, and Periosteum ....................................................................... 74

5.2.2 Periosteum in Bone and Cartilage Repair .............................. 755.3 Differential Survival of Periosteal Progenitor Cells

Versus Chondrocytes .......................................................................... 775.3.1 Introduction ............................................................................ 775.3.2 Cell Labeling and Scaffolds ................................................... 775.3.3 Chondrocytes Survive Transplantation

Better than Periosteal Progenitor ........................................... 785.3.4 Hyaluronan Increases the Number of Viable

Periosteal Progenitors ............................................................ 785.3.5 Discussion .............................................................................. 79

5.4 A Model to Study Periosteal Osteochondrogenesis In Vivo .............. 795.4.1 Introduction ............................................................................ 795.4.2 Periosteal Callus Recapitulates the Sequential

Steps of Endochondral Bone Formation ................................ 805.4.3 HIF-1a Activation and BMP Expression ............................... 815.4.4 Periostin Activation During Periosteal

Callus Formation .................................................................... 835.5 Repair of Osteochondral Defects with Cartilage

from Periosteum ................................................................................. 845.5.1 Introduction ............................................................................ 845.5.2 Improved Repair of Osteochondral Defects ........................... 845.5.3 Discussion .............................................................................. 86

5.6 Evidence That Endochondral Bone Formation Can Be Manipulated in the IVB ......................................................... 875.6.1 Introduction ............................................................................ 875.6.2 Growth Factors ....................................................................... 875.6.3 Hypoxia .................................................................................. 875.6.4 Calcium .................................................................................. 88

5.7 Discussion .......................................................................................... 88References ................................................................................................... 89

6 Bioreactor Systems in Regenerative Medicine ....................................... 956.1 Introduction ........................................................................................ 956.2 Bioreactors in Regenerative Medicine: Key Features ........................ 96

6.2.1 Cell Seeding on Three-Dimensional Matrices ....................... 966.2.2 Maintenance of a Controlled Culture Environment ............... 976.2.3 Physical Conditioning of Developing Tissues ....................... 996.2.4 Predicting Mechanical Functionality

of Engineered Tissues ............................................................ 1006.3 Bioreactor-Based Manufacturing of Tissue

Engineering Products ......................................................................... 1016.3.1 Automating Conventional Cell Culture Techniques .............. 103

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6.3.2 Automating Tissue Culture Processes .................................... 1046.3.3 Streamlining Tissue Engineering Processes........................... 1066.3.4 Different ‘Manufacturing’ Concepts ...................................... 109

6.4 Conclusions and Future Perspectives ................................................. 110References ................................................................................................... 111

7 Biomimetic Approaches to Design of Tissue Engineering Bioreactors ........................................................................... 1157.1 Introduction ........................................................................................ 1167.2 Cardiac Tissue Engineering ............................................................... 116

7.2.1 Myocardium (Cardiac Muscle) .............................................. 1167.2.2 Tissue Engineering ................................................................. 117

7.3 Cartilage Tissue Engineering ............................................................. 1207.3.1 Articular Cartilage ................................................................. 1207.3.2 Tissue Engineering ................................................................. 121

7.4 Conclusions ........................................................................................ 125References ................................................................................................... 126

8 The Nature of the Thermal Transition Influences the Shape-Memory Behavior of Polymer Networks .............................. 1318.1 Introduction ........................................................................................ 1318.2 Architectures of Different Polymer Networks ................................... 136

8.2.1 Synthesis ................................................................................ 1368.2.2 Thermomechanical Properties ............................................... 139

8.3 Shape-Memory Capability ................................................................. 1438.4 Degradation ........................................................................................ 1518.5 Summary ............................................................................................ 1538.6 Outlook .............................................................................................. 154References ................................................................................................... 155

9 Nanoengineered Systems for Regenerative Medicine Surface Engineered Polymeric Biomaterials with Improved Bio-Contact Properties ................................................... 1579.1 Introduction ........................................................................................ 1579.2 Polymeric Materials with Improved

Bio-Contact Properties ....................................................................... 1589.2.1 Strong Hydrophilic “Water-Like” Protein

Repellent Surfaces.................................................................. 1589.2.2 Protein Repelent Plasma Films .............................................. 1649.2.3 Polydimetylesiloxane (PDMS) with Improved

Interactions with Living Cells ................................................ 1649.3 Conclusions ........................................................................................ 172References ................................................................................................... 172

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10 Nanocomposites for Regenerative Medicine ......................................... 17510.1 Perspective ..................................................................................... 17510.2 A Nanophase for the Mechanical Reinforcement

of Tissue Engineering Scaffolds .................................................... 17710.2.1 Introduction ...................................................................... 17710.2.2 Nanocomposite Scaffolds for Hard

Tissue Engineering ........................................................... 17810.2.3 Nanocomposite Scaffolds for Soft

Tissue Engineering ........................................................... 18510.2.4 Conclusion and Future Direction ..................................... 189

10.3 A Nanophase for Drug Delivery Applications ............................... 19110.3.1 Introduction ...................................................................... 19110.3.2 Nanofibrous and Nanoporous Biomaterials ..................... 191

10.4 Conclusions .................................................................................... 201References ................................................................................................. 201

11 Role of Spatial Distribution of Matricellular Cues in Controlling Cell Functions ................................................................. 20711.1 Introduction .................................................................................... 20711.2 Cell-Matrix Interaction .................................................................. 210

11.2.1 Effect of Matrix on Cell Migration .................................. 21211.2.2 Matrix Effect on Embryonic Development ...................... 21811.2.3 Matrix Effect on Angiogenic Processes ........................... 222

11.3 Development of Novel Biofunctional Materials ............................ 22311.4 Conclusions .................................................................................... 228References ................................................................................................. 228

12 Materials Surface Effects on Biological Interactions .......................... 23312.1 Introduction .................................................................................... 233

12.1.1 First Generation ............................................................... 23512.1.2 Second Generation ........................................................... 23612.1.3 Third Generation .............................................................. 23712.1.4 Biomaterials for Substitution, Repair

and Regeneration ............................................................. 23812.1.5 Stem Cells Sources .......................................................... 239

12.2 Surface Modification to Improve Cell–Material Interactions ........ 24012.2.1 Surface Topography ......................................................... 24212.2.2 Surface Chemistry ............................................................ 243

12.3 Oxidation Treatment of NiTi Shape Memory Alloys to Obtain Ni-Free Surfaces and to Enhance Biocompatibility ....................... 244

12.4 Surface Characterisation of Fully Biodegradable Composite Scaffolds for Bone Regeneration ................................. 246

12.5 Micro and Nanopatterned Surfaces for Biomedical Applications .......................................................... 247

References ................................................................................................. 248

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13 Chemical and Physical Modifications of Biomaterial Surfaces to Control Adhesion of Cells................................................... 25313.1 General Introduction ...................................................................... 254

13.1.1 Basics of Cell Adhesion on Material Surfaces ................ 25413.1.2 Short Overview on Techniques to Modify

Surfaces of Biomaterials .................................................. 25713.1.3 Methods to Generate Nanostructured Surface ................. 258

13.2 Self Assembled Monolayers Based on Organosiloxanes ............... 25913.2.1 Background ...................................................................... 25913.2.2 Effect of SAM on Fibroblast Adhesion,

Spreading and Growth ..................................................... 26013.3 Photochemical Immobilization of Polyethylenglycol

on Hydrophobic Biomaterials ........................................................ 26413.3.1 Background ...................................................................... 264

13.4 Effect of Photochemical Immobilization of Poly (Ethylene Glycol) on Adhesion of Cells ........................................ 265

13.5 Application of Layer-by-Layer Technique on Charged Surfaces ...................................................................... 27013.5.1 Background of Layer-by-Layer Technique ...................... 27013.5.2 Application of LbL Technique

to Inorganic Surfaces ....................................................... 27113.5.3 Application of LbL Technique to Poly

(L-Lactide) (PLLA) for Tissue Engineering Applications................................................. 275

13.6 Summary and Conclusions ............................................................ 279References ................................................................................................. 279

14 Results of Biocompatibility Testing of Novel, Multifunctional Polymeric Implant Materials In-Vitro and In-Vivo ............................. 28514.1 Introduction .................................................................................... 286

14.1.1 Regenerative Medicine..................................................... 28614.1.2 Functionalized Implant Materials .................................... 28714.1.3 Clinical Application of Polymer-Based

Implant Materials ............................................................. 28714.2 Materials and Methods ................................................................... 290

14.2.1 Polymer-Based, Biodegradable Implant Materials .......... 29014.2.2 Sterilization Methods ....................................................... 29014.2.3 Investigation of In-Vitro Toxicity .................................... 29014.2.4 In-Vivo Assessment of Tissue Compatibility

of Biomaterials ................................................................. 29114.2.5 Animal Model .................................................................. 29114.2.6 Statistical Evaluation........................................................ 292

14.3 Results ............................................................................................ 29214.3.1 Detailed Evaluation of In-Vitro

Biocompatibility Testing .................................................. 292

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14.3.2 In-Vivo Assessment of Tissue Compatibility of Biomaterials ................................................................. 294

14.4 Discussion ...................................................................................... 296References ................................................................................................. 298

15 UFOs, Worms, and Surfboards: What Shapes Teach Us About Cell–Material Interactions .................................................... 30115.1 Introduction .................................................................................... 30215.2 Particulate Drug Delivery Systems ................................................ 302

15.2.1 Role of Physical Properties in Particle Function ............. 30415.3 Phagocytosis................................................................................... 306

15.3.1 Attachment ....................................................................... 30615.3.2 Internalization .................................................................. 307

15.4 Fabrication of Non-spherical Polymer Particles ............................ 30715.4.1 General Method ............................................................... 30815.4.2 Specific Shapes ................................................................ 309

15.5 Effect of Particle Shape on Phagocytosis ....................................... 31215.5.1 Shape Internalization ....................................................... 31215.5.2 Quantification of Shape ................................................... 31415.5.3 Correlation Between Internalization,

Shape and Size ................................................................. 31515.6 Design of Non-spherical Particles for Drug Delivery .................... 318

15.6.1 Optimal Shapes for Avoiding Phagocytosis ..................... 31815.6.2 Fabrication of Non-spherical Biodegradable

Drug Carriers ................................................................... 31915.7 Conclusions and Future Directions ................................................ 320

15.7.1 Drug Delivery .................................................................. 32015.7.2 Cell–Material Interactions ............................................... 320

References ................................................................................................. 321

16 Nano-engineered Thin Films for Cell and Tissue-Contacting Applications ............................................................................................. 32516.1 Introduction .................................................................................... 32516.2 Resonant Infrared Pulsed Laser Deposition of Thin Films ............ 32616.3 Resonant Infrared Laser Ablation of Thermally

Labile Polymers ............................................................................. 32816.3.1 Poly(Ethylene Glycol) – PEG .......................................... 32816.3.2 Poly(DL-Lactide-Co-Glycolide) – PLGA ....................... 33116.3.3 Proteins and Nucleic Acids .............................................. 33216.3.4 Poly(Tetrafluoroethylene) ................................................ 334

16.4 Deposition of Functional Nanoparticles ........................................ 33616.5 Discussion and Conclusions .......................................................... 338

16.5.1 Evidence for Low-Temperature Character of RIR-PLD ...................................................................... 339

16.5.2 The RIR-PLD Mechanism and Its Consequences ........... 340

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16.5.3 Prospects for Table-Top RIR-PLD Laser Technology ............................................................. 341

16.6 Conclusions .................................................................................... 343References ................................................................................................. 344

17 Injectable Hydrogels: From Basics to Nanotechnological Features and Potential Advances ........................................................... 34717.1 Introduction .................................................................................... 347

17.1.1 The Concept of Scaffold .................................................. 34817.2 Hydrogels ....................................................................................... 350

17.2.1 Methods of Preparation .................................................... 35117.3 Properties Exploitable in Tissue Engineering ................................ 35817.4 Major Issues of Injectable Materials in Tissue Engineering .......... 36417.5 Conclusions .................................................................................... 365References ................................................................................................. 365

18 Polyelectrolyte Complexes as Smart Nanoengineered Systems for Biotechnology and Gene Delivery ..................................... 38318.1 Introduction .................................................................................... 37918.2 Complex Formation and Competitive Reactions

in Solutions of Oppositely Charged Polyelectrolytes .................... 38318.2.1 Polyelectrolyte Complexes and Their Properties ............. 383

18.3 DNA-Containing PECs and Their Properties ................................ 38918.3.1 Stability of DNA-Containing Complexes ........................ 38918.3.2 Selectivity of Competitive Reactions

in DNA Solutions ............................................................. 39018.3.3 Complexing of DNA with Polycations

for Cell Transfection ........................................................ 39118.4 Complexes of Proteins with Oppositely Charged Polyions ........... 393

18.4.1 Soluble Complexes and Competitive Reactions in Their Solutions ............................................ 393

18.4.2 Artificial Chaperones ....................................................... 39618.5 Polyelectrolyte Multilayer Films and Capsules ............................. 397References ................................................................................................. 399