Table of Contents

Contributors
Short biographies of the contributors
Preface

1. General aspects of extracellular vesicles in bone formation and remodeling
Antonio Maurizi and Anna Teti

1. Introduction
2. Communications between bone cells
3. Extracellular vesicles
4. Bone cell overview
5. Bone marrow cells interacting with skeletal cells
6. Extracellular vesicle trafficking in the bone microenvironment
7. Extracellular vesicles in fracture healing
8. Extracellular vesicles and bone cancer
9. Medical applications
10. Conclusions

List of abbreviations
Acknowledgments
References

2. Physiological biomineralization. The properties and role of matrix vesicles in skeletal and dental calcifications
René Buchet, Saida Mebarek, Slawomir Pikula, Agnieszka Strzelecka-Kiliszek, David Magne, Leticia Fernanda Duffles, Thaise Mayumi Taira, Massimo Bottini, Pietro Ciancaglini, José Luis Millán and Sandra Yasuyo Fukada

1. Introduction
2. Biomineralization in teeth
3. Biomineralization in bones
4. Determinants of mineralization support the functions of matrix vesicles
5. Conclusions

List of abbreviations
Acknowledgments
References

3. Pathological biomineralization. Part I: mineralizing extracellular vesicles in cardiovascular diseases
Rene Buchet, Slawomir Pikula, Agnieszka Strzelecka-Kiliszek, Massimo Bottini, Ana Paula Ramos, Pietro Ciancaglini, Jose Luis Millan and Saida Mebarek

1. Introduction
2. Extracellular vesicles: origin and classification
3. Extracellular vesicles related to cardiovascular diseases
4. Extracellular vesicles: biomarkers of cardiovascular diseases
5. Extracellular vesicles as drug delivery vehicles in cardiovascular disease
6. Conclusions

List of abbreviations
Acknowledgments
References

4. Pathologic biomineralization: part II: mineralizing extracellular vesicles in osteoarthritis
Ann K. Rosenthal

1. Introduction
2. Historical perspective
3. Characterization of ACVs
4. ACVs from osteoarthritis cartilage
5. Factors affecting ACV mineralization
6. Conclusions and future directions

List of abbreviations
Acknowledgments
References

5. The biochemistry of mineralizing extracellular vesicles.
Part I: The role of phosphatases
Flavia Amadeu de Oliveira, Cintia Kazuko Tokuhara, Lucas Fabricio Bahia Nogueira, Juçara Gastaldi Cominal, Luiz Henrique da Silva Andrilli, Sonoko Narisawa, Colin Farquharson, Massimo Bottini, Ana Paula Ramos, Pietro Ciancaglini and José Luis Millán

1. Inorganic phosphate (Pi) and pyrophosphate (PPi): a physicochemical balance
2. The molecules regulating the Pi/PPi ratio
3. Tissue-nonspecific alkaline phosphatase
4. Ectonucleotide pyrophosphatase/phosphodiesterase 1 and other ENPPs
5. Nucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-50-nucleotidase (CD73)
6. PHOSPHO1
7. Can Na,K-ATPase act as a phosphatase?
8. Phosphate transporters
9. Genetic diseases caused by altered Pi/PPi ratio
10. Therapeutic approaches to normalizing the Pi/PPi ratio
11. Conclusions

List of abbreviations
Acknowledgments
References

6. The biochemistry of mineralizing extracellular vesicles.
Part II: Annexins 127
Slawomir Pikula, Agnieszka Strzelecka-Kiliszek, René Buchet, Saida Mebarek, Laurence Bessueille, Thierry Granjon, David Magne, Lilianna Weremiejczyk, Agnieszka Kinga Seliga and Joanna Bandorowicz-Pikula

1. Introduction
2. Annexins
3. Annexin knockout animal models
4. Annexins in mineralization-competent cells
5. Annexins in media vesicles and matrix vesicles
6. In vitro properties of annexins
7. Conclusions

List of abbreviations
Acknowledgments
References

7. Calcium and phosphate and their role in matrix vesicles: A biological view
Larwsk Hayann, Pietro Ciancaglini, Ana Paula Ramos and Dobrawa Napierala

1. Calcium (Ca2þ) and inorganic phosphate (PO3/4-/Pi) ions are essential for life
2. Ca2þ and phosphate ions in mineralization physiology and pathology
3. Cellular origins of matrix vesicles
4. Formation of mineralization-competent matrix vesicles requires osteo/chondrogenic molecular phenotype of the cells
5. Conclusions

List of abbreviations
Acknowledgments
References

8. Autophagy in bone metabolism and its possible role on the function of mineralizing extracellular vesicles
Marcos A.E. Cruz, Heitor G. Sebinelli, Pietro Ciancaglini, Massimo Bottini and Ana Paula Ramos

1. Molecular overview of autophagy
2. The canonical role of autophagy on bone maintenance
3. The noncanonical role of autophagy on bone mineralization
4. The role of autophagy on the release of mineralizing extracellular vesicles
5. Induced autophagy decreases smooth muscles cells calcification
6. Conclusions

List of abbreviations
Acknowledgments
References

9. The roles of mineralizing extracellular vesicles in cellecell communication
Tomoko Minamizaki, Shohei Kohono and Yuji Yoshiko

1. Introduction
2. Proteins in mineralizing EVs
3. miRNAs in mineralizing EVs
4. Conclusion
List of abbreviations
Acknowledgments
References

10. Working with mineralizing extracellular vesicles.
Part I: Purification techniques
René Buchet, Slawomir Pikula, Agnieszka Strzelecka-Kiliszek, David Magne, Massimo Bottini and Saida Mebarek

1. Discovery of matrix vesicles
2. Purification of matrix vesicles from the growth plates and epiphyseal cartilage of chicken embryos
3. Purification of matrix vesicles from rodent primary chondrocytes
4. Purification of matrix vesicles from osteoblast cell lines and from primary osteoblasts
5. Conclusion

List of abbreviations
Acknowledgments
References

11. Working with mineralizing extracellular vesicles.
Part II: Proteomic profiling
René Buchet, Saida Mebarek, Agnieszka Strzelecka-Kiliszek, Massimo Bottini and Slawomir Pikula

1. Introduction
2. Essential proteins found prior to the proteomic analysis
3. Proteomic analysis of matrix vesicles extracted from growth plate cartilage of chicken embryo
4. Proteomic analysis of matrix vesicles extracted from osteoblast cell lines
5. Comparative analysis of proteomes of matrix vesicles and articular cartilage vesicles
6. Comparative analysis of matrix vesicles and of extracellular vesicles proteomes
7. Conclusions

List of Abbreviations
Acknowledgments
References

12. Working with mineralizing extracellular vesicles.
Part III: The nucleational core
Ana Paula Ramos, Marcos Antonio Eufrasio Cruz, Pietro Ciancaglini, Saida Mebarek, René Buchet, José Luis Millan and Massimo Bottini

1. Introduction
2. Physicochemical aspects of biomineralization
3. Discovery of the nucleational core
4. Isolation and characterization of the nucleational core
5. Parameters driving the formation of phosphatidylserine complexes and amorphous calcium phosphate
6. Use of the AFM in the characterization of the nucleational core
7. Conclusions

List of abbreviations
Acknowledgments
References

13. Working with mineralizing extracellular vesicles
Part IV: Biomimetic models
Maytê Bolean, Heitor G. Sebinelli, Luiz H.S. Andrilli, Bruno Z. Favarin, Ekeveliny A. Veschi, Marcos A.E. Cruz, Rene Buchet, Saida Mebarek, José Luis Millán, Massimo Bottini, Ana Paula Ramos and Pietro Ciancaglini

1. Introduction
2. Proteoliposomes as biomimetic models to assess the role of TNAP in biomineralization
3. Proteoliposomes as biomimetic models to assess the role of Naþ,Kþ-ATPase and NPP1 in biomineralization
4. Proteoliposomes as biomimetic models to assess the role of annexins in biomineralization
5. Langmuir films as biomimetic models of matrix vesicles
6. Conclusions

List of abbreviations
Acknowledgments
References

14. Working with mineralizing extracellular vesicles.
Part V: Use of Mineralizing extracellular vesicles in bone regeneration
Larwsk Hayann, Maryanne Trafani Melo, Lucas Fabricio Bahia Nogueira, Pietro Ciancaglini, Massimo Bottini and Ana Paula Ramos

1. Introduction
2. Biogenesis of extracellular vesicles
3. Matrix vesicles: A special class of extracellular vesicles
4. Conclusions

List of abbreviations
Acknowledgments
References

Index

About the Author

Massimo Bottini is Associate Professor of Biochemistry at the University of Rome Tor Vergata (Rome, Italy) since 2015. As previous positions, he has been Affiliate Associate Professor at the Sanford Burnham Prebys (La Jolla, USA) from 2015 to 2022, and Visiting Professor at the University of the Chinese Academy of Sciences (Beijing, People’s Republic of China) from 2017 to 2019 and at the University of São Paulo (São Paulo, Brazil) in 2020. He has been the recipient of fellowships/grants from the Juvenile Diabetes Research Foundation, the Arthritis National Research Foundation, the European Commission, the University of Rome Tor Vergata, and the Chinese Academy of Sciences. He leads the Laboratory of Biochemical Nanotechnology at the University of Rome Tor Vergata. His research is mostly focused on the characterization of the biochemical and biophysical properties of the extracellular vesicles released during physiologic and ectopic biomineralization processes. Ana Paula Ramos is an Associate Professor at the University of Paulo-Brazil. She got her first degree in Chemistry in 2004 and a Ph.D. in Physical Chemistry in 2009. She is currently an associate Professor of Physical Chemistry, at the University of São Paulo Brazil and a researcher of the Brazilian National Council for Scientific and Technological Development (CNPq). She is the coordinator of the Physical Chemistry of Colloids and Surfaces Laboratory, with expertise in surfaces modification and characterization by microscopic and spectroscopic techniques. Her main research interests are the synthesis of biomaterials for bone replacement and regeneration. In special, her current projects focus on the use of biomimetic matrices inspired by the interaction of phospholipids and osteogenic proteins found in matrix vesicles as modifiers of metals used for dental implants. Moreover, she is currently investigating the role of strontium on physiological and pathological mineralization.