This document summarizes a presentation on bioinspired strategies for bone regeneration. It discusses how biomimetic calcium phosphate materials can mimic bone's composition, structure, and properties at multiple length scales to promote bone regeneration. Specifically, it describes how biomimetic calcium phosphates with nanostructured features and interconnected macroporosity can enhance osteoinduction, osteogenesis, and osteoimmunomodulation both in vitro and in vivo compared to conventional calcium phosphate ceramics. The document provides examples of how biomimetic calcium phosphate foams and 3D printed scaffolds regenerate bone in preclinical studies better than controls due to their ability to intrinsically induce bone formation through their biomimetic design.

1. Bioinspired strategies for bone
regeneration
Maria-Pau Ginebra
Biomaterials, Biomechanics and Tissue Engineering Group
Dept. Materials Science and Metallurgy
Universitat Politècnica de Catalunya, Barcelona, Spain
Maria.pau.ginebra@upc.edu
B-MRS, Balneario Camboriù, 22-26 September 2019

2. http://biomaterials.upc.edu/en
Department of Materials Science and Metallurgy
Biomaterials, Biomechanics and Tissue Engineering Group

3. Outline
Biomimetic materials for bone regeneration
Porosity: how relevant is material
architecture at different lengthscales?
Osteoinducction and osteogenesis
Nanostructure and osteoimmunomodulation
1
2
3
4

4. 2,000,000 bone grafting procedures are performed annually
worldwide to restore bone function
GreenwaldAS et al. J Bone Joint SurgAm.
2001;83-A, suppl 2, part 2:98-103.
Bone grafting
4

5. Bone grafting
Non-union fracture Comminuted fracture Alveolar ridge defect
Bone tumor/cyst Spinal disc degeneration
5

6. Bone regeneration: the problem
Sedentary lifestyle
Ageing population

7. 7
Bone grafting

8. Bone composition and structure
M. Sadat-Shojai et al. / Acta Biomaterialia 9 (2013) 7591–7621

9. The “form” of biominerals in bone
Fratzl and Weinkamer. Progress in Materials
Science 52 (2007) 1263–1334
Mineral crystal shape/size:
Nanocrystals:
Platelets
(20-60 x 10-20 x 2-5 nm3)
Large specific surface area

10. H.P. Schwarcz, E.A. McNally, G.A. Botton. Journal of Structural Biology 188 (2014) 240–248
Extrafibrillar mineral
The “form” of minerals in bone

11. E.A. McNally, H.P. Schwarcz, G.A. Botton, A.L. Arsenault. PLoS ONE 7(2012) 1 e29258
Extrafibrillar mineral
(40 nm)
(27 nm)
(45 nm Ø)
200 nm
The “form” of minerals in bone

12. The “form” of biominerals in bone
HighHigh reactivity
S. Cazalbou et al., J Mater Chem 2004, 14: 2148-53

13. Bone remodeling
- 2-5% cortical bone remodeled every year
- Trabecular bone 10 times faster

14. C: Bone mineral
phase
A,B: High-T
Synthetic HA
LeGeros, (1991)
HIGH T SINTERED HA
Stoichiometric
Highly crystalline
Low porosity
Low SSA
Non resorbable in vivo
14
High Temperature Calcium Phosphates:
Sintered ceramics

15. Low-T CaPs: Calcium Phosphate CementsBiomimetic Calcum Phosphates
3Ca3(PO4)2 + H2O → Ca9(HPO4)(PO4)5(OH)
α-Tricalcium phosphate Ca-deficient Hydroxyapatite (CDHA)

16. M.P. Ginebra et al., Adv Drug Del Rev (2012) 1μm
1μm
Fine powder
Coarse powder

17. High T CaP ceramics vs low T biomimetic CaP
SSA < 5 m2/g SSA ≈ 20 m2/g SSA ≈ 40 m2/g

18. 250 µm
M. Espanol et al., Acta Biomaterialia (2009), 5: 2752–2762
3 α-Ca3(PO4)2 + H2O → Ca9(HPO4) (PO4)5(OH)
L/P= 0.65 L/P= 0.35
Textural properties of nanostructured CDHA
Liquid/powder ratio

19. 250 µm
M. Espanol et al., Acta Biomaterialia (2009), 5: 2752–2762
3 α-Ca3(PO4)2 + H2O → Ca9(HPO4) (PO4)5(OH)
L/P= 0.65 L/P= 0.35
Textural properties of nanostructured CDHA

20. 250 µm
M. Espanol et al., Acta Biomaterialia (2009), 5: 2752–2762
3 α-Ca3(PO4)2 + H2O → Ca9(HPO4) (PO4)5(OH)
L/P= 0.65 L/P= 0.35
Textural properties of nanostructured CDHA

21. Outline
Biomimetic materials for bone regeneration
Porosity: how relevant is material
architecture at different lengthscales?
Osteoinducction and osteogenesis
Nanostructure and osteoimmunomodulation
1
2
3
4

22. Acidic degradation mimicking osteoclastic environment
CDHA
CO3-CDHA
α-Ca3(PO4)2+ H2O ↑T β-TCP
SHA
Osteoclast
Osteoclast
Hydrochloric acid - HCl
In vitro acidic degradation
A. Diez-Escudero et al., Acta Biomaterialia (2017) 22
Solubility
SHA < CDHA < β-TCP
- logKps 116.8 85.1 28.9
Anna Diez Escudero

23. Acidic degradation
15mL of 0.14M NaCl and 0.01M HCl solution
Medium refresh every hour (8 hours)
Weight loss
Acidic degradation mimicking osteoclastic environment
CDHA
CO3-CDHA
α-Ca3(PO4)2+ H2O ↑T β-TCP
SHA
Osteoclast
Osteoclast
Hydrochloric acid - HCl
In vitro acidic degradation
A. Diez-Escudero et al., Acta Biomaterialia (2017) 23

24. 500 nm
β-TCP
500 nm 500 nm
A. Diez-Escudero et al., Acta Biomaterialia 60 (2017):81-92
In vitro degradation
0.01M hydrochloric acid in 0.14M sodium chloride at 37 oC for 8h.
*
24
In vitro acidic degradation

25. Rizzoli Orthopaedic
Institute, Dr. Baldini
Diez-Escudero, A. Tissue Engineering Part C: Methods, 2017, 23(2), 118–124.
Ciapetti G., et al., Acta Biomaterialia, 50(2017):102–113
Anna Diez Escudero
Osteoclastogenesis and osteoclastic resorption
25

26. F-HA
Osteoclastogenesis and osteoclastic resorption
26

27. F-HA
F-HA
Osteoclastogenesis and osteoclastic resorption
27

28. Cell

29. The biomaterial as a scaffold
The cells
29

30. Cell

31. The foaming agents must essentially be:
• Water soluble
• Present good foamability and foam stability
• Biocompatible
Types of foaming agents:
• LMW synthetic surfactants: Non ionic surfactants approved by the FDA for parenteral use
(Tween, Pluronic)
• Macromolecular surfactants: Protein based foaming agents, with additional biological
functionalities.
• Albumen
• Soy-bean derived polymers
• Gelatine
Montufar et el. Acta Biomater 6 (2010) 876–885
Ginebra et al. JBMRA (2007) 351-361
Perut et el. Acta Biomater 7 (2011) 1780–1787
Montufar et al. Mat Sci Eng C 31(2011):1498-1504
Self-setting Calcium phosphate foams

32. Foaming
.
1 mm 1 mm
(Tween 80)
Montufar EB, et al. Acta Biomater 2010;6:876-85
Pastorino et al. Acta Biomater 12 (2015) 250–59
Kovtun et al. Acta Biomater 12 (2015) 242-49
Self-setting Calcium phosphate foams

33. Foaming
.
1 mm 1 mm
Self-setting Calcium phosphate foams

34. Controlled drug delivery from CPC
David Pastorino
t
Dose Dose Dose
t
Drug delivery from non-swellable matrices
Priya Khurana
Foams: Enhanced fluid circulation
34

35. CaP Foams as Drug delivery systems
Doxycycline Hyclate (0.88-3.52 wt%)
D. Pastorino, et al. Acta Biomaterialia12 (2015) 250–259
G. Mestres et al., Mater Sci & Eng C 97(2019) 84-95
Foaming agent: Tween 80
Tetracyclines chelate Ca2+ ions
35

36. CaP Foams as Drug delivery systems
D. Pastorino, et al. Acta Biomaterialia12 (2015) 250–259 36

37. Espanol, M et al. Biointerphases 7 (2012) 37
Protein retention:
size exclusion chromatography
3 proteins:
• Same chemical affinity,
• Different sizes
Montse Espanol

38. Espanol, M et al. Biointerphases 7 (2012) 37
Protein retention:
size exclusion chromatography

39. Espanol, M et al. Biointerphases 7 (2012) 37
Nanostructured CDHA: protein retention

40. 3D-printing with self-setting CaP inks
Yassine Maazouz
Layer by layer nozzle micro-extrusion Calcium
phosphate
paste

41. 3D-printing with self-setting CaP inks
Classic route
Biomimetic route
Hardening post-treatments
Cesarano, J., Baer, T. A., & Calvert, P. (1997), Proc Solid Freeform Fabr Symp; Smay, J., & III, J. C. (2002), Langmuir;
Michna, S., Wu, W., & Lewis, J. A. (2005), Biomaterials; Miranda, P., Saiz, E., Gryn, K., & Tomsia, A. P. (2006), Acta
Biomater

42. • Injectability
• Pseudoplastic
behaviour
• Self-supporting 3D
structures
3D-printing with self-setting CaP inks
1 mm 1 mm
Yassine Maazouz
Maazouz et al., J. Mater. Chem. B 2 (2014), 5378 – 5386
Maazouz et al., Acta Biomaterialia 49 (2017) 563-574
Raymond et al., Acta Biomaterialia 75 (2018): 451-462

43. Outline
Biomimetic materials for bone regeneration
Porosity: how relevant is material
architecture at different lengthscales?
Osteoinducction and osteogenesis
Nanostructure and osteoimmunomodulation
1
2
3
4

44. Intrinsic osteoinductive biomaterials
o Tunning intrinsic physico-chemical
parameters:
- Chemical Composition
- Macropore architecture
- Microstructre
Barradas AM, et al.2011
Arboleya L, et al. 2013
Janicki P, et al.2011
Engineered osteoinductive biomaterials
o Addition of exogenous growth factors
(BMPs)
o Drawbacks:
- Undesirable side effects
- Variable efficacy
- High costs
Osteoinduction

45. SSA: 0.5-6 m2/g SSA: 20-60 m2/g
Biomimetic Calcium Phosphates

46. Foaming  Interconnected concave macropores
Montufar EB, et al. Acta Biomater 2010;6:876-85.
3D Inkjet printing  Interconnected prismatic macropores
Maazouz Y, et al. Acta Biomaterialia 2018, in press
1 mm 1 mm
1 mm 1 mm
FOAMS
ROBOCASTED
The scaffolds

47. Foams
Macrostructure
Micro/nanostru
cture
3D-Printed
Macrostructure
47
1 µm
BCP
(HA:β-TCP80:20)
Micro/nanostru
cture 2 µm2 µm
2 μm
Macroporosity (%) 52.3 50.7 47.4
SSA (m2/g) 38.5 19.3 0.5
Macroporosity (%) 54.6 54.5
SSA (m2/g) 35.1 17.8

48.  Ectopic implantation (epaxial muscles) in Beagle dog, 6 and 12 weeks
 Orthotopic Implantation (femur) in Beagle dog, 6 and 12 weeks
In vivo study
Albert Barba

49. • μ-CT threshold adjustment
µ-CT BS-SEM µ-CT 3D quantification
S. Lewin et al., Biomedical Materials (2017)
In vivo study

50. Ectopic bone formation
A. Barba et al., ACS Applied Materials and Interfaces 2017, 9, 41722−36)
6 weeks12 weeks
50
Osteoinduction is accelerated in biomimetic nanostructured HA
with concave macroporosity

51. BCP-F : 0/6
Osteoinduction: 6 weeks
BCP-F: 0/6
A. Barba et al., ACS Applied Materials and Interfaces (2017)

52. 1 mm
1 mm 50 μm200 μm
200 μm 50 μm1 mm
F-Foam
A. Barba et al., ACS Applied Materials and Interfaces (2017)
Osteoinduction: 6 weeks

53. Osteoinduction: 12 weeks
CDHA-F: 6/6
1 mm 1 mm
BCP-F: 4/6 CDHA-Rob: 1/6
1 mm
A. Barba et al., ACS Applied Materials and Interfaces (2017)

54. F-Foam
Masson’s Trichrome
Osteoinduction: 12 weeks

55. Fine-CDHACoarse-CDHA
Scaffold degradation
CDHA-F-Rob
55
BCP
CDHA-C-Rob

56. F-Foam
Orthotopic implantation
Monocortical defect in the femur of Beagle dogs
12 weeks

57. Orthotopic implantation: 6 weeks
C-FoamF-RobF-Foam
A. Barba et al., Acta
Biomaterialia 79 (2018):
135-147

58. Orthotopic implantation: 12 weeks
C-
Foam
F-RobF-
Foam
A. Barba et al., Acta
Biomaterialia 79 (2018):
135-147

59. Orthotopic implantation (μ-CT 3D quantification)
<
<
A. Barba et al., Acta Biomaterialia 79 (2018): 135-147

60. Outline
Biomimetic materials for bone regeneration
Porosity: how relevant is material
architecture at different lengthscales?
Osteoinducction and osteogenesis
Nanostructure and osteoimmunomodulation
1
2
3
4

61. (i) Direct trigger of osteogenic differentiation of MSCs through
physicochemical properties or local accumulation/production of
endogenous osteoinductive proteins such as BMP-2
(ii) Osteoimmunomodulation: indirect trigger of osteogenic
differentiation through the inflammatory response or osteoclastogenesis
Mechanisms of osteoinduction
61

62. Fine-CDHA
rMSCs
Coarse-CDHA
A. Barba et al., ACS Applied Materials and Interfaces (2019)
In vitro study:
Effect of nanostructure on MSCs
CO3-CDHA
BCP
62

63. In vitro study:
Effect of nanostructure on MSCs
63

64. POROSITY
TOPOGRAPHY
OSTEOIMMUNOMODULATIONIMMUNOMODULATION
RAW 264.7
RAW 264.7- CDHA
environment
SaOS-2
Nanostructure and osteoimmunomodulation
RAW 264.7
64
Prof. Xiao
Joanna SadowskaF65
C65
4μm
4μm
J. Sadowska et al., Biomaterials 181 (2018): 318-332

65. 65
Gene expression of osteogenic markers
Nanostructure and osteoimmunomodulation
J. Sadowska et al., Biomaterials 181 (2018): 318-332

66. 66
Protein production: Western Blot
Nanostructure and osteoimmunomodulation
J. Sadowska et al., Biomaterials 181 (2018): 318-332

67. 67
Nanostructure and osteoimmunomodulation
J. Sadowska et al., Biomaterials 181 (2018): 318-332
F35 F65 C35 C65 control
-10
0
10
20
30
40
50
ALParea/cellarea[%]
F35 F65 C65C35 SaOS-2 SaOS-2
control+ control-
a
a
b
a
b
*&
200 µm
ALP, F-actin, nuclei
67
Immunohistochemistry: ALP expression
J. Sadowska et al., Biomaterials (2018)

70.  There is room for improvement of synthetic bone grafts
 Nanostructure and macropore geometry are key parameters controlling
osteoinduction and osteogenesis by CaP
 Biomimetic processing allows pushing the material associated
osteoinduction beyond the limits of microstructured CaP ceramics
 Advanced technologies, like 3D printing, open up new possibilities in the
design of patient-specific bone grafts
Summary

71. Acknowledgements
Funding bodies
J. Franch, University of Barcelona
M.C. Manzanares, Univ. Autònoma de Barcelona
C. Persson, Uppsala University, Sweden
P. Layrolle, INSERM, France
C. Aparicio, Univ. Minneapolis, USA
Y. Xiao, Queensland Univ of Technology,Brisbane,
Australia
M. Santin, Univ. Brighton, UK
N. Baldini, G. Ciapetti, Rizzoli Institute, Bolonia, Italy
A. Ignatius, Univ Ulm, Germany
O. Hoffmann, Univ Vienna, Austria
P. Boudeville, Univ. Montpellier, France
Collaborations
71

72. maria.pau.ginebra@upc.edu
https://biomaterials.upc.edu/ca
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