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Nanostructure and osteoimmunomodulation
J. Sadowska et al., Biomaterials 181 (2018): 318-332
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Immunohistochemistry: ALP expression
J. Sadowska et al., Biomaterials (2018)](https://image.slidesharecdn.com/mariapauplenaria-191022131048/75/Bioinspired-strategies-for-bone-regeneration-67-2048.jpg)
Bioinspired strategies for bone regeneration.
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.
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Bioinspired strategies for bone regeneration.
- 1. Bioinspired strategies forbone 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 MaterialsScience and Metallurgy Biomaterials, Biomechanics and Tissue Engineering Group
- 3. Outline Biomimetic materials forbone 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 graftingprocedures 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 fractureComminuted fracture Alveolar ridge defect Bone tumor/cyst Spinal disc degeneration 5
- 6. Bone regeneration: theproblem Sedentary lifestyle Ageing population
- 7.
- 8. Bone composition andstructure M. Sadat-Shojai et al. / Acta Biomaterialia 9 (2013) 7591–7621
- 9. The “form” ofbiominerals 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” ofbiominerals 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: CalciumPhosphate CementsBiomimetic Calcum Phosphates 3Ca3(PO4)2 + H2O → Ca9(HPO4)(PO4)5(OH) α-Tricalcium phosphate Ca-deficient Hydroxyapatite (CDHA)
- 16. M.P. Ginebra etal., Adv Drug Del Rev (2012) 1μm 1μm Fine powder Coarse powder
- 17. High T CaPceramics vs low T biomimetic CaP SSA < 5 m2/g SSA ≈ 20 m2/g SSA ≈ 40 m2/g
- 18. 250 µm M. Espanolet 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. Espanolet 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. Espanolet 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 forbone regeneration Porosity: how relevant is material architecture at different lengthscales? Osteoinducction and osteogenesis Nanostructure and osteoimmunomodulation 1 2 3 4
- 22. Acidic degradation mimickingosteoclastic 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 of0.14M 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 nm500 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 osteoclasticresorption 26
- 27. F-HA F-HA Osteoclastogenesis and osteoclasticresorption 27
- 28.
- 29. The biomaterial asa scaffold The cells 29
- 30.
- 31. The foaming agentsmust 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 1mm (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 1mm Self-setting Calcium phosphate foams
- 34. Controlled drug deliveryfrom CPC David Pastorino t Dose Dose Dose t Drug delivery from non-swellable matrices Priya Khurana Foams: Enhanced fluid circulation 34
- 35. CaP Foams asDrug 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 asDrug delivery systems D. Pastorino, et al. Acta Biomaterialia12 (2015) 250–259 36
- 37. Espanol, M etal. Biointerphases 7 (2012) 37 Protein retention: size exclusion chromatography 3 proteins: • Same chemical affinity, • Different sizes Montse Espanol
- 38. Espanol, M etal. Biointerphases 7 (2012) 37 Protein retention: size exclusion chromatography
- 39. Espanol, M etal. Biointerphases 7 (2012) 37 Nanostructured CDHA: protein retention
- 40. 3D-printing with self-settingCaP inks Yassine Maazouz Layer by layer nozzle micro-extrusion Calcium phosphate paste
- 41. 3D-printing with self-settingCaP 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 forbone regeneration Porosity: how relevant is material architecture at different lengthscales? Osteoinducction and osteogenesis Nanostructure and osteoimmunomodulation 1 2 3 4
- 44. Intrinsic osteoinductive biomaterials oTunning 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/gSSA: 20-60 m2/g Biomimetic Calcium Phosphates
- 46. Foaming Interconnectedconcave 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-TCP80:20) Micro/nanostru cture 2µm2 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 thresholdadjustment 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 mm50 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.
- 55.
- 56. F-Foam Orthotopic implantation Monocortical defectin the femur of Beagle dogs 12 weeks
- 57. Orthotopic implantation: 6weeks C-FoamF-RobF-Foam A. Barba et al., Acta Biomaterialia 79 (2018): 135-147
- 58. Orthotopic implantation: 12weeks C- Foam F-RobF- Foam A. Barba et al., Acta Biomaterialia 79 (2018): 135-147
- 59. Orthotopic implantation (μ-CT3D quantification) < < A. Barba et al., Acta Biomaterialia 79 (2018): 135-147
- 60. Outline Biomimetic materials forbone regeneration Porosity: how relevant is material architecture at different lengthscales? Osteoinducction and osteogenesis Nanostructure and osteoimmunomodulation 1 2 3 4
- 61. (i) Direct triggerof 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 etal., ACS Applied Materials and Interfaces (2019) In vitro study: Effect of nanostructure on MSCs CO3-CDHA BCP 62
- 63. In vitro study: Effectof 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 ofosteogenic markers Nanostructure and osteoimmunomodulation J. Sadowska et al., Biomaterials 181 (2018): 318-332
- 66. 66 Protein production: WesternBlot 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 isroom 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
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