Nanocrystalline apatite: from fundamentals to bone substitute materials
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1 MP1301- NEWGEN WG1 Nanocrystalline apatite: from fundamentals to bone substitute materials Christèle Combes (INPT, CIRIMAT, France) Margarida Almeida (University AVEIRO, CICECO, Portugal) Paulo Quadros (Fluidinova, Portugal) Nantes, 7 th and 8 th May 2014
2 Nanocrystalline apatites: from fundamentals to bone substitute materials Christèle Combes, Christophe Drouet, Sophie Cazalbou, Stéphanie Sarda, Christian Rey University of Toulouse, CIRIMAT, UPS-INPT-CNRS, ENSIACET, Toulouse, France NEWGEN 1st Workshop, 7th-8th may 2014, School of Dentistry, Nantes, France
3 Biological mineralisation properties Mineral elements and structures quite similar Silicates Calcium carbonates Calcium phosphates Adaptable and elaborate architecture ( autorepair (growth and Resistance and lightness Saving energy (low temperatures, recycling, renewal)! scientists A model for material
4 Introduction Characteristics of biological mineralisations (vertebrates) Bone Architecture Composition of tissue Tooth (enamel) % mineral Crystal characteristics Nanocrystals (20-60 x x 2-5 nm) Platelets High specific surface area % mineral large crystals Needles low specific surface area
5 Introduction 16 Å Structure and composition of bone tissue/mineral 40 Å Collagen fibers Compact bone structure [A. Meunier, Galerne 2001] Empty space (HAp location ) Ca 10 (PO 4 ) 6 (OH) 2 CO 3 2- HPO 4 2- Bone mineral: Ca 8,3 r 1,7 (PO 4 ) 4,3 (HPO 4, CO 3 ) 1,7 (OH ) 0,3 r 1,7 X x27 nm (4 y-o bovine) [Kim et al. 1995] [Legros et al. 1986]
6 Introduction A biomimetic approach Formation Synthetic model compounds (nanocrystalline apatites) Evolution Substitution Repair Bone tissue physico-chemical characterisation Bioactive materials
7 Synthesis, properties and applications of nanocrystalline apatites analogous to bone mineral
8 Synthesis Nanocrystalline apatites analogous to bone mineral Synthesis Ca 10-x r x (PO 4 ) 6-y (HPO 4, CO 3 ) y (OH) 2-z r z 2x = y+z; x 2 Ca (NO 3 ) 2, 4 H 2 O (NH 4 ) 2 HPO 4 ambient T physiological ph Nanocrystalline apatites properties:: - Maturation (h, d, m) - Ion exchanges - Protein adsorption 11x19 nm (1d maturation)
9 X-ray diffraction characterisation (002) (310) rat bone (18 months) PCA matured 1 month Theta HAP (JCPDS n 90432) Estimated crystallite size for natural and synthetic apatites [Eichert, PhD thesis, INP Toulouse 2001] Sample L(002) ± 3 Å (length) L(310) ± 3 Å (width/thickness) Chicken bone Rabbit bone 190 Not evaluated Adult human cortical bone Synthetic apatite matured for 3 months
10 Absorbance Spectroscopic characterisation FTIR spectroscopy analysis n 4 PO 4 domain HPO 4 2- = important marker of NCA maturation HAP apatitic HPO 4 Nanocrystalline apatite non-apatitic HPO Wavenumbers (cm -1 ) Wavenumbers (cm-1)
11 IR band intensity IR band intensity Spectroscopic characterisation Apatites maturation HPO 4 2- = important marker of apatite maturation Synthetic apatites Biological apatites (chicken bone) 0,3 0,15 0,2 0,1 0, h 24 h 3 d 9 d 1 m Maturation time 0, d 10 w 30 w 1 year 3 years Embryo Age HPO 4 2- with maturation/ageing [Cazalbou et al., J. Mater. Chem., 2004] [Cazalbou et al., J. Bone Miner. Metab., 2004]
12 Sr/(Sr+Ca) (%) Spectroscopic characterisation and chemical analysis Ion exchange properties of nanocrystalline apatites Cation exchanges Sr 2+ /Ca 2+ Anion exchanges CO 3 2- /HPO 4 - NCA/ 13 CO AC/Sr 10 AC/Sr/Ca NCA/ 13 CO 3 2- / HPO d 3 d 10 d 1 m 2 m 13 m 20 m 32 m Maturation time AC (1day maturation) fast and reversible exchanges exchange ability with maturation Wavenumbers (cm -1 ) 820 Surface divalent ions involved in these ion exchanges [Cazalbou et al., J. Mater. Chem., 2004]
13 Absorbance Drying Spectroscopic characterisation Role of water? FTIR spectroscopy 31 P NMR Wet samples exhibit fine structural details Apatitic PO 4 3- HPO 4 2- which disappear on drying Wet Lyophilized Wavenumber (cm-1) 800 Drying: Loss of water disordered ionic layer alteration of short range order Non-apatitic environments ppm/h 3 PO 4-4 Very fragile surface hydrated layer structure Some analogy but OCP and DCPD [Eichert et al., Bioceramics 16, 2004]
14 (nmol/m2) Affinity constant (ml/mg) Adsorbed quantity (mmol/m 2 ) Amount adsorbed (µmol/m²) Nanocrystalline apatite properties Adsorption properties of nanocrystalline apatites BSA Bisphosphonate Maximum coverage Maturation time (d) ph C Equilibrium Equilibrium concentration (mmol/l) (mmol/l) A B A: nanocrystalline apatite (30 d) B: stoechiometric HAP Adsorption properties vary according to the surface layer characteristics adsorption desorption
15 Specific structural characteristics of apatite nanocrystals Structured hydrated layer at the surface of nanocrystals Apatitic domain core Structured hydrated layer Apatitic Surface (Nonapatitic domain) Solution domain 2-- HPO Ca 2+ 4 Ca CO 3 Protein HCO 3 - Ca 2+ HPO 4 2- H 2 PO 4 - a) Apatite nanocrystal (3D view) b) Apatite nanocrystal in solution (profile) Rey et al., Osteoporosis Int.,
16 Consequences for biomaterials? Apatitic domains Crystal-crystal interactions in aqueous media Hydrated layer by drying Ca 2+ H 3 O + H 2 O Crystal "fusion" (bone, enamel) Setting reactions of biomimetic CaP cements - Ceramics low temperature - Coatings - Composites
17 Nanocrystalline apatites analogous to bone mineral Biocompatibility, biomimetism High surface reactivity Bone tissue engineering (, castinggel, SPS, cements - Low temperature bioceramics (biomimetic Associations with drugs (antibiotic, anticancer, Bioactivity ( ionsactivebiologically, factorgrowth Resorbability for sustained release system - Nanocomposites (nanocrystalline apatites + resorbable polymers) Ceramic prepared by SPS (Cryo SEM-FEG) Human mesenchymatic stem cells on nanostructured coating Nanostructured coatings on implants (ceramics or metal) + association with biologically active molecules Luminescent nanoprobes for medical imaging Histological cross section : neoformed bone (dark blue) in contact with a porous coated ceramic (+BMP-2) implanted in non osseous location (t= 3 months) Colloïdal apatite nanoparticles ( nm), luminescent (rare earth element), associated with a targeting agent towards cancer cellules Nanoparticles (TEM) Internalisation of uminescent nanoparticles l Within a human pancreatic cell
18 Application Biomimetic apatite coating HA-bTCP porous granules (Teknimed) - with or without biomimetic apatite coating - with or without adsorbed rh-bmp2 Raw HA-bTCP ceramic Scale bar = 1 µm Coated HA-bTCP ceramic
19 Application: Biomimetic apatite coating Porosimetry additional porosity corresponding to internanocrystal spaces Adsorption and release properties of rh-bmp2 H. Autefage et al. J. Biomed. Mater. Res. B (2009)
20 Neo-formed bone (%) In vivo study Intramuscular implantation in sheep for 3 months Histological study: after 3 months Application: Biomimetic apatite coating Raw HA-bTCP ceramic without rh-bmp-2 Coated HA-bTCP ceramic Osteoinduction potential of the nanocrystalline coating alone but WHY?!! nanostructure? nanoporosity? composition? Multiple parameters?. a question to investigate within NEWGEN network? Control Coated Control Coated +BMP-2 +BPM-2 See Margarida Almeida talk (effect of [nanoha] on ALP and BMP-2 expression)
21 Biomimetic apatite coating: Technology transfer Coll. Teknimed S.A - CIRIMAT: Ceraform Revolution is a porous bioceramic with a nanocrystalline carbonated apatite coating enhancing surface reactivity leading to better bone reconstruction H. Autefage et al. US patent n 12/487,101 (2009)
22 Conclusion Bone mineral is a very reactive complex structure The evolution of bone apatite with age corresponds to a physical-chemical maturation process Nanocrystalline apatites exhibit specific surface structure in aqueous media This hydrated surface layer is unstable Although the structure of the hydrated surface layer of apatites is unknown it offers ion exchange and protein adsorption capabilities The surface reactivity thus obtained is utilised by living organisms We can take advantage of this surface reactivity for making new bioactive bone substitute materials at low temperature
23 Nantes, 7 th and 8 th May 2014 Tailoring calcium phosphate nanostructured materials for biomedical applications Elizabete Costa (elisabete.costa@ua.pt) Margarida Almeida (margarida@ua.pt) CICECO Centre for Research in Ceramics and Composite Materials Department of Materials and Ceramic Engineering MP1301 NEWGEN WG1 UNIVERSITY OF AVEIRO PORTUGAL
24 MOTIVATION MORPHOLOGY ISSUES DRUG DELIVERY SYSTEMS DDS PARTICLE REQUIREMENTS Morphology accurately engineered is a prerequisite for a predictable performance of DDS Synthesis procedures able to prompt particle design with size and shape surgically tailored MP1301 NEWGEN WG1 M. Ferrari, Nature Nanotech., 2008 Nantes, 7 th and 8 th May 2014
25 MOTIVATION what s the issue? To exploit strategies based on chelant assisted synthesis for producing apatite nanoparticles targeted to biomedical devices (drug delivery systems + bone tissue growth) MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
26 MOTIVATION Chemical precipitation in presence of citrate OH COOH COOH C C C COOH H H H H Citric acid - X H + Citrate ion MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
27 @ the lab... Hydroxyapatite Synthesis Strategy steps 3 Precursor solution Preparation Solution heating HAp particles MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
28 @ the lab... Preparing the precursor solution Citric acidolution [Cit/Ca] ph Precursor Calcium citrate solution Complex Preparation solution Solution heating Calcium Nitrate Solution Ammonium phosphate solution Ammonia Hap particles MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
29 @ the lab... Solution heating Precursor Solution (Ca, P, Cit ) 37ºC (Physiological temperature) or 180ºC (Hydrothermal synthesis, Autoclave) Hap particles MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
30 @ the lab... Characterizing the precipitates Chemical Composition (ICP) and Crystallinity (X-ray Diffraction) Morphology (Electronic Microscopy) Specific surface area (BET) Electrical surface charge (Zeta potencial) Adsorbed species (FTIR) Precipitation of HAp particles MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
31 HAp SYNTHESIS ph= 8.2 T=37ºC ph= 7.7 7m 2 /g 1 mm [1] M.Martins et al, J.Colloid and Interface Sci, 2008; [2] C.Santos et al, Microscopy and Microanalysis, 2009 MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
32 HAp SYNTHESIS T=37ºC ph= mm 20m 2 /g Cit:Ca=3:1 [Citrate] Cit:Ca=5:1 [1] M.Martins et al, J.Colloid and Interface Sci, 2008; [2] C.Santos et al, Microscopy and Microanalysis, 2009 MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
33 HAp SYNTHESIS 55 m 2 /g T=180ºC Rods Hexagonal prisms 100 nm 15 nm Cit:Ca=3:1 5 nm [1] M.Martins et al, J.Colloid and Interface Sci, 2008; [2] C.Santos et al, Microscopy and Microanalysis, 2009 MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
34 HAp SYNTHESIS T=37ºC Cit:Ca=3:1 T=180ºC 55 m 2 /g 100 nm MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
35 HAp SYNTHESIS Rods Hedgehogs T=180ºC Bundle-like 17 m 2 /g 55 m 2 /g 55 m 2 /g 100 nm 43 m 2 /g 1μm 2μm ~ 3:1 4:1 7:1 [Citrate/Calcium] Ratio MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
36 Comparing HAp nanoparticles Intensity (u.a.) ph=8,2 55 m 2 /g HAp nanoparticles : 180ºC versus 37ºC Chemical precipitation O Hydrothermal precipitation O O O O O O O O O O O 180ºC O O 37ºC o-hydroxyapatite Theta (o) [2] C.Santos et al, Microscopy and Microanalysis, 2009 [1] M.Martins et al, J.Colloid and Interface Sci, 2008; MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
37 Comparing HAP properties Zeta Transmittance Potencial (a.u.) (mv) HAp nanoparticles : 180ºC versus 37ºC ph=8,2 FTIR HAP/37 0 ph m 2 /g HAP/180 Santos, C. et al. Journal of the Royal Society Interface, 2012 Wavenumbers (cm -1 ) Electrical surface charge Citrate derived carboxilic groups (COO - ) coordinated to HAp under different configurations MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
38 Interaction with cells reduced MTS formazan product absorbance (λ=490 nm) MG63 osteoblast-like cell response to HAp nanoparticles* (a) 3 days 6 days HAp / 37ºC *Faculty of Dental Medicine, U. Porto MG63 Cell viability (b) 3 days 6 days HAp / 180ºC Low levels do not affect cell viability. Deleterious effects observed for the higher HAp concentrations HAPnanoparticles concentration MP1301 NEWGEN WG1 Santos, C. et al. Journal of the Royal Society Interface, 2012 Nantes, 7 th - 8 th May 2014
39 Interaction with cells MG63 cell cultures exposed to HAP nanoparticles, 50 and 500 μg/ml, 3 days. Control HAP/37ºC- 50 μg/ml HAP/37ºC- 500 μg/ml HAP/180ºC- 5 0 μg/ml HAP/180ºC- 500 μg/ml Low levels (not toxic) increased the expression of ALP and BMP-2 Santos, C. et al. Journal of the Royal Society Interface, 2012 MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
40 Interaction with cells 3h, 50 μg/ml 3h, 500 μg/ml 3h, 50 μg/ml 3d, 50 μg/ml HAP/37ºC HAP/180ºC 24h, 500 μg/ml 3d, 500 μg/ml 24h, 500 μg/ml 3d, 500 μg/ml NanoHap are readily internalized by MG63 cells. Toxic effects observed at high concentrations reflect the vesicular entrapment. Santos, C. et al. Journal of the Royal Society Interface, 2012 MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
41 Concluding HAP nucleation and growth phenomena are strongly dependent on precursor solution chemistry { ph, [cit] } and temperature. Manipulating these variables allows controlling the shape, size and surface properties of HAP nanoparticles (nanohap). nanohap are readily internalized by MG63 osteoblastic cells. Low [nanohap] do not affect cell viability/proliferation and enhance ALP and BMP-2 expression interesting profile for bone tissue applications. MP1301 NEWGEN WG1 Nantes, 7 th - 8 th May 2014
42 Acknowledgments UNIVERSITY OF AVEIRO Thank you
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