PRAMATAROVA Lilyana., Assoc. Prof. Dr.

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1 NANOBIOCOMPOSITES: NOVEL MATERIALS FOR BONE IMPLANTS PRAMATAROVA Lilyana., Assoc. Prof. Dr. Georgi Nadjakov Institute of Solid State Physics, Bulgarian Academy of Sciences 72 Tzarigradsko Chaussee, bulv., 1784 Sofia, Bulgaria tel: /265 fax: Web page:

2 Research area: Development of in vitro systems for studying the process of biomineralization Our work is dedicated to the development of in-vitro systems for research of the main factors contributing to the mechanism of biomineralization the process by which the organisms in nature form minerals. Particularly the aim of our work is to investigate the growth of Hydroxyaptite (HA), the most wellknown bioactive ceramic material used in medicine. to investigate the growth of HA trough Detonation NanoDiamomd (DND) and production of nanobiocomposites HA/DND.

3 PARTICIPANTS BROKERAGE EVENT 1. Georgi Ndjakov Institute of Solid State Physics, Bulgarian Academy of Sciences (ISSP, BAS) Assoc. Prof. Dr. L. Pramatarova, Dr. E. Pecheva and Dr. T.Petrov 72 Tzarigradsko Chaussee blvd., 1784 Sofia, Bulgaria, tel /265, fax web page: 2. Lightsystem Ltd., Bulgaria, Laser systems and laser processing, (SME) MS Ognjn Sabotinov 72 Tzarigradsko Chaussee, blvd., 1784 Sofia, Bulgaria, tel: , fax: web page: 3. Sofia University, Faculty of Chemistry Prof. DS. T. Spassov 1 James Bouchier Str., 1164 Sofia, Bulgaria, tel: , fax: tspassov@chem.uni-sofia.bg 4. Institute of Organic Chemistry, Bulgarian Academy of Sciences Assoc. Prof. Dr. R. Dimitrova BG-1113, Sofia, Bulgaria, +359(2) , +359(2) zeolab@orgchm.acad.bg 5. Space Research Institute, Bulgarian Academy of Sciences Prof. S. Stavrev BG-1113, Sofia, Bulgaria, /885 stavrev@phys.bas.bg

4 In co-operation with Prof. Robert Piticescu National Institute for Non-Ferrous and Rare Metals (INCDMNR), Romania Dr. C. Langu, Prof. I. Mihailesku - National Institute for Lasers, Plasma and Radiation Physics, RAS, Romania Dr. Paul Montgomery - InESS, CNRS, France Prof. Ivo Rangelow- Univerity of Kassel,Germany Dr. P.Laquerrie, University of Reims, France Academic N. Sabotinov, Dr. D. Nesheva, Dr. T. Petrov - ISSP, BAS Prof. D. Dimova-Malinovska- CLSENES, BAS Prof. G. Altankov, Institute of Biophysics, BAS Dr. S. Stanchev - St. Anna Hospital, Bulgaria; Prof. U. Bismayer - University of Hamburg, Germany; Dr. F. Riesz, Dr. A. Toth - Hungarian Academy of Sciences, Hungary, Prof. A. Medvids, Dr. E. Palcevskis - Latvian Academy of Sciences, Latvia Prof. R. Kniep - Max-Plank-Institute for Chemical Physics of Solids, Germany FZ Rossendorf - Dresden, Germany Prof. M. Stutzmann and Dr. Presker - Walter Schottky Institute, Technical University of Munich, Germany

5 AIM OF THE PROJECT To develop A novel technology, based on a process of Laser-Liquid-Solid- Interaction (LLSI) in simulated body fluids (SBF) Optimisation of LLSI method as a model of in vitro system for the controllable production of samples with hierarchically organized micro- and nanometer scale surfaces, typical for nature. To investigate The process of Hydroxyapatite (HA) growth on variously modified substrates The process of HA growth trough Detonation Synthesis Nanodiamand (DND) powders Production of nanobiocomposites HA/DND Application as a coatings of the orthopedic implants

Why production of HA/DND nanobiocomposites BROKERAGE EVENT Bone tissue is an example of a natural, hierarchically organized composite, built from collagen fibres and crystalline hydroxyapatite (HA).

6 Why production of HA/DND nanobiocomposites BROKERAGE EVENT Bone tissue is an example of a natural, hierarchically organized composite, built from collagen fibres and crystalline hydroxyapatite (HA). They are extremely weak, but in the natural state when mixed together they form a tough and flexible material. The fact that this mixing occurs in nature at room temperature without toxic chemicals makes this material all the more fascinating.

7 What we propose BROKERAGE EVENT Use of DND as analog of the collagen (organic part of the bone). * DND can secure architectural plan for mineralization of HA similar to the collagen. * Addition of DND powders to the deposition bath can improve mechanical properties of HA coatings. * DNDs are among the few materials that are really with nano-meter size particles, so its study in the field of implants will apply to the knowledge of understanding of the mechanism and kinetics of the growth of the therapeutic composite layers. Novel LLSI method is based on both * Forming of organized structure with micro design by laser treatment * Nanostructured HA gown through DND. Result: * Controllable production of samples with hierarchically organized micro and nanometer scale surfaces, typical for nature

Detonation Synthesis Nanodiamond Powders (DND) * nanosized DND particles - average diameters of ~5 nm * DND core-covered by graphitic layers and amorphous C.

8 Detonation Synthesis Nanodiamond Powders (DND) * nanosized DND particles - average diameters of ~5 nm * DND core-covered by graphitic layers and amorphous C. * surface of DND is rich in various functional groups * can be further functionalized by chemical treatment. Hydroxyproline (Hyp) BROKERAGE EVENT Collagen - the main protein of connective tissue Glycine (Gly) Proline (Pro) Dolmatov, V. Y., Russian Chem.. Rev., 70, 607, 2001 Hydroxylisine NH2 - CH2 - CH(OH) - CH2 - CH2 - CH(NH2)COOH

9 Hydroxyapatite (HA) - mineral part of the bone Chemical formula: Ca 10 (PO4) 6 (OH) 2 Stoichiometric Ca : P ratio of 1.67:1 Aim is to obtain synthetic HA as much comparable with the natural. unit cell of crystalline HA

10 Method of Simple Soaking Process in SBF Set up is an open system, at 37 C

thermostat plexiglass container solution substrate L. PRAMATAROVA, E. PECHEVA, T.

11 Novel Method of Laser-Liquid-Solid-Interaction (LLSI) Process Set-up for LLSI Design of laser scanning 200μm thermometer substrate holder laser beam thermostat plexiglass container solution substrate L. PRAMATAROVA, E. PECHEVA, T. PETROV, N. MINKOVSKI, A. KONDYURIN, R. PRAMATAROVA, Proceedings of SPIE, v (2004) 46-50

12 LASER PARAMETERS: λ 1 = 511 nm λ 2 = 578 nm P = 4.5 W (average laser power) τ = 35 ns (pulse duration) d = 50 μm (diameter of laser spot) f = Hz (repetition rate) N = pulses/min v = mm/s (velocity of scanner) ARGES scanner head equips the pulsed CBL

13 Design of Laser Scanning

14 Set-up for LLSI BROKERAGE EVENT with Optical Diagnostic of Laser Plasma. L. D. PRAMATAROVA, E. V. PECHEVA, C. P. LUNGU, O. N. SABOTINOV, Proce of the LTL International Symposium, 8-11 October 2005, Plovdiv, Bulgaria, pp (2006)

15 DND preparation DND powder was synthesized from the free carbon of explosives with a negative oxygen balance at high pressure and high temperature produced by the detonation. SBF preparation SBF (Simulated Body Fluid) is a supersaturated aqueous solution that resembles the ion composition, concentrations and ph of human blood plasma.

16 Substrates: Stainless steel, Titanium and Titanium Alloys - materials, widely used in orthopedic and dentistry for medical implants Silicon - widely used material in microelectronics Quartz - typical piezo-electric material Silica glass belongs to the class of insulators Polymers - also find different applications as implants

17 Methods for surface modification of the materials - whole surfase ion implantation (by Ca, P, Na and Si ions); - ion implantation through mask (by Ca, P, Na and Si ions); - laser irradiation of the surface (by LLSI process); - nanostructures (Si and CdSe nanoparticles in SiO x layer); - porous silicon (formed on Si substrate by ECE); - poly silicon (fomed on SG substrate by Al-induced crystallization); - deposition of extracellular matrix proteins; - deposition of detonation nanodiamond

18 Monograph: L. Pramatarova, E. Pecheva, Modified Inorganic Surfaces as a Model for Hydroxyapatite Growth, in Materials Science Foundations, vol. 26, Trans Tech Publications, Switzerland, 2006, pp

19 How can produce artificial bones through nanodiamonds (DND) I. Stage of experiments: Deposition of HA through DND on TiCu alloys Applied Method: soaking process Solution SBF and DND suspension Substrates: Cu 50 Ti 40 Al 10 ; Cu 50 Ti 50 alloys Characterization: by SEM, EDX, CPM and Raman spectroscopy Results: DND produced a stable aqueous suspension in SBF and stimulate the HA growth on the TiCu substrates. L. Pramatarova at all, Proc. International Baltic Sea Region conference Functional materials and nanotechnologies Riga, April 2-4, 2007 (FM&NT-2007)

20 Substrates Cu 50 Ti 40 Al 10 and Cu 50 Ti 50 alloys BROKERAGE EVENT Amorphous ribbons of Cu 50 Ti 40 Al 10 and Cu 50 Ti 50 alloys, with a thickness of 0.02 to 0.03 mm and a width of 2 mm were produced in a He atmosphere (300 mbar) by melt-spinning onto a copper quenching wheel. The substrates were fully amorphous as checked by X-ray diffraction and TEM The roughness (rms), measured by CPM was of the order of 42 nm.

spectrum EDX SEM: 1-sphere-like particles - 200 nm grouped in clusters; 2-particles, reached

21 HA growth on TiCuAl alloys By soaking process (first sample group) : SBF - magnetically stirred (ms) Time of growth - 4h ; at T = 37ºC Substrate - vertical position in SBF Raman spectrum EDX SEM: 1-sphere-like particles nm grouped in clusters; 2-particles, reached of C and Ca; size about 20 nm EDX: 1 and 2 areas Raman: vibrations characteristic of the CaP phase

spectrum EDX SEM: 1 -sphere like particles 2 - particles, reached of C and Ca; size about 10

22 HA through DNDs on TiCuAl alloys By soaking process (second sample group): SBF + DNDs ms DND - suspension Time of growth - 4h ; at T = 37ºC Substrate - vertical position in SBF Raman spectrum EDX SEM: 1 -sphere like particles 2 - particles, reached of C and Ca; size about 10 nm Raman -mixed compound of CaP and graphite CaP vibrational modes - hindered by the C-C vibrations

HA through DNDcs on TiCuAl alloys By soaking process (third sample group): SBF + DND cs DND -

SEM: 1 Ca/P =0.98 sphere like particles; 2- Ca/P =1.34 particles and C 3 Ca/P= 1.

%] C Mg P Ca O Na Cl Si Ca/P TiCu_1 0.00 1.64 9.95 9.76 40.49 1.07 0.03 0.54 0.98 TiCu_2 2.80 0.

23 HA through DNDcs on TiCuAl alloys By soaking process (third sample group): SBF + DND cs DND - clear solution (cs) Time of growth - 24h ; at T = 37ºC Substrate - horizontal position in SBF, EDX SEM: 1 Ca/P =0.98 sphere like particles; 2- Ca/P =1.34 particles and C 3 Ca/P= 1.77 particles and reached of C; 4- bone-like patterning area S/Elements, [at. %] C Mg P Ca O Na Cl Si Ca/P TiCu_ TiCu_ TiCu_ TiCu_

24 Conclusions: Deposition of HA through DND on TiCu alloys by soaking process TiCu alloys induce HA growth. DND could have stimulated the HA growth and inclusion of Mg and Cl in the composites. Adding of the DND suspension and clear solution in SBF influence the grown layer morphology. The used of the DND clear solution apart from the sphere-like particle formats the porous areas similar to those in the structure of bone. The Ca/P ratio and the C concentration in the investigated layers are decreasing with advance of the process.

II. Stage of experiments: HA/DND composites grown by LLSI process Applied Method: LLSI Substrates: SS, S and SG Characterization: SEM/EDX, CPM and Raman spectroscopy and Nnanoscaning Results: The

25 II. Stage of experiments: HA/DND composites grown by LLSI process Applied Method: LLSI Substrates: SS, S and SG Characterization: SEM/EDX, CPM and Raman spectroscopy and Nnanoscaning Results: The mechanical and optical properties of the grown HA/DND composites can be controlled by laser irradiation and in this way to model the situations in nature. L. Pramatarova at all., Proc. ILLA/LTL, pp (2007)

of soaking - 1h at T = 37ºC Substrates: in the horizontal position.

26 HA/DNDs composites on Stainless Steel (SS), Silicon (S), Silica Glass (SG) by LLSI By LLSI process (first sample group): Process of LLSI 1-5 min Process of soaking: SBF + DNDs irradiated by laser Time of soaking - 1h at T = 37ºC Substrates: in the horizontal position. Patterning of micrometer size by laser SEM EDX SEM of SS/HA/DND laser stripes and the typical areas by EDX EDX Ca, P, O and typical peaks for SS

27 Raman spectra of the HA/DNDs composites SG/HA/DND S/HA/DND SS/HA/DND SS/HA/DND composites strongest interaction between the SS and laser process of oxidation 1 - outside the laser stripe 2 - within the laser stripe OH vibration peak at 659 cm -1

Interference microscopy system BROKERAGE EVENT

5 mm Z range = 100 µm Leitz Linnik Microscope

28 Interference microscopy system BROKERAGE EVENT Active anti-vibration table Leica DMR-X Microscope (colour CCD) (x5, x10 et x40) Z res = 1 nm XY res = 0.6 µm XY field = 2x1.5 mm Z range = 100 µm Leitz Linnik Microscope (CCD) (x50, NA = 0.85) Z res = 5 nm XY res = 0,45 µm XY field = 130x100 µm

29 Optical metrology group BROKERAGE EVENT Dr. Paul Montgomery (InESS, CNRS) Fabien Salzenstein (Lecturer) Gyasi Johnson (Ph.D.) Freddy Anstotz (Lecturer) Denis Montaner (Lecturer) Emilia Pecheva (post-doc) Liliana Pramatarova (Prof. Invited ULP, CNRS)

30 The morphology and topographic CPM images on S/HA/DNDs CPM topographic measurements taken over the laser line in two areas Rq=0.01 μm Rq=0.46 μm image size 128 x 40μm 1 - periodic modification of the surface; composite particles 1.6 and 1.8 μm in height.

31 DNDs film deposited on stainless steel (SS) substrate By LLSI process (second sample group): LLSI 1-5 min ; at T = 24ºC DND suspension Substrate - in the horizontal position. Raman spectra EDX elements line scan laser stripe Typical vibration modes for DND 1 a large crystal (shown in the CPM results) OH vibration mode 2 small droplets.

32 Z Y BROKERAGE EVENT Laser assisted growth of DNDs on stainless steel CPM mesearments 1 X 2 Z X Air/HA surface SS surface n eff = ΔZ/d d ΔZ HA/SS interface n eff = 1.45 E. Pecheva at all, Langmuir 23 (7) (2007)

and bio interfaces 2004/05 Challenges : - depth

33 Coherence probe microscopy study of HA Height view 3D view HA Substrate Papers : EU workshops on nano and bio interfaces 2004/05 Challenges : - depth (<30 µm) - small cell size 1-3 µm - translucent R t, R q

34 Nanoscan measurements Results: A notable effect on the value of E of HA/DND composites, grown on silica glass substrate (center). Higher values for E in the center (non irradiated area) compared with those at the edge (laser irradiated area), due to the laser irradiation. BROKERAGE EVENT Nanoscan mapping of the laser spots. e - laser irradiated area (edge) c- non irradiated area (centre)

35 Nanoscan measurements Young s modulus at load 400 μn E = 61.0 GPa - for the samples seeded with HA by LLSI E = 46.0 GPa - for SG/HA/DND/4s (thicker layer) E = 24.0 GPa - for SG/HA/DND/2ms (thinner layer) Conclusion: The decrease in the Young s modulus of the composite material is due to the adding of the DND suspension in the SBF and depend on the thickness of the composite layer.

36 Conclusions: Growth of HA through DND on SS, S and SG by LLSI BROKERAGE EVENT By CPM (developed in InESS), that is an extremely powerful tool for carrying out optical measurements rapidly without contact, and with high precision, newly grown composite materials were characterized. Raman study shows that vibration modes such as C-C, OH, C-O and cage deformations of DND structure are characteristic for composites, obtained only by LLSI. The laser interaction with the SS surface is not so strong when the surface is covered with DND suspension. Line scan measurements along the irradiated areas show a higher carbon content within the laser stripes than outside of them. The laser irradiation influences the value of the Young s modulus of the composites.

37 III. Stage of experiments: Growth of HA/DND composites on substrates with OH groups (SG and CG) Applied Method: simple soaking process Substrates: SG and CG Solution: SBF + DND suspension Characterization: XRD and IR spectroscopy Results: DND surface functional groups interact in a greater extent with the SBF in comparison with the SG and CG functional groups. The DND surface is chemically multifunctional (OH, C-O-H, C=C, C-O-C, C=O), HA is grown both by physical adsorption and chemical interaction. The OH- groups are regarded to play an important role in HA growth on a diamond s surface from SBF, as they charge it negatively and attract Ca2+ ions.

FTIR P-O SEM 4000 3000 2000 1000 0 Wavenumber (cm -1 )

38 SG_HA_18h Absorbtion Units 0,40 0,35 0,30 0,25 0,20 0,15 0,10 SG_HA_18h BROKERAGE EVENT Wates C=O CO-H FTIR P-O SEM Wavenumber (cm -1 ) 20 SG_HA_18h Number Size, nm

SG_HA/DNDcs_18h 8 7 6 BROKERAGE EVENT SG_HADND_18h cross

160 180 200 Size, nm 35 SG_HADND_18h 30 lower layer SEM

39 SG_HA/DNDcs_18h BROKERAGE EVENT SG_HADND_18h cross section lower layer Number Size, nm 35 SG_HADND_18h 30 lower layer SEM Number Size, nm

40 CG_HA CG HA/DNDcs BROKERAGE EVENT 4h SEM 18h

41 FTIR CG_HA Absorbtion Units 0,6 0,5 0,4 0,3 0,2 0,1 C=O straching vibrations O-H ν 3 P-O C=C ν 4 C-O CG_HA/DNDcs CG_HA_2h CG_HA_18h CG_HADNDcs_18h 0,0 CG_HADNDcs_2h Wavenumber (cm -1 )

42 Conclusions: HA through DND on SG and CG by simple soaking process The growth of HA on substrates with OH groups was studied. The applied methods provided evidence that the acidity of the OH groups on DND surface was more effective for HA growth in comparison with the OH groups existing on the SG and CG surface. The observed effect was due to the chemical nonuniformity of the DND surface functional group (presence of C-O-H, C=C, C-O-C and C=O groups) and the OH concentration that ascertained the intensity of the HA growth. At a longer duration of growth, with the increasing of HA surface concentration on the substrates, a chemical reaction proceeded that led to a change in the conformation of HA structure and to an appearance of new HA phase. L. Pramatarova at all., Proc. International Baltic Sea Region conference Functional materials and nanotechnologies Riga, April 2-4, 2007 (FM&NT-2007)

43 Cells Specification Osteoblast-like cells MG-63 (ATCC number CRL 1427) Bladder carcinoma cells T24

44 With bladder carcinoma cells T24 SG_initial x100 SG_HA/4DND x100

45 With bladder carcinoma cells T24 SG_HA (seeded by LLSI) x100 x100 x100 SG_HA (seeded by LLSI) /4DND

46 Conclusions about HA/DND composites BROKERAGE EVENT Through the use of DND it has been shown that it is possible to make HA/DND composites on various substrates. The incorporation of DND in the grown HA has been demonstrated using CPM, Raman spectroscopy, SEM/EDX and Nano-indentation. The refractive index of the DND/SS composite has been found to be 1.45 (using CPM). Addition of the DND suspension to the SBF decreases the elasticity of the grown composite but increases the hardness, depending on the thickness of the layer. It was found that the mechanical and optical properties of the HA/DND composites can be controlled by laser irradiation and in this way to model the situations in nature. A bladder carcinoma cells showed good adhesion and spreading on the composites samples.

47 Biomaterials Applications BROKERAGE EVENT Orthopaedic Applications * Metallic, ceramic and polymeric biomaterials are used in orthopaedic applications. * HA is used for bone bonding applications to assist implant integration Dental Applications * Hydroxyapatite has been used for coatings on metallic pins and to fill large bone voids resulting from disease or trauma. Cardiovascular Applications * For instance, carbon in heart valves and polyurethanes for pace maker leads are used Cosmetic Surgery * Materials such as silicones have been used in cosmetic surgery for applications

48 Ti for medical implants: with specially designed surface for a good contact with the bone (porous surface) or the gingival (smooth surface) artificial hip joint dental implant

49 Acknowledgements Special thanks to my collaborators Dr. E. Pecheva and M.S. M. Dimitrova Assoc.Prof.Dr. R.Dimitrova

50 Profile of the Lightsystems LTD, Laser systems and laser processing, SME, Bulgaria MS. O. Sabotinov, President Address: 72 Tzarigradsko chaussee bulv., 1784 Sofia, Bulgaria tel.: fax: pulslight@issp.bas.bg Abstract : Lightsystems Ltd. is interested in the development and manufacturing of Copper Bromide (CuBr) lasers, which are high power devices that generate pulses of radiation at two wavelengths in the visible region of the spectrum, at 511 and 578 nanometers (nm) as well as in a scientific research and development. Lightsystems Ltd. has scientific collaboration with the Metal Vapor Laser Group at the ISSP, BAS. (Academic N. Sabotinov) and as a result company has high expertise, well developed technology and a good market realization. Pulslight is a partner in two R&D projects (EC funded COPERNICUS Project and NATO funded SCIENCE FOR PEACE Project). The last two years Lightsystems Ltd has scientific collaboration with the Assoc. Prof. Dr L. Pramatarova at the ISSP, BAS, leader of the project Development of in vitro system for growing of hydroxyapatite as an implant coating. The interest for EU project is:. Application of a CuBr laser MOPA (master-oscillator-power amplifier) system for precision processing of various hard materials, drilling of micro-holes, and for the marking and cutting of materials. Application of a CuBr laser in a novel method of laser-liquid-solid-interaction (LLSI) for production of hydroxyapatite coatings on variously modified surfaces of medical implants.

51 BROKERAGE EVENT Theme COOPERATION: NANOSCIENCES, NANOTECHNOLOGIES, MATERIALS AND NEW PRODUCTION TECHNOLOGIES Budget: million ( ) Nanosciences and Nanotechnologies The objective is to create materials and systems with predefined properties and behaviour, based on increased knowledge and experience at the nano scale. This will lead to a new generation of products and services across a range of applications, while minimising any potential adverse environmental and health impacts. Materials Research will focus on developing new multifunctional surfaces and materials with tailored properties and predictable performance for new products and processes as well as for their repair. Experts: We are seeking partners having an experience in following fields: chemistry and online testing of the solutions, growth of thin films and coatings, modification and functionalization of the surfaces, biomaterials, surface engineering, especially partners with well established methodologies for chemical/physical analysis. We are seeking partners with existing projects or new projects that are still looking for suitable partners. Theme PEOPLE: Training and career development of researchers

52 Many thanks for your attention

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