NANOMEDICINE. Luigi Ambrosio & Michele Saviano

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1 NANOMEDICINE Luigi Ambrosio & Michele Saviano Istituto per I Materiali Compositi e Biomedici, Consiglio Nazionale delle Ricerche (IMCB-CNR), Piazzale Tecchio 60, Napoli Istituto di Biostrutture e Bioimmagini. Consiglio Nazionale delle Ricerche (IBB-CNR), Via Mezzocannone, Napoli

2 Definition Nanomedicina applicazione della nanotecnologia nel settore della salute.. utilizzando nuove proprietà fisico-chimiche e biologiche dei materiali a livello nanometrico. La Nanomedicina, ha la potenzialità di ottenere una precoce diagnosi e prevenzione, e un miglioramento del trattamento e monitoraggio delle patologie.

3 Definition Main Topics Regenerative Medicine Biomimetic Biomaterials and Smart Implants Bioactive Signaling Molecules Cell Based Therapies Targeted Delivery Drug Delivery Systems Diagnostics and Imaging In Vitro Applications In Vivo Imaging

4 Programma 25 Settembre, 2009 Medicina Rigenerativa e Drug Delivery I biomateriali nella medicina rigenerativa, Luigi Ambrosio (IMCB-CNR) Approccio biomimetico in medicina rigenerativa Anna Tampieri (ISTEC-CNR) Strutture magnetiche per la rigenerazione dei tessuti, Alek Dediu (ISMN-CNR) Sistemi per il rilascio controllato dei farmaci, Graziella Vecchio (Università di Catania) Bioreattori e sistemi bioibridi per la rigenerazione dei tessuti ed organi Loredana De Bartolo (ITM-CNR) Tecnologie e metodologie di caratterizzazione di materiali per la rigenerazione dei tessuti. Roberto De Santis (IMCB-CNR)

5 Programma 26 Settembre, 2009 Imaging su base molecolare, Identificazione di nuovi target e selezione di nuove molecole naturali o di sintesi biologicamente attive Synthesis and Screening of Synthetic Peptide Libraries. Menotti Ruvo (IBB- CNR) Identificazione ed origine di prodotti naturali marini bioattivi. Angelo Fontana (ICB-CNR) Le SPM come tecniche di imaging e di manipolazione di biomolecole. Francesco Valle (ISMN-CNR) Soft lithography and cell guidance a novel approach for regenerative medicine. Fabio Biscarini (ISMN-CNR) New bioactive peptides by screening synthetic combinatorial libraries: case studies. Menotti Ruvo. (IBB-CNR)

6 RIB I biomateriali nella medicina rigenerativa Luigi Ambrosio Istituto per I Materiali Compositi e Biomedici, Consiglio Nazionale delle Ricerche (IMCB-CNR), Piazzale Tecchio 80, Napoli

7 Tessuti Naturali La natura ha ottimizzato la composizione e la struttura dei tessuti a livello nano-micro micro-macromacro per soddisfare le specifiche funzioni. Eterogeneità Anisotropia

8 Background Esempio: Struttura dell osso Osso Materiale nanocomposito organizzato gerarchicamente a differenti livelli Fibre di Collagene Cristalli di Apatite (dimensioni nanometriche) Acqua Organizzazione gerarchica cm micron Volume % APATITE COLLAGEN WATER nano

9 Background Sistemi Protesici Impianti dentali Protesi d anca Breast Implants Abdominal wall structures Compositi per restauro Sostituti ossei Vascular Grafts Cuore artificiale Legamenti Intraocular lens

10 Background Materiali Biomimetici La nanotecnolgia permette di mimare I tessuti naturali a livello nano-micro-macro. Parametri strutturali possono essere ottimizzati utilizzando opportuni materiali, morfologie, concentrazioni e condizioni dei processo; Interazioni cellule/scaffold/biomateriali e biosintesi cellulare possono essere ottimizzate da specifici segnali chimici e fisici: - Molecole idrofobiche/idrofilliche; - Fattori di crescita, Proteine, Farmaci, etc. - Stimoli meccanici - Cellule

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12 Medicina Rigenerativa Rigenerazione In Vivo Impianto di biomateriali con o senza cellule per facilitare la rigenerazione dei tessuti in vivo. Rigenerazione con cellule

13 Rigenerazione senza cellule PVA/HCement ESTER OF HYALURONIC ACID (HYAFF120P50) GEL IN VIVO RESULTS AFTER 6 WEEKS 4 Weeks IN VIVO RESULTS 12 Weeks

14 Biopsy harvesting cells biopsy TISSUE ENGINEERING APPROACH R. Langer, et al Tissue implantation Expansion Patient Implantation In situ TE Processing3D In vitro TE Cellular culture 2D Cellular construct 3D Cell seeding in 3D

15 INERT SUPPORTS BIOFUNCTIONAL SUPPORTS SUFFICIENT MECHANICAL STRENGTH TO WITHSTAND THE HYDROSTATIC PRESSURES AND THE BEARING LOADS DURING IN VITRO CULTURE. PROPER BIOLOGICAL GUIDE FOR THE NOVEL TISSUE IN FORMATION PROMOTING ADHESION AND PROLIFERATION MECHANISMS POROSITY REQUIREMENTS (PORE GEOMETRICAL SHAPE, SPATIAL DISTRIBUTION, INTERCONNECTION DEGREE) DEGRADATION KINETIC (DEGRADATION RATE COMPARABLE TO TISSUE GROWTH RATE) CELL/MATERIAL RECOGNITION (PROTEIN ADSORPTION) BIOACTIVE SOLID SIGNALS OSTEOCONDUCTION PROPERTIES OSTEOINDUCTION PROPERTIES SOLUBLE AGENT RELEASE (ANTIBIOTICS AND DRUGS)

16 Requirement of a TE Scaffold Degradable Cell guidance Controlled degradation rate Adhesion Growth Differentiation Migration Nutrient supply Appropriate scaffold µ-structure Structural Support Sustain mechanical loading during new tissue growth

17 Morphology of native scaffolds Bone cell Muscle cell Nerve cell Skin cell cells Muscle tissue Nerve Bone Skin

18 Importance of controlled scaffold architectures elongated cubic spherical Pores shape and direction Many tissues (nerve, muscle, tendon, ligament, blood vessel, bone, and teeth) have tubular architectures and anisotropic properties. Need for an oriented network able to promote ordered structures formation. Ma et al. J Biomed Mater Res (2001) Matsuzaka et al. J Biomed Mater Res (2001) 5-15 μm for fibroblast ingrowth 20 μm for hepatocyte ingrowth 35 μm for neovascularization μm for regeneration of adult skin μm for osteoid ingrowth μm for regeneration of bone 500 μm for fibrovascular tissue Nehrer et al. Biomaterials (1997) Ranucci et al. Tissue Eng (1999) F.J. O Brien et al. Biomaterials (2005) Pores size and distribution Little (few nm) Big (hundred of µm) Poorly (~closed) Degree of interconnection The presence of a well-interconnected porosity is important for a good supply of the body fluids with nutrients throughout the scaffold. Vacanti et al. J Pediatr Surg (1988) Mikos et al. Biotechnol Bioeng (1993) Hannah et al. Tissue Engineering 2004 Greatly (totally opened)

19 Mimicking native scaffold structures Processing Techniques 20μm 100μm Gas foaming Solvent casting Salt leaching Phase Separation Microparticles sintering Electro-spinning Composite technology Rapid prototyping, Bio-plotter

20 Extracellular matrix analogue Biopolymers Collagen Collagen/Hyaluronic Acid Composites Hyaluronic Acid

21 Collagen/Hyaluronic Acid Extracellular matrix analogue Materials Hyaluronic Acid (HyA), 155KDa Collagen Type I solution HyA-Collagen Composite Fibrillogenesis of Collagen Type I in Hyaluronic Acid Methods Morphology, SEM, TEM Rheology, Bohlin Rheometer Pig Aortic Endothelial Cells (PAE) Spheroids.

22 MORPHOLOGICAL ANALYSIS B SEM TEM Collagen Collagen-HA Collagen Collagen-HA

23 MATERIAL CONTROLS SPROUTING ANGIOGENESIS RATE Sprouting Angiogenesis in isotropic matrices Sprouting Angiogenesis in isotropic matrices a b c d 24h e f g h Time 48h 72h i j k l C CHA 1 CHA 2.5 Matrix composition CHA 5 HA delays collagen-induced sprouting angiogenic

24 Sprouting angiogenesis in isotropic matrices Collagen gel Collagen-HA SIPNs sprout lent Collagene 1.2 mg/ml Collagene-HA 5mg/ml Matrix composition influences sprouting rate. y = x R 2 = y = x R 2 = time [min] Sprout propagation rate in collagen matrix: 4.3 μm/h Sprout propagation rate in collagen-ha matrix: 0.6 μm/h

25 Sprouting angiogenesis in anisotropic matrices DIRECTIONAL SPROUTING in C-CHA5-C MATRICES Sprout number Sector [ ] HA GRADIENT Anisotropic matrices induce directed and longer sprouts Average sprout length [μm] Sector [ ]

26 Sprouting angiogenesis in anisotropic matrices DIRECTIONAL SPROUTING IN C-CHA5 MATRICES Sprout number HA GRADIENT Anisotropic matrices induce directed and longer sprouts 1 2 Average sprout length[μm] Sector [ ] Sector [ ]

27 CONCLUSION I A spatial gradient of matricellular cues within a scaffold can provide a directional guidance for vessel growth in vitro Gradients can also speed up the process of material vascularization Matrix gradients are means to control and guide morphogenic processes within 3D scaffolds

28 Composite scaffolds for tissue regeneration Scaffolds for bone regeneration Savarino et al. Biomaterials 2007 Scaffolds for ligament regeneration E.C. Contract N. G5RD-CT Scaffolds for meniscus regeneration E. Kohn et al.

29 Composite scaffolds for bone regeneration To define a systematic TE approach to satisfactory clinical outcome, through optimizing the principal Bone TE components: scaffolds, cells and related processes. Difetti ossei Difetti Mandibolari

30 Bone Regeneration MATERIALS POLY ε-caprolactone DIMETHYLACETAMMIDE HYDROXYAPATITE PROCESSING TECNIQUES PHASE SEPARATION SOLVENT CASTING THEIR COMBINATIONS COMPOSITION POLYMER/SOLVENT WEIGHT RATIO 10μm 10μm BIOACTIVATION/MECHANICAL PROPERTIES PCL/HA VOLUME RATIO 10μm 10μm

31 Bone Regeneration MODULATION OF MECHANICAL PROPERTIES % vol HA 20% vol HA 13% vol HA 0% vol HA Vol % HA 0% 13% Elastic Modulus E (Mpa) 8,25 ± 1,10 21,43 ± 1,41 Strain at break (mm/mm mm) 0,240 ± 0,051 0,162 ± 0,051 Max Stress (Mpa) 0,93 ± 0,17 2,19 ± 0,17 Stress (MPa) % 27,90 ± 3,87 0,065 ± 0,018 1,33 ± 0, % 21,96 ± 3,76 0,059 ± 0,012 0,95 ± 0,14 POROSITY FEATURES Strain (mm/mm) Total Porosity (%) Average pore Radius (μm) Bulk Density (g/cm 3 ) PCL/0%vol HA PCL/13%vol HA PCL/20%vol HA PCL/32%vol HA CAUSA et Al. 2006

32 Bone Regeneration IN VITRO RESPONSE (3 weeks) OSTEOBLASTS SAOS-2 PCL 13%vol HA PCL 32%vol HA HUMAN OSTEOBLASTS (HOB) FULLY COVERED SCAFFOLD SURFACES BY CELLS IN CONFLUENCE G. Ciapetti,, N. Baldini. IOR

33 Bone Regeneration MATERIALS POLYCAPROLACTONE (PCL) MW 65 kda BIODEGRADABLE DEGRADATION TIME: MONTHS ESTER OF HYALURONIC ACID BIOABSORBABLE DEGRADATION TIME: 6 MONTHS POLYLACTID ACID (PLA) FIBERS 75DTEX BIODEGRADABLE DEGRADATION TIME: 24 MONTHS α TRI-CALCIUM PHOSPHATES BIOABSORBABLE OSTEOINDUCTIVE PROCESSING TECNIQUES FILAMENT WINDING TECHNIQUE PHASE INVERSION AND SALT LEACHING Pore size: µm D int = 3mm D ext = 7mm WINDING ANGLE φ= = 45 FIBER STEP ξ = 500µm COMPOSITE PREPARATION SOLVENT EXTRACTION SALT LEACHING

34 Bone Regeneration MORPHOLOGICAL ANALYSIS HIGH POROSITY DEGREE (>80%) GOOD PORE INTERCONNECTION PORE SIZE CONTROL BY MODULATION OF NaCl CRYSTAL SIZES AND PROCESS PARAMETERS: MICROPORES ( µm) MACROPORES ( µm) 100μm 1 μm MECHANICAL PROPERTIES IMPROVEMENT OF MECHANICAL RESPONSE OF THE SCAFFOLDS DUE TO REINFORCEMENT SYSTEMS (PLLA FIBERS AND CALCIUM PHOSPHATES) Toe Region Elastic Modulus (MPa) 2,5 2 1,5 1 0,5 0 17% PLLA 0% PLLA TCP0% TCP 13% TCP 20% TCP 26% %(w)tcp

35 Bone Regeneration IN VITRO RESPONSE CELLS LINE: BONE MSC CELLS NUMBER: 4X CM 2 DI A-MEM INTO 10% FETAL BOVINE SERUM (FBS) AND 1% OF ANTIBIOTIC DRUG AT 37 C C IN CONTROLLED ATMOSPHERE (5% CO 2 ) 500x RELATIVE FLUORESCENCE UNIT PCL-PLLA TCPS CELL VIABILITY 1200 x GOOD ADHESION OF MSC CELLS ON SCAFFOLDS SURFACE AND CROSS-SECTION SECTION w eeks G. Ciapetti,, N. Baldini. IOR

36 Bone defect in the sheep metatarsal bone C NB S NB S NB S NB S R. Giardino, M. Fini, E. Kor, IOR, Bologna

37 for bone regeneration generation.. LOW REGENERATION RATE OF PCL-BASED SCAFFOLDS Low initial scaffold recognition into the implant site Not adequate regeneration at the interface of the host tissue Micro or nano-structuration of scaffold surfaces by electrospun fibre coating

38 NANOSTRUCTURED PLATFORMS Electrospinning ηm µm TRADITIONAL PROCESSES Reduced pore spatial homogeneity Limits on the spatial resolution ELECTROSPINNING Random structural organization High resolution technique

39 NANOSTRUCTURED PLATFORMS Electrospinning MATERIAL SELECTION (NATURAL OR SYNTHETIC POLYMERS) 10% (wt/wt) CHCl 3 V = 13 kv 30% in (85/15) H 2 O / CH 2 O 5 V = 10 kv SETTING OF PROCESS PARAMETERS 5% m/v in HexaFluoroPropanolo

40 NANOSTRUCTURED PLATFORMS Electrospinning PCL/Gelatin(50/50) POL/TFE 07/93 V=13 kv ED=12 cm

41 180 NANOSTRUCTURED PLATFORMS hmsc culture - not osteogenic medium: α-mem (Modified( Eagle s s Medium), 10% FBS, 2mM glutamine and antibiotics Electrospinning antibiotics 20µm 20x Cellular Attachment (% of Control) * ** Time in Culture (h) Toluidine 0.30 Blue 0.25 ** A 570 nm * µm Days in Culture

42 The ALP test shows a more pronounced differentiation in the case of culture conditioning by osteogenic medium NANOSTRUCTURED PLATFORMS hmsc culture - osteogenic medium: α-mem (Modified( Eagle s s Medium), 10mM β glycerol phosphate 50µg/ml ascorbic acid M dexomethazone ALP Specific Activity (U/mg prot/min -1 ) ± S.E.M Electrospinning Days in Culture Alizarin Red [μm] Days in Culture Moreover, the staining test by Alizarin Red indicates a higher mineralization (calcium deposits) in the presence of osteogenic medium

43 NANOSTRUCTURED PLATFORMS Electrospinning PCL and gelatin electrospun membranes may be successfully designed to embrace morphological and cellular requirements for replying Bony osteon-like structures

44 natura Evoluzione. biologia - medicina ingegneria L evoluzione dei materiali procede attraverso l incremento: del contenuto di informazione inglobata nel materiale dell integrazione delle prestazioni fornite dal materiale della complessità delle prestazioni dell incidenza e della complessità dei processi di trasformazione Evoluzione - Innovazione informazione integrazione complessità processi materiali quasi viventi materiali funzionalizzati materiali compositi materiali bulk

45 inspiration Evolution a vision for the future self-regeneration strategical analogy molecules regeneration logical analogy integration biocompatibility repair structural analogy regeneration functional analogy mimicking tissue Look deep into nature, and than you will understand everything better Albert Einstein

46 Conclusioni Le nuove conoscenze in biologia molecolare hanno permesso di identificare il linguaggio di base tra cellule e tessuti: codificare segnali biologici in biomateriali è il primo passo verso cellule-tessuti-materiali istruiti per un definito processo di rigenerazione. L integrazione con le necessità cliniche sollecita lo sviluppo di soluzioni innovative.

47 Ringraziamenti Financial Support P.A. Netti A. Borzacchiello C. Borselli F. Causa E. De Rosa R. De Santis A. Gloria V. Guarino A. Luciani L. Mayol F. Mollica M.G. Raucci A. Sannino S. Zeppetelli Campania Region Ministry of University and Research FIRB Ministry of Foreign Affairs National Research of Council V E.C. Framework Program VI E.C. Framework Program Fidia Advanced Biopolymers S.r.l. SVAS-Biosana S.p.A. Ghimas S.p.A. FinCeramica S.r.l. Indaco S.r.l.. Embassy of Italy in Korea Office of Science and Technology

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