Biomaterials: Production, Processing and Application

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1 AITEX infoday - New Textiles. Research and Innovation in the Textile-Clothing- Technical Textiles Industry Brussels, 31st March 2009 Silk-Based Biomaterials: Production, Processing and Application Dr. Giuliano FREDDI Stazione Sperimentale per la Seta, Milan, Italy

2 Summary 11 Properties of 22 Silk as biomaterial 33 Biomedical applications of 44 Conclusions

3 Chemical composition and structure of Silk is a naturally occurring fibrous protein Composite structure of Bombyx mori Sericin Bombyx mori Fibroin Sericin and fibroin are proteins: Sericin: ~25 w% Fibroin: ~75 w% Bombyx mori worm: poly(gly-ala) Spiders and wild worms: poly(ala) Repetitive primary structure (natural block copolymers) of B. mori fibroin Silk Fibroin Heavy Chain: 5263 amino acids 10 Hydrophilic spacers 11 Hydrophobic repetitive blocks: -G-A-G-A-G-S-G-A-A-G-[S-G-(A-G) 2 ] 8 -Y Properties Properties of of 3 3

worms: poly(ala) Repetitive primary structure (natural block copolymers) of B.

4 Self-assembly and hierarchical order Self-assembling into larger fibrous structures (from nano to macro scale) characterized by a high degree of hierarchical molecular order Extended fibroin chains Pleated β-sheet β-sheets crystals Array of interconnected crystalline, laterally-ordered and amorphous domains Nano, micro-fibrils Highly oriented, crystalline fibres Properties Properties of of 3 3

Pleated β-sheet β-sheets crystals Array of interconnected crystalline, laterally-ordered and

5 Structure-function relationship of fibres From structure to properties and function Light weight (1.3 g/m 3 ) Unique mechanical properties: High strength, elasticity, toughness Good environmental, biological, and chemical resistance Biomaterials-related properties Allow versatile processing options to meet tissue-specific needs Be biocompatible to the host immune system and support cell attachment, proliferation and differentiation Provide structural support for cells and neo-tissue formation in the scaffold Be biodegradable at a controlled rate to match the rate of neo-tissue growth Properties Properties of of 3 3

properties Allow versatile processing options to meet tissue-specific needs Be biocompatible to the host immune system and support cell attachment,

6 Processability of fibroin Textile processing (reeling, twisting, weaving, knitting, degumming, dyeingprinting, finishing) Cocoons Fibroin fibres Fibroin solution Casting Freeze-drying Films, Coatings Degumming Dissolution Wet spinning Hydrogels E-spinning Sericin (by-product) Micro-fibres Nano-fibres Silk Silk as as biomaterial biomaterial 3 3

Freeze-drying Films, Coatings Degumming Dissolution Wet spinning Hydrogels E-spinning

7 Biocompatibility and cell interaction in vitro and in vivo Silk fibroin is biocompatible (after sericin removal) Long-standing use as sutures: Ocular, neural, cardiovascular surgery In vitro studies: 1 Contact with human plasma and cells: no adverse interactions with components of the inflammatory system keratinocytes, fibroblasts, and other cells proliferate onto fibroin scaffolds Fibroblasts In vivo studies: 2 Long-term subcutaneous implants (C57BL6 mice): no inflammation/immune/fibrotic response implants well tolerated, integrated into the living tissue Keratinocytes 1. Santin et al., J. Biomed. Mater. Res., 46(1999) Dal Pra et al., Biomaterials, 26(2005)1987 Histology: 6 months Silk Silk as as biomaterial biomaterial 3 3

fibroin scaffolds Fibroblasts In vivo studies: 2 Long-term subcutaneous implants (C57BL6 mice): no inflammation/immune/fibrotic response implants well tolerated, integrated into the

8 Biodegradability of US Pharmacopeia: An absorbable biomaterials is defined as one that loses most of its tensile strength within 60 days post-implantation in vivo Natural fibres: Defined as non-degradable Degradable over a long period of time (months, years) Silk fibres lose the majority of their tensile properties within 1 year in vivo Rate of degradation and absorption depends upon implantation site, mechanical environment, health and physiological status of the patient, type and shape of the material, etc. Regenerated materials (films, gels, etc.): Defined as degradable Degradable over a short period of time (days, weeks) Silk Silk as as biomaterial biomaterial 3 3

Rate of degradation and absorption depends upon implantation site, mechanical environment, health and physiological status of the patient, type and shape of the material,

9 Biomedical applications of fibroin Biomedical applications under study at SSS 3.a 3.a Functional coatings ( fibroin/gelatine blends) 3.b 3.b Scaffold for Reticular Connective Tissue (RCT) bioengineering 3.c 3.c Scaffold for Anterior Cruciate Ligament (ACL) bioengineering 3.d 3.d Electrospun tubular matrices for small vessel bypass grafting Biomedical Biomedical applications applications of of

b Scaffold for Reticular Connective Tissue (RCT) bioengineering 3.c 3.

10 3.a Functional coatings Coating of implantable medical devices (polymers, metals) might become a suitable shortcut to improve biocompatibility and to enhance integration in the host organism and new living tissue regeneration Silk fibroin (SF), collagen, gelatine (G) and other proteins can be used as coatings Advantages of SF and G as coating materials: Both SF and G are biocompatible natural polymers able to promote cell adhesion, growth, and differentiation Both SF and G are endowed with solubility in water and good film forming ability Mechanical properties and biodegradability can be tailored by targeted enzymatic crosslinking with tyrosinase or transglutaminase enzymes Fibroin Gelatine SF/G 30/70 SF/G 70/30 SF/G + Mushroom Tyrosinase Biomedical Biomedical applications applications of of

polymers able to promote cell adhesion, growth, and differentiation Both SF and G are endowed with solubility in water and good film forming ability Mechanical properties and biodegradability can be

11 3.b Scaffold for RCT bioengineering Skin injuries (acute, chronic) may destroy the Reticular Connective Tissue (loss of blood vessels) Current treatments for skin injuries: autologous graft Tissue engineering approach with fibroin: 3D-SF nonwoven sheet manufacture by carding/needling process Activation Functionalization Cell seeding Scaffold for skin regeneration In vivo tests: subcutaneous implantation into mice (up to 6 months) - no inflammation - no immune response - regeneration of a new connective tissue - formation of new blood vessels Biomedical Biomedical applications applications of of

Activation Functionalization Cell seeding Scaffold for skin regeneration In vivo tests: subcutaneous implantation into mice (up to 6 months) - no

12 3.c Scaffold for ACL bioengineering Anterior Cruciate Ligament (ACL) is the most common injured ligament (no spontaneous healing) Current surgical reconstructive techniques: autograft from the patellar/hamstring tendon Tissue engineering approach with fibroin: Knitted, braided structures manufacture Activation Functionalization Cell seeding Construct for ACL regeneration State of the art: 3D knitted structure with mechanical properties adequate for the ACL substitution available (strength up to 2000 N, resistance to fatigue tests) possibility to promote a neo-ligament tissue formation in vitro proved (porosity) structure patented, simulation and animal tests planned Biomedical Biomedical applications applications of of

regeneration State of the art: 3D knitted structure with mechanical properties adequate for the ACL substitution available (strength up to 2000 N, resistance to fatigue tests)

13 3.d Electrospun fibroin tubular grafts Small vessel diseases: stenosis (partial or complete occlusion), aneurysm Current surgical treatment: autograft from saphenous or umbilical vein or mammalian artery Tissue engineering approach with fibroin:production of electrospun tubular matrices Electrospinning system + V Morphology Cell interactions Compliance Øfibres 640 nm % Reduced days Blue: fibroin Red: control ES film control saphenous vein 2. umbilical vein 3. fibroin Biomedical Biomedical applications applications of of

matrices Electrospinning system + V Morphology Cell interactions Compliance Øfibres 640 nm % Reduced 100 90 80 70 60 50 40 30 20 10 0 1 3 7 days

14 Conclusions Biomaterials-related properties and functions of Have versatile processing options to alter the chemical and physical structure and morphology in relation to tissuespecific needs Be biocompatible to the host immune system where it is implanted and support cell attachment, migration, cell-cell interactions, cell proliferation and differentiation Provide structural support for cells and neo-tissue formation in the scaffold during the initial stages of postimplantation Be biodegradable at a controlled rate to match the rate of neo-tissue growth and facilitate the integration of engineered tissue into the surrounding host tissue Conclusions Conclusions

and differentiation Provide structural support for cells and neo-tissue formation in the scaffold during the initial stages of postimplantation Be biodegradable at a

Enzymatic Degradation Properties of Silk Fibroin Film

Journal of Fiber Bioengineering & Informatics 4:1 (2011) 35 41 http://www.jfbi.org doi:10.3993/jfbi04201104 Enzymatic Degradation Properties of Silk Fibroin Film Yamei Xu, Yiyu Wang, Yuhong Jiao, Ceng