Direct‐Write Assembly of Microperiodic Silk Fibroin Scaffolds for Tissue Engineering Applications

Three–dimensional, microperiodic scaffolds of regenerated silk fibroin have been fabricated for tissue engineering by direct ink writing. The ink, which consisted of silk fibroin solution from the Bombyx mori silkworm, was deposited in a layer-by-layer fashion through a fine nozzle to produce a 3D array of silk fibers of diameter 5 µm. The extruded fibers crystallized when deposited into a methanol-rich reservoir, retaining a pore structure necessary for media transport. The rheological properties of the silk fibroin solutions were investigated and the crystallized silk fibers were characterized for structure and mechanical properties by infrared spectroscopy and nanoindentation, respectively. The scaffolds supported human bone marrow-derived mesenchymal stem cell (hMSC) adhesion, and growth. Cells cultured under chondrogenic conditions on these scaffolds supported enhanced chondrogenic differentiation based on increased glucosaminoglycan production compared to standard pellet culture. Our results suggest that 3D silk fibroin scaffolds may find potential application as tissue engineering constructs due to the precise control of their scaffold architecture and their biocompatibility.

[1]  Dietmar W Hutmacher,et al.  Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems. , 2004, Trends in biotechnology.

[2]  Ivan Martin,et al.  Three‐dimensional culture of melanoma cells profoundly affects gene expression profile: A high density oligonucleotide array study , 2005, Journal of cellular physiology.

[3]  David L Kaplan,et al.  Silk as a Biomaterial. , 2007, Progress in polymer science.

[4]  W. H. Goldmann,et al.  MECHANICAL ASPECTS OF CELL SHAPE REGULATION AND SIGNALING , 2002, Cell biology international.

[5]  David L Kaplan,et al.  Sonication-induced gelation of silk fibroin for cell encapsulation. , 2008, Biomaterials.

[6]  David L Kaplan,et al.  Electrospun silk-BMP-2 scaffolds for bone tissue engineering. , 2006, Biomaterials.

[7]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.

[8]  R. Lauster,et al.  Initiation of mesenchymal condensation in alginate hollow spheres--a useful model for understanding cartilage repair? , 2006, Artificial organs.

[9]  G. Whitesides,et al.  Cell shape provides global control of focal adhesion assembly. , 2003, Biochemical and biophysical research communications.

[10]  Chantal Gauvin,et al.  Design and fabrication of 3D‐plotted polymeric scaffolds in functional tissue engineering , 2007 .

[11]  W. An,et al.  Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C‐dependent p38 mitogen‐activated protein kinase signaling , 2003, Journal of cellular biochemistry.

[12]  F. Beier,et al.  RhoA/ROCK Signaling Regulates Sox9 Expression and Actin Organization during Chondrogenesis* , 2005, Journal of Biological Chemistry.

[13]  Robert Langer,et al.  Advances in tissue engineering. , 2004, Current topics in developmental biology.

[14]  S. Bent,et al.  Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix , 2001, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[15]  Shigeo Nakamura,et al.  Physical properties and structure of silk. VI. Conformational changes in silk fibroin induced by immersion in water at 2 to 130°c , 1979 .

[16]  U. Aebi,et al.  The Chondrocyte Cytoskeleton in Mature Articular Cartilage: Structure and Distribution of Actin, Tubulin, and Vimentin Filaments , 2000, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[17]  J. A. Lewis Direct Ink Writing of 3D Functional Materials , 2006 .

[18]  C. Lim,et al.  Mechanical characterization of nanofibers – A review , 2006 .

[19]  Robert L Sah,et al.  Probing the role of multicellular organization in three-dimensional microenvironments , 2006, Nature Methods.

[20]  A I Caplan,et al.  In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. , 1998, Experimental cell research.

[21]  Alexander Augst,et al.  Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors. , 2006, Biomaterials.

[22]  DW Hutmacher,et al.  Concepts of scaffold-based tissue engineering—the rationale to use solid free-form fabrication techniques , 2007, Journal of cellular and molecular medicine.

[23]  Ralph Müller,et al.  Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. , 2007, Biomaterials.

[24]  M. Solursh,et al.  Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton , 1984, The Journal of cell biology.

[25]  G. Vunjak‐Novakovic,et al.  Stem cell-based tissue engineering with silk biomaterials. , 2006, Biomaterials.

[26]  E. Blout,et al.  The Infrared Spectra of Polypeptides in Various Conformations: Amide I and II Bands1 , 1961 .

[27]  Tobias Schmelzle,et al.  Engineering tumors with 3D scaffolds , 2007, Nature Methods.

[28]  Farshid Guilak,et al.  Advanced tools for tissue engineering: scaffolds, bioreactors, and signaling. , 2006, Tissue engineering.

[29]  C A van Blitterswijk,et al.  3D fiber-deposited scaffolds for tissue engineering: influence of pores geometry and architecture on dynamic mechanical properties. , 2006, Biomaterials.

[30]  David L. Kaplan,et al.  Mechanism of silk processing in insects and spiders , 2003, Nature.

[31]  A. Barbero,et al.  Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity. , 2007, Arthritis and rheumatism.

[32]  S. Venyaminov,et al.  Intensities and other spectral parameters of infrared amide bands of polypeptides in the β‐ and random forms , 1973, Biopolymers.

[33]  David L. Kaplan,et al.  Mechanical Properties of Electrospun Silk Fibers , 2004 .

[34]  David L Kaplan,et al.  Silk fibroin microtubes for blood vessel engineering. , 2007, Biomaterials.

[35]  P. Benya,et al.  Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression , 1988, The Journal of cell biology.

[36]  A. U. Daniels,et al.  Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition. , 2005, Biomaterials.

[37]  David L Kaplan,et al.  Porous 3-D scaffolds from regenerated silk fibroin. , 2004, Biomacromolecules.

[38]  David L Kaplan,et al.  Electrospinning Bombyx mori silk with poly(ethylene oxide). , 2002, Biomacromolecules.

[39]  Z. Shao,et al.  Regenerated Bombyx silk solutions studied with rheometry and FTIR , 2001 .

[40]  David L Kaplan,et al.  Nanolayer biomaterial coatings of silk fibroin for controlled release. , 2007, Journal of controlled release : official journal of the Controlled Release Society.

[41]  Y. Lee,et al.  Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-alpha signaling. , 2000, Biochemical and biophysical research communications.

[42]  Seeram Ramakrishna,et al.  Potential of nanofiber matrix as tissue-engineering scaffolds. , 2005, Tissue engineering.

[43]  F. Barry,et al.  Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. , 2001, Experimental cell research.

[44]  Margam Chandrasekaran,et al.  Rapid prototyping in tissue engineering: challenges and potential. , 2004, Trends in biotechnology.

[45]  Young Hwan Park,et al.  Morphology of regenerated silk fibroin: Effects of freezing temperature, alcohol addition, and molecular weight , 2001 .

[46]  R. Sandberg,et al.  Gene expression perturbation in vitro--a growing case for three-dimensional (3D) culture systems. , 2005, Seminars in cancer biology.

[47]  Chris Holland,et al.  Natural and unnatural silks , 2007 .