Silk fibroin/hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering.

The design of new bioactive scaffolds mimicking the physiologic environment present during tissue formation is an important frontier in biomaterials research. Herein, we evaluated scaffolds prepared from blends of two biopolymers: silk fibroin and hyaluronan. Our rationale was that such blends would allow the combination of silk fibroin's superior mechanical properties with the biological characteristics of hyaluronan. We prepared scaffolds with porous microstructures by freeze-drying aqueous solutions of silk fibroin and hyaluronan and subsequent incubation in methanol to induce water insolubility of silk fibroin. Hyaluronan acted as an efficient porogenic excipient for the silk fibroin scaffolding process, allowing the formation of microporous structures within the scaffolds under mild processing conditions. Mesenchymal stem cells were seeded on silk fibroin/hyaluronan scaffolds and cultured for three weeks. Histology of the constructs after cell culture showed enhanced cellular ingrowth into silk fibroin/hyaluronan scaffolds as compared to plain silk fibroin scaffolds. In the presence of tissue-inductive stimuli, in vitro stem cell culture on silk fibroin/hyaluronan scaffolds resulted in more efficient tissue formation when measured by glycosaminoglycan and type-I and type-III collagen gene expression, as compared to plain silk fibroin scaffolds. In conclusion, our data encourages further exploration of silk fibroin/hyaluronan scaffolds as biomimetic platform for mesenchymal stem cells in tissue engineering.

[1]  D. Kaplan,et al.  Porosity of 3D biomaterial scaffolds and osteogenesis. , 2005, Biomaterials.

[2]  Ivan Martin,et al.  Silk matrix for tissue engineered anterior cruciate ligaments. , 2002, Biomaterials.

[3]  石田 治.  Chondrocytes are regulated by cellular adhesion through CD44 and hyaluronic acid pathway , 1997 .

[4]  Gordana Vunjak-Novakovic,et al.  Engineering cartilage‐like tissue using human mesenchymal stem cells and silk protein scaffolds , 2004, Biotechnology and bioengineering.

[5]  G. Vunjak‐Novakovic,et al.  Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells. , 2006, Tissue engineering.

[6]  Ung-Jin Kim,et al.  Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin. , 2005, Biomaterials.

[7]  Carola Cavallo,et al.  Hyaluronan‐based polymer scaffold modulates the expression of inflammatory and degradative factors in mesenchymal stem cells: Involvement of Cd44 and Cd54 , 2006, Journal of cellular physiology.

[8]  K. Leong,et al.  Significance of synthetic nanostructures in dictating cellular response. , 2005, Nanomedicine : nanotechnology, biology, and medicine.

[9]  Jennifer L West,et al.  Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration. , 2005, Biomaterials.

[10]  J. Glowacki,et al.  Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds. , 2001, Journal of biomedical materials research.

[11]  A. Ramamurthi,et al.  Fragment size- and dose-specific effects of hyaluronan on matrix synthesis by vascular smooth muscle cells. , 2006, Biomaterials.

[12]  C. Stanford,et al.  Effects of Implant Microtopography on Osteoblast Cell Attachment , 2003, Implant dentistry.

[13]  K. J. Grande-Allen,et al.  Review. Hyaluronan: a powerful tissue engineering tool. , 2006, Tissue engineering.

[14]  F Grego,et al.  Hyaluronan biodegradable scaffold for small-caliber artery grafting: preliminary results in an animal model. , 2006, European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery.

[15]  R Scapinelli,et al.  Hyaluronan-based biopolymers as delivery vehicles for bone-marrow-derived mesenchymal progenitors. , 2000, Journal of biomedical materials research.

[16]  P. Pavasant,et al.  Hyaluronan contributes to the enlargement of hypertrophic lacunae in the growth plate. , 1996, Journal of cell science.

[17]  D. Beebe,et al.  Hyaluronate in vasculogenesis. , 1983, Science.

[18]  G. Homandberg,et al.  Hyaluronan enhances cartilage repair through low grade tissue remodeling involving cytokines and matrix metalloproteinases , 2004, Inflammation Research.

[19]  Norbert Pallua,et al.  Implantation of preadipocyte-loaded hyaluronic acid-based scaffolds into nude mice to evaluate potential for soft tissue engineering. , 2005, Biomaterials.

[20]  Shin-Ichiro Nishimura,et al.  Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering. , 2005, Journal of biomedical materials research. Part A.

[21]  L. Meinel,et al.  The effect of hyaluronic acid on silk fibroin conformation. , 2008, Biomaterials.

[22]  N. Turner,et al.  A novel hyaluronan-based biomaterial (Hyaff-11) as a scaffold for endothelial cells in tissue engineered vascular grafts. , 2004, Biomaterials.

[23]  Krista L. Niece,et al.  Selective Differentiation of Neural Progenitor Cells by High-Epitope Density Nanofibers , 2004, Science.

[24]  S. Kumar,et al.  The role of hyaluronan in tumour neovascularization (review) , 1995, International journal of cancer.

[25]  B. Toole,et al.  Hyaluronan in morphogenesis. , 2001, Journal of internal medicine.

[26]  J. Wang,et al.  Ectopic Cartilage Formation Induced by Mesenchymal Stem Cells on Porous Gelatin-Chondroitin-Hyaluronate Scaffold Containing Microspheres Loaded with TGF-β1 , 2006, The International journal of artificial organs.

[27]  S. Kumar,et al.  Inverse relationship between hyaluronan and collagens in development and angiogenesis. , 1993, Differentiation; research in biological diversity.

[28]  V. Goldberg,et al.  Hyaluronan‐based polymers in the treatment of osteochondral defects , 2000, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[29]  Brian Ashton,et al.  Effects of high molecular weight hyaluronan on chondrocytes cultured within a resorbable gelatin sponge. , 2004, Tissue engineering.

[30]  R. Midura,et al.  Spatial distribution of CD44 and hyaluronan in the proximal tibia of the growing rat , 1996, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[31]  Ralph Müller,et al.  Effect of scaffold design on bone morphology in vitro. , 2006, Tissue engineering.

[32]  Gordana Vunjak-Novakovic,et al.  Bone Tissue Engineering Using Human Mesenchymal Stem Cells: Effects of Scaffold Material and Medium Flow , 2004, Annals of Biomedical Engineering.

[33]  V. Goldberg,et al.  Repair of osteochondral defects with hyaluronan- and polyester-based scaffolds. , 2005, Osteoarthritis and cartilage.

[34]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[35]  Magnus Lundberg,et al.  Treatment of osteochondral defects with autologous bone marrow in a hyaluronan-based delivery vehicle. , 2002, Tissue engineering.

[36]  L. Lazzeri,et al.  Morphological evaluation of bioartificial hydrogels as potential tissue engineering scaffolds , 2004, Journal of materials science. Materials in medicine.

[37]  S. Nehrer,et al.  Three-year clinical outcome after chondrocyte transplantation using a hyaluronan matrix for cartilage repair. , 2006, European journal of radiology.

[38]  S. Nishimura,et al.  Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering. , 2005, Biomaterials.

[39]  L. De Franceschi,et al.  Down regulation of degenerative cartilage molecules in chondrocytes grown on a hyaluronan-based scaffold. , 2005, Biomaterials.

[40]  M. Morra.  Engineering of biomaterials surfaces by hyaluronan. , 2005, Biomacromolecules.

[41]  S. Tsai,et al.  A study of the influence of polysaccharides on collagen self-assembly: nanostructure and kinetics. , 2006, Biopolymers.

[42]  R. Ian Freshney,et al.  Culture of cells for tissue engineering , 1994 .

[43]  K. Jacobson,et al.  Mapping trajectories of Pgp-1 membrane protein patches on surfaces of motile fibroblasts reveals a distinct boundary separating capping on the lamella and forward transport on the retracting tail. , 1991, Journal of cell science.

[44]  M. Young,et al.  Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro. , 1990, The Journal of biological chemistry.

[45]  G. Vunjak‐Novakovic,et al.  Tissue engineering of bone , 2006 .

[46]  H. Hauner,et al.  Tissue engineering of white adipose tissue using hyaluronic acid-based scaffolds. I: in vitro differentiation of human adipocyte precursor cells on scaffolds. , 2003, Biomaterials.

[47]  David L Kaplan,et al.  The inflammatory responses to silk films in vitro and in vivo. , 2005, Biomaterials.

[48]  Ling Qin,et al.  Cartilage regeneration using mesenchymal stem cells and a PLGA-gelatin/chondroitin/hyaluronate hybrid scaffold. , 2006, Biomaterials.

[49]  David L Kaplan,et al.  Silk-based biomaterials. , 2003, Biomaterials.

[50]  G. Lisignoli,et al.  Analysis of mesenchymal stem cells grown on a three-dimensional HYAFF 11-based prototype ligament scaffold. , 2005, Journal of biomedical materials research. Part A.

[51]  Sang-Hyug Park,et al.  Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation. , 2005, Artificial organs.

[52]  M B McCarthy,et al.  Functionalized silk-based biomaterials for bone formation. , 2001, Journal of biomedical materials research.

[53]  S. Weinstein,et al.  Age‐related changes in hyaluronan, proteoglycan, collagen, and osteonectin synthesis by human bone cells , 1992, Journal of Cellular Physiology.

[54]  Hai-sheng Li,et al.  Stimulation of porcine bone marrow stromal cells by hyaluronan, dexamethasone and rhBMP-2. , 2004, Biomaterials.