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Electrospun Silk Fibroin Nanofibrous Scaffolds with Two-Stage Hydroxyapatite Functionalization for Enhancing the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells Eunkyung Ko,†,‡ Jong Seung Lee,†,‡ Hyunryung Kim,§ Sung Yeun Yang,§ Dasom Yang,§ Kisuk Yang,‡ JiYong Lee,§ Jisoo Shin,‡ Hee Seok Yang,⊥ WonHyoung Ryu,*,§ and Seung-Woo Cho*,‡,∥ ‡
Department of Biotechnology and §School of Mechanical Engineering, Yonsei University, Seoul 03722, Republic of Korea Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Republic of Korea ∥ Center for Nanomedicine, Institute for Basic Science (IBS), Seoul 03722, Republic of Korea ⊥
ABSTRACT: The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZbinding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering. KEYWORDS: electrospun silk fibroin scaffolds, hydroxyapatite particles, polydopamine, human adipose-derived mesenchymal stem cells, osteogenesis
1. INTRODUCTION Several attempts have been made to reconstitute natural bone tissuelike constructs by using various types of materials including polymers, inorganic materials, metals, and their composites. Different fabrication techniques have been used to reconstitute natural extracellular matrix (ECM) structures and mechanical properties of bone tissue. In particular, polymeric scaffolds prepared by electrospinning have been utilized as attractive bone tissue-engineering scaffolds because the generated nanoscale three-dimensional (3D) fibers resemble the natural ECM structures and provide scaffolds with interconnected pores, which increase the efficiency of molecular transport and surface area for attachment of sufficient number of osteogenic cells.1−4 Despite the advantages of electrospinning, challenges still exist in the development of highly osteogenic scaffolds that can provide a favorable physical environment, have sufficient mechanical strength to be useful as bone substitutes,5 and can induce mineralization for functional bone formation during the early stage of osteogenesis.6,7 © XXXX American Chemical Society
The use of silk-based biomaterials has been reported to be advantageous for bone regeneration because of their strong mechanical properties, biocompatibility, low degradation rate, and ease of processing.8−11 In particular, silk fibroin (SF) protein obtained by removing sericin from pure silk to avoid immunogenic responses in vivo10,12 has recently been highlighted as a material for electrospun fibrous scaffolds in bone tissue engineering.2,13,14 It has been reported that electrospun SF fibrous scaffolds enhance the osteogenic differentiation of stem cells and bone formation in bone defect models.15 Despite the osteogenic potential of electrospun SF scaffolds, nanofibrous structures of the scaffolds need reinforcement to Special Issue: 10 Years of Polydopamine: Current Status and Future Directions Received: March 8, 2017 Accepted: April 24, 2017
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DOI: 10.1021/acsami.7b03328 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
the tip of the needle (gauge size, 17 G) and the methanol surface was 18 cm with the ground at the bottom of the bath. A high voltage of 14 kV was applied between the needle and the ground. The electrospinning solution was fed at a speed of 1 mL/h using a precision syringe pump. Electrospun SF fibers suspended in methanol were dialyzed against distilled water for 2 days to replace methanol with water. The dialyzed SF fibers were then transferred to cylinder wells and freeze-dried, resulting in fibrous 3D SF scaffolds. 2.4. Preparation of Electrospun SF Scaffolds with Two-Stage HAp Functionalization. SF scaffolds with HAp on both interior portions and exterior surfaces (HAp-PDA-SF/HAp scaffolds) were prepared by coating the surface of 20% (w/w) SF/HAp scaffolds, first with PDA, and then with HAp. The SF/HAp scaffolds were immersed in DA solution (2 mg/mL in 10 mM Tris-HCl buffer, pH 8.5) for 4 h at room temperature. The PDA-coated scaffold samples were then washed with distilled water to remove the residues and placed in HAp solution (particle size
