A Hybrid Silk/RADA-Based Fibrous Scaffold with Triple Hierarchy for Ligament Regeneration Kelei Chen, B.Eng., 1 Sambit Sahoo, M.D., Ph.D., 2 Pengfei He, B.Eng., 1 Kian Siang Ng, B.Eng., 1 Siew Lok Toh, Ph.D., 1,3 and James C.H. Goh, Ph.D. 1,4 While silk-based microfibrous scaffolds possess excellent mechanical properties and have been used for ligament tissue-engineering applications, the microenvironment in these scaffolds is not biomimetic. We hypothesized that coating a hybrid silk scaffold with an extracellular matrix (ECM)-like network of self-assembling peptide nanofibers would provide a biomimetic three-dimensional nanofibrous microenvironment and enhance liga- ment tissue regeneration after bone marrow-derived mesenchymal stem cell (BMSC)-seeding. A novel scaffold possessing a triple structural hierarchy comprising macrofibrous knitted silk fibers, a silk microsponge, and a peptide nanofiber mesh was developed by coating self-assembled RADA16 peptide nanofibers on a silk microfiber-reinforced-sponge scaffold. Compared with the uncoated control, RADA-coated scaffolds showed enhanced BMSC proliferation, metabolism, and fibroblastic differentiation during the 3 weeks of culture. BMSC- seeded RADA-coated scaffolds showed an increasing temporal expression of key fibroblastic ECM proteins (collagen type I and III, tenascin-C), with a significantly higher tenascin-C expression compared with the con- trols. BMSC-seeded RADA-coated scaffolds also showed a temporal increase in total collagen and glycosami- noglycan production (the amount produced being higher than in control scaffolds) during 3 weeks of culture, and possessed 7% higher maximum tensile load compared with the BMSC-seeded control scaffolds. The results indicate that the BMSC-seeded RADA-coated hybrid silk scaffold system has the potential for use in ligament tissue-engineering applications. Introduction L igaments are strong flexible bands of collagen-rich tissue that connect bone to bone, providing a delicate balance of stability and flexibility to the joints of the body. Anterior cruciate ligament (ACL) injuries are one of the most common injuries affecting about 1 in 3000 of the population. 1 Current treatment consists of using allografts or autografts for reconstructing the functions of ACL and an estimated 75,000–100,000 ACL reconstructions being per- formed every year in the United States. 2 The use of auto- grafts or allografts has, however, been associated with serious complications such as ligament laxity, donor site morbidity, and disease transfer. Tissue engineering offers the possibility of creating functional engineered tissues to treat ACL injuries without the undesirable side effects as- sociated with current reconstructive strategies. The ideal scaffold for ligament regeneration should be biodegradable and biocompatible, exhibit sufficient mechanical strength, and also provide an extracellular matrix (ECM)-like envi- ronment that promotes the formation of new ligament tissue. 3–7 Among the various biomaterials that have been used for ligament tissue engineering, silk, a natural fibrous protein derived from Bombyx mori silk worms, possesses remarkable mechanical properties and a slow degradation rate that is suitable to support the healing ligament/tendon over a pe- riod of 6–12 months. 3,8 Its properties of biocompatibility, morphologic flexibility, environmental stability, and the ability for functionalization via amino-acid side-change modification to immobilize functional groups make it useful as a scaffolding biomaterial. 8–10 Silk-based scaffolds in the form of braided microfibers and porous sponges have al- ready been investigated for ligament regeneration. 4,11,12 However, microfibrous scaffolds often allow only limited cell attachment and result in nonhomogeneous cell distribution within the scaffold. 13 Moreover, silk microfibers (diameter: 10–25 mm) and cells (diameter: 5–30 mm) are of similar Departments of 1 Bioengineering, 3 Mechanical Engineering, and 4 Orthopaedic Surgery, National University of Singapore, Singapore, Singapore. 2 Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio. TISSUE ENGINEERING: Part A Volume 18, Numbers 13 and 14, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2011.0376 1399