Biodegradable Nanofibers-Reinforced Microfibrous Composite Scaffolds for Bone Tissue Engineering Albino Martins, B.Sc., Ph.D., 1,2 Elisabete D. Pinho, B.Eng., 1,2 Vı ´tor M. Correlo, B.Eng., Ph.D., 1,2 Susana Faria, B.Eng., M.Sc., Ph.D., 3 Alexandra P. Marques, B.Sc., Ph.D., 1,2 Rui L. Reis, B.Eng., M.Sc., Ph.D., 1,2 and Nuno M. Neves, B.Eng., M.Sc., Ph.D. 1,2 Native bone extracellular matrix (ECM) is a complex hierarchical fibrous composite structure, resulting from the assembling of collagen fibrils at several length scales, ranging from the macro to the nanoscale. The combination of nanofibers within microfibers after conventional reinforcement methodologies seems to be a feasible solution to the rational design of highly functional synthetic ECM substitutes. The present work aims at the development of bone ECM inspired structures, conjugating electrospun chitosan (Cht) nanofibers within biodegradable polymeric microfibers [poly(butylene succinate)—PBS and PBS/Cht], assembled in a fiber mesh structure. The nanofibers-reinforced composite fiber mesh scaffolds were seeded with human bone marrow mesenchymal stem cells (hBMSCs) and cultured under osteogenic differentiation conditions. These nanofibers-reinforced composite scaffolds sustained ECM deposition and mineralization, mainly in the PBS/Cht-based fiber meshes, as depicted by the increased amount of calcium phosphates produced by the osteogenic differentiated hBMSCs. The oste- ogenic genotype of the cultured hBMSCs was confirmed by the expression of osteoblastic genes, namely Alkaline Phosphatase, Osteopontin, Bone Sialoprotein and Osteocalcin, and the transcription factors Runx2 and Osterix, all involved in different stages of the osteogenesis. These data represent the first report on the biological func- tionality of nanofibers-reinforced composite scaffolds, envisaging the applicability of the developed structures for bone tissue engineering. Introduction T he native extracellular matrix (ECM) is a dynamic and hierarchically organized fibrous nanocomposite. It provides mechanical support for the embedded cells and also interacts with them regulating various cellular functions such as adhesion, migration, proliferation, differentiation, and tissue morphogenesis. 1 The ECM of connective tissues is a complex interconnected nano- and microranged fibrous network of polysaccharides (such as glycosaminoglycans) and proteins (such as collagen and proteoglycans), secreted by the adjacent cells. In the case of bone, the hierarchical organization of the collagen fibrils ensures the multiple functions of this tissue. 2 Besides the structural organization, native collagen fibrils are also covered by hydroxyapatite nanocrystals with their c-axis aligned with the longitudinal axis of the fibrils. Although the basic organization and composition of bone is known, replicating its structure and properties has been very challenging. 3 The understanding that the natural ECM is a multifunc- tional nanocomposite motivated researchers to rationally design synthetic ECM substitutes. To follow the clues pro- vided by the natural ECM, a processing method that is able to fabricate nanofibers from a variety of materials and mix- tures is a prerequisite. Electrospinning allows the production of ECM-mimetics that exhibit a physical structure similar to that of the fibrous proteins in the native ECM, albeit their different chemical composition. 4,5 Submicron electrospun polymer fibers are also good candidates as reinforcing agents in the development of advanced nanocomposites due to their continuity, orientation, inherent flexibility, and potential high compatibility with polymer matrices. However, only a limited number of composites reinforced with electrospun nanofibers have been proposed. The main interest of those composites has been to obtain enhanced physical character- istics, namely optical transparency and also the mechanical properties. 6–17 We recently developed novel biodegradable reinforced fiber-based composites that combine electrospun 1 3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Guimara ˜ es, Portugal. 2 PT Government Associated Laboratory, IBB—Institute for Biotechnology and Bioengineering, Braga, Portugal. 3 Department of Mathematics for Science and Technology, Research Centre Officina Mathematica, University of Minho, Guimara ˜ es, Portugal. TISSUE ENGINEERING: Part A Volume 16, Number 12, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2009.0779 3599