Enzyme-crosslinked Bilayer Composite Scaffold Incorporating with Spatially Controlled Gene Delivery Carriers for the Induction of Mesenchymal Stem Cells in Osteochondral Tissue Regeneration

Y.H. Lee, H.C. Wu, M.H. Shen, J.S. Sun, T.W. Wang
National Tsing Hua University, Taiwan,
Taiwan

Keywords: bilayered scaffold, spatially controlled, enzymatic cross-linking, gene-activated matrix, osteochondral graft substitute

Summary:

Gene delivery from tissue engineering scaffolds has potential to promote localized transgene expression that can induce the formation of functional tissues. The development of osteochondral tissue engineering interfaces would be a novel treatment for traumatic injuries and aging associated diseases that affect joints such as osteoarthritis. However, the different compositions and mechanical properties of cartilage and bone show the complexity of this tissue interface, making it challenging for the design and fabrication of tissue engineering scaffolds. Current osteochondral tissue engineering strategies are hampered by the difficulties inherent in designing a seamless interface between cartilage and subchondral bone. Thus, the bilayered scaffold, which should ideally promote individual growth of both cartilage and bone layers within a single integrated implant has a clinical unmet need. The purpose of this study is to develop the synergistic effect through the combination of biomimetic scaffold, pDNA encoding GAM matrix, and stem cell inoculation to create necessary signals supply and promote the functional osteochondral tissues formation. Specifically, we demonstrate the development of a bilayered gene-activated osteochondral scaffold, in which mesenchymal stem cells (MSCs) are induced by plasmid transforming growth factor-beta 3 (TGF-β3) and plasmid bone morphogenetic protein-2 (BMP-2) in different layers, respectively, to simultaneously support the regeneration of articular cartilage and subchondral bone. In this study, we describe the development of a bilayered scaffold which could ideally promote individual growth of both cartilage and bone layers within a single integrated implant as well as with the capacity to be used as a gene delivery platform to promote transfection of human mesenchymal stem cells (hMSCs). Firstly, for the layer of subchondral bone, the oriented bone matrix with organic (type I collagen, Col) and inorganic (hydroxyapatite, Hap) composite scaffold has been developed through mineralization of hydroxyapatite precursors deposited on collagen fibril using a wet-chemical co-precipitation process. In addition, sponge-like type II collagen scaffold has been developed for the cartilage layer. Secondly, We prepared multi-shell nanoparticles with a calcium phosphate core and DNA/calcium phosphate shells conjugated with polyethyleneimine to act as non-viral vectors for delivery of plasmid DNA encoding bone morphogenetic protein 2 (BMP2) and transforming growth factor-beta 3 (TGF-β3), respectively. The multi-shell nanoparticles were then added into individual layer of composite scaffold. At last, an enzyme, microbial transglutaminase, was used as a cross-linking agent to crosslink and integrate the bilayered scaffold. The ability of this scaffold to act as a gene-activated matrix for BMP-2 and TGF-β3 delivery was demonstrated with successful transfection and stimulate hMSCs differentiation into the osteogenic and chondrogenic lineages by spatial control within the bilayered scaffold. Sustained release of plasmids from scaffolds has shown the ability to promote prolonged transgene expression and stimulate stem cell differentiation in vitro. This improved delivery method should enhance the functionalized composite graft to accelerate healing process in vivo in osteochondral tissue regeneration.