Polymeric scaffolds incorporated with therapeutic genes and growth factors for bone tissue regeneration

Abstract

Applying biological cues regulates cell functions and can lead to the success of new tissue engineering scaffolds for regenerating human body tissues. Biological cues used to activate the scaffolds typically include growth factors and nucleic acids. Tissue engineering scaffolds with specially designed structures and incorporated biological cues should have great potential for tissue regeneration. The research for this project includes the investigation into the fabrication of new polymeric vectors for gene delivery, development of novel gene-activated matrices (GAMs) enabling spatiotemporal delivery of DNA encoding bone morphogens protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) for bone regeneration, and development of composite scaffolds with a vascularized osteon-like structure by encapsulating both osteoblasts and endothelial cells and with the controlled delivery of BMP-2 and VEGF. First, a new non-viral vector was developed by employing poly(D,L-lactic-co-glycolic acid)/polyethyleneimine (PLGA/PEI) nanoparticles which were modified with a cell penetrating peptide, KALA. Their physiochemical properties, including particle size, morphology, zeta potential and gel retardation ability were systematically studied. The PLGA/PEI_pDNA@KALA (PP@KALA) nanocomplexes allowed efficient transfection and exhibited negligible cytotoxicity, showing their superiority to commercial transfection agent PEI. Following the immobilization of new vectors onto electrospun alginate nanofibrous scaffolds via a functional polydopamine layer, an innovative biocompatible and biodegradable GAM was produced. In vitro degradation behavior, DNA release, and transgene expression of the new GAM was investigated. Sustained DNA release, long-term transgene expression and transfection of infiltrated cell were achieved, indicating the potential of newly developed GAMs. To promote bone regeneration by spatiotemporally delivering therapeutic genes, pBMP-2 was immobilized onto fibers of an electrospun nanofibrous tube and pVEGF was incorporated into an alginate hydrogel, which were integrated to form a composite scaffold. Prior to the encapsulation, pBMP-2 and pVEGF were complexed with newly developed PP@KALA to transfect pre-osteoblasts in 2D culture. PP@KALA exhibited high efficiency and lower cytotoxicity as compared with the positive control, PEI; and in comparison to cells transfected via PEI, the cells transfected by PP@KALA showed a more elongated and spread morphology, more well-developed F-actin stress fibers and mature focal adhesions. Cells transfected by PP@KALA exhibited efficient BMP-2 and VEGF production, increased alkaline phosphatase (ALP) activity, and enhanced gene expression of four bone-specific markers, indicating enhanced osteogenesis. After incorporation into 3D composite scaffolds, pBMP-2 and pVEGF could be sequentially released, transported into osteoblasts and induced osteogenesis in vitro. In a bone defect model using rats, the scaffolds providing dual delivery of pBMP-2 and pVEGF genes significantly induced bone healing at 8 week post-surgery. The fiber-hydrogel composite scaffold was also used to construct a new vascularized biomimetic scaffold by encapsulating osteoblasts and endothelial cells. MC3T3-E1s and mouse endothelial cells (MS-1) encapsulated in the composite scaffolds were shown to be spatially distributed in the shell and in the core, respectively, of the scaffolds. Both types of cells showed continuous proliferation over time. For the incorporated growth factors, sequential release of BMP-2 and VEGF from scaffold was achieved, which enhanced osteogenesis, as was evidenced by increased ALP activity, high-level calcium deposition and enhanced gene expression of osteogenesis-related markers.