Nanofibrous scaffolds with both MSC-laden cell fibers and growth factor-loaded fibers for tissue regeneration

Abstract

Tissue engineering scaffolds with biomimetic nanofibrous topography can facilitate the regeneration of tendon and ligament which are fibrous connective tissues composed of specific fibroblasts and aligned nanofibrous extracellular matrix (ECM). As a versatile and effective method to produce nanofibers, electrospinning has been extensively investigated to make nanofibrous scaffolds. Apart from physical cues, biological signaling molecules such as growth factors, are also often used in tissue engineering. Meanwhile, using mesenchymal stem cells (MSCs) for tissue regeneration has many advantages. Among various types of growth factors, basic fibroblast growth factor (bFGF) is a typical biomolecule that upregulate gene expression of tendon and ligamentspecific ECM proteins and hence promote the proliferation and differentiation of MSCs toward fibroblasts. Therefore, the combination of incorporating MSCs in a scaffold and having controlled delivery of bFGF in the scaffold should provide a good strategy for tendon/ligament regeneration. MSCs can be encapsulated in fibers via cell electrospinning developed in our group and the fibers can be aligned parallelly in scaffolds to simulate the cell distribution in native tendon/ligament tissue. In this study, multilayered scaffolds consisting of cell fibers and bFGF-containing nanofibrous membranes were fabricated. Bone marrow-derived MSCs were encapsulated in cell fibers while bFGF was incorporated in nanofibrous membranes for its controlled delivery to promote MSC differentiation. Medical grade poly(lactic-co-glycolic acid) (PLGA) was employed for fabricating bFGF-containing fibers in scaffolds. Cell fibers were made from Na-alginate solutions and contained MSCs. The Na-alginate was crosslinked into Ca-alginate using a CaCl2 solution. In in vitro experiments, the constructs with bFGF-containing PLGA nanofibers and MSC-laden cell fibers were cultured for up to 21 days. In day 1, a sodium citrate solution was dripped onto scaffolds to disrupt the crosslinked Ca-alginate cell fibers to release to MSCs. The viability and proliferation of MSCs were studied using LIVE/DEAD assay and MTT assay. On Day 1 and Day 3, the cell viability was all above 90%. MSCs proliferated well during the culture period up to 21 days. The MSCs in scaffolds with encapsulated bFGF proliferated faster than the control group and elongated notably, indicating that MSCs cultured under bFGF release had fibroblasts-like differentiation. The morphology and structure of scaffolds before and after different culture periods were investigated using SEM. Mechanical properties of the constructs were also examined.