Controlling the Fibroblastic Differentiation of Mesenchymal Stem Cells Via the Combination of Fibrous Scaffolds and Connective Tissue Growth Factor

Abstract

Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, fibroblast-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial fibroblastic differentiation of MSCs in vitro. To this end, connective tissue growth factor (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(e-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindleshaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to 21 days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their fibroblastic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of fibroblast-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC fibroblastic differentiation in vitro.

Dynamic Vibration Cooperates with Connective Tissue Growth Factor to Modulate Stem Cell Behaviors

“…6,15 Interestingly, at the protein level, Col I production was predominantly controlled by CTGF (Fig. 6B) and the stimulatory effects of vibration was not manifested at the protein level.…”

“…6 The vibration frequency and the normal displacement at the center of the scaffold were maintained at 200 Hz and 40 mm, respectively. After 3 days of dynamic culture, cell culture media was replaced with that supplemented with 100 ng/mL CTGF (Biovendor, Asheville, NC) and 50 mg/mL ascorbic acids (Sigma), 15 and MSCs were cultured statically from day 3 to 6 (Fig. 1B).…”

“…14 We have previously demonstrated that high frequency vibrations can alter the gene expression by MSCs cultivated on an electrospun fibrous scaffold. 6,15 On the other hand, vocal fold development and maturation are influenced by various growth factors; higher levels of hormone receptors and/or serum hormones in male vocal folds are, to some extent, correlated with the elevated level of HA found in male vocal folds. 16 Our previous work confirmed the regulatory role of connective tissue growth factor (CTGF) on MSC differentiation toward VFF-like cells.…”

“…16 Our previous work confirmed the regulatory role of connective tissue growth factor (CTGF) on MSC differentiation toward VFF-like cells. 15 CTGF is a multi-domain protein that binds various integrin receptors or heparin sulfate proteoglycans to orchestrate diverse cellular events in embryonic development, angiogenesis, mitogenesis, tissue morphogenesis, and wound healing and fibrosis. 15,17,18 We hypothesize that physiologically relevant vibratory forces and soluble biochemical cues will cooperatively drive MSCs toward the VFF phenotype.…”

“…15 CTGF is a multi-domain protein that binds various integrin receptors or heparin sulfate proteoglycans to orchestrate diverse cellular events in embryonic development, angiogenesis, mitogenesis, tissue morphogenesis, and wound healing and fibrosis. 15,17,18 We hypothesize that physiologically relevant vibratory forces and soluble biochemical cues will cooperatively drive MSCs toward the VFF phenotype. 6,[18][19][20][21] In the present study, we explore the effects of combined vibratory stimulations and soluble CTGF on MSCs in terms of their proliferation, gene expression, and protein synthesis.…”