Collagen Three-Dimensional Hydrogel Matrix Carrying Basic Fibroblast Growth Factor for the Cultivation of Mesenchymal Stem Cells and Osteogenic Differentiation

Abstract

Three-dimensional (3D) collagen hydrogels have been extensively used for cell culture experiments and are more closely representative of in vivo conditions than monolayer (2D) culture. Here we cultured rat bone marrow-derived mesenchymal stem cells (MSCs) in collagen hydrogels containing varying concentrations of basic fibroblast growth factor (bFGF) to examine the effect of bFGF on MSC proliferation and osteogenic differentiation in 3D culture. The optimal bFGF concentration that promoted the greatest degree of cell proliferation and expression of the early osteogenic induction marker alkaline phosphatase was also determined. Subsequent quantitative real-time polymerase chain reaction analysis of gene expression demonstrated that bFGF promoted significant upregulation of the bone-related genes: collagen type I, osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) for periods of up to 21 days. Immunofluorescence staining and fluorescence-activated cell sorting analysis further supported the enhanced osteogenic differentiation of cells as a greater proportion of cells were found to express OPN. Matrix mineralization within the collagen hydrogels was enhanced in the presence of bFGF, as assessed by calcium detection using von Kossa staining. These results clearly demonstrate a positive effect of bFGF on proliferation and osteogenic induction of MSCs in 3D collagen hydrogels when applied at the appropriate concentration. Moreover, collagen hydrogel constructs containing MSCs and appropriate growth factor stimulus might be a potentially useful biological tool for 3D bone tissue engineering.

Evaluation of the Growth Environment of a Hydrostatic Force Bioreactor for Preconditioning of Tissue-Engineered Constructs

“…64,65 The results suggested that gel contraction was due to the interaction of cells with the hydrogel fibers which along with the production of extracellular matrix resulted in the remodeling of fibrous gel structure. [64][65][66] When cells adhered onto the hydrogel fibers through integrin-adhesion complexes, tensile forces are created between cells and the surrounding matrix. 65 Once cells migrate within the hydrogel, fibers are rearranged, resulting in gel contraction.…”

Influence of Initial Seeding Density on Gene Expression during Neuronal Priming

“…The substrate is critical, so for example human pluripotent cells are often plated onto Mouse Embryonic Fibroblast (MEF) feeders [1,2] or are specially adapted for survival and growth on acellular material such as matrigel [3]. In addition to substrate, Initial Cell Seeding Density (ICSD) can have a significant impact on cell behaviour and is reported to alter proliferation and differentiation responses of various adult cells during 2D culture [4] and in 3D culture for tissue engineering purposes [5]. In the case of pluripotent cells, passaging hESC colonies at lower density of 1:8 resulted in a high degree of spontaneous differentiation versus those passaged at high density of 1:3 [6].…”

Sustained knock down of PPARγ and bFGF presentation in collagen hydrogels promote MSC osteogenesis

“…In the past years, several biomaterials were investigated as supporting matrix for bone tissue engineering, such as alginate and nanofibers [9][10][11]. Collagen, a natural extracellular matrix, has a good biocompatibility for stem cells survival and could form hydrogels in situ to retain cells and thus was widely used in tissue engineering, including bone tissue engineering [12][13][14]. For enhancing the therapeutic effects, it is essential to guarantee the osteogenic differentiation of seeding cells within scaffolds [14].…”

“…Collagen, a natural extracellular matrix, has a good biocompatibility for stem cells survival and could form hydrogels in situ to retain cells and thus was widely used in tissue engineering, including bone tissue engineering [12][13][14]. For enhancing the therapeutic effects, it is essential to guarantee the osteogenic differentiation of seeding cells within scaffolds [14]. However, pure collagen itself was limited in guiding osteogenic differentiation of stem cells.…”