Combined effects of chemical priming and mechanical stimulation on mesenchymal stem cell differentiation on nanofiber scaffolds

Abstract

Functional tissue engineering of connective tissues such as the anterior cruciate ligament (ACL) remains a significant clinical challenge, largely due to the need for mechanically competent scaffold systems for grafting, as well as a reliable cell source for tissue formation. We have designed an aligned, polylactide-co-glycolide (PLGA) nanofiber-based scaffold with physiologically relevant mechanical properties for ligament regeneration. The objective of this study is to identify optimal tissue engineering strategies for fibroblastic induction of human mesenchymal stem cells (hMSC), testing the hypothesis that basic fibroblast growth factor (bFGF) priming coupled with tensile loading will enhance hMSC-mediated ligament regeneration. It was observed that compared to the unloaded, as well as growth factor-primed but unloaded controls, bFGF stimulation followed by physiologically relevant tensile loading enhanced hMSC proliferation, collagen production and subsequent differentiation into ligament fibroblast-like cells, upregulating the expression of types I and III collagen, as well as tenasin-C and tenomodulin. The results of this study suggest that bFGF priming increases cell proliferation, while mechanical stimulation of the hMSCs on the aligned nanofiber scaffold promotes fibroblastic induction of these cells. In addition to demonstrating the potential of nanofiber scaffolds for hMSC-mediated functional ligament tissue engineering, this study yields new insights into the interactive effects of chemical and mechanical stimuli on stem cell differentiation.

Keywords: Basic fibroblast growth factor; Bioreactor; Functional tissue engineering; Ligament; Mechanical stimulation; Stem cells.

Fig. 1

Effect of bFGF on hMSC proliferation and matrix production. Treatment with exogenous bFGF resulted in significantly greater cells on scaffolds after 7 days of culture. Growth factor treatment also resulted in a significant increase in matrix deposition after 14 days of culture.

Fig. 2

Effect of bFGF on hMSC differentiation. Growth factor treatment resulted in the upregulation of types I and III collagen, fibronectin and tenascin-C. In contrast, the mean expression of scleraxis and tenomodulin decreased over time.

Fig. 3

Effect of chemical and mechanical stimulation on hMSC proliferation and biosynthesis. Cells remained similarly viable on both unloaded and loaded scaffolds. A significantly greater number of cells was measured on loaded scaffolds as compared to unloaded scaffolds after 28 days. Mechanical stimulation in conjunction with growth factor priming resulted in enhanced collagen deposition after 28 days of loading, as compared to unloaded controls. Histological analysis, via picrosirius red staining of collagen, indicated deeper matrix penetration into loaded scaffolds after 28 days. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Effect of chemical and mechanical stimulation on hMSC differentiation. Mechanical stimulation in conjunction with chemical priming resulted in a significant upregulation of types I and III collagen, tenascin-C and tenomodulin expression.

Fig. 5

Effect of chemical and mechanical stimulation on scaffold mechanical properties. No difference in scaffold mechanical properties was measured between groups over time. A significant decrease in yield strength, ultimate tensile strength (UTS) and ductility were measured 4 weeks after priming in both groups as compared to day 1 (#, p <0.05).