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. 2008 Aug;466(8):1930-7.
doi: 10.1007/s11999-008-0300-x. Epub 2008 Jun 6.

The effects of GDF-5 and uniaxial strain on mesenchymal stem cells in 3-D culture

Eugene Farng  1 , Alfonso R Urdaneta, David Barba, Sean Esmende, David R McAllister
Affiliations

The effects of GDF-5 and uniaxial strain on mesenchymal stem cells in 3-D culture

Eugene Farng et al. Clin Orthop Relat Res. 2008 Aug.

Abstract

Recent endeavors in tissue engineering have attempted to identify the optimal parameters to create an artificial ligament. Both mechanical and biochemical stimulation have been used by others to independently modulate growth and differentiation, although few studies have explored their interactions. We applied previously described fabrication techniques to create a highly porous (90%-95% porosity, 212-300 microm), 3-D, bioabsorbable polymer scaffold (polycaprolactone). Scaffolds were coated with bovine collagen, and growth and differentiation factor 5 (GDF-5) was added to half of the scaffolds. Scaffolds were seeded with mesenchymal stem cells and cultured in a custom bioreactor under static or cyclic strain (10% strain, 0.33 Hz) conditions. After 48 hours, both mechanical stimulation and GDF-5 increased mRNA production of collagen I, II, and scleraxis compared to control; tenascin C production was not increased. Combining stimuli did not change gene expression; however, cellular metabolism was 1.7 times higher in scaffolds treated with both stimuli. We successfully grew a line of mesenchymal stem cells in 3-D culture, and our initial data indicate mechanical stimulation and GDF-5 influenced cellular activity and mRNA production; we did not, however, observe additive synergism with the mechanical and biological stimuli.

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Figures

Fig. 1A–D
Fig. 1A–D
The cultured scaffolds demonstrated successful cell adhesion and growth (Stain, hematoxylin and eosin). (A) A low-power (×10) view of a scaffold treated with only mechanical stimulus shows a large cell colony on the surface of the scaffold. (B) A medium-power (×100) view of a scaffold treated with only GDF-5 also demonstrates a large cell colony on the surface of the scaffold. (C) High-power view (×500) of a scaffold treated with both cyclic strain and GDF-5 shows cell colonies within the substance of the scaffold, spanning scaffold struts. (D) Higher-power view (×1000) of a scaffold treated with both cyclic strain and GDF-5 shows a cell colony within the substance of the scaffold.
Fig. 2
Fig. 2
Cellular proliferation was quantified by MTS reduction. Cultured scaffolds were incubated in the presence of MTS, which undergoes colorimetric change in the presence of cellular reductive agents. Scaffolds treated with both cyclic strain and GDF-5 demonstrated a 70% increase in cellular activity (p = 0.017). Values reported represent increases compared to nonstimulated controls. Error bars indicate 95% confidence interval of the mean.
Fig. 3
Fig. 3
RT-PCR analysis of mRNA gene expression was performed on scaffolds that were either unstimulated or stimulated with uniaxial 10% cyclic strain at 0.33 Hz, GDF-5, or both. Either mechanical stimulation or GDF-5 increased gene expression of collagen I, collagen III, and scleraxis (p < 0.05), without increasing tenascin C (p > 0.05). Combined stimuli only increased expression of collagen III (p < 0.05). We observed no synergy. Values reported represent increases compared to nonstimulated controls. Error bars indicate standard deviation of the mean.
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