Tensile stimulation of murine stem cell-collagen sponge constructs increases collagen type I gene expression and linear stiffness

Abstract

The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 x 10(6) mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h=day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary the mechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.

Fig. 1.

Elongated GFP-T fluorescent cells in (A) nonstimulated constructs and (B) stimulated constructs at day 7 as well as (C) nonstimulated constructs and (D) stimulated constructs at day 14. Original magnification, × 10. Greater fluorescence was observed in the stimulated versus nonstimulated control constructs at 7 days. Color images available online at www.liebertonline.com/ten.

Fig. 2.

Tensile stimulation increased GFP-T expression by RFUs. *Significantly different from nonstimulated (NS) controls at same time interval (p < 0.02). **Significantly different from day 0 (p < 0.00001). ***Significantly different from day 7 stimulated (S) constructs (p < 0.0002). Data represented as mean ± standard deviations; n = 6 for all groups.

Fig. 3.

Tensile stimulation increased type I collagen gene expression by qRT-PCR. *Significantly different from nonstimulated (NS) controls at same time interval (p < 0.02). **Significantly different from day 0 (p < 0.0003). ***Significantly different from day 7 stimulated (S) constructs (p < 0.006). Data represented as means ± standard deviations; n = 6 for all groups.

Fig. 4.

Type I collagen gene expression as measured by RFUs and by qRT-PCR were positively correlated (R² = 0.79). Each point represents the average of six samples (means ±standard deviations). Circle denotes day-0 constructs, triangles denote nonstimulated constructs, and squares denote stimulated constructs.

Fig. 5.

Tensile stimulation increased linear stiffness. *Significantly different from nonstimulated controls at same time interval (p < 0.004). **Significantly different from day 0 (p < 0.0005). ***Significantly different from day 7 stimulated (p < 0.05). Data represented as means ± standard deviations; n = 6 for all groups.