Tensile loading modulates bone marrow stromal cell differentiation and the development of engineered fibrocartilage constructs

Abstract

Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

FIG. 1.

Tensile loading system. The tensile loading system consisted of a linear motor that applied cyclic tensile displacements to the fibrin constructs via a linear sliding table and rake attachment. Motion was controlled by a Trio Motion controller and feedback from an optical encoder. The fibrin constructs (inset) were cast between porous polyethylene end blocks for rigid fixation and connection to the rake attachment. Color images available online at www.liebertonline.com/ten.

FIG. 2.

BMSC chondrogenesis in fibrin gels. Constructs were cultured up to 12 days under free-swelling (FS) conditions in basal or chondrogenic medium and examined for mRNA expression of (A) collagen II, (B) aggrecan, and (C) sox-9 and (D) sGAG accumulation. (E) Cell morphology within the fibrin gels was examined by staining for the f-actin cytoskeleton and imaging with a confocal microscope (representative images from day 4). Levels of mRNA are expressed on a logarithmic scale as nmol of transcript per μg of total RNA. n = 6/condition, *p < 0.05 with basal medium. BMSC, bone marrow stromal cell; sGAG, sulfated glycosaminoglycan. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Matrix synthesis rates during short-term loading. Constructs were cultured in basal or chondrogenic medium for 4 or 12 days and loaded intermittently (3 h on–3 h off) for 24 h. (A) Protein and (B) proteoglycan (PG) synthesis rates were measured during the 24 h loading period by incorporation of 3H-proline and 35S-sulfate, respectively. The total (C) sGAG and (D) DNA contents were also measured in the constructs. n = 6/condition, *p < 0.05 with unloaded.

FIG. 4.

BMSC gene expression. The mRNA expression levels of (A) collagen II, (B) aggrecan, (C) collagen I, and (D) osteocalcin were measured by real time reverse transcription polymerase chain reaction. *p < 0.05 for 10% vs. FS, n = 6/group.

FIG. 5.

Matrix accumulation with long-term loading. Fibrin gel constructs were analyzed for (A) DNA, (C) sGAG, and (D) collagen contents following culture under FS conditions, fixed at the day 7 length (0%), or cyclically loaded (+10%). (B) Conditioned medium samples were analyzed for sGAG content and reported as the cumulative sGAG released. *p < 0.05 for 10% vs. FS, ⁺p < 0.05 for 10% vs. 0%, p < 0.05 for 0% vs. FS. n = 6/group.

FIG. 6.

Extracellular matrix composition and organization. Portions of the fibrin gel constructs were formalin fixed, paraffin embedded, and sectioned to 5 μm. (A) Cross sections from day 21 constructs from FS, unloaded (0%), or cyclically loaded (+10%) groups were stained for sGAG with Safranin-O and counterstained with Fast Green. (B) Transverse sections from day 14 samples were stained with Picrosirius Red. Arrows indicate the long axis and loading direction of the constructs. (C) Type I collagen (green) and type II collagen (red) were detected by immunofluorescence staining of day 21 cross sections, and inset shows the absence of staining without the primary antibody. All scale bars are 50 μm.