Mechanical stimulation alters tissue differentiation and molecular expression during bone healing

Abstract

Further understanding of how mechanical cues modulate skeletal tissue differentiation can identify potential means of enhancing repair following injury or disease. Prior studies examined the effects of mechanical loading on osteogenesis, chondrogenesis, and fibrogenesis in an effort to enhance bony union. However, exploring how mechanical stimuli can divert the bone healing process towards formation of other mesenchymal tissues, as an endpoint, may elucidate new avenues for repair and regeneration of tissues such as cartilage and fibrous tissue. This study investigated the use of mechanical stimulation to promote cartilage rather than bone formation within an osteotomy. Our overall goal was to define skeletal tissue distribution and molecular expression patterns induced by the stimulation. Retired breeder Sprague-Dawley rats (n = 85) underwent production of a mid-diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day 10 and continuing for 1, 2, or 4 weeks, a cyclic bending motion (+35 degrees/-25 degrees at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal bone healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage-related genes (COL2A1 and COL10A1) and downregulation of bone morphogenetic proteins (BMPs) -4, -6 and -7. However, BMP-3 was upregulated with stimulation. These findings illustrate that mechanical cues can selectively modulate osteogenesis and chondrogenesis in vivo, and suggest a potential basis for treatment regimens for injured or diseased cartilaginous tissues.

Figure 1

During the surgical procedure, the femur is exposed (top left panel) and an anodized, aluminum external fixator (bottom panel) is attached using four 1.1 mm, stainless steel bicortical pins (top middle left panel). Following creation of a ∼1.5mm, full thickness osteotomy at the mid-diaphysis (top middle right panel), the surgical site is irrigated with saline and closed with staples (top right panel).

Figure 2

Sagittal sections stained with Safranin-O (red = cartilage) and Fast green (blue = bone) reveal that the mechanical stimulation induces cartilage formation in a distinctive wedge shape at the periphery of the gap and prevents bone formation within the gap. In contrast, control specimens demonstrate bone formation within the gap, and the small amount of cartilage that is present in these specimens is undergoing endochondral ossification. Magnification = 12.5x. Scale bar = 1 cm.

Figure 3

3-D reconstructions of serial histological sections emphasize the differences in spatial distributions of tissues between stimulated (left) and control (right) specimens.

Figure 4

Bending stimulation resulted in differences in volumes of several types of skeletal tissues. Error bars indicate +1 SD. * = significant difference (p<0.05). n=2 for 10-day timepoint, n=3 for 38-day control, n=4 for all other bars.

Figure 5

Bending stimulation altered mRNA expression patterns of COL2A1, COL10A1, BSP, OC, and OPN. Error bars indicate +1 SD. * = significant difference (p<0.05). n=6 for 10-day timepoint, n=5 for 38-day stimulated timepoint, n=4 for all other bars.

Figure 6

Bending stimulation altered mRNA expression patterns of several BMPs. Error bars indicate +1 SD. * = significant difference (p<0.05). n=6 for 10-day timepoint, n=5 for 38-day stimulated timepoint, n=4 for all other bars.

Figure 7

In situ hybridization for collagens type I and II in regions of cartilage at POD 38 (Safranin-O/Fast-green stained slide and corresponding dark-field image). Both COL1A1 and COL2A1 mRNA expression in cartilage tissue were higher in stimulated than control specimens. Magnification = 200x. Bar = 100 microns.