Immunolocalization of myostatin (GDF-8) following musculoskeletal injury and the effects of exogenous myostatin on muscle and bone healing

Abstract

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 µg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury.

Conflict of interest statement

The authors declared no potential conflicts of interest with respect to the authorship and publication of this article.

© The Author(s) 2012

Figures

Figure 1.

(A) General phases of fracture healing and time points examined for immunohistochemical staining of myostatin. (B) Negative control sections of skeletal muscle stained with FITC-conjugated secondary antibody but not primary antibody. Scale bar = 100 µm.

Figure 2.

Immunohistochemistry staining (green fluorescence) of myostatin in transverse section of fracture site. Left-panel figures indicate plane of section and area of interest shown in micrograph. Injured skeletal muscle fibers highly express myostatin at 12 hr (A; scale bar = 25 µm) and 24 hr (B; scale bar = 10 µm) following surgery. Six days postfracture, high myostatin expression (arrow) is restricted to the nuclei of regenerating myotubes (C; scale bar = 25 µm). Four days postfracture, round, mature soft callus chondrocytes (red arrow) and flat proliferating chondrocytes (white arrow) express myostatin (D; scale bar = 50 µm). Sections are counterstained with DAPI, and blue DAPI staining is abundant in the bottom right quadrant of the image (D). f, fibula; MSTN, myostatin; sc, soft callus. Yellow arrows indicate chondroprogenitor cells. Yellow staining represents autofluorescence.

Figure 3.

Endochondral ossification (orange stain, yellow arrows) is apparent at 15 days after fracture and is decreased by local treatment with recombinant myostatin. Light microscopic images of transverse sections in the center of 15-day-old fracture callus of myostatin-treated mice, stained with Safranin O and fast green. Fracture callus outline is indicated by open white arrows and cortical bone of the fractured fibula by the solid black arrow (A; scale bar = 500 µm). Cartilage area relative to total callus area decreased in a dose-dependent manner with myostatin treatment (B; n=5–7 per group). MSTN, myostatin.

Figure 4.

Fracture callus bony union (non-lamellar) is formed at 15 days after fracture and is decreased by local treatment with recombinant myostatin. Microcomputed tomography images of the fracture callus in the mouse fibula, treated with increasing doses of myostatin. Mediolateral (left) and anteroposterior (right) views (A; scale bar = 2 mm). Fracture callus bone volume decreased with myostatin treatment (B; n=9–11 per group). GDF-8, myostatin.

Figure 5.

Fibrous tissue formation in local myostatin-treated mice. Histological sections stained with Masson’s trichrome showing a dose-dependent increase in fibrous tissue formation (blue stain, arrow) during muscle repair with myostatin treatment (A; scale bar = 500 µm). Myostatin treatment (10 or 100 µg/ml) significantly increased the relative fraction of fibrous tissue staining blue in trichrome sections relative to vehicle treatment (B; n=5–7 per group).