Inhibition of Mammalian Target of Rapamycin Signaling with Rapamycin Prevents Trauma-Induced Heterotopic Ossification

Abstract

A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α-positive cells, and α-smooth muscle actin-positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α-positive cells, 4) α-smooth muscle actin-positive blood vessels, and 5) of key extracellular matrix remodeling (CD44, Col1a1, integrins), osteogenesis (Sp7, Runx2, Bmp2), inflammation (Cxcl5, 10, IL6, Ccl2), and angiogenesis (Angpt2) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism.

Figure 1

Quantitative analysis of heterotopic bone formation with and without rapamycin treatment. The total new bone (A) and soft-tissue ectopic bone (nonassociated with cortical margins; B) are quantified at postoperative day 84. Vehicle control (3% dimethyl sulfoxide/saline). ^∗P < 0.05, ^∗∗P < 0.005, and ^∗∗∗P < 0.0005 (two-tailed t-test).

Figure 2

Rapamycin treatment attenuates the formation of ectopic bone in the blast-related extremity injury/methicillin-resistant Staphylococcus aureus infection model of heterotopic ossification (HO). A and E: Representative three-dimensional–rendered micro-computed tomography images of rat femurs at postoperative day 84 treated with vehicle control (3% dimethyl sulfoxide/saline; A) or rapamycin (2.5 mg/kg per day for 14 days, i.p; E). Areas of HO formation (red circles). B–D and F–H: Histologic assessment of hematoxylin and eosin–stained tissue sections from animals treated with vehicle control (B–D) and rapamycin (F–H). Areas of interest in near the distal end of the residual femur (boxed areas) in B, C, F, and G were examined at higher magnification and are shown in C, D, G, and H, respectively. Scale bars: 500 μm (B, C, F, and G); 200 μm (D and H). AS, amputation site; CB, cortical bone; FH, femoral head; FT, fibroblastic tissue; GT, granulation tissue; SM, skeletal muscle; WB, woven bone.

Figure 3

Effect of rapamycin on wound site vascularization/angiogenesis and platelet-derived growth factor receptor (PDGFR)α⁺ osteogenic mesenchymal stromal cell progenitor cell content. Immunofluorescence staining of amputation site from naïve and rapamycin-treated animals for α-smooth muscle actin (α-SMA; A) and PDGFRα⁺ (B) cells. DAPI marks cell nuclei. Data are expressed as the ratio of α-SMA⁺ vessels and PDGFRα⁺ cells in rapamycin-treated versus untreated. ^∗P < 0.05 (two-tailed t-test). Scale bars = 100 μm (A and B). HPF, high-powered field.

Figure 4

Alteration of connective tissue progenitor (CTP) frequency and osteogenic differentiation after rapamycin treatment. Shown is the frequency of CTPs in muscle tissue isolated from naïve, vehicle-, or rapamycin-treated injured rats (minus methicillin-resistant Staphylococcus aureus infection) at postoperative day 7, cultured either in growth (stromal) media (SM) or osteogenic media (OM) for 1 week (A) and early osteogenic differentiation of CTPs in SM or OM, as measured by p-nitrophenyl phosphate–based alkaline phosphatase activity assay (B). ^∗P < 0.05 versus vehicle control (two-tailed t-test).

Figure 5

Effects of rapamycin treatment and vehicle control on expression of extracellular matrix and osteogenic genes (A–D), inflammatory cytokines (E), angiopoietin (F), osteogenic regulators (G), and adipogenic genes (H). The fold-change in gene expression was calculated by normalizing the expression values to those from noninjured, naïve muscle tissue. n = 4 rapamycin-treated (A–H); n = 4 vehicle control (A–H). ^∗P < 0.05, ^∗∗P < 0.005 versus vehicle control (analysis of variance, followed by Tukey's test).