Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model

Abstract

Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent HO in patients at high risk such as those sustaining combat injuries and other forms of blast trauma.

Keywords: Animal model; Bioburden; Blast overpressure exposure; Endochondral ossification; Heterotopic ossification; Prophylaxis; Retinoic acid receptor-γ agonist; Traumatic extremity injury.

Figure 1

Schematic outline of the experimental studies and outcome measures. The number of rats analyzed in each treatment group is indicated.

Figure 2

Palovarotene treatment inhibits heterotopic ossification. Analysis and quantification of (A) total new bone and (B) soft tissue ectopic bone volume using the flattened μ-CT images at 28, 56 and 84 days after blast, extremity injury, amputation ± MRSA (*= p <0.05 using the Welch’s two Sample t-test). Representative 3D reconstructed μCT images of residual amputated limbs from vehicle control (C–D) and Palovarotene (E–F) treated rats at 84 days post injury after blast, extremity injury, and limb amputation with MRSA infection. Red circles indicate soft tissue ectopic bone and yellow circles indicate calcaneal ectopic bone.

Figure 3

Palovarotene treatment inhibits the initial phases of HO. Histologic and microscopic assessment of HO formation in vehicle- control rats at 15 days (A–C), 28 days (D–F) and 84 days (G–I), and in Palovarotene treated rats at 15 day s(J–L), 28 days (M–O) and 84 days (P–R) post injury. CB = cortical bone, AS = amputation site; FT = fibroblastic tissue; HC = immature and hypertrophying cartilage; SM = skeletal muscle; WB = woven bone; V = vascularization and HO = heterotopic ossification. The scale bars in the images (A), (D), (G) (M) and (P) correspond to 500µm. Scale bars in images (B), (E), (H), (K), (N) and (Q) corresponds to 200µm. Scale bars in images (C), (F), (I), (L), (O) and (R) correspond to 50µm.

Figure 4

Effects of Palovarotene treatment (n=4) and vehicle control (n=4) on chondrogenic (A–B) and osteogenic (C–H) related gene targets and matrix metalloproteinase-9 (I) gene expression. The fold change values were calculated by normalizing the values to the samples from non-injured muscle rats. Significantly different changes in gene expression are noted with an asterisk (*) (p < 0.05; Mann-Whitney U-test).

Figure 5

Percentage of wounds that dehisced and were not completely healed by 14 days post-injury. The number of wounds with delayed healing in each treatment group is indicated.