Abstract
Introduction and Objective
Heterotopic ossification is the formation of extraskeletal mineralized tissue commonly associated with either trauma or surgery. While several mouse models have been developed to better characterize the pathologic progression of HO, no model currently exists to study HO of the hip, the most common location of acquired HO in patients. Owing to the unique biological mechanisms underpinning the formation of HO in different tissues, we sought to develop a model to study the post-surgical HO of the hip.
Materials and Methods
Wild-type mice C57BL/6J mice were used to study the procedure outcomes, while Pdgfra-CreERT2;mT/mG and Scx-GFP reporter animals were used for the lineage tracing experiments (total n=16 animals, male, 12 weeks old). An anterolateral approach to the hip was performed. Briefly, a 2 cm incision was made centered on the great trochanter and directed proximal to the iliac crest and distally over the lateral shaft of the femur. The joint was then reached following the intermuscular plane between the rectus femoris and gluteus medius muscles. After the joint was exposed, the articular cartilage was removed using a micropower drill with a 1.2 mm reamer. The medius gluteus and superficial fascia were then re-approximated with Vicryl 5-0 suture (Ethicon Inc, Somerville, NJ) and skin was then closed with Ethilon 5-0 suture (Ethicon Inc). Live high resolution XR imaging was performed every 2 wks to assess the skeletal tissues (Faxitron Bioptics, Tucson, AZ). The images were then scored using the Brooker classification. Ex-vivo microCT was conducted using a Skyscan 1275 scanner (Bruker-MicroCT, Kontich, Belgium). 3D reconstruction and analysis was performed using Dragonfly (ORS Inc., Montreal, Canada). For the histological analysis of specimens, Hematoxylin and Eosin (H&E), modified Goldner's Trichrome (GMT) stainings were performed. Reporter activity was assessed using fluorescent imaging.
Results
Substantial periarticular heterotopic bone was seen in all cases. A periosteal reaction and an initial formation of calcified tissue within the soft tissue was apparent starting from 4 wks after surgery. By XR, progressive bone formation was observed within the periosteum and intermuscular planes during the subsequent 8 weeks. Stage 1 HO was observed in 12.5% of cases, stage 2 in 62.5% of cases, and stage 3 HO in 25% of cases. 3D microCT reconstructions of the treated hip joints demonstrated significant de novo heterotopic bone in several location which phenocopy human disease. Heterotopic bone was observed in an intracapsular location, periosteal location involving the iliac bone and proximal femur, and intermuscular locations. Histological analyses further confirmed these findings. To assess the cells which gave rise to HO in this model, an inducible PDGFRα and constitutive Scx-GFP reporter mice were used. A dramatic increase in mGFP reporter activity was noted PDGFRα within the HO injury site, including in areas of new cartilage and bone formation. Scx-associated reporter activity increased in the soft tissue and periosteal periacetabular areas of injured hips.
Conclusions
HO has a diverse set of pathologies, of which joint associated HO after elective surgery is the most common. Here, we present the first mouse model of hip dislocation and acetabular reaming that mimics elements of human periarticular HO. The diverse locations of HO after acetabular reaming (intracapsular, intermuscular and periosteal) suggests the activation of different and specific HO program after surgery. Such a field effect would be consistent with local trauma and inflammation, which is a well-studied contributor to HO genesis. Not surprisingly, joint-associated HO significantly derives from PDGFRα-expressing cells, which has been shown to similarly give rise to intramuscular and intratendinous HO.
