Scar tissue formation secondary to acute muscle injury, surgical wounding and compartment syndrome can result in significant functional impairment and predispose to further injury. The source of fibroblasts, and the molecular mechanisms driving their activation and persistence in skeletal muscle fibrosis are not known. We hypothesized that cells expressing PDGFRβ become fibroblasts in response to injury and that targeting αv integrins in these cells reduces skeletal muscle fibrosis.

We used double-fluorescent reporter mice to demonstrate that cells expressing PDGFRβ become activated myofibroblasts in response to cardiotoxin (CTX) induced skeletal muscle injury. Following injury, PDGFRβ+ cells moved from perivascular locations into the interstitium in a distribution characteristic of fibroblasts, and showed marked induction of fibroblastic genes including αSMA and collagen1 (all p<0.0001). To confirm that αv integrins present on PDGFRβ cells critically regulate skeletal muscle fibrosis we used Itgavflox/flox;PDGFRβ-Cre mice (transgenic mice in which αv integrins are ‘knocked-down’ in PDGFRβ+ cells). These mice were significantly protected from CTX induced fibrosis (p<0.01). To demonstrate potential clinical utility of targeting αv integrins, we used a small molecule inhibitor of αv integrins (CWHM12). Treatment with CWHM12 significantly reduced fibrosis when delivered from the time of injury (p<0.01) and when delivered after the fibrotic response had become established (p<0.01).

We have identified a core pathway regulating fibrosis in skeletal muscle. Pharmacologic inhibition of αv integrins has potential clinical utility in the treatment and prevention of skeletal muscle fibrosis.

Our unpublished data has indicated that the perivascular stem cells (PSCs) have increased chondrogenic potential compared to mesenchymal stem cells (MSCs) derived in culture. There has been a recent change in the theory that stem cells work by a paracrine effect rather than differentiation. There are minimal data demonstrating the persistence of implanted stem cells when used for engraftment. This study aimed to develop an autologous large animal model for perivascular stem cells as well as to determine if cells were retained in the articular cartilage defects.

The reactivity of anti-human and anti-ovine antibodies was ascertained using immunohistochemistry and fluorescence-activated cell sorting (FACS). A panel of antibodies were combined and used to identify and purify pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-) using FACS. The purified cells were cultured and their identity checked using FACS. These cultured cells demonstrated osteogenic, adipogenic and chondrogenic potential.

Autologous ovine PSCs (oPSCs) were isolated, cultured and transfected using a GFP virus. The transfection rate was 88%. The cells were implanted into an articular cartilage defect on the medial femoral condyle using a hydrogel, four weeks following implantation the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect. Histology did not demonstrate any repair tissue at this early time point.

These data have confirmed the viability our large animal model and that the implanted stem cells were retained in the defect four weeks following implantation.