Poster 120: Understanding the Biological Basis of Contracture Using a Mouse Model of Single Hindlimb Immobilization-Induced Fibrosis

Objectives: Arthrofibrosis is characterized by excessive collagen production and adhesions that result in restricted joint motion (stiffness) pain. The rate of procedural intervention (i.e. manipulation under anesthesia or surgical contracture release) ranges from 0.7% to 35% after anterior cruciate ligament (ACL) reconstruction surgery, up 57% a knee dislocation event. Current treatments are limited early range injury, physical therapy, dynamic splinting, release, but at present there no non-surgical therapeutic interventions can reliably reduce prevent stiffness. To better understand the mechanisms underlying development stiffness, we have developed novel mouse model formation based on single hindlimb immobilization using 3D-printed clamshell casts. Previous work has demonstrated produces contractures ankle vary reversibility depending age duration immobilization. current study seeks biological lead immobilization-induced through analysis gene expression histopathology. Methods: Female male C57BL/6J mice aged 14-19 weeks underwent (SHLI) custom, cast for 2 3 prior release (see Figure 1 design, n=4 each gender, time point). Casts were changed was assessed weekly custom measurement system. Mice sacrificed day 4 days (3-week only) tissues harvested histology immunohistochemistry (IHC). Mouse joints fixed 4% formaldehyde, decalcified EDTA embedded paraffin sectioning. Serial 7mm sections stained with hematoxylin eosin (H&E), Masson’s Trichrome, Picrosirius Red Safranin O/Fast Green histological analyses. Sections additionally analyzed identify markers myofibroblasts (α-smooth muscle actin, αSMA) macrophages (F4/80). For analysis, fatpad adjacent synovium immobilized followed (n=9 male, n=9 female). Tissues immediately placed RNAlater high-quality RNA (RIN>7, 260/280>1.8) isolated established protocols. Changes NanoString Fibrosis panel confirmed RTqPCR. Histological quantification done QuPath, statistics completed Graphpad Prism. Results: Single sustained both female mice, an average loss 31.9 ± 6.5 degrees 37.3 15.6 consistent previously reported results mice. There deposition excess extracellular matrix, including collagen, fat pad posterior capsule joints. These changes more pronounced compared 2-weeks group. 3-weeks showed fibrosis free cage release. Additionally, identified increases αSMA F4/80- immunopositive cells limbs casting Analysis persistent periarticular fibrosis. analyses than 230 differentially expressed genes between control / contralateral limbs. Specifically, pathway uncovered robust increase involved biosynthesis modification, matrix synthesis (ECM), ECM degradation, transforming growth factor beta (TGFβ) signaling epithelial mesenchymal transition (EMT) pathways. Differential selected RTqPCR Conclusions: Here provided in-depth characterization histologic occur SHLI. Our demonstrates SHLI provides simple effective preclinical immobilization-based peri-articular This reproducible, cost effective, permits events be readily employed transgenic animals, will allow evaluation interventions. Similar human phenotype, found structures infrapatellar pad, as well F4/80 positive immunostaining, suggesting macrophages, respectively. Importantly, consistency our sequencing studies post-surgical arthrofibrosis subjects, which includes upregulation degradation TGF-β signaling. We also observe immobilization, may opportunity intervention. several potential targets Future should aim test preventative therapies specifically target these pathways non- SHLI-driven