Mechanisms of reducing joint stiffness by blocking collagen fibrillogenesis in a rabbit model of posttraumatic arthrofibrosis

Posttraumatic fibrotic scarring is a significant medical problem that alters the proper functioning of injured tissues. Current methods to reduce posttraumatic fibrosis rely on anti-inflammatory and anti-proliferative agents with broad intracellular targets. As a result, their use is not fully effective and may cause unwanted side effects. Our group previously demonstrated that extracellular collagen fibrillogenesis is a valid and specific target to reduce collagen-rich scar buildup. Our previous studies showed that a rationally designed antibody that binds the C-terminal telopeptide of the α2(I) chain involved in the aggregation of collagen molecules limits fibril assembly in vitro and reduces scar formation in vivo. Here, we have utilized a clinically relevant arthrofibrosis model to study the broad mechanisms of the anti-scarring activity of this antibody. Moreover, we analyzed the effects of targeting collagen fibril formation on the quality of healed joint tissues, including the posterior capsule, patellar tendon, and subchondral bone. Our results show that blocking collagen fibrillogenesis not only reduces collagen content in the scar, but also accelerates the remodeling of healing tissues and changes the collagen fibrils’ cross-linking. In total, this study demonstrated that targeting collagen fibrillogenesis to limit arthrofibrosis affects neither the quality of healing of the joint tissues nor disturbs vital tissues and organs.


Target
Band position  Figure S1. Crucial surgical steps to generate knee injury in a rabbit model of arthrofibrosis. A, Getting an access to the knee cavity. B, Drilling a hole in the tibia (Tb) to install a Kirschner wire (Kw). C, Placing a loop of the Kw over a femur (Fe), D, Fixing the Kw with a nut placed over the tibia. E, Installing a pump (P) connected to the knee cavity via a silicone tube (St). F, An X-ray image of the operated leg immobilized in the flexed position with a Kw.
Supplementary Figure S2. A schematic of the experimental design of the study.
Supplementary Figure S3. A schematic depicting the premises of assays of collagen metabolites. A, A procollagen I molecule with intact N propeptides (NP) and C propeptides (CP); hydroxyproline residues present in the triple-helical domain are also indicated (HP). B, A collagen I molecule generated by extracellular cleavage of the NP and the CP; here, we will analyze the CP of procollagen I. C, A typical staggered arrangement of collagen molecules incorporated into a fibril. Collagen cross-links (XL) that link these collagen molecules are indicated with X. D, Collagen fragments formed due to turnover/degradation of collagen fibrils. Note that all collagen triple helix-derived fragments will include HP, and some will retain the collagen cross-links. Supplementary Figure S13. A graphic representation of differences in mineralization of osteochondral defects in the ACA-treated (□ and ■) and the CA-treated (○ and •) rabbits from the 8wk group. The left column shows results of measurements of uninjured (Un) and injured (In) legs. The GMs and 95% confidence intervals are indicated. The left panels show the Un/In ratios of analyzed parameters.

Supplementary
Supplementary Figure S14. A graphic representation of differences in mineralization of osteochondral defects in the ACA-treated (□ and ■) and the CA-treated (○ and •) rabbits from the 12wk group. The left column shows results of measurements of uninjured (Un) and injured (In) legs. The GMs and 95% confidence intervals are indicated. The left panels show the Un/In ratios of analyzed parameters.
Supplementary Figure S18. Histology of the lung tissue and intestines. Symbols: Ig; intestinal glands. Because the lungs were not inflated prior to the fixation, the lung tissue appears compact.
Supplementary Figure S21. Histology of the sciatic nerve and the Achilles tendon. Symbols: Nf; neural filaments, Cf; collagen fibers.