Phenotypic characterization of canine cranial cruciate ligament associated synoviocytes

Abstract

Identification of synoviocytes surrounding the canine cranial cruciate ligament (CrCL) has not been investigated. Objectives 1) Develop and validate a technique to identify and quantify normal canine CrCL associated synoviocytes. 2) Compare synoviocyte phenotype proportions surrounding normal and abnormal canine CrCLs Design In vitro experimental Animals Cranial cruciate ligaments from 4 intact female and 6 intact adult male mixed-breed dogs (objective 1) and from 8 adult female hound dogs (objective 2) Methods Objective 1) Normal CrCLs - CD18 and HSP25 epitopes were colocalized using immunohistochemistry. Sagittal sections were quantified in the proximal, middle, and distal aspects of each section. Western blot, RT-PCR and immunoelectron microscopy was used to confirm the presence of CD18 and HSP25 in the canine CrCL. Objective 2) Normal, artificially stretched and naturally partially disrupted canine CrCLs - CD18 and HSP25 epitopes were colocalized using fluorescent immunohistochemistry. Sagittal sections were prepared from the central aspect of each CrCL and phenotypes were quantified in the proximal, middle, and distal aspects of each section. Results Objective 1) Synoviocyte populations stained positive for CD18 (CD18+) or HSP25 (HSP25+), and a small population of cells stained for both epitopes (DS+). The proportion (mean ± SEM) of HSP25 + synoviocytes (57 ± 7.5 %) was significantly greater than the proportion of CD18 + synoviocytes (27 ± 8.2 %), which was significantly greater than the proportion of DS+ synoviocytes (16 ± 3.5%). Western blot, RT-PCR and immunoelectron microscopy confirmed the presence of CD18 and HSP25 epitopes in the canine CrCL. Objective 2) The pixel count for HSP25 + synoviocytes (57 ± 7.5 %) was significantly greater than the proportion of CD18 + synoviocytes (27 ± 8.2 %), which was significantly greater than the proportion of DS+ synoviocytes (16 ± 3.5%) in all groups. There was no significant difference in the proportions of each of the phenotypes between CrCLs. Conclusion Three synoviocyte phenotypes were identified using immunohistochemical staining. Synoviocyte phenotype proportions did not differ between normal and abnormal CrCLs, however the HSP25+ synoviocytes were the predominant phenotype in all CrCLs.