Abstract
Introduction: Hip femoroacetabular impingement (FAI) is a pre-arthritic hip disease and has an etiologic role in up to 50% of hip OA. Synovial macrophages and fibroblasts have been reported to play a significant role in the pathophysiology of synovitis and OA. Isolation of these cells from synovial tissue obtained from hip arthroscopy procedures has not been previously established. This study aims to establish a protocol to harvest and digest hip synovial tissue to isolate the synovial macrophages and fibroblasts, especially to standardize the procedure to obtain synovium during hip arthroscopy and total hip replacement (THR).
Methods: Synovium tissue was obtained from patients undergoing hip arthroscopy for the treatment of hip FAI (pre-arthritic group) and patients undergoing THR for the treatment of hip OA (arthritic group). The tissue was fixed for cryosection to confirm synovial histology. The synovial tissue was minced and enzymatically digested by 100 µg/ml of DNase I and 100 µg/ml of Liberase TL. Antibodies used for cell sorting were anti-podoplanin (PDPN), anti-CD45, and anti-CD14. Fluorescence-activated cell sorting (FACS) was performed with BD FACSAriaII to isolate macrophages and fibroblasts.
Results: A total of 16 patients were included in this study. Eight synovial samples (male/female 1/7, age 45 ± 5.76y) were obtained from the arthritic group, among which 7 samples showed typical synovial histology composed of a lining layer and sublining layer. One sample was excluded as it was not synovium tissue. Regarding FAI synovial samples, a total of 9 samples were collected from 8 FAI patients (male/female 2/6, age 37.25 ± 5.95y). Five samples obtained from the anterior synovial fold were synovial tissue; the other 4 samples obtained from the head-neck were not synovium samples, excluding this location as a good anatomic location for synovial harvest. The antibody dilution ratios were optimized and set as CD45 1:100, CD14 1:50, PDPN 1:50. CD45+CD14+ cells were identified as macrophages/monocytes, while PDPN+CD45- cells were identified as fibroblasts.
Summary: In hip arthroscopy, the anterior synovial fold is a more feasible location to harvest synovium compared to the head-neck area. The enzymatic protocol of 100 µg/ml of DNase I and 100 µg/ml of Liberase TL is appropriate to digest hip synovial tissue for FACS. The established tissue collection, digestion, and staining workflow for hip FAI/OA synovium will be used for FACS, bulk RNA sequence, and single-cell RNA sequence for our future study.