Characterisation of synovial fluid cells of early osteoarthritis patients

Purpose: The synovial fluid (SF) of early stage osteoarthritis (OA) patients harbours a population of cells that may be involved in pathogenesis and/or attempted repair of cartilage, but remain poorly characterised. Recent research has revealed the involvement of inflammatory processes in the degenerative joint, as well as potential mechanisms of attempted repair to damaged joint tissues. In this study, the SF of patients receiving cell therapy for the treatment of cartilage injury were investigated to determine the phenotypes of the various subsets of adherent cells infiltrating the joint at an early stage of the disease. Methods: Knee SF was obtained from 15 patients (8 males, 7 females, mean age 34 years ± 11) receiving cell therapy to treat early OA. After centrifugation, the adherent cells in SF were culture in monolayer. At passage three (P3), cell surface immunoprofiling using flow cytometry was employed to determine the positivity of markers indicative of mesenchymal stem/stromal cells (MSCs) and chondrogenic cells. Gene expression of key chondrogenic (SOX9, ACAN, COL2A1, and FRZB) and hypertrophy markers (COL10A1 and ALK1) was also determined in SF cells (P3) by qRT-PCR. After chondrogenic differentiation, the production of glycosaminoglycan (GAG) by SF cells was quantified using biochemical assays (DMMB) and histological scoring (modified Bern Score). Statistical analyses were used to correlate surface marker positivity and gene expression to chondrogenic potency of the cells. Results: Cell surface immunoprofiling revealed a heterogeneous population of cells in the SF samples tested as demonstrated by the positivity for the monocyte/macrophage marker (CD14+, 53.8%±35.8), a haematopoietic, endothelial or adipose derived cell marker (CD34+, 28.4%±28.9) and mesenchymal stem/stromal cell markers (CD90+, 89.9%±16.3) (CD105+, 98.9%±1), (CD73+, 88.9%±17.8). All SF cells tested were highly positive for chondrogenic potency markers CD151 (99.5%±0.6) and CD44 (98.8%±1.4), but showed variable positivity for CD39 (26.8%±20.5), CD49c (26.7%±18) and CD166 (23.4%±23). We found a strong correlation, not only between CD14+ and cells positive for the leukocyte adhesion molecule CD106 (r=0.81, p=0.0003), but also between CD14 and CD34 (r=0.69, p=0.031), and between CD34 and CD106 (r=0.72, p=0.023).The genetic and surface makers assessed did not correlate with the generally low production of GAG/DNA (3.6μg/μg±2.7) and a mean Bern scores of 3.1±2.7 (Range of score: 0 (Poor) to 9 (good)). Conclusions: The cellular contents of the SF from early stage OA patients is comprised of both inflammatory and potentially reparative cells that may have originated from various joint tissues as part of an inflammatory response. These cell subsets could include monocyte/macrophages, endothelial, haematopoietic, mesenchymal stem/stromal cells and chondrocytes. The correlations between the positivity of CD14, CD34 and CD106 warrant further studies to explore the theory of inflammatory cells being recruited into the SF at an early stage of OA, potentially through increased angiogenic activity. SF cells are able to undergo chondrogenic differentiation, albeit with weak matrix production. Additional investigations are needed to determine the timeline of cellular and molecular events that result in these cell populations being present in joint SF.