We investigated whether mesenchymal stem cells (MSCs) in synovial fluid (SF) increased in the knee with degenerated cartilage and osteoarthritis. SF was obtained from the knee joints of 22 patients with anterior cruciate ligament (ACL) injury during ACL reconstruction, and cartilage degeneration was evaluated arthroscopically. SF was also obtained from the knee joints of 6 healthy volunteers, 20 patients with mild osteoarthritis, and 26 patients with severe osteoarthritis, in which the grading was evaluated radiographically. The cell component in the SF was cultured for analyses. Synovium (SYN) and bone marrow (BM) were also harvested during total knee arthroplasties. The MSC number in SF was correlated with the cartilage degeneration score evaluated by arthroscopy. The MSC number in the SF was hardly noticed in normal volunteers, but it increased in accordance with the grading of osteoarthritis. Though no significant differences were observed regarding surface epitopes, or differentiation potentials, the morphology and gene profiles in SF MSCs were more similar to those in SYN MSCs than in BM MSCs. We listed 20 genes which were expressed higher in both SYN MSCs and SF MSCs than in BM MSCs, and 3 genes were confirmed by quantitative RT-PCR. MSCs in SF increased along with degenerated cartilage and osteoarthritis. ß

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a non enzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44 + cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

BackgroundSynovial fluid was collected prior to and at 3 to 4 days after ACL reconstruction to investigate the correlation between inflammatory cytokine levels in the acute phase after surgery and physical functional recovery at 3 months postoperatively.Methods For this purpose, 79 patients with ACL reconstruction using semitendinosus tendons were included in the study. Median days from injury to surgery were 80 days (13–291 days). Synovial fluid was obtained just before surgery and at 3 to 4 days after surgery. Physical activity of each patient was evaluated at 3 months postoperatively, and scored from 0 (hard to walk) to 5 (run). Patients able to jog (score 4) or run (score 5) were considered as the “quick recovery” group and others (scores 1–3) as the “delayed recovery” group.ResultsPhysical activity recovery scores in the early surgery group (preoperative period less than 60 days; Group I) were significantly better than those in the delayed surgery group (Group II). Among the cytokines tested, TNF-alpha and IL10 levels in synovial fluid were significantly higher in Group II at 3 to 4 days postoperatively, while levels of these cytokines were quite comparable preoperatively between the groups. Increased IL1-beta expression was noted in the delayed recovery group at 3 to 4 days postoperatively. In addition, levels of IL6, IL10 and IFN-gamma also tended to increase in patients with delayed recovery.ConclusionDelayed ACL reconstruction increases levels of inflammatory cytokines in synovial fluid after surgery and correlates with a prolonged recovery of short-period physical activity of the patients.

BackgroundAlthough osteoarthritis (OA) is a multifactorial disease, little has been reported regarding the cooperative interaction among these factors on cartilage metabolism. Here we examined the synergistic effect of ovariectomy (OVX) and excessive mechanical stress (forced running) on articular cartilage homeostasis in a mouse model resembling a human postmenopausal condition.MethodsMice were randomly divided into four groups, I: Sham, II: OVX, III: Sham and forced running (60 km in 6 weeks), and IV: OVX and forced running. Histological and immunohistochemical analyses were performed to evaluate the degeneration of articular cartilage and synovitis in the knee joint. Morphological changes of subchondral bone were analyzed by micro-CT.ResultsMicro-CT analyses showed significant loss of metaphyseal trabecular bone volume/tissue volume (BV/TV) after OVX as described previously. Forced running increased the trabecular BV/TV in all mice. In the epiphyseal region, no visible alteration in bone morphology or osteophyte formation was observed in any of the four groups. Histological analysis revealed that OVX or forced running respectively had subtle effects on cartilage degeneration. However, the combination of OVX and forced running synergistically enhanced synovitis and articular cartilage degeneration. Although morphological changes in chondrocytes were observed during OA initiation, no signs of bone marrow edema were observed in any of the four experimental groups.ConclusionWe report the coordinate and synergistic effects of extensive treadmill exercise and ovariectomy on articular cartilage degeneration. Since no surgical procedure was performed on the knee joint directly in this model, this model is useful in addressing the molecular pathogenesis of naturally occurring OA.

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