Artikel
Calcification during Osteogenic Differentiation of articular Chondrocytes is amplified by Anaphylatoxins
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Veröffentlicht: | 25. Oktober 2022 |
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Gliederung
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Objectives: Previous studies indicate an involvement of the complement system in posttraumatic osteoarthritis (OA) pathogenesis. So far, less attention is given to the anaphylatoxins C3a and C5a, which are released after cleavage of the complement components C3 and C5. The induction of the respective anaphylatoxin receptors C3aR and especially C5aR is described during osteogenic differentiation (OD) of human mesenchymal stem cells (MSC). Therefore, our aim was to investigate the influence of C3a and C5a on chondrogenic differentiation (CD) and OD of cartilage derived cells, which might be relevant in regards of posttraumatic cartilage regeneration.
Methods: Macroscopically intact human cartilage was obtained from donors undergoing knee joint replacement (n=22) and cartilage slices were either cultured to enable cartilage stem/progenitor cells (CSPC) outgrowth or digested for human articular chondrocyte (hAC) isolation. Human MSCs were isolated via gradient centrifugation of bone marrow, received during triple osteotomy (n=6). C3aR and C5aR expression was analysed by flow cytometry. Cells were cultured in pellets (CD) for 28 days or monolayer (OD) for 21 days in basal medium (negative control) or the respective differentiation media (positive control) with or without C3a (50, 1000 ng/mL) or C5a (50 ng/mL). Safranin O staining and immunostaining against collagen type II was used to evaluate CD, while Alizarin Red S staining was applied to assess OD. Statistical analysis: one-way ANOVA.
Results and conclusion: C3aR and C5aR were detected on the cell surface of MSC (C3aR: 29.67%; C5aR: 52.73%), CSPC (C3aR: 4.25%; C5aR: 17.47%) and hAC (C3aR: 12.94%; C5aR: 28.92%). Overall, MSCs expressed significantly more C3aR ([vs.CSPC]: p=0.0007; [vs.hAC]: p=0.006) and C5aR ([vs.CSPC]: p<0.0001; [vs.hAC]: p<0.0001). The addition of C3a or C5a did not significantly influence the CD of dedifferentiated hAC. In contrast, an increased calcium deposition could be observed during OD of hAC in the presence of 50 ng/mL C5a (+36%; p=0.018), while an inductive trend was found in the case of 1000 ng/mL C3a (+17%; p=0.234). Calcification during OD of MSC and CSPC was not affected in the presence of anaphylatoxins.
Our results indicate an increased calcium deposition during OD of hAC by the addition of C3a or C5a. High levels of C3a and the C5aR are both known to be associated with vascular calcification. Thus, we assume that C3a and C5a, released after cartilage trauma and subsequent complement activation, might contribute to the initiation of cartilage calcification. This could promote cartilage degeneration and consequently OA development. Future studies need to show whether inhibition of C3aR and C5aR represents an effective strategy against cartilage calcification.