gms | German Medical Science

Objectives: Endochondral ossification, a key bone formation process, involves the differentiation of mesenchymal stem cells into chondrocytes after a fracture, initiating sequential stages including hypertrophic chondrocyte formation, bone matrix deposition and mineralization, ultimately transforming cartilage into bone tissue. The osteoimmune system has emerged as a focus for exploring the involvement of the innate immune system in chondrogenesis, including the study of neutrophils. Recent recognition of N1 (pro-inflammatory) and N2 neutrophil (anti-inflammatory) phenotypes has been extensively documented in various fields. However, this distinction remains unexplored in relation to cartilage. The current study aims to investigate the distinct effects of N1 and N2 neutrophils on the process of chondrogenesis during endochondral ossification.

Methods: Neutrophils were isolated from the peripheral blood of a healthy donor using Magnetic-Activated Cell Sorting (MACS). The isolated neutrophils were individually stimulated with N0 (unstimulated phenotype)/N1/N2 cocktails in a 37°C, 5% CO2 incubator for 24 hours. A part of neutrophils in each stimulation subset was analyzed using flow cytometry to assess their correct phenotypes. Each phenotype of neutrophils was washed twice with PBS and subsequently cultured without altering the cell concentration using the media with apoptosis inhibitor for an additional 4 hours. Subsequently, the conditioned media from N0/N1/N2 neutrophil phenotypes were collected and mixed with chondrogenic differentiation media for culturing ADTC5 cells. After 24 hours, the media mixture was replaced with chondrogenic differentiation media only following a PBS wash. On day 7 and 14, ALP activity, as well as the expression of several chondrocyte related genes (SOX9, Col2a1, Runx2, Col10a1, Col1a1, and MMP13), were assessed.

Results and conclusion: N1 and N2 neutrophils were successfully polarized. In the context of gene expression in ADTC5 cells, Sox 9 was significantly higher in the N2 group than N0 group on day 7. Runx2 was expressed at higher levels in both N1 and N2 groups on day 7. Col10a1 expression was lower in each neutrophil phenotype group on day 7. Col1a1 was expressed at higher levels in the N1 group on both day 7 and 14. MMP13 exhibited increased expression in the N1 group but decreased in the N2 group on day 7. Regarding ALP activity, the N1 group exhibited significantly enhanced ALP activity on both day 7 and 14. N1 and N2 neutrophils play distinct roles in chondrogenesis. The N1 neutrophil phenotype contributes to promoting the early osteogenesis of ADTC5. Conversely, the N2 neutrophil phenotype promotes anti-inflammation, chondrogenic, and hypertrophic effects.

Figure 1 [Fig. 1]