Exploration of the roles of N0, N1, and N2 neutrophils on bone and cartilage regeneration in vitro from blood samples drawn from healthy volunteers

Neutrophils, comprising 60-70% of white blood cells, play a pivotal role as
the predominant cell type in human blood and are a key component of the innate
immune system. Functioning as the frontline defenders against PAMPs and DAMPs,
neutrophils migrate to inflammatory sites by deformation, adhesion,
infiltration, and migration. Subsequently, neutrophils carry out essential
functions through various mechanisms, such as the formation of NETs and the
production of cytokines and granular proteins.
Nonetheless, an expanding body of research is revealing the diverse
functions of neutrophils, moving beyond their singular roles and attempting to
uncover their potential mechanisms. A notable example is the work of Fridlender
et al., who initially proposed the N1 (pro-inflammatory) and N2
(anti-inflammatory) neutrophil phenotypes based on their distinct functions in
cancer, drawing an analogy with the M1 and M2 macrophage phenotypes.
Subsequently, the "N1/N2" terminology gained widespread usage in various
fields, including cardiology, clinical immunology, neurology, and infectious
diseases, to denote the multifaceted effects of neutrophils. Building on this
understanding, Ohms et al. developed a protocol to polarize human neutrophils
into N1 and N2 phenotypes in vitro, facilitating further exploration of the
characteristics of N1 and N2 neutrophils. In subsequent studies, researchers
have employed similar methods to identify distinct neutrophil phenotypes based
on their surface biomarkers. Neutrophil subsets, designated as N1
(characterized by CD54 and CD95) and N2 (defined by CD182), were thoroughly
investigated. These investigations have unveiled the diverse roles played by
these distinct neutrophil phenotypes across a wide array of fields.
Bone and cartilage healing is an intricate process involving the active
participation of neutrophils. The manifold roles of neutrophils in bone
regeneration have been widely acknowledged, encompassing diverse mechanisms
such as the formation of NETs, modulation of angiogenesis, influence on the
extracellular matrix, and interaction with macrophages, among others.
Nevertheless, there is a paucity of studies linking varied effects of
neutrophils with N1/N2 neutrophil phenotypes. Cai et al. have elucidated the
beneficial role of N2 neutrophils in mouse bone healing, highlighting the
involvement of Stromal cell-Derived Factor 1 (SDF-1)/CXCR4 signaling. However,
it remains unclear whether N1 and N2 neutrophil phenotypes can be attributed to
the diverse roles observed in human bone regeneration. Furthermore, the
recognition of N1 and N2 neutrophils is still an empty in the field of
cartilage regeneration.
In this study, we utilize the polarization of N1 and N2 neutrophils to
explore potential differential effects on the osteogenesis of human BMSCs and
the chondrogenesis of chondrocytes. We hypothesize that N1 neutrophils inhibit,
while N2 neutrophils promote both osteogenesis and chondrogenesis.

Primary Objective: The objective of this trial is to examine the distinctive
surface biomarkers and cytokines found in the medium of N1 and N2 neutrophils.
Furthermore, this study aims to investigate the varying effects of N1 and N2
neutrophils on bone and cartilage regeneration. We hypothesize that N1
neutrophils display inflammatory properties (elevated levels of CD54, CD95,
Tumour Necrosis Factor α (TNF-α), IL-8, Monocyte Chemoattractant Protein 1
(MCP-1)), leading to the inhibition of bone and cartilage regeneration.
Conversely, we anticipate that N2 neutrophils exhibit anti-inflammatory
characteristics (elevated levels of CD182, SDF-1α), thereby promoting bone and
cartilage regeneration.

Our study 'The effect of N1-N2 neutrophils on bone/cartilage regeneration':
This study is an in vitro study in which blood will be drawn from healthy
control volunteers by venipuncture. Neutrophils will be isolated from healthy
donor blood in the laboratory and stimulated in vitro using a specialized
culture medium. Following stimulation, the neutrophils anticipated to exhibit
the N0, N1, and N2 phenotypes will be examined by flow cytometry and ELISA
technique, subsequently directed to stimulate BMSCs or chondrocytes.
Subsequently, the osteogenic and chondrogenic potential of BMSCs and
chondrocytes, respectively, will be systematically analyzed. Furthermore, N0,
N1, and N2 neutrophils will artificially adhere to slides for MALDI-MSI to
analyze the unique metabolites and their spatial distribution associated with
the N0, N1, and N2 phenotypes.

The venipuncture will be done by competent and authorized technicians
(according to the Dutch law, *voorbehouden handeling, wet BIG* and the NVKC
guideline *veneuze bloedafname*). Blood sample collection is a relatively minor
invasive procedure, from which we do not foresee complications other than the
formation of small hematomas or minor hemorrhage at the site of venipuncture.
Subjects do not have to be sober at the time of blood sample collection.
Therefore, we anticipate only minor discomfort as a consequence of
participation. Our proposed research will not directly benefit the subjects
included in our study. However, our study can lead to new insights from which
patients may benefit in the future.