GPNMB~+ Macrophages Enhancing Osteogenic Differentiation Of Nucleus Pulposus Cells,Facilitating Intervertebral Disc Degeneration

Background and Purpose:Intervertebral disc degeneration is considered a major cause of low back pain,imposing a significant burden on healthcare and socio-economic fields worldwide.However,there is currently no definitive treatment strategy to reverse or halt the progression of disc degeneration.Therefore,in-depth research into the biological pathogenesis of disc degeneration is crucial.Regardless of the pathogenic mechanisms,lumbar disc degeneration presents as a heterogeneous pathological phenomenon,including fibrosis,ectopic calcification,protrusion,or mixed phenotypes across a wide spectrum.The differences between these phenotypes are currently unclear and subject to some controversy,with calcification being perhaps the least understood.Disc calcification is closely associated with aging,high degeneration levels,and increased expression of osteogenic markers within the disc.Calcified discs may progress degeneration due to abnormal stress concentration and more severe immune reactions,leading to more pronounced symptoms and presenting greater challenges during treatment.Indeed,several chronic degenerative diseases have elucidated the upregulation of osteogenic markers mediating tissue calcification and ossification as a significant contributor to tissue dehydration sclerosis and resultant degeneration outcomes.However,research into this mechanism in the context of intervertebral disc degeneration is still in its infancy,and understanding the reasons for the appearance of calcification phenotype in lumbar disc degeneration and its specific impact on degeneration,exploring its molecular mechanisms,may represent a novel direction with important clinical and translational value in understanding the pathogenesis of lumbar disc degeneration.Therefore,this study aims to explore the cell differentiation trajectory of nucleus pulposus cells in the degeneration process using single-cell sequencing technology,elucidate the tendency of osteogenic cell differentiation as an endpoint outcome in nucleus pulposus degeneration,demonstrate that intervening in nucleus pulposus cell osteogenic differentiation fate can alleviate disc degeneration progression through in vivo and in vitro models,identify the composition of the immune microenvironment in the nucleus pulposus tissue,clarify that GPNMB⁺macrophages are a key immune subset promoting nucleus pulposus cell osteogenic differentiation fate,and investigate the specific molecular mechanisms by which GPNMB⁺macrophages regulate nucleus pulposus cell osteogenic differentiation fate,validating therapeutic effects through targeted small molecule inhibitors in in vivo and in vitro models.Methods:Firstly,single-cell transcriptome sequencing was performed on human nucleus pulposus tissue samples from different degeneration grades.Bioinformatics analysis methods such as quality control,dimensionality reduction clustering,pseudo-temporal analysis,functional enrichment analysis,and cell communication analysis were used to identify characteristic cell subgroups,differentiation fates of nucleus pulposus cells during degeneration,key immune cell subgroups in the immune microenvironment,and the specific molecular mechanisms by which these immune cells promote nucleus pulposus cell differentiation fate leading to disc degeneration.Next,experiments including conditional gene knockout mouse models,in vitro lentiviral transfection of nucleus pulposus cells,tissue staining,etc.,were conducted to confirm the correlation between osteogenic calcification ability in nucleus pulposus cells and the severity of disc degeneration,as well as the role of nucleus pulposus cell osteogenic differentiation fate in degeneration progression.Furthermore,experiments such as human tissue immunofluorescence,cell co-culture experiments,and in vivo and in vitro administration of small molecule targeted receptor inhibitors were performed to confirm the impact of GPNMB⁺macrophages on nucleus pulposus cell osteogenic differentiation fate and the specific molecular mechanisms,as well as to explore the potential therapeutic effects of these drugs.Results:Firstly,we identified a subgroup of nucleus pulposus cells with high expression of osteogenic markers and found that these osteoblast-like nucleus pulposus cells are an endpoint outcome of phenotype transformation during degeneration,resembling a pathological process similar to endochondral ossification from stable nucleus pulposus chondrocytes to osteoblast-like nucleus pulposus cells.Secondly,through in vitro cell experiments,we confirmed that primary human nucleus pulposus cells themselves have osteogenic differentiation potential,and inducing osteogenesis in vitro is accompanied by a stronger degenerative phenotype.Subsequently,we conducted functional experiments and found that intervention in the osteogenic differentiation of nucleus pulposus cells by downregulating the key osteogenic differentiation transcription factor RUNX2 in Col2a1^Runx2 mice and in vitro lentivirus knockdown models of nucleus pulposus cells has a clear alleviating effect on nucleus pulposus degeneration.We then identified GPNMB⁺nucleus pulposus cells as a key immune subset in the nucleus pulposus immune microenvironment and validated the expression of this cell subset in different degeneration grades in human nucleus pulposus tissue pathological samples.Through bioinformatics analysis,we elucidated that this immune cell subset can promote nucleus pulposus cell osteogenic differentiation fate and degeneration progression via the PDGF pathway.Subsequently,from a clinical prevention perspective,we further validated this through in vitro co-culture experiments and in vivo and in vitro administration of the PDGF receptor-targeted small molecule inhibitor,clarifying that inhibiting the PDGF receptor in nucleus pulposus cells can effectively intervene in disc degeneration.Conclusion:The phenotypic transformation of nucleus pulposus cells is the cellular basis of disc degeneration,and understanding their differentiation fate is of great significance in understanding the underlying logic of degeneration.Based on dry and wet experimental methods,this study discovered that nucleus pulposus cells have an inherent tendency toward osteogenic differentiation during disc degeneration,with osteoblast-like nucleus pulposus cells being an endpoint outcome of degeneration,elucidating the cellular biological mechanism of tissue dehydration sclerosis and calcification during degeneration,and revealing the promoting effect of osteogenic differentiation fate of nucleus pulposus cells on disc degeneration.Based on the important role of the immune microenvironment in cell osteogenic differentiation and nucleus pulposus degeneration,we uncovered the key immune cell subset and specific molecular mechanisms that promote nucleus pulposus cell osteogenic differentiation fate.This study enriches the theoretical foundation of disc degeneration,clarifies the significant impact of nucleus pulposus cell osteogenic differentiation ability on promoting disc degeneration,provides new concepts for future non-surgical precision treatments,and offers a novel therapeutic target for developing targeted drugs in the future.