Analysis of the influence of Heparan sulfate proteoglycan Syndecan-1 on osteoclastogenesis and neoangiogenesis in chondro-osseous remodeling during fracture healing and potential therapeutic strategies

骨折延迟愈合和假关节的发展至今尚未得到很好的理解，尽管由于这些患者的长期持续和成本密集型治疗，其具有高度的临床相关性。骨代谢和再生的新见解可以帮助使用特定的信号通路和骨折愈合过程，以开发新的治疗方法，诊断和治疗骨折愈合并发症。本项目的重点是在骨痂中的软骨-骨边界处的软骨内骨化和新血管生成的过程。在初步调查中，我们发现，硫酸乙酰肝素蛋白聚糖Syndecan-1的影响破骨细胞在体外和Syndecan-1的缺乏导致延迟的软骨骨痂的重塑过程中，由于破骨细胞数量减少。单细胞mRNA测序数据指出，可能受Syndecan-1影响的进一步细胞-细胞相互作用。此外，我们发现Syndecan-1对血管的发育很重要，血管驱动软骨向骨的重塑。本项目的目的是表征Syndecan-1在骨折骨痂重塑过程中破骨细胞生成和新血管生成过程中的功能，并研究重组Syndecan-1或Synstatin肽作为新的治疗策略应用后愈合过程中的差异。（=脱落的）Syndecan-1在野生型和Sydnecan-1缺陷小鼠中的愈伤组织重塑期间增加破骨细胞生成。此外，我们将通过体外共培养和3D类器官培养（如软骨细胞和成骨细胞或内皮细胞）进一步探索Syndecan-1影响的细胞-细胞相互作用。Syndecan-1在骨折愈合过程中对新血管生成的作用将在体外使用原代内皮细胞以及在我们的骨折模型中的体内进行分析。Synstatin肽的应用将允许我们在体外和体内调节Syndecan-1依赖性VEGFR 2/VEGF对血管生成的激活。在一种转化方法中，我们将确定骨折后出现愈合障碍的患者中Syndecan-1的血清浓度，以评估Syndecan-1作为预后标志物的可能功能，从而识别愈合并发症。

The development of delayed fracture healing and pseudarthroses is not well understood to date, although it is of high clinical relevance due to long lasting and cost intensive treatment of these patients. New insights in bone metabolism and regeneration could help to use specific signaling pathways and processes of fracture healing to develop new therapeutic approaches in diagnostic and treatment of fracture healing complications. This project focusses on the process of enchondral ossification and neoangiogenesis at the chondro-osseous border in the callus. In preliminary investigations, we showed that the heparan sulfate proteoglycan Syndecan-1 influences osteoclastogenesis in vitro and deficiency of Syndecan-1 leads to a delay in the remodeling process of the cartilaginous callus due to a decreased osteoclast number. Single cell mRNA sequencing data pointed to further cell-cell interactions that might be influenced by Syndecan-1. Furthermore, we showed that Syndecan-1 was important for the development of blood vessels that drive the remodeling of cartilage to bone. The aim of this project is to characterize the function of Syndecan-1 during osteoclastogenesis and neoangiogenesis during fracture callus remodeling and to investigate differences in the healing process after application of recombinant Syndecan-1 or Synstatin peptides with regard as a new therapeutic strategy.Using a well-established femur shaft fracture model in mice we will investigate the application of recombinant (=shedded) Syndecan-1 to increase osteoclastogenesis during callus remodeling in wild type and Sydnecan-1 deficient mice. Additionally, we will explore further cell-cell interaction influenced by Syndecan-1 via in vitro co-culture and 3D organoid culture like chondrocytes and osteoblasts or endothelial cells. The role of Syndecan-1 for neoangiogenesis during fracture healing will be analyzed in vitro using primary endothelial cells as well as in vivo in our fracture model. The application of Synstatin peptides will allow us to modulate Syndecan-1 dependent VEGFR2/VEGF activation of angiogenesis in vitro and in vivo. In an translational approach we will determine the serum concentration of Syndecan-1 in patients after fracture, that develop disturbed healing to evaluate a possible function of Syndecan-1 as a prognostic marker to identify healing complications.