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

延迟骨折愈合和假关节的发展至今尚未得到很好的了解，尽管由于这些患者的长期持续和高成本治疗，它具有很高的临床相关性。对骨代谢和再生的新认识有助于利用骨折愈合的特定信号通路和过程，为骨折愈合并发症的诊断和治疗开发新的治疗方法。本项目主要研究骨痂软骨-骨交界处软骨内成骨和新生血管生成的过程。在初步研究中，我们发现硫酸肝素蛋白多糖Syndecan-1影响体外破骨细胞的形成，Syndecan-1缺乏导致软骨愈伤组织的重塑过程因破骨细胞数量减少而延迟。单细胞mRNA测序数据表明，Syndecan-1可能会进一步影响细胞间的相互作用。此外，我们发现Syndecan-1对于驱动软骨向骨重塑的血管发育很重要。本项目旨在研究Syndecan-1在骨折骨痂重塑过程中破骨细胞生成和新生血管生成过程中的功能，并探讨重组Syndecan-1或Synstatin肽作为一种新的治疗策略在愈合过程中的差异。通过建立小鼠股骨骨干骨折模型，我们将研究重组（=脱落）Syndecan-1在野生型和Sydnecan-1缺失小鼠愈伤组织重塑过程中增加破骨细胞生成的应用。此外，我们将通过体外共培养和三维类器官培养（如软骨细胞、成骨细胞或内皮细胞）进一步探索Syndecan-1对细胞间相互作用的影响。Syndecan-1在骨折愈合过程中对新生血管生成的作用将在体外用原代内皮细胞和体内骨折模型中进行分析。Synstatin肽的应用将使我们能够在体外和体内调节Syndecan-1依赖性VEGFR2/VEGF的血管生成激活。在一项转化方法中，我们将测定骨折后患者的血清Syndecan-1浓度，这些患者的愈合受到干扰，以评估Syndecan-1作为识别愈合并发症的预后标志物的可能功能。

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.