Gtn-HPA as a new biomaterial in dental reconstructive surgery - the effect of the injectable hydrogel on the vitality, on the cytokine expression, on the differentiation and on the vascularization of endothelial progenitor cells and osteoblasts in 2D and

新型骨替代材料(BSM)的发展在口腔外科领域具有很高的科学和临床意义，因为它们代表了一种有吸引力的替代自体骨移植的方法。BSM的早期血管化是骨再生的重要前提。根据这一过程优化BSM被广泛视为未来可能改进以优化愈合的关键领域之一。关于血管形成和成骨细胞分化，初步研究已经证明内皮祖细胞(EPC)和成骨细胞(OB)具有高度的相关性。此外，研究表明，BSM的机械性能会影响这些细胞的功能、增殖和分化。在这种背景下，基质的硬度似乎是骨再生的关键成分之一。水凝胶已经被广泛用作组织工程的支架，因为它通过其含水率基质对氧气、营养物质和其他水溶性代谢物具有很高的渗透性。这种基质是细胞生长和组织再生的极佳环境。然而，现有的可注射水凝胶系统的一个主要缺点是它们不能控制凝胶化速度，这导致硬水凝胶中的血管形成不佳或软水凝胶中的机械稳定性不佳。最近，我们的合作伙伴从哈佛-麻省理工学院引进了可注射水凝胶明胶-羟基苯丙酸(GTN-HPA)，用于神经缺陷的治疗。在交联过程中，酶(辣根过氧化物酶，HRP)和氧化剂(过氧化氢，过氧化氢)的浓度可以独立控制，以改变水凝胶的凝胶化速度。因此，这种水凝胶的凝胶化速度可以第一次微调。本研究旨在将GTN-HPA作为一种新的牙科重建基质材料应用于牙科重建手术，探讨GTN-HPA作为EPC和OB在二维和三维培养中共培养的基质材料。GTN-HPA的细胞相容性将通过活性测定进行评估。此外，GTN-HPA最大限度的血管形成和成骨的最佳硬度将被检测。因此，我们将对EPC和OB在共培养条件下的增殖、分化和细胞因子的表达进行分析。此外，还将通过共聚焦激光扫描显微镜(CLSM)和CD34免疫组织化学对细胞形态和血管形成进行检查。通过这些分析，一个具有可调力学性能的水凝胶系统将在2D和3D培养中考察EPC和OB的相互作用，以描述BSM的维度和硬度对骨再生的影响，并具有预期的临床应用前景。

The development of new bone substitute materials (BSM) is of high scientific and clinical interest in dental surgery as they represent an attractive alternative to autogenous bone grafts. Early vascularization of the BSM is an important precondition for bone regeneration. Optimizing the BSM with respect to this process is widely seen as one of the key areas for possible future improvement to optimize healing. Regarding vascularization and osteoblast differentiation, preliminary studies have proven the high relevance of endothelial progenitor cells (EPC) and osteoblasts (OB). In addition, studies have shown that mechanical properties of BSM influence the function, proliferation and differentiation of these cells. In this context, the stiffness of the matrix seems to be on of the key components for bone regeneration.Hydrogels are already widely used as scaffolds for tissue engineering, because of their high permeability for oxygen, nutrients and other water-soluble metabolites through their water-content matrix. This matrix is an excellent environment for cell growth and tissue regeneration. However, a major drawback of existing injectable hydrogel systems is their missing control of the gelation rate which leads to suboptimal vascularization in hard hydrogels or missing mechanical stability in soft hydrogels. Recently, the injectable hydrogel gelatin-hydroxyphenylpropionic acid (Gtn-HPA) has been introduced by our cooperation partner from Harvard-MIT in the context of neural defects. During crosslinking, the concentrations of the enzyme (horseradish peroxidase, HRP) and the oxidant (hydrogen peroxide, H2O2) can independently be controlled to modify the gelation rate of the hydrogel. Thus, the gelation rate of this hydrogel can be fine-tuned for the first-time. With the vision to establish Gtn-HPA as a new BSM in reconstructive dental surgery, it is the aim of this study to evaluate Gtn-HPA as a matrix for the coculture of EPC and OB in 2D and 3D culture. The cytocompatibility of Gtn-HPA will be assessed with a viability assay. Furthermore, optimal stiffness of Gtn-HPA for maximal vascularization and osteogenesis will be examined. Therefore, an analysis of proliferation, differentiation and cytokine expression of EPC and OB in coculture will be performed. In addition, an examination of cell morphology and vascularization will be done by confocal-laser-scanning-microscopy (CLSM) and CD34 immunohistochemistry. By these analyses, a hydrogel system with tunable mechanical properties will be examined regarding the interaction of EPC and OB in 2D and 3D culture, to describe the effects of dimensionality and stiffness of BSM for bone regeneration for the first time and for prospective clinical use.