掺锶钙磷材料介导的T细胞免疫微环境调控颅颌面骨组织再生及机理研究

It is still difficult to repair and reconstruct the function of craniomaxillofacial bone defects in clinic. Due to the limited osteogenic/angiogenic activity, calcium-phosphate materials cannot meet the clinical needs. Recent studies confirmed that the regulation of osteoimmunology could promote osteogenesis. Based on the promotion of osteogenic/angiogenic activity for calcium-phosphate materials by strontium doping in our group, we further found that T cell-mediated adaptive immune response regulated by strontium doping could apparently enhance osteogenic differentiation of BMSCs. However, the exact effects of the strontium doping mediated T cell immune response on osteogenesis and angiogenesis, the underlying mechanisms and the influence of the related processes on bone regeneration are still unclear. The present project aims to investigate the regulation of T cell-mediated immune response induced by strontium doping on craniofacial bone regeneration at molecular-cell-overall animal level, via the study of the cytological effects of conditioned media co-cultured with strontium-doped calcium-phosphate materials and CD4+ T cells on BMSCs, combining wild-type and T-cell immunodeficient cranial defect mice models. IL-17A is selected as a potential key factor to reveal the intrinsic regulating mechanism of strontium-doped calcium-phosphate materials on CD4+ T cells secreting IL-17A and IL-17A on osteogenic differentiation of BMSCs. This project is expected to provide new concept for biological properties of strontium-doped calcium-phosphate materials, and guide the design of bone repair materials with T cell-mediated immune response to regulate bone formation.

颅颌面骨缺损修复及功能重建是临床治疗的难点，临床大量应用的钙磷材料因成骨/成血管诱导活性有限无法满足临床需求。研究证实调控骨免疫反应可促进成骨。申请人在锶掺杂促进钙磷材料成骨/成血管活性基础上，进一步发现锶掺杂可调控T细胞介导适应性免疫反应促进BMSCs成骨分化。然而，锶掺杂介导T细胞免疫反应调控成骨/成血管规律、机制及其该过程对骨修复的影响仍然未知。本项目拟通过掺锶钙磷材料与CD4+ T细胞共培养条件培养基对BMSCs细胞学效应研究，结合野生型和T细胞免疫缺陷小鼠颅骨缺损动物模型，从分子-细胞-整体动物水平阐述锶掺杂诱导T细胞介导免疫反应对颅颌面骨再生调控作用；以IL-17A作为关键因子，研究掺锶钙磷材料调控CD4+ T细胞分泌IL-17A及IL-17A调控BMSCs成骨分化内在机理。本项目可望为掺锶钙磷材料生物学性能提供新观点，为设计具有T细胞介导免疫反应调控成骨的修复材料提供指导。

炎症、外伤、肿瘤、先天性疾病均可导致的颅颌面骨组织缺损，目前临床大量应用的钙磷材料因成骨/成血管诱导活性有限，无法满足大块骨缺损修复的临床需求。近年，骨免疫调节的作用提示调节良好的免疫环境对成功的材料介导骨再生的重要性。然而，以往研究主要集中在先天性免疫反应介导的骨再生。最近的研究表明，适应性免疫系统作为骨再生的一个重要调节因素受到广泛关注。然而，生物材料的化学信号是否能通过适应性免疫反应的免疫调节功能来调节骨再生尚待探索。本课题应用纳米棒羟基磷灰石材料为基础，研究不同形貌及掺锶羟基磷灰石纳米材料对T淋巴细胞为代表的骨免疫微环境的调控作用及机制。本项目通过纳米材料与T淋巴细胞共培养条件培养基，对骨髓基质细胞细胞学效应研究，结合野生型和T淋巴细胞免疫缺陷TCRβ-/-小鼠颅骨、下颌骨缺损动物模型，运用Micro-CT、组织切片染色、流式细胞术、Luminex细胞因子检测、Western blot、实时荧光定量PCR等技术，从分子-细胞-整体动物水平阐述不同形貌及锶掺杂的羟基磷灰石纳米材料诱导T细胞介导免疫反应对颅颌面骨再生调控作用。研究指出长径比45-70nm/10-15nm的羟基磷灰石纳米棒及掺锶羟基磷灰石纳米材料植入野生型小鼠颅面部骨后成骨效应明显；同时，TCRβ-/-小鼠的下颌骨缺损模型实验发现，其成骨效应明显低于野生型。进一步发现两种材料分别诱导T淋巴细胞产生的IL-22及IL-17A是调节骨免疫再生的关键因子，促进小鼠颅颌面部骨缺损区早、中期成骨的再生功能，JAK/STAT3 通路在T细胞表达Th17相关转录因子（Stat3、RORγt）以及IL-22介导骨髓基质细胞成骨分化中均发挥重要作用。本项目可为不同形貌及掺锶钙磷材料生物学性能提供新观点，为设计具有T淋巴细胞介导免疫反应调控成骨的修复材料提供指导。