氟化生物源羟基磷灰石激活单核-巨噬细胞M1/M2极化调控BMSCs成骨功能的机制研究

Bone substitute mediated immune response is the initial step for the repairing of bone defects. The occurrence, development and prognosis of this immune response directly affects the final outcome of bone repair. Monocyte derived macrophages are the earliest immune cells arriving at the bone repair area. The transformation and balance of M1 and M2 polarization phonotypes of macrophages was decided by both the physiochemical properties of the implant material and the local microenvironment. On the other hand, macrophages could regulate the body's inflammatory response and tissue repair through the secretion of various cytokines. Therefore, macrophages should be involved for in vitro evaluation of bone graft material. However, the immune response of material was commonly ignored in most previous studies, and only osteoblasts were involved, which may further lead to the inconsistencies of evaluation on ostegenic efficacy of bone substitute in vivo and in vitro. Thus, the fluorinated biological hydroxyapatite with confirmed good osteogenic efficacy was set as the start point in this study. The effect of fluoride substitution to biological hydroxyapatite on activation of the M1/M2 polarization of macrophages would be evaluated. The co-culture model of macrophages and bone marrow mesenchymal stem cells (BMSCs) would be developed to evaluate the effect of activation of macrophages on the chemotaxis and oteoblastic function of BMSCs. Meanwhile, the molecular mechanism on regulation the osteogenic differentiation of BMSCs would also be explored. The results would help to better understand how implant material regulate the bone remodeling through the new perspective of osteoimmunology, and to provide basic data and theory for the development of fluoride substituted bone graft material.

骨替代材料介导的免疫应答是骨缺损修复的初始步骤，其发生、发展和转归直接影响骨缺损最终修复效果。单核巨噬细胞是材料植入后最早趋化到达骨修复区域的免疫细胞，其M1与M2两种极化表型间的转换与平衡受植入材料理化性能与局部微环境诱导，并可通过分泌不同细胞因子调控机体炎症反应与组织修复。因此，将巨噬细胞引入材料体外成骨性能评估具有重要意义。但过往研究常忽略了免疫应答因素，而仅仅关注材料与成骨细胞的相互作用，导致材料体内外成骨性能评估结果的差异。鉴于此，本研究拟以我们已成功制备的氟化生物源羟基磷灰石为切入点，明确氟植入对巨噬细胞M1/M2极化的诱导作用；构建巨噬细胞与BMSCs共培养模型，研究材料激活巨噬细胞极化对BMSCs的趋化作用及成骨功能的影响，并探索其调控BMSCs成骨分化的分子机制。研究结果有望从骨免疫的新视角对材料影响骨改建的机理加深认识，并为新型含氟骨替代材料的应用提供基础参数和理论依据

生物骨源羟基磷灰石BHA是口腔种植临床治疗骨缺损最常用的骨替代材料，其保留了生物骨组织天然的孔隙结构，具有良好的骨引导性，但由于缺乏骨诱导性，材料成骨效能有待进一步改性提高。前期研究中，我们将氟植入猪骨羟基磷灰石，制备获得氟化生物骨羟基磷灰石FBHA，并证实FBHA能促进早期成骨，但其促成骨机制尚不明确。本研究中，我们通过较完善的体外细胞学与动物体内实验对FBHA材料促成骨机制进行探究。.我们完成了BHA与FBHA骨替代材料的制备与表征，检测了BHA与FBHA对巨噬细胞增殖活性及M1/M2极化功能的影响：发现FBHA抑制巨噬细胞在LPS刺激下的M1极化，促进巨噬细胞在IL-4刺激下的M2极化。为了明确氟对巨噬细胞极化的影响，我们测试了含不同浓度氟离子的细胞培养液对单核巨噬细胞极化方向的影响，研究结果显示: 2.4-24μM氟能促进单核巨噬细胞M2极化方向表达，抑制炎症发展，促进骨组织修复与成血管；高于120μM氟能促进单核巨噬细胞M1极化方向表达，促进炎症发展。氟与巨噬细胞共培养的条件培养基对BMSC成骨功能具有调节作用：2.4-24μM氟与巨噬细胞共培养的条件培养基能促进BMSC成骨分化及矿化，RT-PCR及Western-Blot证实BMSC成骨活性增强与Wnt/β-catenin相关通路激活相关。动物实验中，我们利用大鼠骨缺损模型检测FBHA修复骨缺损的效果并反向验证FBHA促进成骨的机制：不脱钙硬组织切片结合组织计量分析提示FBHA组促进早期新骨形成，免疫组化染色提示FBHA组与BHA组均仅少量炎症细胞存在植骨材料颗粒周围，免疫荧光结果同样证实FBHA促进成骨与β-catenin相关通路激活相关。同时，比格犬下颌骨缺损实验中，不脱钙硬组织切片组织计量学分析和显微CT结果进一步证实了FBHA能促进骨缺损的修复。.上述研究结果表明，FBHA的骨免疫调节作用主要与氟离子介导的骨修复微环境有关：骨修复微环境的氟离子浓度处于2.4-24μM时，具有一定的免疫调节性能，可降低炎症水平并促进血管化与新生骨组织修复；当氟离子浓度高于或低于上述范围时候，则会引发不同程度的炎症反应和破骨活动，伴随成骨性能降低，这些因素可能阻碍新生骨生成，导致骨缺损修复最终以炎性纤维包裹为结局。将FBHA骨替代材料释氟浓度控制在上述范围将有利于发挥材料促进骨缺损修复的作用。