氟化生物源性羟基磷灰石溶解平衡与促进成骨的机制研究

Bio-derived hydroxyapatite is the most widely used bone substitute clinically in dentistry. However, due to the lack of osteoinductivity, there is still neccessity to further improve its osteogenesis capability to meet the clinical needs of reconstruction of bone defects. Ions incorporation is one of the popular methods for apatite modification. Fluorine is the trace element involved in bone remodeling in promoting osteogenesis. And therefore, fluoride ion incorporation to hydroxyapatite has been suggested to adjust the physicochemical properties of bone substitute material and promote osteogenesis. In our previous study, the high temperature chemical substitution reaction was explored to incorporate fluoride ions into bio-derived hydroxyapatite(porcine bone), and the fluorinated bio-derived hydroxyapatite(FBHA) was successfully prepared. In the follow-up study, FBHA has been confirmed to show good biocompatibility and promote osteogenic differentiation of osteoblast. However, the mechanism how FBHA promoted osteogenesis was still not understood. Based on preliminary studies, the solubility isotherm of FBHA would be determined with our innovative method, solid titration and laser-scattering end-point detection approach. The dynamic changes of ions would be analyzed with ion chromatography. The osteogenesis capability of FBHA would be evaluated both by cell culture in vitro and by animal studies in vivo. And the mechanism how dissolved fluoride ions from FBHA affect cellular microenvironment and further initiate cell signaling pathways to promote osteogenic differentiation would be investigated. This study aims to further discuss the mechanism how the equilibruim of FBHA affect promotion of osteogenesis, which could help to better understand the mechanism how inorganic materials promoted osteogenesis.

生物源性羟基磷灰石是口腔临床应用最广泛的骨组织替代材料，但由于其缺乏骨诱导性，学者们试图对其改性以更好地满足骨缺损修复的临床需求。离子掺杂是磷灰石材料改性研究的热点，氟是参与骨改建中促进成骨的微量元素，因此氟离子植入被认为可能提高磷灰石材料的成骨性能。我们在过往的研究中以高温化合置换法对生物源性羟基磷灰石(猪骨)进行氟离子植入，制备完成氟化生物源性羟基磷灰石（FBHA），后续研究发现FBHA具有良好生物相容性且能促进细胞成骨分化，但其促进成骨的机制尚不明确。本项目拟在前期研究基础上，运用固体滴定和激光散射监测技术测定FBHA的等温溶解线并监测溶解平衡过程中离子动态变化；评估材料表面细胞行为和动物体内成骨性能；研究FBHA溶解平衡中氟离子影响细胞微环境并启动促进细胞成骨分化的相关信号通路机制。本课题是基于前期研究基础的深入性机制探讨，将加深对磷灰石无机材料促进成骨作用机制的理解。

生物源性羟基磷灰石是口腔临床应用最广泛的骨组织替代材料，但由于其缺乏骨诱导性，学者们试图对其改性以更好地满足骨缺损修复的临床需求。离子掺杂是磷灰石材料改性研究的热点，氟是参与骨改建中促进成骨的微量元素，因此氟离子植入被认为可能提高磷灰石材料的成骨性能。我们在过往的研究中以高温化合置换法对生物源性羟基磷灰石(猪骨)进行氟离子植入，制备完成不同掺氟量梯度浓度的氟化生物源性羟基磷灰石（FBHA），后续研究发现FBHA具有良好生物相容性且能促进细胞成骨分化，但其促进成骨的机制尚不明确。本项目在前期研究基础上，运用固体滴定和激光散射监测技术测定FBHA的等温溶解线发现，随着掺氟量的提高，FBHA的等温溶解线会整体压低，表明FBHA的溶解度会随着掺氟量的提高而下降，尤其是在低pH的环境中（pH3.0-4.0）；抗压能力结果显示，氟离子掺杂有利于提高BHA的抗压能力；蛋白吸附实验发现，FBHA的蛋白吸附能力远高于BHA，然而随着氟含量增高，反而会下降。在后续的体外细胞和体内实验中，我们进一步比较了低氟含量FBHA和BHA的性能。低氟含量FBHA和BHA在培养基中浸泡24h后，检测其浸提液中的钙、磷、镁、氟含量发现，BHA和FBHA的浸提液中，钙和磷的含量下降了，镁含量提高了，而在FBHA的浸提液中，可以检测到1ppm浓度的F离子；用BHA和FBHA的浸提液体外培养大鼠骨髓间充质干细胞（rBMSC），ALP活性和成骨相关基因、蛋白的表达结果显示，FBHA浸提液有利于提高rBMSC的成骨相关指标的表达；免疫荧光实验显示FBHA浸提液可促进rBMSC中β-catenin的核内转移，WB结果显示FBHA浸提液可促进rBMSC中RUNX2、β-catenin以及p-GSK-3β的表达，而加入Wnt/β-catenin通路抑制剂DKK-1后可阻断FBHA浸提液对上述指标的促进作用，证实FBHA是通过Wnt/β-catenin通路促进细胞成骨分化的；通过大鼠颅骨缺损模型评价低氟浓度FBHA和BHA的体内成骨性能，结果显示植入骨缺损6和12周后FBHA组的新生骨形成率高于BHA组，提示FBHA具有良好的体内成骨性能。本课题是基于前期研究基础的深入性机制探讨，加深了对磷灰石无机材料促进成骨作用机制的理解。