Gene Expression Profiling of Peri-implant Healing of PLGA-Li+ Implants Suggests an Activated Wnt Signaling Pathway in vivo

Bone development and regeneration is associated with the Wnt signaling pathway that, according to literature, can be modulated by lithium ions (Li+). The aim of this study was to evaluate the gene expression profile during peri-implant healing of poly(lactic-co-glycolic acid) (PLGA) implants with incorporated Li+, while PLGA without Li+ was used as control, and a special attention was then paid to the Wnt signaling pathway. The implants were inserted in rat tibia for 7 or 28 days and the gene expression profile was investigated using a genome-wide microarray analysis. The results were verified by qPCR and immunohistochemistry. Histomorphometry was used to evaluate the possible effect of Li+ on bone regeneration. The microarray analysis revealed a large number of significantly differentially regulated genes over time within the two implant groups. The Wnt signaling pathway was significantly affected by Li+, with approximately 34% of all Wnt-related markers regulated over time, compared to 22% for non-Li+ containing (control; Ctrl) implants. Functional cluster analysis indicated skeletal system morphogenesis, cartilage development and condensation as related to Li+. The downstream Wnt target gene, FOSL1, and the extracellular protein-encoding gene, ASPN, were significantly upregulated by Li+ compared with Ctrl. The presence of ß-catenin, FOSL1 and ASPN positive cells was confirmed around implants of both groups. Interestingly, a significantly reduced bone area was observed over time around both implant groups. The presence of periostin and calcitonin receptor-positive cells was observed at both time points. This study is to the best of the authors’ knowledge the first report evaluating the effect of a local release of Li+ from PLGA at the fracture site. The present study shows that during the current time frame and with the present dose of Li+ in PLGA implants, Li+ is not an enhancer of early bone growth, although it affects the Wnt signaling pathway.骨发育与骨再生密切关联Wnt信号通路（Wnt signaling pathway），现有文献显示该通路可被锂离子（Li+）调控。本研究旨在探究负载锂离子的聚乳酸-羟基乙酸共聚物（poly(lactic-co-glycolic acid), PLGA）种植体在种植体周围愈合过程中的基因表达谱，以未负载锂离子的PLGA作为对照，并重点关注Wnt信号通路的变化。 将种植体植入大鼠胫骨，分别于术后7天和28天取材，采用全基因组微阵列分析（genome-wide microarray analysis）检测基因表达谱，并通过定量聚合酶链反应（qPCR）与免疫组织化学（immunohistochemistry）对结果进行验证。采用组织形态计量学（histomorphometry）评估锂离子对骨再生的潜在影响。 微阵列分析结果显示，两组种植体随时间推移均存在大量显著差异表达的基因。Wnt信号通路受锂离子调控的影响显著：随时间变化，约34%的Wnt相关标志物发生表达调控，而未负载锂离子的对照组（Ctrl）仅为22%。功能聚类分析表明，与锂离子相关的生物学过程包括骨骼系统形态发生、软骨发育与软骨凝集。与对照组相比，锂离子可显著上调下游Wnt靶基因FOS样抗原1（FOSL1）以及细胞外基质蛋白编码基因饰胶蛋白聚糖（ASPN）的表达。两组种植体周围均检测到β-连环蛋白（β-catenin）、FOSL1及饰胶蛋白聚糖阳性细胞的存在。 值得注意的是，随时间推移，两组种植体周围的骨面积均出现显著降低。在两个时间点均观察到骨膜蛋白（periostin）与降钙素受体（calcitonin receptor）阳性细胞的存在。据作者所知，本研究是首次报道从PLGA载体局部释放锂离子在骨折部位的作用效果。 本研究结果表明，在当前实验周期与PLGA种植体的锂离子负载剂量下，尽管锂离子可调控Wnt信号通路，但并未促进早期骨生长。