基于多功能普鲁士蓝/KGN纳米缓释系统调控间充质干细胞分化行为及在骨软骨组织工程中的应用

Scaffolds, growth factors, and cells are the three main elements of tissue engineering, which provides a promising therapeutic option for osteochondral regeneration. In osteochondral tissue engineering, mesenchymal stem cells (MSCs) are the most commonly used seed cells because of their several advantages, i.e., facile extraction, immunoregulation capacity, and differentiation into almost any type of end-stage lineage cells. However, disadvantages like limited chondrogenic differentiation ability and hypertrophic chondrocyte differentiation tendency of MSCs restrict the repair effect of this technique in repairing hyaline cartilage. Recently, kartogenin (KGN) has attracted much attention as a small-molecule chondrogenesis-inducing agent that can stimulate the repair of damaged cartilage. However, KGN is generally poorly soluble in water, thereby making its efficient intracellular delivery difficult. Furthermore, the sustained exposure of optimal dosage of the inductive agents is also necessary to effectively induce the differentiation of the MSCs and to minimize the off-target effect. And, hypertrophic chondrocyte differentiation tendency of MSCs can be inhibited by intracellularly scavenge of reactive oxide species (ROS). It showed prussian blue nanoparticles as multienzyme mimetics and ROS scavengers are excellent drug delivery carriers which are biodegradable and approved by US FDA for clinical use. Hence, in this study, we propose a new osteochondral repair strategy involving a novel KGN-release system on the use of hyaluronic acid and polyethylenimine dual-functionalized prussian blue nanoparticles (PHAPB@KGN), a drug-delivery system that can efficiently deliver and controlled release of KGN into MSCs for enhanced differentiation efficacy. Besides, it can inhibit hypertrophic chondrocyte differentiation tendency by ROS scavenge. Minipigs are used as experimental animals for this purpose. Osteochondral defects are created in the femoral trochlea, which are followed by biomimetic osteochondral scaffolds implantation. Then, PHAPB@KGN is intra-articularly injected into the model joints. This strategy overcomes the disadvantages of limited chondrogenic differentiation ability and hypertrophic chondrocyte differentiation tendency of MSCs, which may enable better repair of hyaline cartilage.

组织工程技术是修复关节骨软骨损伤较为理想的方法，但作为种子细胞的间充质干细胞（MSCs）成软骨分化能力欠佳及成肥大软骨细胞分化是导致透明软骨修复效果欠佳的关键因素。研究提示小分子药物Kartogenin（KGN）具有促MSCs成软骨分化效应，但其生物利用效率较差；而普鲁士蓝纳米材料可通过其模拟酶学活性清除活性氧自由基（ROS）进而抑制细胞肥大分化。据此，项目组提出构建多功能普鲁士蓝/KGN纳米缓释系统（PHAPB@KGN）以增加KGN药物递送效率及胞内持续性药物浓度，提高KGN生物利用效率，促进MSCs成软骨分化；同时，通过清除ROS抑制细胞肥大分化，以期获得更为优良的透明软骨修复效果。本项目拟对PHAPB@KGN的制备技术、材料属性及生物学效应进行研究；以小香猪为实验动物并结合项目组前期构建的仿生支架修复关节骨软骨损伤，以验证该策略的可行性和有效性，为骨软骨组织工程提供新材料、新策略。

关节骨软骨损伤在人群中十分常见，目前尚无理想修复策略。通过调控间充质干细胞（MSCs）成软骨分化是骨软骨损伤修复策略之一。但MSCs成软骨分化能力欠佳及成肥大软骨细胞分化是导致软骨修复效果欠佳的关键因素。Kartogenin (KGN) 分子可促进MSCs成软骨分化从而促进损伤软骨修复。本研究中，项目组构建了多孔普鲁士蓝纳米颗粒，并利用相转化材料（PCM）进行KGN分子装载。同时，对普鲁士蓝纳米颗粒表面进行透明质酸（HA）修饰（HPBNs-PCM@KGN），以改善材料的稳定性及生物相容性，并可起到封装KGN分子的作用，防止KGN的无序释放。该材料具有以下特点：① 普鲁士蓝纳米颗粒具有良好的光热效应，通过近红外光（NIR）照射，可控制PCM材料发生可逆相变转换，从而实现KGN分子的可控释放，进而促进MSCs成软骨分化；② 普鲁士蓝纳米颗粒具有活性氧自由基（ROS）清除效应，可抑制MSCs成肥大软骨细胞分化；③ HPBNs-PCM@KGN材料表面的HA可与间充质干细胞CD44受体结合，介导材料的跨膜转运，提高材料的细胞摄入效率，进而提高KGN分子生物利用率。基于以上特性，HPBNs-PCM@KGN可促进MSCs成软骨分化并抑制其肥大化，预期具有良好的损伤软骨修复效应。研究中项目组成功制备了HPBNs-PCM@KGN，表征分析发现该材料具有良好的稳定性、均一性、分散性及生物安全性。同时，研究结果提示该材料具有浓度依赖的ROS清除效应及循环可控的KGN光热控释效应。而体外研究发现：MSCs可时间依赖性摄入该材料；在体外共培养条件下可促进MSCs成软骨分化并抑制肥大标志物的表达。体内研究发现：该材料具有良好的体内光热效应，对大鼠膝关节骨软骨缺损模型具有良好的修复效应。本项目研究表明：通过普鲁士蓝纳米材料负载KGN分子，利用其光热效应及ROS清除效应，可光热控释KGN分子以促进MSCs成软骨分化并抑制其肥大分化，具有良好的关节骨软骨损伤修复效应。本研究提示：通过多功能纳米材料促进MSCs成软骨分化并抑制其肥大分化具有一定的可行性和有效性，可为关节骨软骨缺损修复提供新材料、新策略。

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