含富血小板血浆的纳米纤维支架的构建和对软骨缺损的修复作用

Platelet-rich plasma (PRP) has been widely used for decades in the clinic, since an abundance of growth factors can be released when it is activated. However, its clinical use is limited because this release is temporal and PRP lacks mechanical strength. The aim of this study was to incorporate PRP- derived growth factors into PCL/gelatin nanofibers using the emulsion electrospinning method to determine how growth factors are released from the scaffolds and how the presence of these factors enhances the bioactivity of the scaffolds. Scaffolds with or without PRP were prepared and characterized. Release of proteins from scaffolds over time and rabbit BMSC chemotaxis, proliferation, and chondrogenic induction were quantified in vitro. The in vivo restoring effect of the scaffolds was also evaluated by transplanting the scaffolds into a cartilage defect in an animal model, and the outcomes were determined by histological assessment, micro-CT scanning, and IL-1 measurement. The results showed that the mechanical properties of the scaffolds were mildly compromised by the addition of PRP, and that sustained release of growth factors from PRP-containing scaffolds occurred up to ~ 30 days in culture. Scaffold bioactivity was enhanced, as BMSCs demonstrated increased proliferation and notable chemotaxis in the presence of PRP. The chondrogenesis of BMSCs was also promoted when the cells were cultured on the PRP scaffolds. Furthermore, the PRP scaffolds showed better restorative effects on cartilage defects, as well as anti-inflammatory effects in the joint cavity (the IL-1 level was decreased). In conclusion, the results of the current study indicate the potential for using a PRP-containing electrospun nanofibrous scaffold as a bioactive scaffold, which is beneficial for optimizing the clinical application of PRP.

富血小板血浆(platelet-rich plasma,PRP)是通过离心自体全血而得到的血小板浓缩物,含有多种高浓度的生长因子、纤维蛋白和白细胞等。已证实,PRP可显著加速软组织和骨组织的修复。但PRP的生长因子释放时间相对较短,同时其机械强度较低，难以直接应用于软骨修复。因此我们采用静电纺丝技术,将 PRP 与可降解纳米纤维支架结合,形成一种既能提供足够的机械强度,又能保持生物活性的缓释系统,即含有PRP的PCL/GE纳米纤维支架,并对支架进行力学、亲水性、降解性等表征。通过ELISA法测定生长因子的缓释曲线。将支架在体外与 BMSC 共培养,通过 SEM、CCK-8、免疫荧光、qPCR 和 Western blot 等方法研究支架对细胞的增殖、分化的影响。体内将支架植入兔膝关节软骨缺损区,并通过组织学、Micro-CT 和 IL-1β 浓度检测的方法对其修复软骨缺损的效果进行评价。

富血小板血浆(platelet-rich plasma,PRP)是通过离心自体全血而得到的血小板浓缩 物,含有多种高浓度的生长因子、纤维蛋白和白细胞等。已证实,PRP可显著加速软组织和 骨组织的修复。但PRP的生长因子释放时间相对较短,同时其机械强度较低，难以直接应用 于软骨修复。因此我们采用静电纺丝技术,将 PRP 与可降解纳米纤维支架结合,形成一种 既能提供足够的机械强度,又能保持生物活性的缓释系统,即含有PRP的PCL/GE纳米纤维支 架,并对支架进行力学、亲水性、降解性等表征。通过ELISA法测定生长因子的缓释曲线。 将支架在体外与 BMSC 共培养,通过 SEM、CCK-8、免疫荧光、qPCR 和 Western blot 等 方法研究支架对细胞的增殖、分化的影响。体内将支架植入兔膝关节软骨缺损区,并通过 组织学、Micro-CT 和 IL-1β 浓度检测的方法对其修复软骨缺损的效果进行评价。经过研究，已能制备出稳定的电纺纳米纤维支架，以及均匀分布的含PRP的电纺纳米纤维支架。支架具备足够的机械强度，且与人类关节软骨的杨氏模量相近。支架与兔BMSC亲和力良好，且能提升细胞的活力及增殖。复合PRP的支架能促进干细胞的成软骨分化，并能增加细胞外软骨基质的合成。体内实验发现，复合PRP的支架可见成簇分布的软骨细胞，细胞周围软骨基质沉积，II型胶原、蛋白多糖和Sox9表达上升。通过将 PRP 与电纺纳米纤维复合，实现了生长因子随纤维降解而发生缓释， 从而克服了 PRP 中生长因子释放快、作用时间短的缺点，可以在动物体内长时间作用于软骨缺损处，大大提升了修复的效果。同时，二者的复合还解决了 PRP 力学性能差，空间支撑性能不足的缺点，为其后续实验或扩展应用提供了理论依据。