Evaluation of the influence of platelet-rich plasma (PRP), platelet lysate (PL) and mechanical loading on chondrogenesis in vitro.
Evaluation of the influence of platelet-rich plasma (PRP), platelet lysate (PL) and mechanical loading on chondrogenesis in vitro.
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DOI：
10.1038/s41598-021-99614-0
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发表时间：
2021-10-12
影响因子：
4.6
通讯作者：
Kubosch EJ
中科院分区：
文献类型：
--
作者：
Pötter N;Westbrock F;Grad S;Alini M;Stoddart MJ;Schmal H;Kubosch D;Salzmann G;Kubosch EJ
本研究的目的是探讨PRP作为自体软骨细胞植入（ACI）的辅助治疗结合多轴负荷对软骨再生的影响。关节软骨的修复能力差，软骨缺损的治疗方法仍然缺乏。成熟的手术治疗包括ACI，越来越多的证据表明PRP的有益效果。血小板含有许多生长因子，其中包括转化生长因子β（TGF-β）。已知动态机械载荷对于组织形成是必不可少的，从而改善细胞外基质（ECM）的产生。对于我们的ACI模型，将单层扩增的人软骨细胞接种到聚氨酯支架中，并包埋在纤维蛋白（hChondro）、PRP-凝胶（PRP）或具有血小板裂解物（PL）的纤维蛋白中，所述血小板裂解物以50体积%的浓度每周一次添加到培养基中。将各组暴露于静态条件或球关节生物反应器中的多轴力，每天1小时，持续2周，模拟生理负荷下的ACI。收集培养基并分析糖胺聚糖（GAG）、亚硝酸盐和转化生长因子β 1（TGF-β1）含量。收集细胞支架构建体用于DNA和GAG定量;使用qPCR分析软骨形成基因、TGF-β和相关受体以及炎性基因的表达。加载条件显示出比静态条件优越的上级软骨形成分化（COL 2A 1、ACAN、COMP和PRG 4表达的上调）。PRP和PL组结合机械负荷显示COL 2A 1、ACAN和COMP上调。PL组中总TGF-β1的量最高。各负荷组均出现潜伏性TGF-β1的激活，PL组激活量最高。负荷增加TGFBR 1/TGFBR 2 mRNA的比例，进一步增加响应补充剂。一般来说，加载增加亚硝酸盐释放到介质中。然而，随着时间的推移，与对照组相比，PL组的培养基亚硝酸盐含量较低。基于这些实验，我们得出结论，软骨形成分化是最强的，当模拟ACI与动态机械负荷和PRP-凝胶或PL补充组合进行。PRP和PL可减轻炎症反应，这可能是主要的治疗效果之一。推测负载可增强TGF-β1的作用，其在负载PL组中主要被激活。负荷和PRP的组合代表了关于基于软骨细胞的软骨修复的有效且有前景的协同作用。
The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-β). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-β1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-β and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-β1 was quantified in the PL group. Latent TGF-β1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-β1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.
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