Lückgen Janine, Diederichs Solvig, Raqué Elisabeth, Renkawitz Tobias, Richter Wiltrud, Buchert Justyna
Department of Orthopaedics, Experimental Orthopaedics, Research Centre for Molecular and Regenerative Orthopaedics, Heidelberg University Hospital, Heidelberg, Germany.
Department of Orthopaedics, Research Centre for Molecular and Regenerative Orthopaedics, Heidelberg University Hospital, Heidelberg, Germany.
J Cell Physiol. 2024 Dec;239(12):e31430. doi: 10.1002/jcp.31430. Epub 2024 Sep 5.
Abnormal mechanical loading is one of the major risk factors for articular cartilage degeneration. Engineered mesenchymal stromal cell (MSC)-derived cartilage holds great promise for cell-based cartilage repair. However, physiological loading protocols were shown to reduce matrix synthesis of MSC-derived neocartilage in vitro and the regulators of this undesired mechanoresponse remain poorly understood. Parathyroid hormone-related protein (PTHrP) is involved in cartilage development and can affect extracellular matrix (ECM) production during MSC chondrogenesis opposingly, depending on a continuous or transient exposure. PTHrP is induced by various mechanical cues in multiple tissues and species; but whether PTHrP is regulated in response to loading of human engineered neocartilage and may affect matrix synthesis in a positive or negative manner is unknown. The aim of this study was to investigate whether dynamic loading adjusts PTHrP-signaling in human MSC-derived neocartilage and whether it regulates matrix synthesis and other factors involved in the MSC mechanoresponse. Interestingly, MSC-derived chondrocytes significantly upregulated PTHrP mRNA (PTHLH) expression along with its second messenger cAMP in response to loading in our custom-built bioreactor. Exogenous PTHrP(1-34) induced the expression of known mechanoresponse genes (FOS, FOSB, BMP6) and significantly decreased glycosaminoglycan (GAG) and collagen synthesis similar to loading. The adenylate-cyclase inhibitor MDL-12,330A rescued the load-mediated decrease in GAG synthesis, indicating a direct involvement of cAMP-signaling in the reduction of ECM production. According to COL2A1-corrected hypertrophy-associated marker expression, load and PTHrP treatment shared the ability to reduce expression of MEF2C and PTH1R. In conclusion, the data demonstrate a significant mechanoinduction of PTHLH and a negative contribution of the PTHrP-cAMP signaling axis to GAG synthesis in MSC-derived chondrocytes after loading. To improve ECM synthesis and the mechanocompetence of load-exposed neocartilage, inhibition of PTHrP activity should be considered for MSC-based cartilage regeneration strategies.
异常机械负荷是关节软骨退变的主要风险因素之一。工程化间充质基质细胞（MSC）来源的软骨在基于细胞的软骨修复方面具有巨大潜力。然而，生理负荷方案在体外显示会降低MSC来源的新软骨的基质合成，而这种不良机械反应的调节因子仍知之甚少。甲状旁腺激素相关蛋白（PTHrP）参与软骨发育，并且在MSC软骨形成过程中，根据持续或短暂暴露情况，会以相反方式影响细胞外基质（ECM）的产生。PTHrP在多种组织和物种中由各种机械信号诱导产生；但PTHrP是否会因人类工程化新软骨的负荷而受到调节，以及是否会以正向或负向方式影响基质合成尚不清楚。本研究的目的是调查动态负荷是否会调节人MSC来源的新软骨中的PTHrP信号，以及它是否会调节基质合成和其他参与MSC机械反应的因子。有趣的是，在我们定制的生物反应器中，MSC来源的软骨细胞在负荷刺激下，PTHrP mRNA（PTHLH）表达及其第二信使cAMP显著上调。外源性PTHrP(1 - 34)诱导了已知机械反应基因（FOS、FOSB、BMP6）的表达，并且与负荷刺激类似，显著降低了糖胺聚糖（GAG）和胶原蛋白的合成。腺苷酸环化酶抑制剂MDL - 12,330A挽救了负荷介导的GAG合成减少，表明cAMP信号直接参与了ECM产生的减少。根据COL2A1校正的肥大相关标志物表达，负荷和PTHrP处理都具有降低MEF2C和PTH1R表达的能力。总之，数据表明负荷刺激后，PTHLH存在显著的机械诱导，并且PTHrP - cAMP信号轴对MSC来源的软骨细胞中的GAG合成有负面作用。为了改善ECM合成和负荷暴露的新软骨的机械性能，在基于MSC的软骨再生策略中应考虑抑制PTHrP活性。
