Abstract：
Human tissue-engineered matrices (hTEMs) have been proposed as a promising approach for in situ tissue engineered heart valves (TEHVs). However, there is still a limited understanding on how ECM composition in hTEMs develops over tissue culture time. Therefore, we performed a longitudinal hTEM assessment by 1) multiscale evaluation of hTEM composition during culture time (2, 4, 6-weeks), using (immuno)histology, biochemical assays, and mass spectrometry (LC-MS/MS); 2) analysis of protein pathways involved in ECM development using gene set enrichment analysis (GSEA); and 3) assessment of hTEM mechanical characterization using uniaxial tensile testing. Finally, as a proof-of-concept, TEHVs manufactured using 6-weeks hTEM samples were tested in a pulse duplicator. LC-MS/MS confirmed the tissue culture time-dependent increase in ECM proteins observed in histology and biochemical assays, revealing the most abundant collagens (COL6, COL12), proteoglycans (HSPG2, VCAN), and glycoproteins (FN, TNC). GSEA identified the most represented protein pathways in the hTEM at 2-weeks (mRNA metabolic processes), 4-weeks (ECM production), and 6-weeks (ECM organization and maturation). Uniaxial mechanical testing showed increased stiffness and stress at failure, and reduction in strain over tissue culture time. hTEM-based TEHVs demonstrated promising in vitro performance at both pulmonary and aortic pressure conditions, with symmetric leaflet coaptation and no stenosis. In conclusion, ECM protein abundance and maturation increased over tissue culture time, with consequent improvement of hTEM mechanical characteristics. These findings suggest that longer tissue culture impacts tissue organization, leading to an hTEM that may be suitable for high-pressure applications. STATEMENT OF SIGNIFICANCE: It is believed that the composition of the extracellular matrix (ECM) in the human tissue engineered matrices (hTEM) may favor tissue engineered heart valve (TEHV) remodeling upon implantation. However, the exact protein composition of the hTEM, and how this impacts tissue mechanical properties, remains unclear. Hence, we developed a reproducible rotation-based tissue culture method to produce hTEM samples. We performed a longitudinal assessment using different analytical techniques and mass spectrometry. Our data provided an in-depth characterization of the hTEM proteome with focus on ECM components, their development, and how they may impact the mechanical properties. Based on these results, we manufactured functional hTEM-based TEHVs at aortic-like condition in vitro. These outcomes pose an important step in translating hTEM-based TEHVs into clinics and in predicting their remodeling potential upon implantation.
摘要：
已经提出人类组织工程基质(hTEMs)作为原位组织工程心脏瓣膜(TEHVs)的有希望的方法。然而,关于hTEMs中ECM组成如何随组织培养时间发展的理解仍然有限。因此,我们通过1）在培养时间（2、4、6周）期间对hTEM组成进行了纵向hTEM评估，使用（免疫）组织学，生化化验,和质谱(LC-MS/MS);2)使用基因集富集分析(GSEA)分析参与ECM发育的蛋白质途径;和3)使用单轴拉伸测试评估hTEM机械表征。最后,作为概念证明，在脉冲复制器中测试使用6周hTEM样品制造的TEHV。LC-MS/MS证实了组织学和生化测定中观察到的ECM蛋白的组织培养时间依赖性增加，揭示了最丰富的胶原蛋白（COL6，COL12），蛋白聚糖（HSPG2，VCAN），和糖蛋白(FN，跨国公司）。GSEA在2周（mRNA代谢过程）时在hTEM中鉴定出最具代表性的蛋白质途径，4周（ECM生产），和6周（ECM组织和成熟）。单轴力学测试表明，破坏时的刚度和应力增加，随着组织培养时间的推移，菌株减少。基于hTEM的TEHVs在肺动脉和主动脉压力条件下都表现出了有希望的体外性能，对称小叶接合，无狭窄。总之,ECM蛋白丰度和成熟度随组织培养时间增加，从而改善了hTEM的力学特性。这些发现表明，更长的组织培养会影响组织组织，导致可能适用于高压应用的hTEM。重要声明::据信,人组织工程基质(hTEM)中的细胞外基质(ECM)的组成可有利于植入后的组织工程心脏瓣膜(TEHV)重塑。然而,hTEM的确切蛋白质组成，以及这如何影响组织的机械性能，尚不清楚。因此，我们开发了一种可重复的基于旋转的组织培养方法来生产hTEM样品。我们使用不同的分析技术和质谱进行了纵向评估。我们的数据提供了hTEM蛋白质组的深入表征，重点是ECM成分，他们的发展,以及它们如何影响机械性能。基于这些结果,我们在体外主动脉样条件下制造了基于hTEM的功能性TEHV。这些结果在将基于hTEM的TEHV转化为临床和预测其植入后的重塑潜力方面提出了重要步骤。
