Department of Bioengineering, Temple University, Philadelphia, Pennsylvania 19122, USA.
Analyst. 2022 Apr 11;147(8):1730-1741. doi: 10.1039/d1an02351a.
Tissue engineering of cartilage for tissue repair has many challenges, including the inability to assess when the developing construct has reached compositional maturity for implantation. The goal of this study was to provide a novel analytical approach to nondestructively assess tissue engineered cartilage (TEC) during development. We applied attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to establish a quick and straightforward method to evaluate consumption of glucose and secretion of the metabolite lactate in the culture media, processes that are associated with tissue development. Using a series of standards, we showed by principal component analysis (PCA) that ATR-FTIR data was able to distinguish culture media with varying amounts of glucose and lactate. The 2 derivative spectra displayed specific peaks of glucose at 1035 cm and lactate at 1122 cm, and both the spectral first principal component (PC-1) scores and the 1122/1035 peak ratio very strongly correlated with the concentration of these components. TEC was prepared using chondrogenic cells grown in hydrogels, and analyzed for cell viability, distribution, and formation of proteoglycan (PG, a major cartilage protein). ATR-FTIR data of the cell culture media harvested during TEC development showed that the spectral PC-1 and the 1122/1035 peak ratio could significantly distinguish cultures with different amounts of constructs (1, 3 or 5 constructs per well) or with constructs at different developmental stages (3 or 5 weeks of culture). Interestingly, we also found that the PG content of the TEC was significantly correlated with both spectral PC-1 ( = -0.79) and the 1122/1035 peak ratio ( = 0.80). Therefore, monitoring relative glucose and lactate concentrations in cell culture media by ATR-FTIR provides a novel nondestructive approach to assess development of TEC.
组织工程软骨用于组织修复存在诸多挑战，其中包括无法评估发育中的构建体何时达到植入的组成成熟度。本研究的目的是提供一种新的分析方法，以非破坏性地评估组织工程软骨（TEC）的发育过程。我们应用衰减全反射傅里叶变换红外（ATR-FTIR）光谱法建立了一种快速而直接的方法，用于评估培养物中葡萄糖的消耗和代谢产物乳酸的分泌，这些过程与组织发育有关。通过主成分分析（PCA），我们使用一系列标准表明，ATR-FTIR 数据能够区分具有不同葡萄糖和乳酸浓度的培养基。二阶导数光谱显示出葡萄糖在 1035cm 处和乳酸在 1122cm 处的特定峰，并且光谱第一主成分（PC-1）得分和 1122/1035 峰比与这些成分的浓度非常强相关。我们使用在水凝胶中生长的软骨细胞制备 TEC，并分析其细胞活力、分布和糖胺聚糖（PG，软骨的主要蛋白质）的形成。在 TEC 发育过程中收集的细胞培养物培养基的 ATR-FTIR 数据表明，光谱 PC-1 和 1122/1035 峰比可以显著区分具有不同数量构建体（每个孔 1、3 或 5 个构建体）或具有不同发育阶段构建体（3 或 5 周培养）的培养物。有趣的是，我们还发现 TEC 的 PG 含量与光谱 PC-1（= -0.79）和 1122/1035 峰比（= 0.80）均显著相关。因此，通过 ATR-FTIR 监测细胞培养物培养基中的相对葡萄糖和乳酸浓度为评估 TEC 的发育提供了一种新的非破坏性方法。
