Nondestructive assessment of tissue engineered cartilage based on biochemical markers in cell culture media: application of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy.
Nondestructive assessment of tissue engineered cartilage based on biochemical markers in cell culture media: application of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy.
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DOI：
10.1039/d1an02351a
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发表时间：
2022-04-11
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影响因子：
--
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用于组织修复的软骨的组织工程具有许多挑战，包括无法评估发育中的构造何时达到用于植入的组成成熟度。本研究的目的是提供一种新的分析方法，非破坏性地评估组织工程软骨（TEC）在体外开发。我们应用衰减全反射傅里叶变换红外（ATR-FTIR）光谱法建立一个快速和简单的方法来评估葡萄糖的消耗和代谢产物乳酸在培养基中的分泌，与组织发育相关的过程。使用一系列的标准，我们表明，主成分分析（PCA），ATR-FTIR数据能够区分培养基与不同量的葡萄糖和乳酸。二阶导数光谱在1035 cm−1处显示葡萄糖的特定峰，在1122 cm−1处显示乳酸盐的特定峰，并且光谱第一主成分（PC-1）分数和1122/1035峰比率都与这些组分的浓度非常强相关。使用在水凝胶中生长的软骨形成细胞制备TEC，并分析细胞活力、分布和蛋白聚糖（PG，一种主要软骨蛋白）的形成。在TEC发育期间收获的细胞培养基的ATR-FTIR数据显示光谱PC-1和1122/1035峰比率可以显著区分具有不同量的构建体（1、3或5个构建体/孔）或具有处于不同发育阶段的构建体（培养3或5周）的培养物。有趣的是，我们还发现TEC的PG含量与光谱PC-1（r=-0.79）和1122/1035峰比（r=0.80）显著相关。因此，通过ATR-FTIR监测细胞培养基中的相对葡萄糖和乳酸浓度提供了一种新的非破坏性方法来评估TEC的发展。
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 in vitro 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 2nd derivative spectra displayed specific peaks of glucose at 1035 cm−1 and lactate at 1122 cm−1, 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/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 (r=−0.79) and the 1122/1035 peak ratio (r=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.
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影响因子：
18.2
作者：
Bergholt, Mads S.;St-Pierre, Jean-Philippe;Offeddu, Giovanni S.;Parmar, Paresh A.;Albro, Michael B.;Puetzer, Jennifer L.;Oyen, Michelle L.;Stevens, Molly M.
通讯作者：
Stevens, Molly M.
影响因子：
3.3
作者：
Sophia Fox AJ;Bedi A;Rodeo SA
通讯作者：
Rodeo SA
影响因子：
14
作者：
Kim, Iris L.;Mauck, Robert L.;Burdick, Jason A.
通讯作者：
Burdick, Jason A.
DOI：
10.1016/0304-4165(86)90306-5
发表时间：
1986-09-04
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
FARNDALE, RW;BUTTLE, DJ;BARRETT, AJ
通讯作者：
BARRETT, AJ
影响因子：
3
作者：
Kim, Minwook;Foo, Li F.;Pleshko, Nancy
通讯作者：
Pleshko, Nancy