Bergholt Mads S, Albro Michael B, Stevens Molly M
Department of Materials, Imperial College London, London SW7 2AZ, United Kingdom; Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom; Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, United Kingdom.
Department of Materials, Imperial College London, London SW7 2AZ, United Kingdom; Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom; Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, United Kingdom.
Biomaterials. 2017 Sep;140:128-137. doi: 10.1016/j.biomaterials.2017.06.015. Epub 2017 Jun 14.
Tissue engineering (TE) has the potential to improve the outcome for patients with osteoarthritis (OA). The successful clinical translation of this technique as part of a therapy requires the ability to measure extracellular matrix (ECM) production of engineered tissues in vitro, in order to ensure quality control and improve the likelihood of tissue survival upon implantation. Conventional techniques for assessing the ECM content of engineered cartilage, such as biochemical assays and histological staining are inherently destructive. Raman spectroscopy, on the other hand, represents a non-invasive technique for in situ biochemical characterization. Here, we outline current roadblocks in translational Raman spectroscopy in TE and introduce a comprehensive workflow designed to non-destructively monitor and quantify ECM biomolecules in large (>3 mm), live cell TE constructs online. Diffuse near-infrared fiber-optic Raman spectra were measured from live cell cartilaginous TE constructs over a 56-day culturing period. We developed a multivariate curve resolution model that enabled quantitative biochemical analysis of the TE constructs. Raman spectroscopy was able to non-invasively quantify the ECM components and showed an excellent correlation with biochemical assays for measurement of collagen (R = 0.84) and glycosaminoglycans (GAGs) (R = 0.86). We further demonstrated the robustness of this technique for online prospective analysis of live cell TE constructs. The fiber-optic Raman spectroscopy strategy developed in this work offers the ability to non-destructively monitor construct growth online and can be adapted to a broad range of TE applications in regenerative medicine toward controlled clinical translation.
组织工程学（TE）有潜力改善骨关节炎（OA）患者的治疗效果。作为治疗手段的一部分，该技术的成功临床转化需要具备在体外测量工程组织细胞外基质（ECM）生成的能力，以确保质量控制并提高植入后组织存活的可能性。评估工程软骨ECM含量的传统技术，如生化分析和组织学染色，本质上具有破坏性。另一方面，拉曼光谱法是一种用于原位生化表征的非侵入性技术。在此，我们概述了TE中转化拉曼光谱法当前面临的障碍，并介绍了一种全面的工作流程，旨在对大型（>3毫米）活细胞TE构建体中的ECM生物分子进行非侵入性在线监测和定量。在56天的培养期内，从活细胞软骨TE构建体中测量了漫反射近红外光纤拉曼光谱。我们开发了一种多元曲线分辨率模型，能够对TE构建体进行定量生化分析。拉曼光谱法能够非侵入性地定量ECM成分，并且在测量胶原蛋白（R = 0.84）和糖胺聚糖（GAGs）（R = 0.86）方面与生化分析显示出极好的相关性。我们进一步证明了该技术在活细胞TE构建体在线前瞻性分析中的稳健性。这项工作中开发的光纤拉曼光谱策略提供了在线非侵入性监测构建体生长的能力，并且可以适用于再生医学中广泛的TE应用，以实现可控的临床转化。
