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. 2017 Sep;140:128-137.
doi: 10.1016/j.biomaterials.2017.06.015. Epub 2017 Jun 14.

Online Quantitative Monitoring of Live Cell Engineered Cartilage Growth Using Diffuse Fiber-Optic Raman Spectroscopy

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Free PMC article

Online Quantitative Monitoring of Live Cell Engineered Cartilage Growth Using Diffuse Fiber-Optic Raman Spectroscopy

Mads S Bergholt et al. Biomaterials. .
Free PMC article

Abstract

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 (R2 = 0.84) and glycosaminoglycans (GAGs) (R2 = 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.

Keywords: Articular cartilage; Fiber-optic Raman spectroscopy; Live cell Raman spectroscopy; Online biomedical Raman spectroscopy; Tissue-engineering.

Figures

Fig. 1
Fig. 1
(A) Schematic of the fiber-optic Raman spectroscopy system for monitoring and biochemical quantification in live cell tissue-engineered (TE) constructs. (B) Workflow of Raman spectroscopic monitoring of live cell TE constructs. Also shown is the developed computational processing framework for online evaluation of TE constructs.
Fig. 2
Fig. 2
(A) Images of live cells (green) and dead cells (red) after an initial exposure to 785 nm laser for continuous 0, 2, 5, or 15 min duration. (B-C) GAG and collagen contents of TE constructs 2 weeks after initial exposure to 785 nm laser probe for 0, 2, 5, or 15 min. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
(A–B) Biochemically measured GAG and collagen contents of TE constructs (TGF-β free and TGF-β supplemented) after 0, 14, 28, 42, and 56 days of culture. (C) Mean Raman spectra ± 1 standard deviation (SD) of native cartilage and TE constructs after varying culture durations. (D) Multivariate curve resolution (MCR) developed from native and tissue-engineered (TE) constructs (TGF-β free and TGF-β supplemented). The MCR pure components correlate well with laboratory grade water, GAG, and collagen (water R2 = 0.62, GAG: R2 = 0.80, and collagen: R2 = 0.80) (E–F) Correlations between the Raman prediction (in arbitrary units) and biochemically measured GAG and collagen, respectively, with linear fits to the data. Raman spectroscopy showed an excellent correlation with biochemical assays for measurement of collagen (R2 = 0.84) and GAG (R2 = 0.86). The correlation for prediction of water content was poor (R2 = 0.16) due to inherently weak signals in the range 800–1800 cm−1 and is therefore not shown.
Fig. 4
Fig. 4
(A) Mechanical properties (compressive Young’s modulus) of TE constructs (TGF-β free and TGF-β supplemented) at various time points (i.e. 0, 14, 28, 42, and 56 days). *p < 0.05: represents statistical difference between TGF-β free and TGF-β supplemented groups at corresponding time point. (B-C) Correlation curves between mechanical properties and biochemically-measured GAG and collagen contents for combination of both TGF-β supplemented and TGF-β free data points. Dashed curves represent exponential fits.
Fig. 5
Fig. 5
(A) Mean Raman difference spectra ±1 SD between native tissues and TE constructs at various time points (native – day 0, native – day 14, native – day 28, native – day 42, and native – day 56). (B) Principal component analysis (PCA) loadings developed from native articular cartilage. (C) Application of PCA model to TGF-β free TE constructs: Q-residuals plotted against the Hotelling T2 values for native articular cartilage tissues and TGF-β free TE constructs at day 0 (n = 8), day 14 (n = 8 spectra), day 28 (n = 8 spectra), day 42 (n = 8 spectra), day 56 (n = 8 spectra).
Fig. 6
Fig. 6
Online prospective application of the developed MCR model for online quantification of relative concentrations (arbitrary units) of collagen and GAG in live TE constructs at various time points (i.e. 0, 14, 28, and 42 days). Due to inherent difficulty in getting adequate signal to noise ratio at day 0 (since the majority of signal originates from water), only two data-points were measured.

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References

    1. O’Connell G.D., Lima E.G., Bian L., Chahine N.O., Albro M.B., Cook J.L., Ateshian G.A., Hung C.T. Toward engineering a biological joint replacement. J. Knee Surg. 2012;25(3):187–196. - PMC - PubMed
    1. Johnstone B., Alini M., Cucchiarini M., Dodge G.R., Eglin D., Guilak F., Madry H., Mata A., Mauck R.L., Semino C.E., Stoddart M.J. Tissue engineering for articular cartilage repair–the state of the art. Eur. Cell Mater. 2013;25:248–267. - PubMed
    1. Byers B.A., Mauck R.L., Chiang I.E., Tuan R.S. Transient exposure to transforming growth factor beta 3 under serum-free conditions enhances the biomechanical and biochemical maturation of tissue-engineered cartilage. Tissue Eng. Part A. 2008;14(11):1821–1834. - PMC - PubMed
    1. Cigan A.D., Nims R.J., Albro M.B., Vunjak-Novakovic G., Hung C.T., Ateshian G.A. Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs. J. Biomech. 2014;47(16):3847–3854. - PMC - PubMed
    1. Cigan A.D., Roach B.L., Nims R.J., Tan A.R., Albro M.B., Stoker A.M., Cook J.L., Vunjak-Novakovic G., Hung C.T., Ateshian G.A. High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties. J. Biomech. 2016;49(9):1909–1917. - PMC - PubMed
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