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Short-term follow-up of disc cell therapy in a porcine nucleotomy model with an albumin–hyaluronan hydrogel: in vivo and in vitro results of metabolic disc cell activity and implant distribution

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Abstract

Purpose

Cell therapy would be favorably performed immediately after nucleotomy, to restore intervertebral disc functionality and to slow down disc degeneration. Promising results were reported from small animal models but remaining problems, especially in larger animals, include loss of vital cells due to annular damage at the injection site and detrimental intradiscal conditions. The aim of the present study was to optimize cell-based disc therapy using a new albumin–hyaluronan hydrogel together with bone marrow-derived mesenchymal stem cells in a large porcine disc model.

Methods

Luciferase cell labeling was evaluated to follow-up stem cells metabolically up to 7 days in 3D cell cultures mimicking the harsh disc environment with low oxygen and glucose concentrations. As a pilot in vivo study, the implant was injected into porcine discs after removal of ~10 % of nucleus volume and animals were killed immediately after surgery (n = 6) and 3 days later (n = 6). 24 discs were analyzed. Implant persistence and cell activity (luciferase + WST assay) were observed simultaneously.

Results

In vitro cell culture with reduction of glucose (20, 5, 0.5, 0 mM) and oxygen (21, 5, 2 %) significantly decreased metabolic cell activity and luciferase activity after 3 days, with no recovery and a further decrease after 7 days, establishing luciferase activity as a metabolic sensor. During 3 days of 3D culture with disc-like conditions, luciferase activity decreased to 8 %. In vivo, initial implant volume shrank to 61 % at day 3 with evidence for hydrogel compression. Luciferase activity in vivo at day 3 was 2 % without referencing but 23 % after referencing to in vitro cell adaptation, and 38 % after additional consideration of detected implant volume loss.

Conclusion

In vitro analysis up to 7 days established for the first time luciferase activity as a metabolic sensor for mesenchymal stem cells used in regenerative disc therapy. Under the present protocol, short-term in vivo analysis after 3 days suggests improved implant retainment inside the disc and persistence of metabolically active cells; however, further studies will have to prove long-term in vivo outcome.

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Acknowledgments

We would like to thank S. Gantz for statistical support, J. Mollenhauer and K. Gaissmaier from TETEC AG for technical support, providing us with samples of the hydrogel implant and critically reading the manuscript, and V. Kraemer from Friatec AG for samples of Degussit. The study was funded by BioProfile Grant 0313755 and Grant 0315579 from the Ministry of Education and Research, Germany. Also parts of this work were financially supported by the Orthopaedic University hospital.

Conflict of interest

None of the authors has any potential conflict of interest.

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Author notes

  1. K. Kleinschmitt & J. Holschbach

    Present address: Merck KGaA, Frankfurter Str. 250, 64293, Darmstadt, Germany

Authors and Affiliations

  1. Clinic for Orthopaedics and Trauma Surgery, Heidelberg University Hospital, Schlierbacher Landstr. 200a, 69118, Heidelberg, Germany

    G. W. Omlor

  2. Division of Experimental Orthopaedics, University of Heidelberg, Schlierbacher Landstr. 200a, 69118, Heidelberg, Germany

    J. Fischer, K. Kleinschmitt, J. Holschbach, K. Brohm & W. Richter

  3. NMI Natural and Medical Sciences Institute, University of Tübingen, Markwiesenstr. 55, 72770, Reutlingen, Germany

    K. Benz

  4. Institute of Experimental Oncology, Klinikum Rechts der Isar TU München, 81675, Munich, Germany

    M. Anton

  5. BG Trauma Hospital Ludwigshafen, University of Heidelberg, 67071, Ludwigshafen, Germany

    T. Guehring

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G. W. Omlor and J. Fischer share first authorship.

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Omlor, G.W., Fischer, J., Kleinschmitt, K. et al. Short-term follow-up of disc cell therapy in a porcine nucleotomy model with an albumin–hyaluronan hydrogel: in vivo and in vitro results of metabolic disc cell activity and implant distribution. Eur Spine J 23, 1837–1847 (2014). https://doi.org/10.1007/s00586-014-3314-y

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