Cryopreserved intervertebral disc with injected bone marrow–derived stromal cells: a feasibility study using organ culture

Chan, Samantha C.W.; Gantenbein-Ritter, Benjamin; Leung, Victor Y.L.; Chan, Danny; Cheung, Kenneth M.C.; Ito, Keita

doi:10.1016/j.spinee.2009.12.019

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Volume 10, Issue 6 , Pages 486-496, June 2010

Cryopreserved intervertebral disc with injected bone marrow–derived stromal cells: a feasibility study using organ culture

Samantha C.W. Chan, MPhil
- AO Research Institute, CH-7270 Davos Platz, Switzerland
- Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong
- ARTORG Center for Biomedical Engineering Research, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland
Benjamin Gantenbein-Ritter, PhD
- AO Research Institute, CH-7270 Davos Platz, Switzerland
- ARTORG Center for Biomedical Engineering Research, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland
- Corresponding author. ARTORG Center for Biomedical Engineering Research, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland. Tel.: (41) 31-6315926; fax: (41) 31-6315960.
Victor Y.L. Leung, PhD
- Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong
Danny Chan, PhD
- Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong
Kenneth M.C. Cheung, MD
- Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong
Keita Ito, ScD
- AO Research Institute, CH-7270 Davos Platz, Switzerland
- Department of Biomedical Engineering, Eindhoven University of Technology, NL-5600MB, Eindhoven, The Netherlands

Received 29 January 2009; received in revised form 7 November 2009; accepted 25 December 2009. published online 22 February 2010.

Abstract

Background Context

A recent clinical study demonstrated that cryopreserved allogeneic intervertebral disc transplantation relieved pain and preserved motion, thus opening up a new treatment option for degenerative disc disease. However, these transplanted discs continued to degenerate, possibly due to a lack of viable cells. Bone marrow–derived stromal cell (BMSC) implantation has been shown to delay disc degeneration.

Purpose

This study examined the viability over time of endogenous and injected BMSCs in cryopreserved disc under simulated-physiological loading conditions.

Study Design/ Setting

An in vitro study of BMSCs injected into cryopreserved bovine caudal discs.

Methods

Bovine caudal discs were harvested and cryopreserved at −196°C. After thawing, PKH-26–labeled BMSCs embedded in peptide hydrogel carrier were injected into the nucleus pulposus. Two BMSC injection quantities, that is, 1×10⁵ and 2.5×10⁵ were examined. Discs with injected cells were maintained in a bioreactor for 7 days under simulated-physiological loading. Cell viability (staining), gene expression (reverse transcription-polymerase chain reaction) profile, and proteoglycan content (histologically) were evaluated.

Results

Forty percent of endogenous cell viability was maintained after freeze thawing. Over the 7-day culture, this did not change further. However, there was upregulation of Col1a2 and Mmp-13 and downregulation of Col2a1gene expression. Sixty percent of BMSCs survived the initial injection procedure, and only 20% remained alive after 7 days of culture. Bone marrow–derived stromal cell implantation did not alter the viability of the endogenous cells, but discs injected with 1×105 BMSCs showed significantly higher ACAN expression than sham discs.

Conclusions

Although only 40% of cells survived cryopreservation, these endogeneous cells continued to survive over 7 days if maintained under simulated-physiological loading conditions. Although only a small portion of injected BMSCs survived, they did have some effect on the matrix protein gene expression profile. Their influence on native cells requires long-term evaluation.

Keywords: Intervertebral disc, Cryopreservation, Bone marrow–derived mesenchymal stromal cells, Bovine, Cell viability, Gene expression