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

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 x 10(5) and 2.5 x 10(5) 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 Colla2 and Mmp-13 and downregulation of Col2algene 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 x 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. (C) 2010 Elsevier Inc. All rights reserved.