AO Research Institute, CH-7270 Davos Platz, Switzerland.
Spine J. 2010 Jun;10(6):486-96. doi: 10.1016/j.spinee.2009.12.019. Epub 2010 Feb 19.
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.
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.
Bovine caudal discs were harvested and cryopreserved at -196 degrees C. After thawing, PKH-26-labeled BMSCs embedded in peptide hydrogel carrier were injected into the nucleus pulposus. Two BMSC injection quantities, that is, 1x10(5) and 2.5x10(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.
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 1x105 BMSCs showed significantly higher ACAN expression than sham discs.
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.
最近的一项临床研究表明，冷冻保存同种异体椎间盘移植可以缓解疼痛并保持运动，从而为退行性椎间盘疾病开辟了新的治疗选择。然而，这些移植的椎间盘仍在继续退化，可能是由于缺乏有活力的细胞。骨髓基质细胞（BMSC）的植入已被证明可以延缓椎间盘的退化。
本研究在模拟生理负荷条件下，研究冷冻保存椎间盘内内源性和注射 BMSC 的存活时间。
研究设计/地点：在冷冻保存的牛尾椎间盘内注射 BMSC 的体外研究。
采集牛尾椎间盘并在-196°C 下冷冻保存。解冻后，将 PKH-26 标记的 BMSCs 嵌入肽水凝胶载体中，注入髓核。检查了两种 BMSC 注射量，即 1x10(5)和 2.5x10(5)。将带有注射细胞的椎间盘在生物反应器中在模拟生理负荷下培养 7 天。评估细胞活力（染色）、基因表达（逆转录-聚合酶链反应）谱和糖胺聚糖含量（组织学）。
冷冻解冻后，内源性细胞活力保持在 40%。在 7 天的培养过程中，这一数值没有进一步变化。然而，Col1a2 和 Mmp-13 的基因表达上调，Col2a1 的基因表达下调。60%的 BMSC 在前注射过程中存活下来，只有 20%在 7 天的培养后仍然存活。骨髓基质细胞的植入并没有改变内源性细胞的活力，但与 sham 椎间盘相比，注射 1x105 BMSC 的椎间盘的 ACAN 表达明显更高。
尽管只有 40%的细胞在冷冻保存中存活下来，但如果在模拟生理负荷条件下保存，这些内源性细胞可以继续存活 7 天以上。尽管只有一小部分注射的 BMSC 存活下来，但它们确实对基质蛋白基因表达谱有一定的影响。它们对天然细胞的影响需要长期评估。
