Department of Orthopedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA.
Am J Phys Med Rehabil. 2013 May;92(5):420-9. doi: 10.1097/PHM.0b013e31825f148a.
Back pain associated with symptomatic disc degeneration is a common clinical condition. Intervertebral disc (IVD) cell apoptosis and senescence increase with aging and degeneration. Repopulating the IVD with cells that could produce and maintain extracellular matrix would be an alternative therapy to surgery. The objective of this study was to determine the potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as a novel cell source for disc repair. In this study, we intended to confirm the potential for hUCB-MSCs to differentiate and display a chondrocyte-like phenotype after culturing in micromass and after injection into the rabbit IVD explant culture. We also wanted to confirm hUCB-MSC survival after transplantation into the IVD explant culture.
This study consisted of micromass cultures and in vitro rabbit IVD explant cultures to assess hUCB-MSC survival and differentiation to display chondrocyte-like phenotype. First, hUCB-MSCs were cultured in micromass and stained with Alcian blue dye. Second, to confirm cell survival, hUCB-MSCs were labeled with an infrared dye and a fluorescent dye before injection into whole rabbit IVD explants (host). IVD explants were then cultured for 4 wks. Cell survival was confirmed by two independent techniques: an imaging system detecting the infrared dye at the organ level and fluorescence microscopy detecting fluorescent dye at the cellular level. Cell viability was assessed by staining the explant with CellTracker green, a membrane-permeant tracer specific for live cells. Human type II collagen gene expression (from the graft) was assessed by polymerase chain reaction.
We have shown that hUCB-MSCs cultured in micromass are stained blue with Alcian blue dye, which suggests that proteoglycan-rich extracellular matrix is produced. In the cultured rabbit IVD explants, hUCB-MSCs survived for at least 4 wks and expressed the human type II collagen gene, suggesting that the injected hUCB-MSCs are differentiating into a chondrocyte-like lineage.
This study demonstrates the abiity of hUBC-MSCs to survive and assume a chondrocyte-like phenotype when injected into the rabbit IVD. These data support the potential for hUBC-MSCs as a cell source for disc repair. Further measures of the host response to the injection and studies in animal models are needed before trials in humans.
与症状性椎间盘退变相关的背痛是一种常见的临床病症。椎间盘（IVD）细胞凋亡和衰老随着年龄和退变而增加。用能够产生和维持细胞外基质的细胞来再生 IVD 将是一种替代手术的治疗方法。本研究的目的是确定人脐带血间充质干细胞（hUCB-MSCs）作为一种新型椎间盘修复细胞来源的潜力。在这项研究中，我们打算证实 hUCB-MSCs 在微团培养后和注射到兔 IVD 外植体培养物中后具有分化并表现出类软骨细胞表型的潜力。我们还想证实 hUCB-MSC 移植到 IVD 外植体培养物后的存活情况。
这项研究包括微团培养和体外兔 IVD 外植体培养，以评估 hUCB-MSC 的存活和分化为类软骨细胞表型。首先，将 hUCB-MSCs 培养在微团中并用阿尔辛蓝染料染色。其次，为了确认细胞存活，在将 hUCB-MSCs 注射到整个兔 IVD 外植体（宿主）之前，用红外染料和荧光染料对其进行标记。然后将 IVD 外植体培养 4 周。通过两种独立的技术确认细胞存活：器官水平检测红外染料的成像系统和细胞水平检测荧光染料的荧光显微镜。通过用细胞膜通透性示踪剂 CellTracker green 对外植体进行染色来评估细胞活力，该示踪剂特异性标记活细胞。通过聚合酶链反应评估人 II 型胶原基因表达（来自移植物）。
我们已经表明，在微团中培养的 hUCB-MSCs 用阿尔辛蓝染料染色呈蓝色，这表明富含蛋白聚糖的细胞外基质正在产生。在培养的兔 IVD 外植体中，hUCB-MSCs 至少存活了 4 周，并表达了人 II 型胶原基因，这表明注射的 hUCB-MSCs 正在分化为类软骨细胞谱系。
这项研究表明，hUBC-MSCs 具有在注射到兔 IVD 后存活并表现出类软骨细胞表型的能力。这些数据支持 hUBC-MSCs 作为椎间盘修复细胞来源的潜力。在进行人体试验之前，还需要进一步评估宿主对注射的反应和动物模型研究。
