Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds
Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds
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DOI：
10.1089/ten.2006.0278
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发表时间：
2007-06-01
影响因子：
--
通讯作者：
Carson, Polly
中科院分区：
文献类型：
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作者：
Falanga, Vincent;Iwamoto, Satori;Carson, Polly
骨髓的非造血成分包括能够分化为脂肪、骨、肌肉、软骨和内皮的多能间充质干细胞（MSC）。在本报告中，我们描述了细胞培养和表征、输送系统以及局部应用自体 MSC 加速人类和实验小鼠伤口愈合的成功应用。从皮肤癌手术造成的急性伤口患者 (n = 5) 和长期、长期不愈合的下肢慢性伤口患者 (n = 8) 中采集 35-50 mL 的单次骨髓抽吸物。细胞在有利于 MSC 增殖的条件下在体外生长，流式细胞术和免疫染色显示其谱（CD29+、CD44+、CD105+、CD166+、CD34-、CD45-）与已发表的人类 MSC 报告高度一致。功能诱导研究证实间充质干细胞可以分化为骨、软骨和脂肪组织。使用带有双管注射器的纤维蛋白聚合物喷雾系统将培养的自体 MSC 涂抹到伤口上最多四次。纤维蛋白原（含有 MSC）和凝血酶均经过稀释，以最佳方式递送聚合凝胶，该凝胶立即粘附在伤口上，不会流失，但仍允许 MSC 保持活力并从凝胶中迁移。应用 MSC 后，创面活检标本的连续相邻切片显示细长的梭形细胞，与体外对应物类似，对 MSC 标记进行免疫染色。通过特殊染色剂和人类弹性蛋白抗体可以明显看出新弹性纤维的生成。培养细胞的应用是安全的，没有与治疗相关的不良事件。研究发现，所应用的细胞数量（每 cm(2) 伤口面积大于 1 x 10(6) 个细胞）与随后慢性伤口尺寸的减小之间存在很强的直接相关性 (p = 0.0058)。局部应用自体 MSC 还刺激糖尿病小鼠全层伤口的闭合 (db/db)。对小鼠伤口中绿色荧光蛋白 (GFP)+ MSC 的追踪显示出 GFP+ 血管，这表明所应用的细胞可能会持续存在并刺激伤口修复过程。这些发现表明，使用纤维蛋白喷雾系统可以将自体骨髓来源的 MSC 安全有效地递送至伤口。
The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 mL was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD105+, CD166+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than 1 x 10(6) cells per cm(2) of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system.