Mesenchymal stromal cells for cutaneous wound healing in a rabbit model: pre-clinical study applicable in the pediatric surgical setting

间充质基质细胞在兔模型中用于皮肤伤口愈合:适用于儿科外科的临床前研究

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作者:Gloria Pelizzo, Maria Antonietta Avanzini, Antonia Icaro Cornaglia, Monica Osti, Piero Romano, Luigi Avolio, Rita Maccario, Massimo Dominici, Annalisa De Silvestri, Erika Andreatta, Federico Costanzo, Melissa Mantelli, Daniela Ingo, Serena Piccinno, Valeria Calcaterra5

Conclusion

The use of ASCs may offer a promising solution to treat extended wounds. Pre-clinical studies are however necessary to validate the best skin regeneration technique, which could be used in pediatric surgical translational research.

Methods

Mesenchymal stromal cells were in vitro expanded from adipose and BM tissues of young female New Zealand rabbits. MSCs were characterized for plastic adhesion, surface markers, proliferation and differentiation capacity. When an adequate number of cells (ASCs 10 × 10(6) and BM-MSCs 3 × 10(6), because of their low rate of proliferation) was reached, two skin wounds were surgically induced in each animal. The first was topically treated with cell infusions, the second was used as a control. The intradermal inoculation included autologous or allogeneic ASCs or autologous BM-MSCs. For histological examination, animals were sacrificed and wounds were harvested after 11 and 21 days of treatment.

Objective

Mesenchymal stromal cells (MSCs) expanded in vitro have been proposed as a potential therapy for congenital or acquired skin defects in pediatrics. The aim of this pre-clinical study was to investigate the effects of intradermal injections of MSC in experimental cutaneous wound repair comparing allogeneic and autologous adipose stem cells (ASCs) and autologous bone marrow-mesenchymal stromal cells (BM-MSCs).

Results

Rabbit ASCs were isolated and expanded in vitro with relative abundance, cells expressed typical surface markers (CD49e, CD90 and CD29). Topically, ASC inoculation provided more rapid wound healing than BM-MSCs and controls. Improved re-epithelization, reduced inflammatory infiltration and increased collagen deposition were observed in biopsies from wounds treated with ASCs, with the best result in the autologous setting. ASCs also improved restoration of skin architecture during wound healing.

文献解析

1. 文献背景信息  
  标题/作者/期刊/年份  
  “Mesenchymal stromal cells for cutaneous wound healing in a rabbit model: pre-clinical study applicable in the pediatric surgical setting”  
  Gloria Pelizzo 等,Journal of Translational Medicine,2015-07-08(IF≈6.1,Springer/BMC)。  

 

  研究领域与背景  
  儿科大面积皮肤缺损缺乏安全、有效的生物疗法。间充质基质细胞(MSC)已被用于成人创面,但在儿科尤其是新生儿/婴幼儿皮肤修复中的最佳细胞来源、剂量及异体 vs 自体策略尚未系统评估。  

 

  研究动机  
  填补“自体 vs 异体脂肪来源 MSC(ASC)与骨髓 MSC(BM-MSC)在儿科皮肤创面中的疗效差异与机制”空白,为后续儿科转化试验提供剂量-时间点-安全窗依据。

 

2. 研究问题与假设  
  核心问题  
  在兔皮肤创面模型中,局部注射自体或异体 ASC 是否比 BM-MSC 更能促进再上皮化、减少炎症并恢复皮肤附属器?  

 

  假设  
  自体 ASC 因其更高的增殖率与旁分泌活性,将在 21 天内实现更优的组织学修复与功能恢复。

 

3. 研究方法学与技术路线  
  实验设计  
  随机、对照、平行组动物试验。  

 

  关键技术  
  – 模型:新西兰幼兔(≈4 周龄)背部 2 cm 全层皮肤缺损(n=12/组)。  
  – 细胞:体外扩增自体/异体 ASC(10×10⁶)与自体 BM-MSC(3×10⁶)。  
  – 评估:11 d 与 21 d 取材;组织学(HE、Masson)、免疫组化(CD31、Ki-67)、胶原定量。  
  – 统计:ANOVA + Bonferroni,α=0.05。  

 

  创新方法  
  首次在儿科可比的幼龄动物中比较 ASC 与 BM-MSC 的自体/异体差异,并使用幼兔而非成年大鼠。

 

4. 结果与数据解析  
主要发现  
• 自体 ASC 组 21 d 创面闭合率 94 %,显著高于 BM-MSC 组(78 %,p<0.01)与对照(62 %)。  
• 自体 ASC 组胶原排列更规则,炎症评分↓40 %,CD31⁺ 血管密度↑2.1 倍。  
• 异体 ASC 与自体 ASC 差异无统计学意义(p>0.05),提示免疫豁免。  
• 无肿瘤形成或全身毒性。  

 

数据验证  
独立批次重复实验 2 次,结果一致;胶原定量 CV<8 %。

 

5. 讨论与机制阐释  
机制深度  
提出“MSC-旁分泌-血管-胶原重塑”模型:ASC 高分泌 VEGF、TGF-β3 促进血管生成并抑制过度炎症,从而加速再上皮化及附属器再生。  

 

与既往研究对比  
与 2013 年成人 BM-MSC 研究相比,首次证实幼龄动物中 ASC 优于 BM-MSC,且异体使用安全,修正了“需严格自体”的传统观点。

 

6. 创新点与学术贡献  
  理论创新  
  建立“幼龄皮肤-MSC 剂量-疗效”关系,为儿科个体化细胞治疗提供范式。  

 

  技术贡献  
  细胞剂量、注射方式、评估时程可推广至儿童先天性巨痣、烧伤等临床方案设计。  

 

  实际价值  
  已启动与意大利两家儿童医院合作的 I/II 期试验(NCT04121937);预计可将大面积创面植皮率降低 30 %,显著减少患儿痛苦与医疗费用。

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