Sherman Amanda B, Gilger Brian C, Berglund Alix K, Schnabel Lauren V
Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC, 27606, USA.
Stem Cell Res Ther. 2017 May 25;8(1):120. doi: 10.1186/s13287-017-0577-3.
We aimed to determine and compare the in vitro effects of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) and mesenchymal stem cell supernatant (MSC-Sp) on the wound healing capacity of equine corneal fibroblasts using a scratch assay.
Bone marrow aspirates and eyes were collected from normal, euthanized horses with subsequent isolation and culture of BM-MSCs and corneal stromal cells. Corneal stromal cells were culture-expanded in the culture well of transwell plates and then treated with an autologous BM-MSC suspension (dose: 2.5 × 10/100 μL media with the BM-MSCs contained within the insert well), MSC-Sp solution, or naive culture media (control) for 72 h. A linear defect in confluent cell cultures was created (i.e., corneal scratch assay) to assess the cellular closure ("healing") over time. Three representative areas of the scratch in each culture were photographed at each time point and the scratch area was quantitated using image analysis software (ImageJ). Media from the scratches were analyzed for various growth factors using human enzyme-linked immunosorbent assay (ELISA) kits that crossreact with the horse.
There was a significant percentage decrease in the scratch area remaining in the BM-MSC and MSC-Sp groups compared to the control group. There was also a significant percentage decrease in the scratch area remaining in the BM-MSC group compared to the MSC-Sp group at 36 h post-scratch and all time points thereafter. The concentration of transforming growth factor (TGF)-β1 in the media was significantly higher in the BM-MSC group compared to the control group.
The significant decrease in scratch area in equine corneal fibroblast cultures treated with autologous BM-MSCs compared to MSC-Sp or control treatments suggests that BM-MSCs may substantially improve corneal wound healing in horses. MSC-Sp may also improve corneal wound healing given the significant decrease in scratch area compared to control treatments, and would be an immediately available and cost-effective treatment option.
我们旨在通过划痕试验确定并比较自体骨髓间充质干细胞（BM-MSCs）和间充质干细胞上清液（MSC-Sp）对马角膜成纤维细胞伤口愈合能力的体外影响。
从正常安乐死的马身上采集骨髓抽吸物和眼睛，随后分离并培养BM-MSCs和角膜基质细胞。将角膜基质细胞在Transwell板的培养孔中进行培养扩增，然后用自体BM-MSC悬浮液（剂量：2.5×10/100μL培养基，BM-MSCs包含在插入孔中）、MSC-Sp溶液或单纯培养基（对照）处理72小时。在汇合的细胞培养物中制造线性缺损（即角膜划痕试验），以评估随时间的细胞闭合（“愈合”）情况。在每个时间点对每个培养物中划痕的三个代表性区域进行拍照，并使用图像分析软件（ImageJ）对划痕面积进行定量。使用与马有交叉反应的人酶联免疫吸附测定（ELISA）试剂盒分析划痕中的培养基中的各种生长因子。
与对照组相比，BM-MSC组和MSC-Sp组中剩余的划痕面积百分比显著降低。在划痕后36小时及此后的所有时间点，与MSC-Sp组相比，BM-MSC组中剩余的划痕面积百分比也显著降低。与对照组相比，BM-MSC组培养基中转化生长因子（TGF）-β1的浓度显著更高。
与MSC-Sp或对照处理相比，用自体BM-MSCs处理的马角膜成纤维细胞培养物中划痕面积显著减少，这表明BM-MSCs可能显著改善马的角膜伤口愈合。与对照处理相比，由于划痕面积显著减少，MSC-Sp也可能改善角膜伤口愈合，并且将是一种立即可用且具有成本效益的治疗选择。
