Xia Xiaopeng, Li Jing, Xia Bo, Yang Huilin, Zhang Dongmei, Zhou Bin, Zhang Jie, Zhou Man, Liu Fan
Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215000, P.R. China; Department of Orthopedics, Nantong Hospital of Traditional Chinese Medicine, Nantong, Jiangsu 215000, P.R. China.
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Exp Ther Med. 2017 Feb;13(2):542-550. doi: 10.3892/etm.2016.3972. Epub 2016 Dec 13.
The aim of this study was to explore an effective method for the repair of cartilage defects using chitosan/glycerophosphate (C/GP) gel- and Matrigel-engineered human bone morphogenetic protein 7 (hBMP7)-expressing chondrocytes. Rabbit chondrocytes were obtained, cultured and transfected with an adenovirus containing hBMP7 and green fluorescent protein (Ad-hBMP7-GFP). The expression of hBMP7 in the transfected cells was tested by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The phenotype of the transfected cells was evaluated by detecting the yields of collagen II and hyaluronic acid using RT-PCR and enzyme-linked immunosorbent assay (ELISA). The growth of chondrocytes in the C/GP gel and Matrigel was accessed by measuring the cell growth rate, hematoxylin and eosin (H&E) staining and observation under a scanning microscope. Twelve adult male New Zealand white rabbits were randomly divided into three groups. Two cartilage defects were created in the rabbits' knees by aseptic surgery. Group A (n=4) did not receive any treatment, group B (n=4) were treated with C/GP gel and Matrigel-engineered Ad-mock-GFP-transfected chondrocytes, and group C (n=4) were treated with C/GP gel and Matrigel-engineered Ad-hBMP7-GFP-transfected chondrocytes. Rabbits were sacrificed at 4 weeks after transplantation, and the repair effect was measured by the Wakitani scoring method. On the basis of the RT-PCR and western blot results, hBMP7 was efficiently overexpressed in the Ad-hBMP7-GFP-transfected chondrocytes. The ELISA results showed that the yields of collagen II and hyaluronic acid in Ad-hBMP7-GFP-transfected chondrocytes were significantly higher than those in Ad-mock-GFP-transfected chondrocytes. Chondrocytes have a better morphology and arrangement in a Matrigel scaffold than in C/GP, as assessed by H&E staining and scanning microscopy. According to the Wakitani score, Matrigel combined with Ad-hBMP7-GFP-transfected chondrocytes successfully promoted the repair of cartilage defects in rabbit knees.
本研究的目的是探索一种使用壳聚糖/甘油磷酸酯（C/GP）凝胶和基质胶构建的表达人骨形态发生蛋白7（hBMP7）的软骨细胞修复软骨缺损的有效方法。获取兔软骨细胞，进行培养并用含hBMP7和绿色荧光蛋白的腺病毒（Ad-hBMP7-GFP）转染。通过逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测转染细胞中hBMP7的表达。通过RT-PCR和酶联免疫吸附测定（ELISA）检测Ⅱ型胶原蛋白和透明质酸的产量来评估转染细胞的表型。通过测量细胞生长速率、苏木精和伊红（H&E）染色以及在扫描显微镜下观察来评估软骨细胞在C/GP凝胶和基质胶中的生长情况。将12只成年雄性新西兰白兔随机分为三组。通过无菌手术在兔膝关节制造两个软骨缺损。A组（n = 4）不接受任何治疗，B组（n = 4）用C/GP凝胶和基质胶构建的Ad-mock-GFP转染软骨细胞治疗，C组（n = 4）用C/GP凝胶和基质胶构建的Ad-hBMP7-GFP转染软骨细胞治疗。移植后4周处死兔子，采用Wakitani评分法测量修复效果。基于RT-PCR和蛋白质印迹结果，hBMP7在Ad-hBMP7-GFP转染的软骨细胞中高效过表达。ELISA结果显示，Ad-hBMP7-GFP转染软骨细胞中Ⅱ型胶原蛋白和透明质酸的产量显著高于Ad-mock-GFP转染软骨细胞。通过H&E染色和扫描显微镜评估，软骨细胞在基质胶支架中的形态和排列比在C/GP中更好。根据Wakitani评分，基质胶联合Ad-hBMP7-GFP转染软骨细胞成功促进了兔膝关节软骨缺损的修复。
