Zhang Zi-Qi, Wang Chun-Sheng, Yang Pei, Wang Kun-Zheng
Department of Orthopaedic Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Orthop Surg. 2018 Nov;10(4):328-336. doi: 10.1111/os.12411.
To evaluate the therapeutic effects of mesenchymal stem cells induced by microencapsulated chondrocytes on repairing of intervertebral disc degeneration.
Rabbit chondrocytes and bone marrow-derived mesenchymal stem cells (MSC) were derived. Chondrocytes were microencapsulated by a microcapsule generator to produce microencapsulated chondrocytes (MEC). MSC were then co-cultured with MEC (MSC-MEC) and the properties and the therapeutic effects on repairing of intervertebral disc degeneration were studied. For the in vitro study, cell proliferation, type II collagen, and glycosaminoglycan (GAG) were studied. The MSC induced by chondrocytes in the Transwell system (MSC-MLC) and pure MSC were used as the control group. For the in vivo studied, MSC-MEC were implanted into the intervertebral disc degenerated (IDD) models, and the radiological images, biomechanical properties, collagen II, and histology of the discs were studied. The IDD, MSC, and MSC-MLC groups were used as the control group.
In the in vitro study, no significant differences were found among the three groups, indicating that the microcapsule co-culture system will not affect the proliferation of MSC. The type II collagen quantity secreted by MSC-MEC was 23.57 ± 2.46 ng/μL, which was more than for MSC-MLC (15.14 ± 2.31 ng/μL) and MSC groups (4.17 ± 1.23 ng/μL, all P < 0.025). GAG secreted by MSC-MEC was 0.184 ± 0.006 mg/well, which was more than for the MSC-MLC (0.151 ± 0.011 mg/well) and MSC groups (0.023 ± 0.002 mg/well, all P < 0.025). In the in vivo study, no obvious degenerative or protrusive disc was found in the MSC-MEC group, while protrusive discs could be found in the MSC-MLC group, and both degenerative and protrusive discs were found in MSC and IDD groups, which indicated that the reparative effects of MSC-MEC on degenerated discs were better than for the control groups. Biomechanical properties of discs in the MSC-MEC group were maintained at all four time points (2nd, 4th, 8th, and 16th week after implantation). The compressive strength (CS) and the elastic modulus (EM) of MSC and IDD groups were consistently decreased. The CS of the MSC-MLC group was increased in the 4th week but decreased again in the 8th week, while the EM of the MSC-MLC group consistently decreased. Western blot results indicated that discs of the MSC-MEC group had more collagen II, which is an important component of discs. Histology staining showed that the nucleus pulposus of MSC-MEC was complete; no obvious fragment of component loss was found, while those of MSC-MLC, MSC, and IDD groups were widened, broken, and hollow.
The microencapsulation method for half-contact co-culturing improves the differentiation extent of MSC, and MSC induced by chondrocytes could also be used for treatment of IDD.
评估微囊化软骨细胞诱导的间充质干细胞对椎间盘退变修复的治疗效果。
获取兔软骨细胞和骨髓来源的间充质干细胞（MSC）。用微囊发生器对软骨细胞进行微囊化，制备微囊化软骨细胞（MEC）。然后将MSC与MEC共培养（MSC-MEC），研究其特性及对椎间盘退变修复的治疗效果。体外研究中，检测细胞增殖、Ⅱ型胶原蛋白和糖胺聚糖（GAG）。将Transwell系统中软骨细胞诱导的MSC（MSC-MLC）和单纯MSC作为对照组。体内研究中，将MSC-MEC植入椎间盘退变（IDD）模型，研究椎间盘的影像学图像、生物力学特性、Ⅱ型胶原蛋白和组织学情况。将IDD、MSC和MSC-MLC组作为对照组。
体外研究中，三组之间无显著差异，表明微囊共培养系统不影响MSC的增殖。MSC-MEC分泌的Ⅱ型胶原蛋白量为23.57±2.46 ng/μL，高于MSC-MLC组（15.14±2.31 ng/μL）和MSC组（4.17±1.23 ng/μL，均P<0.025）。MSC-MEC分泌的GAG为0.184±0.006 mg/孔，高于MSC-MLC组（0.151±0.011 mg/孔）和MSC组（0.023±0.002 mg/孔，均P<0.025）。体内研究中，MSC-MEC组未发现明显退变或突出的椎间盘，而MSC-MLC组可发现突出的椎间盘，MSC组和IDD组均发现退变和突出的椎间盘，这表明MSC-MEC对退变椎间盘的修复效果优于对照组。MSC-MEC组椎间盘的生物力学特性在植入后的第2、4、8和16周这四个时间点均得以维持。MSC组和IDD组的抗压强度（CS）和弹性模量（EM）持续下降。MSC-MLC组的CS在第4周增加，但在第8周再次下降，而MSC-MLC组的EM持续下降。蛋白质印迹结果表明，MSC-MEC组的椎间盘有更多的Ⅱ型胶原蛋白，这是椎间盘的重要组成部分。组织学染色显示，MSC-MEC组的髓核完整；未发现明显的成分丢失碎片，而MSC-MLC组、MSC组和IDD组的髓核则变宽、破裂和中空。
半接触共培养的微囊化方法提高了MSC的分化程度，软骨细胞诱导的MSC也可用于治疗IDD。
