Laboratory of Stem Cell Biology and Orthopaedic Tissue Engineering, Department of Surgery, Division of Orthopaedic Surgery, University of Alberta, Edmonton, AB, T6G 2R3, Canada.
Arthritis Res Ther. 2012 Jun 22;14(3):R153. doi: 10.1186/ar3889.
The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.
Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.
Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.
Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.
本研究的主要目的是确定半月板内、外区（MCO 和 MCI）的半月板细胞与间充质基质干细胞（MSCs）相互作用是否相似或不同。先前的研究表明，与半月板细胞和 MSCs 的单核培养相比，半月板细胞与骨髓来源的 MSCs 的共培养导致基质形成增强。然而，该研究并未检查半月板不同区域的细胞是否与 MSCs 相似或不同地相互作用。
从接受全膝关节置换术的四名患者中采集半月板。来自外区和内区的组织分别取自半月板的三分之一和三分之二的径向距离。胶原酶处理后从半月板中释放半月板细胞。从两名患者的髂嵴获得骨髓 MSCs，经过塑料贴附和体外培养至第 2 代。来自半月板外（MCO）或内（MCI）区的原代半月板细胞分别与 MSCs 在三维（3D）球状体培养物中以 1:3 的比例共培养 3 周，在含有 TGF-β1 的无血清软骨形成培养基中。MCO、MCI 和 MSCs 的单核培养作为实验对照组。在第 3 周后，通过生化、组织化学和定量 RT-PCR 评估形成的组织。
与 MSCs 的共培养增加了内（MCI）或外（MCO）半月板细胞形成的新组织中糖胺聚糖（GAG）基质的含量（高达 2.2 倍），与 MSCs、MCI 和 MCO 的单核培养形成的组织相比。MCI 或 MCO 与 MSCs 的共培养在形成的组织中产生相同量的基质。然而，聚集蛋白聚糖的表达水平在 MSC 的单核培养中最高，但在其他四个组中相似。细胞共培养组织的 DNA 含量与 MSCs、MCI 和 MCO 的单核培养组织无统计学差异。与 MCO 和 MCI 的单核培养相比，细胞共培养中Ⅰ型胶原（COL1A2）mRNA 的表达增加，但与 MSC 的单核培养相比则没有增加。COL2A1 mRNA 的表达在 MCO 和 MCI 与 MSCs 的共培养中显著增加，与各自的对照（MCO 和 MCI 的单核培养）相比，但只有 MCO:MSC 的共培养与 MSC 对照单核培养相比显著增加。COL2A1 蛋白表达通过 COL2A1 免疫组织化学可见增加。所有球状体培养物中 Sox9 的 mRNA 表达水平相似。COL10A1 mRNA 的表达在 MCI:MSC 的共培养中是 MCO:MSC 的共培养的 2 倍。此外，COL10A1 mRNA 的表达在 MCI:MSC 的共培养中是 MCO:MSC 的共培养的 2 倍。另外，其他肥大基因 MMP-13 和 Indian Hedgehog（IHh）的表达分别增加了 4 倍和 18 倍。
与 MSCs 的共培养增加了基质的形成。MCI 和 MCO 与 MSCs 共培养后基质形成相似。然而，与 MCI 相比，MCO 更能抑制 MSCs 的肥大分化。这些发现表明，半月板内、外血管区的半月板细胞可以与 MSCs 一起补充，以构建功能性移植物来重建内血管半月板。
