背景:半月板部分切除术是半月板损伤最常见的手术策略之一,但有时候,患者抱怨膝盖疼痛,由于在消融室超负荷。在这些情况下,可植入组织工程支架。目前,两个商业脚手架,基于胶原蛋白或聚己内酯-聚氨酯(PCL-PU),可用于弯月形脚手架。在短期后续评估中,均显示临床改善和组织形成。然而,在PCL-PU中进行的长期研究表明,新组织体积减少并呈现不规则形状.此外,在某些情况下,脚手架被完全重新吸收,没有新的组织形成。间充质干细胞(MSC)与支架结合,由于其多能性和自我更新性,可以代表治疗半月板缺损的有希望的方法。在这项工作中,我们的目的是比较MSCs和软骨细胞在PCL-PU支架上的体外行为。MSCs表达与纤连蛋白(FN)结合的整合素,因此,我们还研究了FN涂层对支架生物活性的影响。
方法:我们从两只骨骼成熟的新西兰白兔中分离兔骨髓间充质干细胞(rBM-MSCs),并建立了最佳培养条件以扩增它们。然后,将它们接种在未涂覆和FN涂覆的支架上,并在软骨形成条件下培养。要评估单元格功能,我们进行了MTS分析以比较两种条件下的细胞增殖.最后,进行了组织学研究以评估两种样品中的细胞外基质(ECM)产生,并将它们与接种在非涂层支架中的兔软骨细胞(rCH)获得的细胞进行比较。
结果:将基于低FBS浓度的培养方案设定为rBM-MSC扩增的最佳方案。MTS测定揭示,与接种在未涂覆的支架上的rBM-MSC相比,接种在FN涂覆的支架上的rBM-MSC具有更多的细胞增殖(145%;95%CI:107%-182%)。最后,组织学研究表明,接种在非涂层支架上的rCHs显示出最高的ECM产量,随后是接种在FN涂覆的支架上的rBM-MSC。此外,两种细胞类型产生了相当的ECM模式。
结论:这些结果表明,MSCs对PCL-PU支架的附着能力较低,但是MSCs中整合素α5β1(FN受体)的存在使它们能够与FN涂层的支架相互作用。这些结果可以应用于支架的设计,并可能在半月板损伤的骨科手术中具有重要的临床意义。
BACKGROUND: Partial meniscectomy is one of the most common surgical strategy for a meniscal injury, but sometimes, patients complain of knee pain due to an overload in the ablated compartment. In these cases, implantation of tissue engineering scaffold could be indicated. Currently, two commercial scaffolds, based on collagen or polycaprolactone-polyurethane (PCL-PU), are available for meniscus scaffolding. In short term follow-up assessments, both showed clinical improvement and tissue formation. However, long-term studies carried out in PCL-PU showed that the new tissue decreased in volume and assumed an irregular shape. Moreover, in some cases, the scaffold was totally reabsorbed, without new tissue formation.Mesenchymal stem cells (MSCs) combined with scaffolds could represents a promising approach for treating meniscal defects because of their multipotency and self-renewal. In this work, we aimed to compare the behaviour of MSCs and chondrocytes on a PCL-PU scaffold in vitro. MSCs express integrins that binds to fibronectin (FN), so we also investigate the effect of a FN coating on the bioactivity of the scaffold.
METHODS: We isolated rabbit bone marrow MSCs (rBM-MSCs) from two skeletally mature New Zealand white rabbits and stablished the optimum culture condition to expand them. Then, they were seeded over non-coated and FN-coated scaffolds and cultured in chondrogenic conditions. To evaluate cell functionality, we performed an MTS assay to compare cell proliferation between both conditions. Finally, a histologic study was performed to assess extracellular matrix (ECM) production in both samples, and to compare them with the ones obtained with rabbit chondrocytes (rCHs) seeded in a non-coated scaffold.
RESULTS: A culture protocol based on low FBS concentration was set as the best for rBM-MSCs expansion. The MTS assay revealed that rBM-MSCs seeded on FN-coated scaffolds have more cells on proliferation (145%; 95% CI: 107%-182%) compared with rBM-MSCs seeded on non-coated scaffolds. Finally, the histologic study demonstrated that rCHs seeded on non-coated scaffolds displayed the highest production of ECM, followed by rBM-MSCs seeded on FN-coated scaffolds. Furthermore, both cell types produced a comparable ECM pattern.
CONCLUSIONS: These results suggest that MSCs have low capacity attachment to PCL-PU scaffolds, but the presence of integrin alpha5beta1 (FN-receptor) in MSCs allows them to interact with the FN-coated scaffolds. These results could be applied in the design of scaffolds, and might have important clinical implications in orthopaedic surgery of meniscal injuries.