Potential of Centrifugal Seeding Method in Improving Cells Distribution and Proliferation on Demineralized Cancellous Bone Scaffolds for Tissue-Engineered Meniscus

摘要

Tissue-engineered meniscus offers a possible solution to the regeneration and replacement problem of meniscectomy. However, the nonuniform distribution and declined proliferation of seeded cells on scaffolds hinder the application of tissue-engineered meniscus as a new generation of meniscus graft. This study systematically investigated the performances of different seeding techniques by using the demineralized cancellous bone (DCB) as the scaffold. Static seeding, injection seeding, centrifugal seeding, and vacuum seeding methods were used to seed the meniscal fibrochondrocytes (MFCs) and mesenchymal stem cells (MSCs) to scaffolds. Cell-binding efficiency, survival rate, distribution ability, and long-term proliferation effects on scaffolds were quantitatively evaluated. Cell adhesion was compared via cell-binding kinetics. Cell viability and morphology were assessed by using fluorescence staining. Combined with the reconstructed three-dimensional image, the distribution of seeded cells was investigated. The Cell Counting Kit-8 assay and DNA assay were employed to assess cell proliferation. Cell-binding kinetics and cell survival of the MFCs were improved via centrifugal seeding compared to injection or vacuum seeding methods. Seeded MFCs by centrifugation showed a more homogeneous distribution throughout the scaffold than cells seeded by other methods. Moreover, the penetration depth in the scaffold of seeded MFCs by centrifugation was 300-500 mu m, much higher than the value of 100-300 mu m by the surface static and injection seeding. The long-term proliferation of the MFCs in the centrifugal group was also significantly higher than that in the other groups. The results of the MSCs were similar to those of the MFCs. The centrifugal seeding method could significantly improve MFCs or MSCs distribution and proliferation on the DCB scaffolds, thus providing a simple, cost-effective, and effective cell-seeding protocol for tissue-engineered meniscus.