Abstract
Summary
The findings demonstrate that culture expanded human mesenchymal stem cells (MSCs) incorporated and proliferated in clinically relevant cell scaffolds better than freshly isolated bone marrow mononucleated cells (MNCs); in fact, only in MSC cultures were cells present for longer term chondrogenic inductions.
Introduction
The treatment of chondral defects poses a significant clinical problem and a variety of cell sources and techniques have been studied and practiced to regenerate cartilage. Preclinical and clinical evidence suggests that MSCs can help regenerate cartilage when transplanted into cartilage lesions. However, the uptake of MSCs for cell therapies is limited due to the need for their culture expansion to generate subsequent numbers for transplantation. An alternative is to use minimally manipulated MNCs, which avoids the costs and regulatory implications of culture expansion and would enable the treatment of cartilage defects in a one-step procedure. Therefore, this study has focused on comparing these two cell types within three different scaffolds that can currently be used as cell delivery systems.
Methods
Culture expanded human MSCs and MNCs freshly isolated from bone marrow were seeded at a density of 50,000 cells in 3mm2 scaffolds of Chondro-Gide® (type I/III collagen), Alpha Chondro Shield® (polyglycolic acid) and Hyalofast™ (hyaluronic acid). The cell-seeded scaffolds were incubated for 2 hours to permit initial cell adhesion and then treated with or without chondrogenic inducers (100nM dexamethasone, 10ng/ml TGF-β1, 37.5µg/ml ascorbic acid and ITS-X in DMEM/10% serum) for 28 days at 37°C. The Cell incorporation, growth and viability was assessed using Live/Dead staining and confocal microscopy, along with histological stains of the sectioned scaffolds. Proteoglycan synthesis was measured using DMMB assay of glycosaminoglycan (GAG) into the harvested culture medium.
Results
MSCs adhered to the scaffolds to a much greater extent than the MNCs. In fact, the low number of MNCs initially incorporated into the scaffolds diminished over time such that no viable MNCs were seen during long term cultures and in all cases. MSCs incorporated into the Chondro-Gide® scaffold better than into the Alpha Chondro Shield® or Hyalofast™, and during long term cultures the MSCs in Chondro-Gide® proliferated to become significantly greater in number than those in the other two scaffolds. There was no clear matrix deposition. However, the MSCs in Hyalofast™ were rounded in shape, which is consistent with the morphology of chondrocytes, in the presence of chondrogenic inducers only. Furthermore, a significantly greater level of GAG was detected in the medium harvested from Chondro-Gide® and Hyalofast™ cultures under chondrogenic conditions compared with non chondrogenic conditions.
Discussion/Conclusion
This study has shown that human MSCs incorporated, adhered and proliferated better in clinically utilised cell scaffolds compared to MNCs, enabling the induction of chondrogenesis in the longer term. Freshly isolated MNCs from bone marrow contain only 0.01–0.001% of MSCs in addition to non-adherent cell types, e.g. hematopoietic cells, which may account for their low cellular incorporation and decreased cell proliferation in the scaffolds. This outcome for MNCs may be improved using prospective MSC isolation techniques, where in vivo studies are also required to properly examine the chondrogenic potential. Nonetheless, our initial work suggests that culture expanded MSCs are a better option than minimally manipulated cells for cartilage repair.
