Mesenchymal stem cell isolation and characterization from ligamenta flava, interspinous ligaments and facet joints

Abstract

Mesenchymal stem cells (MSCs) are among the most prominent adult progenitor cells and are interesting for their potential in tissue engineering applications as well as their involvement in various diseases. Hypertrophy and calcifications of spinal ligaments are progressive conditions were MSCs are believed to play a role and the existence of MSCs has been demonstrated within the ligamentum flavum (LF) and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within the interspinous ligaments (ILs) and facet joints (FJs), if they were comparable to LF-derived MSCs and wherein these three tissues cells were localized. Samples were harvested from 10 patients undergoing spinal surgery and cells isolated using direct tissue explant technique. Surface antigen profiling for CD29, CD34, CD44, CD45, CD73, CD90 and CD105 was performed via flow cytometry and to evaluate tri-lineage potential cells were grown in osteogenic, adipogenic and chondrogenic induction medium for 21 days. Histological staining on tissue samples were performed to visualize wherein the tissue cells were localized. Flow cytometry analysis revealed both cell lines expressed the MSCs markers CD29, CD44, CD73, CD90 and CD105 and were negative for the haematopoietic markers CD34 and CD45. All cell lines were capable of osteogenic, adipogenic and chondrogenic differentiation confirmed with histochemical staining of calcium (Alizarin Red S stain), lipids (Oil Red O stain) and polysaccharides (Toluidine blue and Alcian Blue stain). RT-PCR gene expression analysis also confirmed the expression of differentiation related markers. Moreover, a very strong osteogenic response was observed from individual colonies subjected to induction media. Vascular cell rich areas surrounded the avascular dense collagenous areas in LF, FJs and ILs tissues. To the best of our knowledge, this study is the first to demonstrate that MSCs can be found within both the interspinous ligaments and facet joints. Our findings show that they are positive of MSCs markers, capable of tri-lineage differentiation and show a strong osteogenic response suggesting that they might be directly involved in the calcifications of soft tissues in the vertebral column.