Abstract
Introduction
Degeneration of the nucleus pulposus (NP), part of the intervertebral disk (IVD), is a major reason for back pain. Stem cell therapy is one approach for treatment. Therefore, it is necessary to differentiate the human mesenchymal stem cells (hMSC) into NP-cells. Differentiation requires a 3D environment in a biocompatible matrix in which the cells were embedded. Beside biocompatibility, pressure resistance is a necessary requirement on the matrices. In vivo the IVD is exposed to pressures up to 3 MPa (30 bar). With regard to minimal invasive treatment of patients later on, the matrices have to be injectable and solidify after injection. In this work, different compositions of potential matrices for the regeneration of the NP were analyzed. hMSC-TERT (- telomerase reverse transcriptase) have been embedded in gelatin and a “gelatin-porcine nucleus pulposus” matrix. The enzyme transglutaminase was found used for cross-linking of the matrices. The results were compared with work previously done on agarose matrix.
Materials and Methods
Cells were seeded in different matrices at a density of 4 × 106 cells per cm3, equal to the density in the NP. The immobilized cells were cultivated for 21 days in media, containing growth and differentiation factors. Viability of the cells in the different matrices was proofed by using a tetrazolium salt (WST-1). At certain times, RNA isolation was done following the phenol/guanidine isothiocyanate protocol. RNA was transformed into cDNA followed by a RT-PCR. Different primers were used to analyze the differentiation of hMSC-TERT.
Results
Via WST1-test the survival of hMSC-TERT in gelatine was proofed. The differentiation status of hMSC-TERT in the two different matrices (gelatine, NP extract) was analyzed. The PCR analysis showed a differentiation in a chondrogenic type, less with the NP extract. In agarose a differentiation in a NP cell type could be obtained.
Conclusion
The results lead to the conclusion that the influence of matrices on cell differentiation is greater than recently surmised. Therefore, it is necessary to find a “close to nature”- matrix for the need in cell therapy.
Yes
None declared