Prostorová proliferace buněk nucleus pulposus na kultivačních podmínkách

Abstract

Autologous disc chondrocyte transplantation (ADCT) is the only currently available medical treatment which aims to regenerate diseased intervertebral discs. Reaching appropriate number of cells fast enough as well as retention of right phenotype are main limitations of ADCT. Objective of this study was to investigate effect of FGF-2 and TGF-?3 growth factors on nucleus pulposus (NP) cells in ADCT. We investigated the effect of expansion condition on bovine NP cells cultured in alginate beads. Expansion included four different conditions varying presence of oxygen and FGF-2. After NP cells were encapsulated in 1.5% alginate hydrogel beads, they were cultured in hypoxic conditions for 21 days with and without presence of TGF-?3, while portion of cells were initially encapsulated without expansion to act as control group. Additionally, expanded cells of the four groups were used to measure proliferation kinetics over a period of 7 days. Presence of FGF-2 increased the ability of cells to proliferate in 2D culture, showing significant difference between groups expanded in the presence or absence of FGF-2. Presence of TGF-?3 during cultivation in alginate beads induced significantly bigger proliferation in respect to the groups cultivated without TGF-?3. Fresh NP cells showed greater ability to produce sulphated glycosaminoglycans (sGAG) without TGF in respect to the other groups (23.47?1.29 vs 7.82?2.09 ?g/?g of DNA, against best group of expanded cells). Groups cultivated in presence of TGF-?3 showed significant increase in sGAG production in all cases. Results imply that FGF-2 can be beneficial in order to accelerate the expansion rate for ADCT treatments. Proliferation in 3D appears to be promoted by TGF-?3 in all conditions, as well as in the case of fresh cell group. Accumulation of sGAG in alginate beads is improved with addition of TGF-?3 which demonstrates importance of this growth factor on restoration of phenotype of cells in 3D condition.

Autologous disc chondrocyte transplantation (ADCT) is the only currently available medical treatment which aims to regenerate diseased intervertebral discs. Reaching appropriate number of cells fast enough as well as retention of right phenotype are main limitations of ADCT. Objective of this study was to investigate effect of FGF-2 and TGF-?3 growth factors on nucleus pulposus (NP) cells in ADCT. We investigated the effect of expansion condition on bovine NP cells cultured in alginate beads. Expansion included four different conditions varying presence of oxygen and FGF-2. After NP cells were encapsulated in 1.5% alginate hydrogel beads, they were cultured in hypoxic conditions for 21 days with and without presence of TGF-?3, while portion of cells were initially encapsulated without expansion to act as control group. Additionally, expanded cells of the four groups were used to measure proliferation kinetics over a period of 7 days. Presence of FGF-2 increased the ability of cells to proliferate in 2D culture, showing significant difference between groups expanded in the presence or absence of FGF-2. Presence of TGF-?3 during cultivation in alginate beads induced significantly bigger proliferation in respect to the groups cultivated without TGF-?3. Fresh NP cells showed greater ability to produce sulphated glycosaminoglycans (sGAG) without TGF in respect to the other groups (23.47?1.29 vs 7.82?2.09 ?g/?g of DNA, against best group of expanded cells). Groups cultivated in presence of TGF-?3 showed significant increase in sGAG production in all cases. Results imply that FGF-2 can be beneficial in order to accelerate the expansion rate for ADCT treatments. Proliferation in 3D appears to be promoted by TGF-?3 in all conditions, as well as in the case of fresh cell group. Accumulation of sGAG in alginate beads is improved with addition of TGF-?3 which demonstrates importance of this growth factor on restoration of phenotype of cells in 3D condition.