Effects of Mechanical Stimulation and Scaffolds for Nucleus Pulposus Replacement of Intervertebral Disc / 대한정형외과연구학회지

PURPOSE: In this study, we investigated the potential of injectable hydrogel scaffolds for the regeneration of nucleus pulposus. MATERIALS AND METHODS: We prepared injectable hydrogels [Chitosan-Pluronic (CP), CP/Osteogenic Protein-1 (CP/OP-1), CP/Gly-Arg-Gly-Asp-Ser (CP/GRGDS), CP/GRGDS/OP-1] for this study. One of the four potential materials was selected through the cell viability tests. For each material, primary cultured nucleus pulposus (NP) cells from New Zealand rabbits were seeded onto each material. For the investigation of the effects of mechanical stimulation, the commercially available bioreactor was used. 0.2 MPa of intermittent hydrostatic pressure was imposed for 3 days after 7th day of seeding with the pattern of 2 min and 15 min for stimulating and resting, respectively. The specimens were harvested at 1, 10, 14 day after seeding for analyses. RESULTS: The MTT assay for 5 days revealed that CP/OP-1 group showed significant increase. The other two groups (CP/GRGDS and CP/GRGDS/OP-1) showed that the proliferation rate increased until 3 days after culture, while it decreased on day 5. The mechanical stimuli induced higher amounts of DNA measured in CP/OP- 1 on day 5 after culture. However, no significant difference was observed between two groups. CONCLUSION: We came to the conclusions that the biochemical environment as well as mechanical stimulation may play an important role in regenerating nucleus pulposus matrix, especially in CP/OP-1 in this study. However, further study are recommended in relation to mechanical effects as well as biochemical conditions.

Phenotypical Stability and Matrix Synthesis of Human Intervertebral Disc Cells in Response to Dexamethasone and Transforming Growth Factor-beta1 / 대한척추외과학회지

STUDY DESIGN: An in vitro experiment. OBJECTIVES: To evaluate the mRNA expressions of matrix components, and analyze the cellular proliferation and proteoglycan synthesis of human intervertebral disc cells in response to dexamethasone and TGF-beta1 SUMMARY OF LITERATURE REVIEW: Corticosteroids are responsible for the regulation of a diverse range of biological processes through modulation of the expression of target genes. The direct injection of methylprednisolone to the intervertebral disc (IVD) has been shown to cause degeneration and calcification of the disc in rabbits. Systemic administration of hydrocortisone induced degeneration of notochordal cells, which accelerated the aging process of the disc in mice. Transforming growth factor beta-1 (TGF-beta1) is known as a potent agent for the proliferation, differentiation and matrix synthesis of IVD. MATERIALS AND METHODS: IVD cells were isolated from ten patients, and subsequently cultured. Various doses of dexamethasone (DEX) and/or TGF-beta1 were administered to the IVD cultures. DNA and proteoglycan syntheses were measured by the incorporation of [3H]-thymidine and [35S]-sulfate, respectively. RT-PCRs were performed for the expressions of aggrecan, collagen types I and II, and osteocalcin mRNA. RESULTS: Cultures with DEX showed increased cellular proliferation and decreased proteoglycan synthesis (p<0.05). TGF-beta1 potentiated the proliferative effect of DEX, but failed to stimulate proteoglycan synthesis in the cultures containing DEX. There were no recognizable changes in the mRNA expressions of aggrecan, collagen types I and II, and osteocalcin in response to DEX and TGF-beta1. CONCLUSIONS: DEX demonstrated a proliferative effect on human IVD cells, with the combination of DEX and TGF-beta1 showing potentiation of the proliferative effect, while at high doses(100 and 1000nM, the DEX was shown to down-regulate the proteoglycan synthesis. Caution should be exercised in the use of corticosteroid in the therapeutic approaches for the treatment of disc disease or in the regenerative matrix of the IVD.

The Effect of Pulsed Electromagnetic Field in Human Intervertebral Disc Cell / 대한척추외과학회지

STUDY DESIGN: An in vitro experiment. OBJECTIVES: To assess the effect of pulsed sinusoidal EMF on human intervertebral disc (IVD) cells. LITERATURE REVIEW SUMMARY: Electromagnetic field (EMF) is known to modify some relevant physiological parameters of cells cultured in vitro, such as proliferation, synthesis, secretion of growth factors and transcription. EMF induces bone formation in delayed, non union and spinal fusion models. Also, the exposure of EMF has been shown to protect against the hazardous effect of smoking in the rabbit IVD. MATERIALS AND METHODS: Human IVD cells were three-dimensionally cultured in alginate beads and exposed to a 650 omega, 1.8millitesla magnetic flux density, 60Hz sinusoidal wave of EMF. The cultures were divided into the control and EMF groups, with various exposure times. The cytotoxicity, and DNA and proteoglycan syntheses were measured by the MTT assay, and [3H]-thymidine and [35S]-sulfate incorporation, respectively. RT-PCRs were performed for aggrecan, and collagen types I and II mRNA expressions. RESULTS: There was no recognizable cytotoxicity in the EMF group, but cellular proliferation was stimulated (p<0.05). Newly synthesized proteoglycan, normalized by DNA synthesis, was decreased in the EMF group (p<0.05) as were the expressions of aggrecan (48hour exposure) and type II collagen (72 hours exposure) mRNA compared to the control group. CONCLUSIONS: EMF seems to be hazardous in the synthesis of the chondrogenic matrix, while marginally beneficial in the cellular proliferation of human IVD cells.

The Effect of Pulsed Electromagnetic Field in Human Intervertebral Disc Cell / 대한척추외과학회지

STUDY DESIGN: An in vitro experiment. OBJECTIVES: To assess the effect of pulsed sinusoidal EMF on human intervertebral disc (IVD) cells. LITERATURE REVIEW SUMMARY: Electromagnetic field (EMF) is known to modify some relevant physiological parameters of cells cultured in vitro, such as proliferation, synthesis, secretion of growth factors and transcription. EMF induces bone formation in delayed, non union and spinal fusion models. Also, the exposure of EMF has been shown to protect against the hazardous effect of smoking in the rabbit IVD. MATERIALS AND METHODS: Human IVD cells were three-dimensionally cultured in alginate beads and exposed to a 650 omega, 1.8millitesla magnetic flux density, 60Hz sinusoidal wave of EMF. The cultures were divided into the control and EMF groups, with various exposure times. The cytotoxicity, and DNA and proteoglycan syntheses were measured by the MTT assay, and [3H]-thymidine and [35S]-sulfate incorporation, respectively. RT-PCRs were performed for aggrecan, and collagen types I and II mRNA expressions. RESULTS: There was no recognizable cytotoxicity in the EMF group, but cellular proliferation was stimulated (p<0.05). Newly synthesized proteoglycan, normalized by DNA synthesis, was decreased in the EMF group (p<0.05) as were the expressions of aggrecan (48hour exposure) and type II collagen (72 hours exposure) mRNA compared to the control group. CONCLUSIONS: EMF seems to be hazardous in the synthesis of the chondrogenic matrix, while marginally beneficial in the cellular proliferation of human IVD cells.