The effects of exposure of primary bovine chondrocytes to a 60 Hz magnetic field alone and in combination with mechanical stimulation on gene expression and extracellular matrix production should be investigated in view of a therapeutic use.
Articular cartilage has a very low regenerative potential and tissue engineering seems to be a promising therapeutic tool in cartilage repair. Seeding of chondrocytes on 3-D matrices (in this study, a combined hydrogel polyurethane scaffold), mechanical stimulation (compression and shear forces) and EMF exposure have shown to have a positive effect on in vitro chondrogenesis and cartilage maturation. However, a combination of these factors had not been tested before.
Cells were seeded on matrices and divided into the following groups after 7 days: exposure to a magnetic field of 1) 1 mT, 2) 2 mT, 3) 3 mT), 4) mechanical stimulation, 5) co-exposure to a 1 mT MF and mechanical stimulation, 6) co-exposure to a 2 mT MF and mechanical stimulation, 7) co-exposure to a 3 mT MF and mechanical stimulation, 8) control group.
Four independent experiments with cells derived from different calves were performed. All scaffolds were cut into equal halves for later analysis, resulting in a total of 24 scaffold halves per group. Nine of those halves were used for gene expression analysis, 8 for biochemical analysis and 7 for histological analysis.
| Exposure | Parameters |
|---|---|
|
Exposure 1:
60 Hz
Exposure duration:
intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
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Exposure 2:
60 Hz
Exposure duration:
intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
|
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|
Exposure 3:
60 Hz
Exposure duration:
intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
|
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In co-exposure groups, mechanical stimulation and MF exposure were conducted on alternating days; in exclusive MF exposure and mechanical stimulation groups, respective treatments were conducted every other day; on respective days, mechanical stimulation was conducted for 1 h twice a day with 8 h rest in between
| Frequency | 60 Hz |
|---|---|
| Type | |
| Waveform | |
| Exposure duration | intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours) |
| Exposure source |
|
|---|---|
| Chamber | bioreactors covered with sterile glass bodies were placed in an incubator at 37°C, 95% air, 5% CO2 and 95% humidity |
| Setup | exposure system (similar to a Helmholtz system) consisted of 2 coils of 120 turns each and 12 cm diameter; distance between the coils was 8 cm. Both coils were wrapped onto a non-magnetic body entirely made of polyethylene together with the cooling system consisting of turns of a polysiloxane tube connected to a water bath in which water at 37°C was circulating to guarantee a constant temperature inside the incubator; between exposure cycles, cells remained outside the coil system |
| Additional info | there were three incubators, one dedicated to MF exposure, one containing the mechanical stimulation system and one used to keep the cells between stimulation/exposure cycles |
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| magnetic flux density | 1 mT | - | measured | - | - |
| Frequency | 60 Hz |
|---|---|
| Type | |
| Waveform | |
| Exposure duration | intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours) |
| Exposure source |
|
|---|
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| magnetic flux density | 2 mT | - | measured | - | - |
| Frequency | 60 Hz |
|---|---|
| Type | |
| Waveform | |
| Exposure duration | intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours) |
| Exposure source |
|
|---|
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| magnetic flux density | 3 mT | - | measured | - | - |
Gene expression was significantly different between all co-exposure groups (groups 5-7) and the control group, with a significantly increased collagen 2/collagen 1 ratio and a significantly decreased metalloproteinase-13 gene expression. There was no statistical significant difference for other genes, however, co-exposure groups also showed a non-significant tendency towards higher gene expression of proteoglycan-4 and a lower gene expression of metalloproteinase-3 compared to the control group.
The extracellular matrix production was significantly increased in all treatment groups (groups 1-7) compared to the control group, with the highest value in the 3 mT co-exposure group (group 7).
Immunohistochemistry showed a tendency (no significance specifications) towards decreased collagen 1 and increased collagen 2 and aggrecan levels in all treatment groups compared to the control group. Co-exposure groups showed the largest differences to the control group with supreme values in the 3 mT co-exposure group.
The authors conclude that exposure of primary bovine chondrocytes to a 60 Hz magnetic field in combination with mechanical stimulation might improve the extracellular matrix production. Exposure to the magnetic field alone showed only a slight and non-significant positive effect.
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