Study type: Medical/biological study (experimental study)

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering. med./bio.

Published in: Bioelectromagnetics 2014; 35 (2): 116-128

Aim of study (acc. to author)

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.

Background/further details

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.

Endpoint

Exposure

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)
Exposure 2: 60 Hz
Exposure duration: intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
Exposure 3: 60 Hz
Exposure duration: intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)

General information

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

Exposure 1

Main characteristics
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 setup
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
Parameters
Measurand Value Type Method Mass Remarks
magnetic flux density 1 mT - measured - -

Exposure 2

Main characteristics
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 setup
Exposure source
Parameters
Measurand Value Type Method Mass Remarks
magnetic flux density 2 mT - measured - -

Exposure 3

Main characteristics
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 setup
Exposure source
Parameters
Measurand Value Type Method Mass Remarks
magnetic flux density 3 mT - measured - -

Exposed system:

Methods Endpoint/measurement parameters/methodology

Investigated system:
Time of investigation:
  • before exposure
  • after exposure

Main outcome of study (acc. to author)

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.

Study character:

Study funded by

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