研究のタイプ: 医学/生物学の研究 (experimental study)

[軟骨組織工学のための軟骨3D構造に対する超低周波、低エネルギーの電磁界および機械的刺激の組み合せの影響] med./bio.

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

掲載誌: Bioelectromagnetics 2014; 35 (2): 116-128

この研究は、機械的ストレス電磁界ストレスの組み合わせがインビトロ軟骨細胞に与える影響を調べた。ウシの球節関節から採取した関節軟骨細胞初代培養株を3次元(3D)のポリウレタン足場材の中に播き、それを用いて7種類の刺激実験を行った。機械的刺激単独群、3つの磁界刺激単独群(1mT、2 mT、3mT:60 Hz)、3つの組み合わせばく露群である。機械的刺激には関節専用に製作された機械的負荷バイオリアクタ、電磁界刺激にはヘルムホルツコイルをそれぞれ培養器内で用いた。足場材内での細胞増殖グリコサミノグリカン(GAG)量およびDNA量、組織学遺伝子発現(collagen-1、collagen-2、aggrecan、血清軟骨オリゴメリック・ マトリクス・プロテイン、Sox9、roteoglycan-4、マトリクスメタロプロテイナーゼ(MMP-3、-13)など)で分析した。その結果、各刺激群と対照群との間でGAG/DNA 量に有意差があり、3mTの組み合わせばく露群で最大であった;遺伝子発現については、組み合わせばく露群で、対照群に比べ、collagen 2/ collagen 1の比が高くなり、MMP-13が低くなった;その他の遺伝子では、軟骨形成との関係が強い表現型発現する傾向刺激群で見られた、と報告している。

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研究目的(著者による)

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.

詳細情報

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.

影響評価項目

ばく露

ばく露 パラメータ
ばく露1: 60 Hz
ばく露時間: intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
ばく露2: 60 Hz
ばく露時間: intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
ばく露3: 60 Hz
ばく露時間: 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

ばく露1

主たる特性
周波数 60 Hz
タイプ
  • magnetic field
波形
  • sinusoidal
ばく露時間 intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
ばく露装置
ばく露の発生源/構造
チャンバの詳細 bioreactors covered with sterile glass bodies were placed in an incubator at 37°C, 95% air, 5% CO2 and 95% humidity
ばく露装置の詳細 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 information 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
パラメータ
測定量 種別 Method Mass 備考
磁束密度 1 mT - 測定値 - -

ばく露2

主たる特性
周波数 60 Hz
タイプ
  • magnetic field
波形
  • sinusoidal
ばく露時間 intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
ばく露装置
ばく露の発生源/構造
  • E1と同じ装置
パラメータ
測定量 種別 Method Mass 備考
磁束密度 2 mT - 測定値 - -

ばく露3

主たる特性
周波数 60 Hz
タイプ
  • magnetic field
波形
  • sinusoidal
ばく露時間 intermittent exposure for 3 h/day every other day for 2 weeks (a total of 24 hours)
ばく露装置
ばく露の発生源/構造
  • E1と同じ装置
パラメータ
測定量 種別 Method Mass 備考
磁束密度 3 mT - 測定値 - -

ばく露を受けた生物:

方法 影響評価項目/測定パラメータ/方法

研究対象とした生物試料:
調査の時期:
  • ばく露前
  • ばく露後

研究の主なアウトカム(著者による)

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.

研究の種別:

研究助成

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