壓電刺激對軟骨組織及軟骨細胞極性的影響

Abstract

壓電刺激可以誘導體外培養的軟骨細胞重新排列 (Lim et al., 2022)。本研究欲針對先前觀察到的現象做進一步探討。首先，壓電刺激是否也能影響小鼠關節軟骨細胞？其次，壓電刺激能否促進小鼠軟骨損傷的修復？第三，細胞分布是否與1 MHz或3 MHz超音波刺激形成的壓電場分布相關？最後，軟骨細胞是通過電壓門控離子通道還是酸敏感離子通道(ASIC3)調控壓電刺激？ 在小鼠刺激實驗中，我們使用低強度脈衝超音波刺激關節軟骨產生壓電刺激；在細胞實驗中壓電刺激由實驗室開發的超音波刺激裝置mini-LIC產生。軟骨細胞取自崽鼠關節軟骨並種植在石英片上，使用低強度連續超音波在一天內進行四次壓電刺激，每次刺激5分鐘，刺激後一小時進行免疫螢光染色程序。為了研究刺激頻率對細胞分布的影響，本研究比較了1MHz和3MHz超音波刺激下的細胞分布。我們使用電壓門控鈣離子通道抑制劑和電壓門控鉀離子通道抑制劑，以及ASIC3+/+與ASIC3-/-崽鼠軟骨細胞進行通道測試實驗。 結果顯示，壓電刺激能夠誘導體內和體外培養之軟骨細胞分布改變，並加速關節軟骨損傷的恢復，且細胞分布間距與超音波頻率成反比，壓電刺激後初級纖毛方向改變，在抑制電壓門控鈣離子通道和電壓門控鉀離子通道後，壓電刺激對纖毛的影響受到抑制，而在ASIC3-/-的崽鼠軟骨細胞中仍然有觀察到壓電刺激對纖毛的改變。 壓電刺激同時改變纖毛方向與影響細胞分布，電壓門控鈣離子通道和電壓門控鉀離子通道參與調控纖毛的改變。本研究的發現可能對組織工程和再生醫學具有重要意義。

Piezoelectric stimulation can induce chondrocyte cell rearrangement (Lim et al., 2022). This study aims to answer several questions arising from these previous observations. First, can cell rearrangement also be observed in mouse articular cartilage upon piezoelectric stimulation? Second, does piezoelectric stimulation facilitate the repair of cartilage injury? Third, are cell distributions strictly correlated to the nodes of piezoelectrical field upon either 1 MHz or 3 MHz ultrasound stimulation? Finally, is the piezoelectric stimulation transduced by voltage-gated ion channels or acid-sensing ion channel (ASIC3)? In vivo, we used low-intensity pulsed ultrasound (LIPUS) to generate piezoelectric stimulation in articular cartilage. In vitro, piezoelectric stimulation was provided by mini-LIC, an ultrasound stimulation chamber developed in the laboratory. The stimulation was low-intensity continuous ultrasound (LICUS) and last for 5 minutes. In the experiment, chondrocytes were isolated from the articular cartilage of mice and seeded on quartz coverslips. To investigate the differences influenced by the frequency of stimulation, we compared the distances between the cell distributions formed under 3MHz and 1MHz ultrasound stimulation. We tested ASIC3 by using knockout cells. We tested voltage-gated calcium channel (CaV3.2) and voltage-gated potassium channel (KV1.6) by using chemical inhibitors to block these channels. Results showed that the stimulation could influence cell distribution both in vivo and in vitro. It could also accelerate the recovery of injuries made in articular cartilage. We discovered that the distances between the cell distributions were inversely correlated with the ultrasound frequencies. The stimulation may be mediated by CaV3.2 and KV1.6, but not by ASIC3. Piezoelectric stimulation changes the orientation of cilia and cell distribution, which may impact on chondrocyte proliferation and regeneration. This process is possibly mediated via VGCC and KV. This finding may have implications for tissue engineering and regenerative medicine.