超音波介導的壓電刺激對軟骨細胞外基質及軟骨組織修復之影響

Abstract

關節軟骨因缺乏血管與神經供應，且細胞密度低，一旦受損後自我修復能力有限，臨床上至今仍缺乏具潛力的非侵入性再生療法。低強度脈衝超音波（Low-Intensity Pulsed Ultrasound, LIPUS）作為一種安全且可穿透組織的物理刺激，近年廣泛應用於促進組織修復與再生。有研究指出，人體多種組織具備壓電特性，而軟骨因富含膠原蛋白，亦被視為具有生物壓電潛力的組織之一。本研究以超音波作用於生物壓電材料而誘發的壓電效應為切入點，探討其調控細胞行為與促進軟骨修復的潛力。 為了更精確模擬LIPUS作用於軟骨組織的微環境，首先於體外建立以具壓電性的石英片作為細胞培養基材之刺激模型，評估超音波介導的壓電刺激對初代軟骨細胞之影響。結果顯示，使用LIPUS刺激軟骨細胞4天後，相較於種植在普通玻璃片的組別(純LIPUS組)，種植在石英片上的軟骨細胞，其SOX9和Collagen II 的表現有更顯著的上升。顯示LIPUS與壓電效應之複合刺激，有更能促進細胞表行穩定與基質合成能力之可能性。 其次進行動物實驗，進一步於小鼠全層膝軟骨缺損模型中評估LIPUS治療成效。其治療參數設定為頻率1 MHz、600 mVpp、占空比5%、聲強度(ISATA)為61.4mW/cm2，每次刺激5分鐘，每週三次（週一、三、五），並於第2、4與6週進行組織取樣與分析。透過組織修復半定量評分與免疫螢光染色評估修復情形與關鍵蛋白表現，並進一步將軟骨劃分為損傷周圍區域（Peri-lesional region）與整體區域（Entire region）進行空間層級分析，以確認LIPUS對損傷的治療效果。 結果顯示，第4週LIPUS治療組的修復評分顯著高於對照組，且在損傷周圍區域SOX9與Aggrecan表現比例顯著提升，Collagen II亦呈回升趨勢；整體區域呈相似表現。至第6週，LIPUS治療組於損傷周圍區域觀察到Collagen X表現下降，顯示病理性肥大分化受到抑制，整體組織修復品質獲得改善。 綜合細胞與動物實驗結果，本研究顯示超音波介導的壓電效應能優化超音波調節軟骨細胞的能力，促進軟骨細胞SOX9與基質蛋白的表現，並有效抑制病理性肥大變化。區域間的表現差異進一步突顯LIPUS對損傷區域修復潛力，為未來非侵入性軟骨再生療法之發展提供潛在機轉與應用依據。然而，壓電效應所涉之細胞內分子傳遞機制(Cell Signaling Pathway)仍有待後續研究進一步釐清。

Articular cartilage has limited self-repair capacity due to its lack of vasculature and innervation, as well as its low cell density. Clinically, there is still a lack of promising non-invasive regenerative therapies. Low-Intensity Pulsed Ultrasound (LIPUS), a safe and tissue-penetrating physical stimulus, has been widely applied in recent years to promote tissue repair and regeneration. Studies have shown that various human tissues possess piezoelectric properties. Cartilage, rich in collagen, is also considered a biologically piezoelectric tissue. This study explores the potential of LIPUS-induced piezoelectric effects to modulate cell behavior and enhance cartilage repair. To better simulate the microenvironment of LIPUS acting on cartilage, an in vitro stimulation model was first established using a piezoelectric quartz substrate for cell culture. The effects of ultrasound-mediated piezoelectric stimulation on primary chondrocytes were evaluated. After 4 consecutive days of stimulation, the expression levels of SOX9 and Collagen II were significantly increased, showing a clear difference compared to the group treated with LIPUS alone. This suggests that piezoelectric effects may enhance the ability of LIPUS to promote chondrocyte phenotype stability and extracellular matrix (ECM) synthesis. Subsequently, the therapeutic effects of LIPUS were evaluated in a full-thickness cartilage defect model in the mouse knee joint. The treatment was applied at an intensity of 61.4 mW/cm² for 5 minutes per session, three times per week. Tissue samples were collected at weeks 2, 4, and 6 for histological and molecular analyses. Cartilage repair was assessed using semi-quantitative scoring and immunofluorescence staining for key markers. Spatial analysis was further performed by dividing cartilage into the peri-lesional region and the entire region to confirm the localized effect of LIPUS. The results showed that by week 4, the LIPUS-treated group exhibited significantly higher repair scores compared to the control group. SOX9 and Aggrecan expression levels were significantly elevated in the peri-lesional region, with Collagen II also showing an increasing trend. Similar patterns were observed in the entire region. By week 6, a reduction in Collagen X expression was observed in the peri-lesional region of the LIPUS group, suggesting suppression of pathological hypertrophic differentiation and improved overall tissue repair quality. In summary, results from both in vitro and in vivo experiments demonstrate that ultrasound-mediated piezoelectric stimulation may regulate chondrocyte behavior, promoting phenotype stability and ECM synthesis while effectively suppressing pathological hypertrophy. The regional differences in expression further highlight the potential of LIPUS to enhance cartilage repair at the injury site, providing mechanistic insight and application potential for future non-invasive cartilage regeneration therapies. However, the exact cellular signaling pathways involved in the piezoelectric effects remain to be elucidated in future studies.