慢性傷口如褥瘡(Pressure Sore)、下肢潰瘍(Leg Ulcers)、糖尿病足部症候群(Diabetic Foot Syndrome)等是人們亟須解決的問題，雖然目前有許多治療方法，例如抗生素治療、高壓氧療法、負壓療法等，但欠缺一個有效且便宜的解決方法。而近年來治療性超音波被發現對於傷口癒合擁有一定的效果，且可藉由調整不同的強度、頻率等參數去促進或抑制細胞的生長。然而目前對於非接觸式低頻超音波(Noncontact Low-frequency Ultrasound,NLFUS)在慢性傷口癒合上的應用並沒有許多研究探討。因此，為了瞭解NLFUS對於傷口所造成的生長因子改變之生物效應，本研究建構頻率40 kHZ的超音波換能器。利用劃痕分析細胞實驗，使用不同的NLFUS參數如佔空比、強度、照射時間來評估纖維母細胞(3T3) 受外力破壞後接受NLFUS治療後增生的影響。此外，我們建構慢性傷口的糖尿病大鼠動物模型，來評估最有效的NLFUS參數在糖尿病大鼠傷口上的治療效果。同時將大鼠分成五組不同的治療條件，不接受NLFUS治療(對照組)、每天接受一次50 mW/cm2治療(A組)、每天接受50 mW/cm2兩次治療(B組)、一個禮拜接受50 mW/cm2三次治療(C組)、每天接受100 mW/cm2一次治療(D組)以及每天接受一次50 mW/cm2治療(E組,間斷波)。除了觀察不同NLFUS的治療條件對於傷口癒合的狀況，並研究潛在的生長因子如膠原蛋白(collagen)、纖連蛋白(Fibronectin)、Rac-1蛋白、血管內皮生長因子(VG-1)等在開創後第三天、第七天，及傷口完全癒合之後的含量變化，以評估NLFUS所帶來的幫助。
實驗結果顯示：40kHz超音波換能器在離傷口1.5公分以內，並且不會因為造成劇烈的溫度改變而引起熱效應及空穴效應。在細胞研究中，經過NLFUS治療之後的12小時，超音波可幫助細胞比對照組多增生大約20%的增生面積，其中以連續波(100%佔空比)強度50mW/cm2、照射時間3分鐘的參數效果相對較好，因此可初步判斷NLFUS可幫助細胞增生。而體內研究發現超音波組在前九天比起對照組擁有明顯的效果，而在第七天時差異最大，在第七天時，對照組、A組、B組、C組、D組的傷口縮合率分別為-8.57±24.93%、51.36±18.49%、53.38±8.31%、24.15±13%、15.06±16.55%。並且各組傷口平均癒合時間為17.2±0.98天、14.83±0.9天、14.7±0.47、14.4±0.8天、16±1天。而在經過蘇木精-伊紅染色(H&E)及免疫組織化學染色法(IHC)分析之後，H&E結果顯示超音波組在第三天及第七天擁有較好的癒合效果，比對照組也擁有較多的毛囊表現。超音波組相比對照組在第三天及第七天時擁有較多的膠原蛋白一型、纖連蛋白、Rac-1蛋白、以及血管內皮生長因子表現，證明NLFUS可以刺激生長因子的產生並幫助傷口癒合。實驗結果證實NLFUS在前期對於傷口癒合擁有一定的癒合效果，幫助傷口縮合以達到快速癒合，並減少大約三天的癒合時間。且在比較連續波(A組)及間斷波(E組)在傷口癒合的差異，發現連續波與間斷波擁有差不多的癒合時間及組織回復狀況，然而連續波在傷口癒合之後擁有較多的膠原蛋白一型表現，因此可初步判斷連續波比起間斷波在傷口癒合中擁有較好的癒合效果。

Chronic wounds such as pressure sores, leg ulcers, and diabetic foot syndrome are critical problems that require solutions. Although there are many methods for treating chronic wounds, including antibiotic treatment, hyperbaric oxygen therapy, and negative pressure therapy, there is still a lack of an effective, cheap solution for treating chronic wounds. Recently, ultrasound therapy was found to have a specific effect in wound healing. It can be used to promote or restrain the proliferation of cells by adjusting different parameters such as frequency and intensity. However, not many studies have explored the application of noncontact low-frequency ultrasound (NLFUS) in wound healing up to the present time. Therefore, for the purpose of evaluating the biological effect of changes in growth factors caused by NLFUS, in this study, transducers with 40kHz were developed, and the scratch assay was used in the in vitro experiments. We then evaluated whether there was damage during the proliferation of 3T3 fibroblasts and then subjected them to ultrasound therapy using different parameters, such as duty cycle, intensity, and treatment duration. In addition, we developed chronic wound animal models by inducing diabetes in rats in order to evaluate the healing effect of the optimal NLFUS parameters on wounds in diabetic rats. At the same time, the rats were divided into 5 groups with different treatment conditions, including the sham group (non-treated), the A group (50 mW/cm2, once/day), the B group (50 mW/cm2, twice/day), the C group (50 mW/cm2, thrice/7 days), the D group (100 mW/cm2, once/day), and the E group (50. mW/cm2, once/day, pulse wave). In addition to observing the status of wound healing under the different treatment conditions, we researched changes in underlying growth factors, including collagen, fibronectin, rac-1, and VEGF on day 3 and day 7 after the surgery and after the wounds had healed completely to measure the benefits of NLFUS.
The results showed that the 40kHz transducers wouldn’t cause excessive changes in temperature that led to a thermal effect and cavitation when the distance between the wound and the transducer was below 1.5cm. In in vitro experiments, NLFUS could improve cell proliferation in more than 20% of the healing area after ultrasound treatment for 12 hours. The best parameter was the continuous wave (100% duty cycle) with an intensity of 50 mW/cm2 with a treatment duration of 3 min. It was thus initially evidenced that NLFUS could help with cell proliferation. In the in vivo experiments, the ultrasound groups exhibited more improvement in the first 9 days in comparison with the sham group. The most obvious difference occurred on day 7. On day 7, the wound closure of the sham group, A group, B group, C group, and D group were, respectively, -8.56±24.93%, 51.36±18.49%, 53.38±8.31%, and 24.15±13%, and 15.06±16.55%, and the average healing time of each group was 17.2±0.98 days, 14.83±0.9 days, 14.7±0.47 days, 14.4±0.8 days, and 16±1 days, respectively. After the hematoxylin & eosin (H&E) stain and immunohistochemistry (IHC) analyses, the results of H&E stain indicated that the ultrasound groups had the best healing effects on day 3 and day 7. The ultrasound groups were also found to have more follicles than the sham group. After analyzing the IHC results, the ultrasound groups were found to have more collagen type 1, fibronectin, rac-1, and VEGF in comparison with the sham group on day 3 and day 7. This proved that NLFUS could stimulate the generation of growth factors and in turn help wound healing. The results of the experiments indicated that NLFUS had the specific healing effect in wound healing in the early period. It helped with the condensation of wounds to achieve rapid healing and reduced the healing time to approximately 3 days. After comparing the difference between the continuous wave and the pulse wave in wound healing, it was found that the continuous wave and the pulse wave had similar healing time and tissue recovery. However, the continuous wave had more expression of collagen-1 and fibronectin after the wounds were healed. Therefore, it could be inferred that the continuous wave had a better healing effect in wound healing in comparison with the pulse wave.

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