Dissertations / Theses on the topic 'Liput'

Low Intensity Pulsed Ultrasound (LIPUS) is used clinically to promote wound healing. In vivo studies show that LIPUS is effective in a wide range of tissue types, and in vitro experiments show that multiple cell types respond to LIPUS stimulation. Despite this, there is no unifying mechanism of how LIPUS stimulation is sensed by cells, and it is unknown what the early signalling events are. The LIPUS signal is a mechanical one; therefore I hypothesised that mechanosensitive organelles, called focal adhesions, would be essential for the induction of cellular signalling events in response to this type of stimulation. Proteins within these structures (such as vinculin and talin) link the actin cytoskeleton to the extracellular matrix via integrins, and are known to be sensitive to mechanical forces, capable of generating intracellular signalling events in response to mechanical stimulation. The purpose of this work was to identify the early signalling events occurring within minutes of LIPUS stimulation; determine the molecular mechanisms behind such events; and to investigate whether such events require integrin-mediated adhesions. In the first part of the work, I established the use of live-cell imaging together with LIPUS stimulation to directly observe the cellular response. I determined rapid reorganizations of the actin cytoskeleton, which led to increased cell velocity. These effects were found to be Rac dependent, and, using FRET-based probes, I measured rapid increases in Rac activity occurring within minutes of LIPUS stimulation. The second part of this work identified an increase in the number of early endosomes in cells stimulated with LIPUS. This phenotype was also Rac dependent, as well as requiring the early endosomal regulator protein Rab5. In this chapter, I observed an increase in the association between Rac and Rab5 in response to LIPUS stimulation, and this contributes to Rac activation. Using substrates to block integrin-mediated adhesion, I determined that cell-matrix adhesions are required for the effects of LIPUS stimulation. Using vinculin-deficient cells, I determined that this mechanosensitive protein is vital for co-ordinating Rac activation in response to LIPUS. In particular, the actin binding tail is needed for mechanosensing of this LIPUS signal. In the final chapter I established the use of photoactivatable (PA) GFP to assess adhesion protein turnover. This technique was used to show that LIPUS stimulation directly affects the turnover of vinculin. Overall, this work shows that the mechanosensitive protein vinculin is crucial for sensing the mechanical stimulation provided by LIPUS, orchestrating downstream Rab5-mediated Rac activation to enhance cell motility.

La popolazione di testuggine palustre europea Emys orbicularis, un tempo comunemente diffusa in tutto il territorio planiziale e litoraneo del Veneto, è ora ridotta e molto frammentata, confinata soprattutto nelle aree planiziali prossime all’Adriatico e in poche zone umide palustri dell’entroterra. In questo contesto, l’Oasi LIPU Cave di Gaggio Nord (VE), ospita una consistente popolazione di questa specie e della sua potenziale competitrice Trachemys scripta ssp., accertata attraverso un precedente monitoraggio a vista (Desiderà I., 2014). Ciò ha permesso attraverso un monitoraggio cattura-marcatura-ricattura di stimare l’abbondanza e valutare lo stato di conservazione e le problematiche ambientali della popolazione di E. orbicularis, anche in relazione alle testuggini alloctone. In particolare il monitoraggio ha consentito lo studio della popolazione presente nell'oasi attraverso la caratterizzazione biometrica, struttura di popolazione, stima dell'abbondanza e caratterizzazione degli habitat utilizzati. Sono state inoltre analizzate le problematiche di competizione e conflittualità con Trachemys scripta e altre specie alloctone ai fini di delineare possibili indicazioni gestionali per la conservazione e salvaguardia della specie autoctona e indirizzare azioni locali mirate per la gestione delle specie invasive che interagiscono con essa. Ai fini della caratterizzazione dell’habitat è stata monitorata anche la qualità delle acque attraverso misure di temperatura, OD e concentrazione di anioni (solfati, nitrati, nitriti, cloruri, fluoruri, fosfati e bromuri).

Submitted by Jaqueline Bastos (keca78@yahoo.com.br) on 2016-04-26T17:00:34Z No. of bitstreams: 1 TESE JAQUELINE S BASTOS - finalizando 180316.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5)
Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-28T17:50:25Z (GMT) No. of bitstreams: 1 bastos_js_dr_guara.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5)
Made available in DSpace on 2016-04-28T17:50:25Z (GMT). No. of bitstreams: 1 bastos_js_dr_guara.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5) Previous issue date: 2016-01-20
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O objetivo desse estudo in vivo foi verificar a resposta óssea de scaffolds porosos revestidos processados pela técnica de Sinterização Direta de Metais a laser (DMLS) associado à terapia de ultrassom de baixa intensidade. Os scaffolds foram processados empregando a técnica DMLS e tratados termicamente a 1000°C por 24 horas. Três tipos de tratamento de superfície foram avaliados: Alcalino, biomimético e imobilização de alendronato de sódio. Para o tratamento alcalino, as amostras foram imersas na solução de NaOH (5M) a 60ºC por 24 horas. O tratamento biomimético consistiu na imersão dos scaffolds em solução SBF (SimulatedBodyFluid) enquanto a imobilização do alendronato foi realizada a partir da imersão dos scaffolds em uma solução formada por SBF e medicamento durante 5 dias. As superfícies dos scaffolds foram avaliadas para cada etapa empregando microscopia eletrônica de varredura (MEV) e análise por difração de raios-X. Os scaffolds foram implantados na tíbia direita de 85 ratos machos da raça wistar com idade média de 12 semanas. A microtomografia computadorizada (µCT) e análise histológica foram realizadas para avaliar o reparo ósseo no defeito. As micrografias das superfícies obtidas mostraram mudanças no aspecto da superfície e composição química de acordo com o tratamento. O tratamento biomimético promoveu o crescimento da apatita sobre a superfície enquanto a imobilização com alendronato suprimiu sua formação. As imagens obtidas na microtomografia mostraram elevado valor de densidade óssea para o último grupo. No entanto, análises histológicas mostraram a formação de cápsula fibrosa em torno dos scaffolds a qual foi minimizada usando ultrassom pulsado de baixa intensidade. No entanto, mais estudos precisam ser realizados para avaliar a influência da geometria dos scaffolds na incorporação de medicamentos.
The objective of this in vivo study was to verify the bone response of coated Ti6Al4V porous scaffolds processed by Direct Metal Laser Sintering (DMLS) technique associated to low intensity pulsed ultrasound therapy. Scaffolds were processed by using Direct Metal Laser Sintering technique (DMLS) and heat treated at 1000 °C for 24 hours. Three types of surface treatments were evaluated: alkaline, biomimetic and sodium alendronate immobilization. For alkaline treatment, samples were immersed in a NaOH (5M) solution at 60ºC for 24 hours. Biomimetic treatment consisted in the immersion of the scaffolds into Simulated Body Fluid solution while for sodium alendronato immobilization the scaffolds were immersed in the solution formed by SBF plus drug during 5 days. The scaffolds surfaces were evaluated after each step employing SEM (Scanning Electron Microscopy)and X-rays diffraction analysis(XRD). Scaffolds were implanted into right tibia of 85 male Wistar rats with average age of 12 weeks. X-rays micro-computed tomography (µCT) and histological analysis were carried out to evaluate the bone repair on the defect. Micrographs analysis showed that the aspect of the surfaces and chemical composition changed according treatment. Biomimetic treatment promoted the growth of the apatite on the surface; in contrast the immobilization of alendronate suppressed apatite formation. Micro CT images showed higher value of bone density for the last group. However, histological analysis showed the formation of encapsulation fibrous around the scaffolds. This formation was minimized by using low intensity pulsed ultrasound technique, however, more studies can be carried out to evaluate the influence of scaffolds geometry in the drug incorporation.

Orientadora: Ana Paula Rosifini Alves Claro
Banca: André Luiz Jardini Munhoz
Banca: Ricardo Tadeu Lopes
Banca: Orivaldo Lopes da Silva
Banca: Ana Lia Anbinder
Resumo: O objetivo desse estudo in vivo foi verificar a resposta óssea de scaffolds porosos revestidos processados pela técnica de Sinterização Direta de Metais a laser (DMLS) associado à terapia de ultrassom de baixa intensidade. Os scaffolds foram processados empregando a técnica DMLS e tratados termicamente a 1000°C por 24 horas. Três tipos de tratamento de superfície foram avaliados: Alcalino, biomimético e imobilização de alendronato de sódio. Para o tratamento alcalino, as amostras foram imersas na solução de NaOH (5M) a 60ºC por 24 horas. O tratamento biomimético consistiu na imersão dos scaffolds em solução SBF (SimulatedBodyFluid) enquanto a imobilização do alendronato foi realizada a partir da imersão dos scaffolds em uma solução formada por SBF e medicamento durante 5 dias. As superfícies dos scaffolds foram avaliadas para cada etapa empregando microscopia eletrônica de varredura (MEV) e análise por difração de raios-X. Os scaffolds foram implantados na tíbia direita de 85 ratos machos da raça wistar com idade média de 12 semanas. A microtomografia computadorizada (µCT) e análise histológica foram realizadas para avaliar o reparo ósseo no defeito. As micrografias das superfícies obtidas mostraram mudanças no aspecto da superfície e composição química de acordo com o tratamento. O tratamento biomimético promoveu o crescimento da apatita sobre a superfície enquanto a imobilização com alendronato suprimiu sua formação. As imagens obtidas na microtomografia mostraram elevado valor de densidade óssea para o último grupo. No entanto, análises histológicas mostraram a formação de cápsula fibrosa em torno dos scaffolds a qual foi minimizada usando ultrassom pulsado de baixa intensidade. No entanto, mais estudos precisam ser realizados para avaliar a influência da geometria dos scaffolds na incorporação de medicamentos
Abstract: The objective of this in vivo study was to verify the bone response of coated Ti6Al4V porous scaffolds processed by Direct Metal Laser Sintering (DMLS) technique associated to low intensity pulsed ultrasound therapy. Scaffolds were processed by using Direct Metal Laser Sintering technique (DMLS) and heat treated at 1000 °C for 24 hours. Three types of surface treatments were evaluated: alkaline, biomimetic and sodium alendronate immobilization. For alkaline treatment, samples were immersed in a NaOH (5M) solution at 60ºC for 24 hours. Biomimetic treatment consisted in the immersion of the scaffolds into Simulated Body Fluid solution while for sodium alendronato immobilization the scaffolds were immersed in the solution formed by SBF plus drug during 5 days. The scaffolds surfaces were evaluated after each step employing SEM (Scanning Electron Microscopy)and X-rays diffraction analysis(XRD). Scaffolds were implanted into right tibia of 85 male Wistar rats with average age of 12 weeks. X-rays micro-computed tomography (µCT) and histological analysis were carried out to evaluate the bone repair on the defect. Micrographs analysis showed that the aspect of the surfaces and chemical composition changed according treatment. Biomimetic treatment promoted the growth of the apatite on the surface; in contrast the immobilization of alendronate suppressed apatite formation. Micro CT images showed higher value of bone density for the last group. However, histological analysis showed the formation of encapsulation fibrous around the scaffolds. This formation was minimized by using low intensity pulsed ultrasound technique, however, more studies can be carried out to evaluate the influence of scaffolds geometry in the drug incorporation
Doutor

Le Diagnostic Expérimental des Pulsions, aussi connu comme le « Test de Szondi », est une méthode projective conçue par Léopold Szondi afin d'explorer les aspirations pulsionnelles inconscientes chez le sujet. Les aspects théorico-cliniques développés par Szondi ou ses successeurs ne sont plus à démontrer, mais il est une zone d'ombre qui est présente et qui n'a toujours pas trouvé de réponse objective, qui est la suivante : « Comment ce test fonctionne-t-il, dans une compréhension et une démarche scientifique ? ». C'est donc pour cela qu'à l'aide des outils et des moyens modernes, nous avons pu mettre à l'épreuve ce qu'il se cachait derrière ces visages, derrière ces fameux « caractères évocateurs » que nous décrivait Szondi lui-même dans son ouvrage Diagnostic Expérimental des Pulsions. Notre recherche nous a amené à étudier chaque visage sous l'angle de la reconnaissance émotionnelle, afin de comprendre l'émotion qui y est présente sur chacun d'entre-eux, pour ainsi établir des ponts théorico-empiriques satisfaisants du point de vue scientifique. Au-delà de cette reconnaissance émotionnelle de chaque visage, nous avons mis au point un modèle, que nous avons appelé la Dialectique Emotionnelle, qui est un modèle multidimensionnel permettant de rendre compte de la théorie szondienne à la lumière de sa compréhension scientifique, modèle reprenant les travaux de Szondi ainsi que ceux de ses successeurs, afin de rendre clair ce point d'obscurité qui est venu entacher la compréhension de l'oeuvre szondienne, et qui a fortement participé à ce que cette oeuvre soit presque oubliée. Dans l'objectif de comprendre toutes les facettes et la richesse de l'oeuvre szondienne, nous avons aussi réalisé certaines expérimentations relevant de la méthode des jugements libres ainsi que l'élaboration d'un protocole de cotation relevant du quantum émotionnel et non pas du quantum pulsionnel, afin d'apporter une compréhension plus fine du Diagnostic Expérimental des Pulsions et de ses possibilités.La compréhension moderne du Diagnostic Expérimental des Pulsions trouve aussi son corollaire dans l'oeuvre première szondienne, qui est l'Analyse du Destin, où il y sera proposé une réflexion croisée entre la pensée szondienne de la première moitié du XXe siècle et la pensée moderne qui vient apporter une compréhension scientifique à l'intuition szondienne, notamment au regard de l'épigénétique et de la psychogénéalogie.La clinique szondienne a aussi été mise à l'épreuve, repensée, remaniée ou augmentée d'autres tests ou pratiques (comme le Test de Rorschach, le Test de la Maison ou même la Psychoboxe), et où nous avons pu constater qu'elle se révélait d'une grande efficacité, et d'un intérêt clinique des plus intéressants mais aussi des plus pertinents.Cette thèse, dans son aspect à rendre compréhensible la théorie szondienne ainsi que la méthode projective qui la sous-tend sur des fondements scientifiques, a pour objectif de rendre hommage à une pensée féconde et toujours d'actualité, mais qui est arrivée trop tôt dans l'Histoire des sciences humaines
The Experimental Diagnosis of Drives, also known as the "Szondi Test", is a projective method created by Léopold Szondi to explore the subject's unconscious drive aspirations. The theoretical-clinical aspects developed by Szondi or his successors are no longer to be demonstrated, but there is a gray area which is present and which has still not found an objective answer, which is the following: "How does this test work, in a scientific understanding and approach? ". This is why, using modern tools and means, we were able to test what was hidden behind these faces, behind these famous "evocative characters" that Szondi himself described to us in his book Experimental Diagnosis of Drives. Our research led us to study each face from the angle of emotional recognition, in order to understand the emotion that is present on each of them, in order to establish satisfactory theoretical-empirical bridges from the scientific point of view. Beyond this emotional recognition of each face, we have developed a model, which we have called the Emotional Dialectic, which is a multidimensional model making it possible to account for the Szondian theory in the light of its scientific understanding, a model taking up the works of Szondi as well as those of his successors, in order to make clear a point of obscurity which has come to taint the understanding of the work of Szondi, and which has strongly contributed to this work being almost forgotten. With the aim of understanding all the facets and the richness of the Szondian work, we also carried out certain experiments relating to the method of free judgments as well as the development of a rating protocol relating to the emotional quantum and not the drive quantum, in order to provide a finer understanding of the Experimental Diagnosis of Drives and its possibilities.The modern understanding of the Experimental Diagnosis of Drives also finds its corollary in the first Szondian work, which is the Analysis of Destiny, where a cross-reflection between Szondian thought of the first half of the 20th century and modern thought will be proposed, which comes to bring a scientific understanding to the Szondian intuition, in particular with regard to epigenetics and psychogenealogy.The Szondian clinic has also been put to the test, redesigned or augmented with other tests or practices (such as the Rorschach Test, the House Test or even Psychoboxing), and where we have been able to see that it proved to be very effective.This thesis, in its aspect of making understandable the Szondian theory as well as the projective method which underlies it on scientific foundations, aims to pay homage to a fruitful and still current thought, but which arrived too early in the history of the human sciences

碩士
義守大學
生物醫學工程學系
106
Porous scaffold can provide space for cell adhesion, migration, growth and differentiation, and the pore geometry and interconnection play an important role on the cellular behaviors. In this present study, we prepared a collagen scaffold with concentration of 7 mg/mL and 0.5 w% hyaluronan (HA) to mimetic natural ECM to evaluate the growth and differentiation of ADSCs. As known, HA molecules have binging cites for CD44 and RHAMM, can activate the signal transduction of angiogenesis. Furthermore, low intensity pulsed ultrasound stimulus (LIPUS) can stimulate angiogenesis by path of PI3K-Akt-eNOS signal transduction. In this study, we utilized HA and LIPUS simulative factors to induce the differentiation of ADSCs and evaluate their effects on the live repairs. From SEM and H&E stains observations, ADSCs grew, proliferate well on the collagen scaffold containing 0.5% HA. The cellular growth significantly improved after extra LIPUS stimulus. From immunofluorescence stains, co-cultured ADSCs with live cells or HUVECs and LIPUS stimulus, the ADSCs expressed higher CD45 expression, however, the ADSCS expressed lower albumin expression. Higher CD31 expressions were observed from immunofluorescence stains in animal angiogenetic study, indicated that the angiogenesis of ADSCs on the collagen scaffolds.

碩士
義守大學
生物醫學工程學系
103
Ultrasound has been shown to be a noninvasive treatment method. It produces mechanical waves through soft tissues to change intracellular responses. Mechanical forces, cavitation and microstreaming accompanied with ultrasound exposure could serve as extracellular information, which may directly affect stem cell proliferation and differentiation. Many animal and clinical studies have shown that low-intensity pulsed ultrasound (LIPUS) can promote healing in the fracture model and enhance bone growth. However, less study investigates the effects of LIPUS stimulation on mesenchymal stem cells (MSCs). The purpose of this study is to investigate the effects of ultrasonic stimulation on the differentiation of MSCs into cardiomyocytes. In this study, we accomplished an ultrasonic exposure system to drive ultrasound transducers to generate LIPUS energy. An in-vitro study was employed to investigate the performance of ultrasound exposure on the differentiation of MSCs. Cells were isolated from the Femurs of Sprague-Dawley rats. Following flow cytometry analysis, MSCs express the expression of surface markers which CD90 expression is 97.48% and CD34 is not expressed, which demonstrates the high purity of the isolated MSCs. The MSCs of the second passage are cultured in a 75T-flask (cell numbers is 105 cells/well). And we use pre-culture method to let more MSCs to attach on the 75T-flask. Then cells received the exposure of a 1 MHz ultrasound 10 minutes/day. Acoustic parameters studied include power level (145mW, 304.5mW and 449.5mW) and duty cycle (20% and 100%) of LIPUS. Cell proliferation and specific protein markers of cardiomyocytes are examined. at 0th, 7th and 14th day after MSCs have received ultrasonic exposure. Results show that pulsed ultrasound (duty cycle 20%) has a positive effect on the increase of cell numbers, and the maximum cell proliferation is 39 % at 14th day (acoustic power 449.5mW). On the contrary, continuous ultrasound (duty cycle 100%) makes the cell number decreased. In addition, both continuous and pulsed ultrasound have significant effects on cellular differentiation at 32.6% ~ 51.9% at 7th days. At 14th day, continuous ultrasound shows a better cellular differentiation effects than pulsed ultrasound with a differentiation ratio of 23.6% (acoustic power 304.5mW). Base on the experimental results, it shows that ultrasonic stimulation could affect the differentiation of MSCs into cardiomyocytes.

O assédio moral é um fenómeno antigo, que remonta ao começo do trabalho e infringe o princípio da dignidade humana. Pode-se atribuir várias designações mas a mais consensial e antiga foi a utilizada por Leymann na década de 80, que designou este fenómeno como mobbing, tendo igualmente definido este fenómeno como um conjunto de condutas hostis, repetidas e persistentes contra um ou mais indivíduos numa relação entre o ofensor e a vítima. O objetivo principal deste estudo foi a aferição para a população portuguesa da Escala de Mobbing de Leymann (LIPT-60), tendo por base o LIPT (Leymann Inventory of Psychological Terrorization), mais concretamente o Cuestionario de estratégias de acosso en el trabajo – el LIPT-60 modificado de González de Rivera & Rodriguez Abuín (2005), constituído por 60 questões. Procedeu-se a um estudo quantitativo, de carácter correlacional, de corte transversal e exploratório, onde a amostra é constituída por 294 participantes. Apesar das diversas dificuldades e limitações encontradas ao longo da elaboração desta tese, os resultados deste estudo demonstraram que é válida a utilização da Escala de Mobbing de Leymann (LIPT-60). Este estudo permitiu, deste modo, uma melhor compreensão da temática do mobbing, sendo a utilização desta escala importante porque o assédio moral é uma realidade presente em muitos locais de trabalho, que além de trazer consequências para a vítima, traz igualmente consequências para a própria organização e para a sociedade.
Mobbing is an old phenomenom that goes back to the beginning of the work and violates the principle of human dignity. It can be attributed several names but the most consensial and old one was the one used by Leymann in the decade of 80, that designated this phenomenon like mobbing, having aldo defined this phenomenon like a set of hostile, repeated and persistente conducts against one or more individuals in a relationship between the ofender and the victim. The main objective of this study was to assess the Leymann Mobbing Scale (LIPT-60) for the Portuguese population, based on the Leymann Inventory of Psychological Terrorization (LIPT), more specifically the Workplace Harassment Strategies Questionnaire LIPT-60 modified by González de Rivera & Rodriguez Abuín (2005), consisting of 60 questions. We proceeded to a quantitative study of correlation character and exploratory cross section, where the sample consists of 294 participants. Despite the various difficulties and limitations encountered during the development of this thesis, the results of this study demonstrated that the Leymann Mobbing Scale (LIPT-60) is valid. This study allowed a better understanding of the subject of mobbing, and the use of this scale is importante because bullying is a reality presente in many places of work, which in addition to having consequences for the victim, also has consequences for the organization and society.

碩士
中原大學
生物醫學工程研究所
98
Ultrasound had been shown to be a potent method for bone fracture healing acceleration and induction in vitro, in vivo and in clinical for many years. Different frequency of ultrasound has different effects on different fracture healing period and cell types. In our previous data, it showed that varying frequencies pattern of ultrasound would have better effects on bone fracture healing acceleration than the single frequency stimulation. Stem cells are the key factors in tissue regeneration process. Recently, the concept of ultrasound stimulation for the stem cell differentiation and proliferation has also been proved. Two kinds of mediums (basal and bone differentiation medium) were used to mimic different growth environments and stages of stem cells (C3H10T1/2) under different frequencies of low-intensity pulsed ultrasound (LIPUS) treatments. The optimum stimulation frequency of C3H10T1/2 proliferation and osteogenesis in different growth stages were evaluated by trypan blue exclusion method, alkaline phosphotase activity, Alizarin Red stain and ALP stain. The results showed that higher frequency (3 MHz) low-intensity pulsed ultrasound had better stimulating effects on C3H10T1/2 cell proliferation than lower frequency treatments. On the other hand, lower frequency (1 MHz) ultrasound had better stimulating effects on C3H10T1/2 cell differentiation than higher frequency treatments. It means that choosing a suitable frequency at different periods for stem cell proliferation or differentiation stimulating might be a new feasible way to accelerate fracture healing.

临床及动物实验的文献报告表明, 低能量脉冲超声波 (LIPUS) 能促进骨折愈合。 可是, 不同研究小组针对LIPUS的功效所提供的数据结果往往并不一致。为了找出导致数据结果不一致的原因, 以及提升LIPUS的生物功效, 科研人员正致力于测定超声波在骨折治疗中的最佳信号参数。 在临床运用上, LIPUS对骨折的治疗一般是以经皮方式应用的。 故此, 不同层次深度的骨折会暴露在不同的超声波场区里。 超声波场有两个不同的区域, 就是近场区 (接近超声波换能器的区域) 及远场区 (远离超声波换能器的区域)。 在我们早期的临床研究中, 我们曾使用超声波的近场区治疗胫骨的复杂性骨折。 我们发现, 当超声波换能器置于胫骨骨折处的前方, 骨痂集中生成于胫骨骨折处的背面。 这研究结果显示, 近场区以外的超声波场或许更具促进骨痂形成的功效。 再者, 针对LIPUS声场仿真分析的结果显示, 近场区内的声压分布是不稳定的, 而远场区内的声压则远比近场区的均及稳定。由于声压的稳定性会大大影响超声波于组织内的能量透射, 我们相信在超声波场中, 骨折的深度会影响LIPUS的生物效应。
本研究采用大鼠闭合性股骨骨折模型及细胞培养实验, 探究不同的超声波场区对骨折愈合的影响。本研究作出了以下三个科研假设: (1) LIPUS 的远场区在促进骨折愈合的应用上有着更高的生物效应; (2) LIPUS 的远场区能透过促使骨细胞产生旁分泌调节因子, 从而提升成骨细胞的成骨活性; (3) 通过换能器直径的调制而产生的LIPUS远场区能有效地促进骨折愈合。
在第一部分的实验里, 股骨骨折的SD大鼠被随机分为对照组 (control), 近场区超声波治疗组 (near field; 伤肢跟换能器相距0mm), 中近场区超声波治疗组 (mid-near field; 伤肢跟换能器相距60mm), 远场区超声波治疗组 (far field; 伤肢跟换能器相距130mm)。在伤肢及超声波换能器之间安放了跟软组织具有同一超声波衰减系数的凝胶 (长度: 0mm, 60mm, 130mm)。LIPUS每天治疗20分钟, 每周治疗5天。 我们研究结果显示, 治疗后的第四周, 远场治疗组的骨痂组织具有各组中最高的相对骨体积及组织矿密度, 这造就远场治疗组相比对照组具有更强的力学属性。我们的研究结果表明, LIPUS的远场区治疗能通过提升骨痂的骨体积及骨矿化, 进一步促进骨折的愈合。
在第二部分的实验里, 我们把骨细胞株(MLO-Y4) 暴露在三種不同的超声波場中: 0 mm, 60 mm 及130 mm 。 经过不同的LIPUS处理后, 我们把条件培养基(CM) 收集, 并将其用于培养成骨前趋细胞株(MC3T3-E1)。 这部分的实验共有5组: Non组(非条件培养基处理组), Con组(骨细胞条件培养基处理组), 0mm组(条件培养基处理组; 条件培养基收集自经过LIPUS近场区刺激后的骨细胞), 60mm组(条件培养基处理组; LIPUS中近场区刺激后的骨细胞), 以及 130mm组(条件培养基处理组; LIPUS远场区刺激后的骨细胞)。我们测试了各超声波场对骨细胞的直接影响, 以及成骨前趋细胞经过各类骨细胞条件培养基培养后的成骨活性。 免疫染色的结果显示近场区以后的超声波场 (130mm 及 60mm) 能进一步诱导β-catenin 于骨细胞的入核作用。 另外, 远场区的骨细胞条件培养基 (130mm CM) 的处理促进了成骨前趋细胞的: (1) 细胞迁移的能力 (反映自细胞伤口愈合测试) ; (2) 细胞分化成熟的机制 (BrdU细胞增殖检验及ALP活性分析): (3) 基质钙化 (Alizarin red 钙化结节染色)。
在第三部分的实验里, 我们把换能器的直径缩减致一半, 以致换能器跟LIPUS远场区之间的距离从130 mm被拉近至30 mm。 当LIPUS以经皮的方式应用, 位于皮下大约40 mm的大鼠股骨骨折因此暴露在LIPUS的远场区。 相对于在换能器及伤肢之间安置130 mm凝胶, 以调制换能器直径而直接让骨折暴露于LIPUS远场区是更具临床应用性的方法。 在这部分, 股骨骨折的SD大鼠被随机分为对照组 (control), LT-Near30 (正常的换能器直径; 近场超声处理; I[subscript SATA] = 30 mW/cm²), ST-Far30 (缩减后的换能器直径; 远场超声处理; I[subscript SATA] = 30 mW/cm²), ST-Far150 (缩减后的换能器直径; 远场超声处理; I[subscript SATA] = 150 mW/cm²)。 研究结果证实, 以调制换能器直径而产生的远场LIPUS (ST-Far30)能透过提升骨痂的生成及力学属性, 进一步促进骨折愈合。 同时, 我们的结果显示, 相对高强度 (150 mW/cm²) 的远场LIPUS治疗不能更有效地促进骨折愈合。
综上所述, 动物及细胞培养实验结果证明, LIPUS的远场区在促进骨折愈合上更具功效。由于LIPUS的远场区放射稳定的超声波束, 骨痂中的骨细胞受引发释放可溶性因子, 从而进一步激发成骨样细胞的成骨活性。 这些细胞的生物效应造就LIPUS的远场区在促进骨折愈合上更具治疗效果。最后, 我们亦把以上的研究发现转化成具临床应用性的LIPUS应用方法。 这应用方法能让超声波换能器以紧贴皮肤的方式直接使骨折暴露于LIPUS远场区, 从而达成促进骨折愈合的功效。
Low-intensity Pulsed Ultrasound (LIPUS) has been confirmed to enhance fracture healing in many clinical and animal studies. However, the evidences from literatures to support the applications of LIPUS on fracture healing were inconsistent. Therefore, scientists have been studying various ultrasound parameters aiming to find out the factors resulting in the inconsistent outcomes among research groups, and to further enhance the efficacy of LIPUS. Clinically, LIPUS is usually applied onto fracture sites transcutaneously, hence, fractures at different depths are exposed to different zones of ultrasound beam. There are two characteristic zones of ultrasound beam: the near field (close to the transducer) and far field (farther from the transducer). In our previous clinical study, direct transcutaneously applied LIPUS (near field LIPUS exposure) was used to treat human tibial complex fractures. We found that callus usually formed on the posterior side when the transducer was placed on the anterior side. This finding implied that ultrasound beam beyond near field bears higher potential in promoting callus formation. Moreover, beam mapping measurement of LIPUS shows a variable spatial pressure in near field; while a more uniform pressure profile was found beyond it (far field). As the stability of pressure profile influences the ultrasound energy transmission in tissue, we postulate that the biological effects of LIPUS are affected by the fracture depths within the ultrasound field.
This study aims to address the research question of how ultrasound fields influence the fracture healing through testifying the following hypotheses in animal and cell culture studies: (1) Far field LIPUS bears higher biological effect in facilitating fracture healing; (2) Far field LIPUS could enhance the osteogenic activities of osteoblastic cells via paracrine factors secreted from osteocytes; (3) Far field LIPUS setup by transducer diameter modulation could facilitate fracture healing.
In part one study, femoral fractured Sprague-Dawley (SD) rats were randomized into control, near-field (fractures placed at 0mm away from transducer), mid-near field (60mm away from transducer) or far-field (130mm away from transducer) groups. Rubber gel block (lengths: 0mm, 60mm and 130mm) with attenuation coefficient equivalent to soft tissue was interposed between the transducer and the fractured limb. LIPUS was given 20min/day and 5days/week. We found the callus in 130mm group was the highest in bone volume fraction and tissue mineral density at week 4. These advancements mutually contributed to its significantly stronger mechanical properties than the control group. Our results indicated that far field LIPUS could further facilitate fracture healing by promoting bone volume increase and callus mineralization, which led to enhanced mechanical properties.
In part two study, LIPUS was applied to osteocyte cell line (MLO-Y4) at three distances: 0mm, 60mm and 130mm. The conditioned medium (CM) collected from different LIPUS treatment regimens were used to culture pre-osteoblast cell line (MC3T3-E1). There were 5 groups in the CM treatment: Non group (plain α-MEM treatment), Con group (osteocyte CM), 0mm group (Near field LIPUS treated osteocyte CM), 60mm group (Mid-near field LIPUS treated osteocyte CM) and 130mm group (Far field LIPUS treated osteocyte CM). The effect of ultrasound fields on osteocytes and the osteogenic activities of the pre-osteoblasts after different CM treatments were assessed. The immunostaining results indicated that beyond near field LIPUS (LIPUS at 130mm and 60mm) could further promote β-catenin nuclear translocation in osteocytes. The far field LIPUS osteocyte-CM (130mm group) caused the highest biological effect on (1) pre-osteoblasts migration (reflected by wound healing assay); (2) maturation of pre-osteoblasts: transition of cell proliferation into osteogenic differentiation (BrdU cell proliferation assay and ALP activity assay); and (3) matrix calcification (Alizarin red calcium nodule staining).
In part three study, the transducer diameter was reduced by half in order to draw the far field location closer to the transducer (i.e. from 130 mm to 30 mm). As the femoral shaft fractures of rats are located at around 40 mm beneath the skin, fractures were directly exposed to far field LIPUS transcutaneoulsy. It is a more clinically applicable approach than the method of physical separation (i.e. 130 mm separation between transducer and fractured limb). Femoral fractured SD rats were randomized into control, LT-Near30 (conventional transducer diameter, near field, I[subscript SATA] = 30 mW/cm²), ST-Far30 (small transducer, far field, I[subscript SATA] = 30 mW/cm²) and ST-Far150 (small transducer, far field, I[subscript SATA]=150 mW/cm²). Our results confirmed that the far field LIPUS emitted from the transducer diameter reduction setup (ST-Far30) could further facilitate fracture healing process by enhancing callus formation and mechanical properties. Our findings also indicated that fractures exposed to far field LIPUS with relatively higher intensity (150 mW/cm²) did not heal better.
In summary, our in vivo and in vitro findings reinforce each other to confirm the positive effects of far field LIPUS on promoting fracture healing. As far field LIPUS radiates a stable ultrasound beam, osteocytes inside the callus are triggered to secrete soluble factors to promote the osteogenic activities of osteoblastic cells. This contributes to the higher therapeutic effects of far field LIPUS on fracture healing. We also translated these findings to establish a clinically applicable LIPUS device, which directly radiates far field LIPUS to subcutaneous fracture site without any distance separation needed.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Fung, Chak Hei.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 186-207).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Abstract --- p.i
中 文 摘 要 --- p.v
Publications --- p.ix
Acknowledgements --- p.xii
List of Abbreviations --- p.xiii
Index for Figures --- p.xvi
Index for Tables --- p.xviii
Chapter Chapter 1. --- Introduction and Literature Review --- p.1
Chapter 1.1 --- Long Bone Fracture - A Growing Global Challenge --- p.2
Chapter 1.2 --- Long-Bone Fracture - Current Management and Limitations --- p.5
Chapter 1.3 --- Cellular Biology of Fracture Healing --- p.7
Chapter 1.3.1 --- Stage 1: Inflammation --- p.7
Chapter 1.3.2 --- Stage 2: Soft callus formation --- p.8
Chapter 1.3.3 --- Stage 3: Hard callus formation --- p.9
Chapter 1.3.4 --- Bone Remodeling --- p.10
Chapter 1.4 --- Biophysical Stimulation to Bone --- p.13
Chapter 1.5 --- Low-intensity Pulsed Ultrasound --- p.14
Chapter 1.5.1 --- Application of LIPUS on Fracture Healing --- p.14
Chapter 1.5.2 --- Physics of Ultrasound --- p.16
Chapter 1.5.3 --- Ultrasound Parameters --- p.20
Chapter 1.5.3.1 --- Ultrasound Frequency --- p.20
Chapter 1.5.3.2 --- Duty Cycle --- p.22
Chapter 1.5.3.3 --- Intensity --- p.22
Chapter 1.5.3.4 --- Angle of Incidence --- p.24
Chapter 1.5.3.5 --- Ultrasound Field --- p.25
Chapter 1.5.4 --- Possible Mechanism of LIPUS on Tissue --- p.31
Chapter 1.5.4.1 --- Thermal Effect --- p.31
Chapter 1.5.4.2 --- Cavitation --- p.31
Chapter 1.5.4.2 --- Acoustic Streaming --- p.32
Chapter 1.5.4.3 --- Frequency Resonance Hypothesis --- p.32
Chapter 1.5.4.4 --- Micromotion --- p.33
Chapter 1.6 --- Possible Cellular and Molecular Mechanotransduction Mechanism of LIPUS --- p.34
Chapter 1.6.1 --- Osteocyte: Potential Mechanosensor --- p.34
Chapter 1.6.3 --- Osteocyte-osteoblast mechanotransduction --- p.39
Chapter 1.7 --- Hypothesis --- p.39
Chapter 1.8 --- Study Plan and Objectives --- p.40
Chapter 1.8.1 --- Study Plan --- p.40
Chapter 1.8.2 --- Objectives --- p.42
Chapter Chapter 2. --- Characterization of Ultrasound Field Distances on Rat Fracture Model --- p.43
Chapter 2.1 --- Introduction --- p.44
Chapter 2.2 --- Material & Methods --- p.47
Chapter 2.2.1 --- Closed Femoral Shaft Fracture Model in Rat --- p.47
Chapter 2.2.2 --- Ultrasound Field Distances Setup --- p.51
Chapter 2.2.3 --- Animal Grouping & LIPUS Treatment Protocol --- p.53
Chapter 2.2.4 --- Assessments --- p.56
Chapter 2.2.4.1 --- Radiological Analysis --- p.56
Chapter 2.2.4.2 --- Micro-computed Tomography --- p.61
Chapter 2.2.4.3 --- Histomorphometry --- p.64
Chapter 2.2.4.4 --- Mechanical Testing --- p.66
Chapter 2.2.4.5 --- Statistical Analysis --- p.66
Chapter 2.3 --- Results --- p.68
Chapter 2.3.1 --- Radiological Analysis --- p.71
Chapter 2.3.2 --- MicroCT --- p.77
Chapter 2.3.3 --- Histomorphometry --- p.82
Chapter 2.3.4 --- Mechanical Testing --- p.85
Chapter 2.4 --- Discussion --- p.87
Chapter 2.4.1 --- Far Field LIPUS Enhances Mechanical Properties of Healing Callus --- p.88
Chapter 2.4.2 --- Mid-near field and Near field LIPUS Enhances Woven Bone Formation --- p.90
Chapter 2.4.3 --- The Biological Effects of LIPUS with Different Ultrasound Field Exposure --- p.94
Chapter 2.5 --- Conclusion --- p.97
Chapter Chapter 3. --- The Effect of Ultrasound Field Distances on Bone Cells --- p.100
Chapter 3.1 --- Introduction --- p.101
Chapter 3.2 --- Material & Methods --- p.102
Chapter 3.2.1 --- Cell Culture --- p.102
Chapter 3.2.2 --- Ultrasound Field Distances Setup & Treatment Protocol --- p.102
Chapter 3.2.2 --- Immunostaining of β-catenin --- p.106
Chapter 3.2.3 --- Wound Healing Assay --- p.109
Chapter 3.2.4 --- BrdU Cell proliferation Assay --- p.111
Chapter 3.2.5 --- Alkaline phosphatase activity assay --- p.112
Chapter 3.2.6 --- Alizarin calcium nodule staining --- p.113
Chapter 3.2.7 --- CM characterization - PGE₂ ELISA assay --- p.114
Chapter 3.2.8 --- CM characterization - nitrite assay --- p.114
Chapter 3.2.9 --- Statistical Analysis --- p.115
Chapter 3.3 --- Results --- p.116
Chapter 3.3.1 --- Immunostaining of β-catenin --- p.116
Chapter 3.3.2 --- Wound healing assay --- p.119
Chapter 3.3.3 --- BrdU Cell proliferation Assay --- p.119
Chapter 3.3.4. --- Alkaline phosphatase activity assay --- p.122
Chapter 3.3.5 --- Alizarin calcium nodule staining --- p.122
Chapter 3.3.6. --- CM characterization - PGE₂ ELISA assay --- p.125
Chapter 3.3.7 --- CM characterization - nitrite assay --- p.125
Chapter 3.4 --- Discussion --- p.128
Chapter 3.4.1 --- The Osteogenic Effect of Far Field LIPUS-Osteocyte Conditioned Medium --- p.128
Chapter 3.4.2. --- Mechanisms of Mechanotransduction between Osteocyte and Osteoblast --- p.131
Chapter 3.5 --- Conclusion --- p.136
Chapter Chapter 4. --- Rat Fracture Exposed to Far Field LIPUS by Modulating Ultrasound Transducer Diameter --- p.139
Chapter 4.1 --- Introduction --- p.140
Chapter 4.2 --- Material & Methods --- p.143
Chapter 4.2.1 --- Closed Femoral Shaft Fracture Model in Rat --- p.143
Chapter 4.2.2 --- Ultrasound Field Distances Setup & Treatment Protocol --- p.143
Chapter 4.2.3 --- Assessments --- p.148
Chapter 4.2.3.1 --- Radiological Analysis --- p.148
Chapter 4.2.3.2 --- Micro-computed Tomography --- p.148
Chapter 4.2.3.3 --- Histomorphometry --- p.149
Chapter 4.2.3.4 --- Mechanical Testing --- p.151
Chapter 4.2.3.5 --- ex vivo Temperature Measurements --- p.151
Chapter 4.2.3.6 --- Statistical Analysis --- p.151
Chapter 4.3 --- Results --- p.152
Chapter 4.3.1 --- Radiological Analysis --- p.154
Chapter 4.3.2 --- MicroCT --- p.157
Chapter 4.3.3 --- Histomorphometry --- p.160
Chapter 4.3.4 --- Mechanical Testing --- p.166
Chapter 4.3.5 --- ex vivo Temperature Measurement --- p.168
Chapter 4.4 --- Discussion --- p.170
Chapter 4.4.1 --- Far field LIPUS Setup by Transducer Diameter Modulation Enhanced Fracture Healing --- p.170
Chapter 4.4.2 --- Fractures Exposed to Far Field LIPUS with Higher Intensity Did Not Heal Better --- p.174
Chapter 4.4.3 --- Biphasic Effect of LIPUS Intensities on Fracture Healing --- p.176
Chapter 4.5 --- Conclusion --- p.178
Chapter Chapter 5. --- Conclusion --- p.179
Chapter 5.1 --- Differential Biological Effects of Ultrasound Fields --- p.180
Chapter 5.2 --- Far Field LIPUS exposure can be achieved by transducer diameter modulation --- p.181
Chapter 5.3 --- Biphasic Effect of Ultrasound Intensities on Fracture Healing --- p.181
Chapter 5.4 --- Mechanotransduction between Osteocyte and Osteoblastic cells --- p.182
Chapter 5.5 --- Clinical Implications --- p.183
Chapter 5.6 --- Future Investigations --- p.184
Chapter 5.7 --- Limitations --- p.184
Bibliography --- p.186
Appendix --- p.208

The objective of these experiments is to explore the effect of LIPUS on the ultraexpansion and osteogenic differentiation of harvested passage-4 HUCPV-SCs. HUCPV-SCs were divided into two groups: a treatment group that received LIPUS for 10 minutes for 1, 7, and 14 days and a control group that received a sham treatment utilizing both basic and osteogenic media. The results in basic media and osteogenic media demonstrated nonsignificant differences in cell count, ALP, DNA content, and CD90. Statistically significant expression of OSP and PCNA was observed on day 14 in LIPUS treated group. Nucleostemin expression in the LIPUS-treated group was insignificant on days 1 and 7. However, a selective increase in osteogenic markers was obtained on day 7 for ALP and OCN and on day 14 for OPN. Future experiments are required to explore the effects of different application times and/or techniques of LIPUS on the behaviour of HUCPV-SCs.
Medical Science

Le applicazioni degli ultrasuoni(US) in campo medico per i trattamenti terapeutici sono da almeno 2 decenni sotto la lente di ingrandimento da parte di numerosi gruppi di ricerca che hanno messo a punto numerosi setup sperimentali ed attraverso i quali hanno individuato diversi protocolli promettenti che inducono effetti biologici e biomeccanici sulle cellule dei tessuti trattati: le divergenze delle sperimentazioni che si possono individuare in letteratura risiedono principalmente nella dimensione della sonda ad US, il range di frequenze utilizzate, l’intensità del campo acustico, il tempo di esposizione, il tipo di tessuto trattato, lo stadio di oncogenesi delle cellule trattate e la modalità di stimolazione (contatto/distanza). Una chiara distinzione è però individuabile in base all’intensità degli US ed al conseguente andamento della temperatura durante il trattamento, infatti se quest’ultima rimane in un range di ± 2°C si può ragionevolmente ritenere che gli effetti indotti sulla struttura cellulare sono di tipo meccanico. La sonoporazione o la sonicazione sono due particolari effetti che possono condurre la cellula ad apoptosi e sono generati da un processo di cavitazione acustica (stabile o instabile) attraverso l’applicazione di un trattamento a Low Intensity (Continuous/Pulsed) Ultrasound (LICUS/LIPUS). Nel caso alternativo in cui l’aumento di temperatura sia l’effetto predominante con incrementi che possono raggiungere gli 80-100°C si parla di trattamenti ipertermici, identificabili in letteratura come High Intensity Focused Ultrasound (HIFU). Questo lavoro di tesi si focalizza sui trattamenti ad US a bassa intensità ponendosi come obiettivo finale l’individuazione di un protocollo di stimolazione efficace per indurre un processo di apoptosi/lisi/disgregazione di un agglomerato di cellule (sferoide) tumorali. Per raggiungere tale obiettivo ed associare ai risultati una correlazione tra gli effetti biofisici e biomeccanici indotti dal trattamento ad US nella cultura cellulare bersaglio e le sue proprietà costitutive meccaniche sono stati esplorati più metodi di analisi microscopica. Dai risultati delle analisi condotte è stato possibile acquisire le informazioni necessarie alla costituzione di un modello tridimensionale robusto e condurre su di esso delle analisi agli elementi finiti: simulazione di campo di pressione acustica transitorio a diverse frequenze (1,2,3,5 MHz) per la valutazione dello stato di stress generato. Al termine della fase di simulazione del protocollo da testare in vitro si è proceduto con la fase di validazione dei risultati di simulazione attraverso una sperimentazione in vitro che ha richiesto la progettazione e messa in opera di un setup ad US ad immersione dedicato alla stimolazione di culture cellulari 3D(sferoidi) di tumorali pancreatici (Panc-1). Questa fase estremamente delicata ha richiesto il costante coordinamento di un team multidisciplinare (Biologi, Ingegneri, Medici) ed interdipartimentale (Dipartimento di Ingegneria Industriale DIEF, Dipartimento di Ingegneria Dell’Informazione DINFO, Dipartimento di Medicina Sperimentale e Clinica DMSC): da questa collaborazione è nato un robusto protocollo operativo sperimentale che ha permesso di raggiungere ragguardevoli risultati di ricerca nonostante il difficile periodo pandemico. In questo elaborato saranno enucleati in modo più chiaro possibile i passi che sono stati effettuati per raggiungere gli obiettivi prefissi, questo al fine di rendere ripetibile la sperimentazione condotta anche su altre tipologie di cellule con la speranza che questa attività di ricerca possa aprire la strada verso la realizzazione e la sperimentazione di un dispositivo ad US selettivo per il trattamento percutaneo oncologico. ____________________________________________________________________________________________________________________________________________ The applications of ultrasound (US) in the medical field for therapeutic treatments have been under the magnifying glass for at least 2 decades by numerous research groups that have developed numerous experimental setups and through which they have identified several promising protocols that induce biological and biomechanical effects on the cells of the treated tissues: the divergences of the experiments that can be identified in the literature mainly reside in the size of the US probe, the range of frequencies used, the intensity of the acoustic field, the exposure time, the type of treated tissue, the stage of oncogenesis of the treated cells and the modality of stimulation (contact / distance). However, a clear distinction can be identified based on the intensity of the US and the consequent temperature trend during treatment, in fact if the latter remains in a range of ± 2 ° C it can reasonably be assumed that the effects induced on the cellular structure are of mechanical type. Sonoporation or sonication are two particular effects that can lead the cell to apoptosis and are generated by a process of acoustic cavitation (stable or unstable) through the application of a Low Intensity (Continuous / Pulsed) Ultrasound (LICUS / LIPUS) treatment. In the alternative case in which the temperature increase is the predominant effect with increases that can reach 80-100 ° C we are talking about hyperthermic treatments, identifiable in the literature as High Intensity Focused Ultrasound (HIFU). This thesis focuses on low-intensity US treatments with the ultimate goal of identifying an effective stimulation protocol to induce a process of apoptosis / lysis / disintegration of an agglomeration of tumor cells (spheroid). To achieve this objective and to associate the results with a correlation between the biophysical and biomechanical effects induced by the US treatment in the target cell culture and its mechanical constitutive properties, several methods of microscopic analysis were explored. From the results of the analysis it was possible to acquire the information necessary to build a robust three-dimensional model and conduct finite element analysis on it: simulation of a transient acoustic pressure field at different frequencies (1,2,3,5 MHz) for the evaluation of the state of stress generated. At the end of the simulation phase of the protocol to be tested in vitro, we proceeded with the validation phase of the simulation results through an in vitro experimentation which required the design and implementation of a US immersion setup dedicated to the stimulation of cultures. 3D cells (spheroids) of pancreatic tumors (Panc-1). This extremely delicate phase required the constant coordination of a multidisciplinary team (Biologists, Engineers, Doctors) and interdepartmental (DIEF, DINFO, DMSC): from this collaboration a robust experimental operational protocol was born which allowed to achieve remarkable research results despite the difficult pandemic period. In this paper the steps that have been taken to achieve the objectives will be enucleated as clearly as possible, in order to make the experimentation conducted on other types of cells repeatable with the hope that this research activity can pave the way towards the realization and experimentation of a selective US device for percutaneous oncological treatment.