Academic literature on the topic 'Paracrine signalling'

Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Angiogenesis is maintained by a balance of pro-and antiangiogenic factors. Hence, concomitantly with the upregulation of proangiogenic factors, antiangiogenic proteins ADAMTS1 and regulator of calcineurin 1 (RCAN1) were upregulated by P CM treatment of HUVECs. They were subsequently shown to limit endothelial cell network formation and proliferation in response to P CM. Finally, the role of PGF2α in angiogenesis was investigated using two in vivo models. PGF2α treatment did not increase angiogenesis in a sponge matrigel mouse model. In a xenograft mouse model, PGF2α-FP signalling increased expression of angiogenic factors in human epithelial cells and mouse stroma but this did not enhance microvessel density. Taken together, this thesis had highlighted that PGF2α-FP receptor signalling stimulates expression of pro-and antiangiogenic factors that in turn regulate endothelial cell function. However, in vivo studies demonstrate that PGF2α-FP receptor interaction does not impact on the level of angiogenesis but may control other aspects of vascular function.

Introduction: Cancer cells are affected by paracrine signalling from surrounding stromal cells. Here we investigate the role of kinases in this signalling using a model breast cancer (BC) co-culture system to identify novel paracrine signalling mechanisms supporting the growth and survival of tumour cells and potentially modulating epithelial to mesenchymal transition (EMT). Methods: The influence of paracrine signalling of the MSCs in the growth of luminal and basal-like breast cancer cells after the knock-down of human kinases, selected through a screen of a siRNA library, was investigated using a co-culture system that involves the culture of these transfected cells with or without human bone marrow-derived MSCs. An in silico analysis was also performed to investigate potential clinical relevance of these molecules. Results: The screen of the human kinase siRNA library in the MCF-7 cells co-cultured with MSCs revealed a number of kinases that seemed to be involved in the regulation of tumour growth. The knock-down of a subset, including GKAP1, CALM2, NEK7, MAPK7 and PI3KC2G, in the MCF-7, MDA-MB-231 and BT-549 cells growing alone or with MSCs resulted in the modulation of growth through autocrine or paracrine pathways and some may also be involved in the activation of the EMT pathways. Conclusion: There is a need for novel cancer biomarkers and targets to treat some forms of tumours such as the triple-negative BC, lacking a targeted therapy, or those that are resistant to the available ones. Here, the importance of the tumour microenvironment (TME) in the response to the inhibition of the targets in the cells was demonstrated. Potential therapeutic targets and pathways were presented as novel candidates for new treatments that need further investigation.

The mammalian testis is divided into two distinct compartments which carry out its principal functions. Spermatogenesis occurs within the seminiferous tubules and androgen biosynthesis primarily occurs in the interstitial space. Both these processes are entirely dependent upon the two major testicular somatic cell populations - the Sertoli and Leydig cells respectively. In human males, testicular spermatogenic and endocrine function declines during the ageing process. Of particular significance is the reported age-related decrease in Leydig cell androgen production as androgens have been suggested to play a crucial role in supporting lifelong general health in men, with low circulating testosterone linked to an increased risk of developing chronic age-related cardiometabolic diseases. However, the relationship between ageing, testicular function and disease is not fully understood, impeding the development of novel therapeutic strategies to treat age-related testicular dysfunction. In one set of studies undertaken herein, a series of novel mouse models of premature ageing were utilised to begin to dissect the process of age-related testicular degeneration. Firstly, a novel knockout-first conditional allele of a previously reported premature-ageing model driven by Cisd2 (CDGSH Iron Sulphur Domain 2) deficiency was validated and the testicular phenotype characterised and compared to that of naturally aged mice at 18-months of age. Histological analyses revealed premature testicular atrophy at 6-months of age in CISD2 deficient mice, consistent with observations of the naturally aged testis. Circulating testosterone was significantly lower in CISD2-deficient mice compared to wild-type controls at 6-months of age and the luteinising hormone/testosterone ratio was significantly elevated, indicative of compensated Leydig cell failure. mRNA expression of key genes involved in androgen production were also significantly reduced in the CISD2-deficient testis, pointing to Leydig cell dysfunction in this model of premature aging. Next, Cre/LoxP technology was used to delete Cisd2 from specific testicular cell populations to determine which cell types control/support Leydig cell function during the ageing process. Testosterone production was unaffected when Cisd2 was disrupted in either the Leydig cell population or Sertoli cell population. These observations suggest that disruption to the testicular microenvironment in which Leydig cells reside, rather than intrinsic Leydig cell ageing, may play a significant role in age-associated Leydig cell dysfunction. A second set of studies were carried out to investigate the role of leukemia inhibitory factor (LIF) signalling in the maintenance of testicular function. LIF is a pleiotropic cytokine belonging to the interleukin-6 family. In the rodent testis, LIF is expressed in fetal life and adulthood; the peritubular myoid cells thought to be the main site of production. Given their anatomical location within the testis, LIF produced by peritubular myoid cells may act on both intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, LIFR is expressed in germ cells, Sertoli cells, Leydig cells as well as testicular macrophages suggesting that LIF may be a key paracrine regulator of testicular function. However, the precise role of LIF/LIFR signalling in the testis is largely unknown. As such, models of testicular cell-specific Lifr deletion were generated using Cre/LoxP technology. Analysis of these novel models of conditional LIFR ablation revealed that LIFR is dispensable in germ cells for normal spermatogenesis. However, LIFR ablation from Sertoli cells resulted in a progressive degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules. In a final set of studies, a rat model of Leydig cell ablation-regeneration was used to determine the regenerative capacity of human adipose-derived perivascular stem cells (hAd-PSC) as a potential therapy for testicular dysfunction. Following ethane dimethanesulphonate (EDS) mediated Leydig cell ablation, primary hAd-PSCs, cultured with or without LH, IGF-1, PDGFBB, T3 and ITS supplement, were transplanted into the rat testis and Leydig cell regeneration was monitored via serial measurements of circulating luteinising hormone (LH) and testosterone. Overall, hAd- PSCs had no impact on the recovery of circulating testosterone levels. However, when pre-cultured with the cocktail of hormone/growth factor supplements, the LH spike induced by the removal of testosterone negative feedback was dampened, suggesting the transplanted cells may promote Leydig cell regeneration. Whether these cells differentiate into Leydig cells, or simply provide paracrine support to the regenerating Leydig cells remains to be determined. Although Ad-PSCs may enhance regeneration kinetics, the transplanted cells were undetectable in the testis 5 weeks post transplantation suggesting they may not survive in the context of long term xenogeneic transplantation.

Macrophages are the largest cells in the immune system and they regulate inflammatory signalling and inform cell fate decisions. Many signals, including those mediated by Tumor Necrosis Factor alpha (TNFα) converge on a few key intracellular signalling pathways, including the Nuclear Factor kappa B (NFκB) network. The NFκB signalling pathway plays a vital role in the regulation of many different cellular responses, including the production of TNFα itself, which is required to sustain and propagate immune responses to, for example, infection or tissue damage. In this thesis we report on studies-both experimental and theoretical-of the NFκB signalling pathway in macrophages. Our collaborators stimulated these cells with various doses of Lipopolysaccharide (LPS), a molecule that forms the major component of the outer membrane of Gram-negative bacteria: in these experiments it serves as a proxy for bacterial infection. The macrophages, studied in vitro, respond as they are believed to do in tissues, by secreting certain signalling molecules called cytokines: the level of secretion proved to depend on the strength of the LPS stimulus. Further, heterogeneity of macrophage signalling was observed in response to a range of LPS doses. Within individual macrophages LPS stimulation results in oscillatory behaviour of NFκB localisation-NFκB shuttles in and out of the nucleus-with an amplitude (peak nuclear concentration) that also depends on the LPS dose. Heterogeneity was also observed in cells that were stimulated with the same dose intensity. This raises an important question about how immune cells coordinate inflammatory activity in the presence of this variability. In this thesis we aim to achieve an understanding of the system through the qualitative analysis of mathematical models of it. This work explores both the parametric sensitivity and bifurcation analyses for two mathematical models of NFκB in macrophages. Parametric sensitivity analysis is used to investigate the role of parameters on the model's output, especially on certain features of the signal-peak amplitudes, inter-peak intervals and areas beneath curves-that are commonly measured in single-cell experiments. Local bifurcation analysis is conducted in order to show all the possible behaviours produced when varying parameters.

Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM). Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome. To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins. This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2). Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models. The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.

The brain-derived neurotrophic factor (BDNF) is a neurotrophin that binds to the TrkB full-length receptor (TrkB-FL), triggering cascades responsible for neuroprotection. This BDNF/TrkB-FL system is known to be impaired in Alzheimer’s disease (AD)due to an amyloid-β-mediated TrkB-FL cleavage, leading tothe formation of two fragments,a membrane-bound truncated receptor (TrkB-T’) and an intracellular domain fragment (TrkB-ICD). TrkB-ICD hastyrosine kinase activity, promotescognitive impairments,and modifiesgene expression.Notwithstanding, its intracellular clearance mechanisms remain elusive. Importantly, TrkB-ICD has been detected in cerebrospinal fluid of humans, questioning its release pathway. Interestingly, AD pathological features may be disseminated by extracellular vesicles (EVs) such as exosomes and microvesicles, which by incorporatingharmful mediators, candrive intercellular propagation of inflammatory, misfolded proteinsand toxic factors. Although AD remains as an untreated disorder, our lab designed a peptide, TAT-TrkB, capable of preventing TrkB-FL cleavage.As such,this project aimed at:i)exploreTrkB-ICD cellular clearance mechanisms;and ii)evaluate the efficacy ofTAT-TrkB in 5xFAD, an AD mouse model.Data withTrkB-ICD-expressingSH-SY5Y cells corroborated thatTrkB-ICD isa stable fragment with a half-life of approximately 6-8h. Studies evaluating the contributionof both the proteasome and autophagy pathways in TrkB-ICD clearance did not completely elucidate its degradation mechanisms,so far. We pioneeredthe discoveryof TrkB-ICD in microvesicles,and exosomesreleased by SH-SY5Y cells. This is a remarkable observation that may imply that TrkB-ICD can be disseminated among cells. propagatingits toxicity. The experiments performed to evaluate TAT-TrkB efficacyin the 5xFAD AD mouse modelwere done in the context of a pilot study and did not provide a coherent conclusion, prompting usto redesignthisin vivo study infuture work. Nevertheless, the obtained data strongly suggest that TAT-TrkB mayhave a beneficial role in learning and memory,asevaluated by the Morris Water Mazetest.

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。<br>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。<br>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。<br>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)。<br>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。<br>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。<br>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.<br>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.<br>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.<br>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).<br>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.<br>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.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Fung, Chak Hei.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 186-207).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.<br>Abstract --- p.i<br>中 文 摘 要 --- p.v<br>Publications --- p.ix<br>Acknowledgements --- p.xii<br>List of Abbreviations --- p.xiii<br>Index for Figures --- p.xvi<br>Index for Tables --- p.xviii<br>Chapter Chapter 1. --- Introduction and Literature Review --- p.1<br>Chapter 1.1 --- Long Bone Fracture - A Growing Global Challenge --- p.2<br>Chapter 1.2 --- Long-Bone Fracture - Current Management and Limitations --- p.5<br>Chapter 1.3 --- Cellular Biology of Fracture Healing --- p.7<br>Chapter 1.3.1 --- Stage 1: Inflammation --- p.7<br>Chapter 1.3.2 --- Stage 2: Soft callus formation --- p.8<br>Chapter 1.3.3 --- Stage 3: Hard callus formation --- p.9<br>Chapter 1.3.4 --- Bone Remodeling --- p.10<br>Chapter 1.4 --- Biophysical Stimulation to Bone --- p.13<br>Chapter 1.5 --- Low-intensity Pulsed Ultrasound --- p.14<br>Chapter 1.5.1 --- Application of LIPUS on Fracture Healing --- p.14<br>Chapter 1.5.2 --- Physics of Ultrasound --- p.16<br>Chapter 1.5.3 --- Ultrasound Parameters --- p.20<br>Chapter 1.5.3.1 --- Ultrasound Frequency --- p.20<br>Chapter 1.5.3.2 --- Duty Cycle --- p.22<br>Chapter 1.5.3.3 --- Intensity --- p.22<br>Chapter 1.5.3.4 --- Angle of Incidence --- p.24<br>Chapter 1.5.3.5 --- Ultrasound Field --- p.25<br>Chapter 1.5.4 --- Possible Mechanism of LIPUS on Tissue --- p.31<br>Chapter 1.5.4.1 --- Thermal Effect --- p.31<br>Chapter 1.5.4.2 --- Cavitation --- p.31<br>Chapter 1.5.4.2 --- Acoustic Streaming --- p.32<br>Chapter 1.5.4.3 --- Frequency Resonance Hypothesis --- p.32<br>Chapter 1.5.4.4 --- Micromotion --- p.33<br>Chapter 1.6 --- Possible Cellular and Molecular Mechanotransduction Mechanism of LIPUS --- p.34<br>Chapter 1.6.1 --- Osteocyte: Potential Mechanosensor --- p.34<br>Chapter 1.6.3 --- Osteocyte-osteoblast mechanotransduction --- p.39<br>Chapter 1.7 --- Hypothesis --- p.39<br>Chapter 1.8 --- Study Plan and Objectives --- p.40<br>Chapter 1.8.1 --- Study Plan --- p.40<br>Chapter 1.8.2 --- Objectives --- p.42<br>Chapter Chapter 2. --- Characterization of Ultrasound Field Distances on Rat Fracture Model --- p.43<br>Chapter 2.1 --- Introduction --- p.44<br>Chapter 2.2 --- Material & Methods --- p.47<br>Chapter 2.2.1 --- Closed Femoral Shaft Fracture Model in Rat --- p.47<br>Chapter 2.2.2 --- Ultrasound Field Distances Setup --- p.51<br>Chapter 2.2.3 --- Animal Grouping & LIPUS Treatment Protocol --- p.53<br>Chapter 2.2.4 --- Assessments --- p.56<br>Chapter 2.2.4.1 --- Radiological Analysis --- p.56<br>Chapter 2.2.4.2 --- Micro-computed Tomography --- p.61<br>Chapter 2.2.4.3 --- Histomorphometry --- p.64<br>Chapter 2.2.4.4 --- Mechanical Testing --- p.66<br>Chapter 2.2.4.5 --- Statistical Analysis --- p.66<br>Chapter 2.3 --- Results --- p.68<br>Chapter 2.3.1 --- Radiological Analysis --- p.71<br>Chapter 2.3.2 --- MicroCT --- p.77<br>Chapter 2.3.3 --- Histomorphometry --- p.82<br>Chapter 2.3.4 --- Mechanical Testing --- p.85<br>Chapter 2.4 --- Discussion --- p.87<br>Chapter 2.4.1 --- Far Field LIPUS Enhances Mechanical Properties of Healing Callus --- p.88<br>Chapter 2.4.2 --- Mid-near field and Near field LIPUS Enhances Woven Bone Formation --- p.90<br>Chapter 2.4.3 --- The Biological Effects of LIPUS with Different Ultrasound Field Exposure --- p.94<br>Chapter 2.5 --- Conclusion --- p.97<br>Chapter Chapter 3. --- The Effect of Ultrasound Field Distances on Bone Cells --- p.100<br>Chapter 3.1 --- Introduction --- p.101<br>Chapter 3.2 --- Material & Methods --- p.102<br>Chapter 3.2.1 --- Cell Culture --- p.102<br>Chapter 3.2.2 --- Ultrasound Field Distances Setup & Treatment Protocol --- p.102<br>Chapter 3.2.2 --- Immunostaining of β-catenin --- p.106<br>Chapter 3.2.3 --- Wound Healing Assay --- p.109<br>Chapter 3.2.4 --- BrdU Cell proliferation Assay --- p.111<br>Chapter 3.2.5 --- Alkaline phosphatase activity assay --- p.112<br>Chapter 3.2.6 --- Alizarin calcium nodule staining --- p.113<br>Chapter 3.2.7 --- CM characterization - PGE₂ ELISA assay --- p.114<br>Chapter 3.2.8 --- CM characterization - nitrite assay --- p.114<br>Chapter 3.2.9 --- Statistical Analysis --- p.115<br>Chapter 3.3 --- Results --- p.116<br>Chapter 3.3.1 --- Immunostaining of β-catenin --- p.116<br>Chapter 3.3.2 --- Wound healing assay --- p.119<br>Chapter 3.3.3 --- BrdU Cell proliferation Assay --- p.119<br>Chapter 3.3.4. --- Alkaline phosphatase activity assay --- p.122<br>Chapter 3.3.5 --- Alizarin calcium nodule staining --- p.122<br>Chapter 3.3.6. --- CM characterization - PGE₂ ELISA assay --- p.125<br>Chapter 3.3.7 --- CM characterization - nitrite assay --- p.125<br>Chapter 3.4 --- Discussion --- p.128<br>Chapter 3.4.1 --- The Osteogenic Effect of Far Field LIPUS-Osteocyte Conditioned Medium --- p.128<br>Chapter 3.4.2. --- Mechanisms of Mechanotransduction between Osteocyte and Osteoblast --- p.131<br>Chapter 3.5 --- Conclusion --- p.136<br>Chapter Chapter 4. --- Rat Fracture Exposed to Far Field LIPUS by Modulating Ultrasound Transducer Diameter --- p.139<br>Chapter 4.1 --- Introduction --- p.140<br>Chapter 4.2 --- Material & Methods --- p.143<br>Chapter 4.2.1 --- Closed Femoral Shaft Fracture Model in Rat --- p.143<br>Chapter 4.2.2 --- Ultrasound Field Distances Setup & Treatment Protocol --- p.143<br>Chapter 4.2.3 --- Assessments --- p.148<br>Chapter 4.2.3.1 --- Radiological Analysis --- p.148<br>Chapter 4.2.3.2 --- Micro-computed Tomography --- p.148<br>Chapter 4.2.3.3 --- Histomorphometry --- p.149<br>Chapter 4.2.3.4 --- Mechanical Testing --- p.151<br>Chapter 4.2.3.5 --- ex vivo Temperature Measurements --- p.151<br>Chapter 4.2.3.6 --- Statistical Analysis --- p.151<br>Chapter 4.3 --- Results --- p.152<br>Chapter 4.3.1 --- Radiological Analysis --- p.154<br>Chapter 4.3.2 --- MicroCT --- p.157<br>Chapter 4.3.3 --- Histomorphometry --- p.160<br>Chapter 4.3.4 --- Mechanical Testing --- p.166<br>Chapter 4.3.5 --- ex vivo Temperature Measurement --- p.168<br>Chapter 4.4 --- Discussion --- p.170<br>Chapter 4.4.1 --- Far field LIPUS Setup by Transducer Diameter Modulation Enhanced Fracture Healing --- p.170<br>Chapter 4.4.2 --- Fractures Exposed to Far Field LIPUS with Higher Intensity Did Not Heal Better --- p.174<br>Chapter 4.4.3 --- Biphasic Effect of LIPUS Intensities on Fracture Healing --- p.176<br>Chapter 4.5 --- Conclusion --- p.178<br>Chapter Chapter 5. --- Conclusion --- p.179<br>Chapter 5.1 --- Differential Biological Effects of Ultrasound Fields --- p.180<br>Chapter 5.2 --- Far Field LIPUS exposure can be achieved by transducer diameter modulation --- p.181<br>Chapter 5.3 --- Biphasic Effect of Ultrasound Intensities on Fracture Healing --- p.181<br>Chapter 5.4 --- Mechanotransduction between Osteocyte and Osteoblastic cells --- p.182<br>Chapter 5.5 --- Clinical Implications --- p.183<br>Chapter 5.6 --- Future Investigations --- p.184<br>Chapter 5.7 --- Limitations --- p.184<br>Bibliography --- p.186<br>Appendix --- p.208