Saudi Cultural Missions Theses & Dissertations
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Item Restricted Bioengineering of transcriptional elements driving MMP13 gene in skeletal development(University of Liverpool, 2024-08) AlSalhi, Sara Ibrahim; Bou-Gharios, GeorgeMmp13 is a primary catabolic factor involved in cartilage degradation through its ability to cleave collagen type II and other cellular components. In addition to being necessary for the formation of various cells, organs, and tissues, Mmp13 expression is regulated transcriptionally by two main elements: the proximal promoter and distal enhancers. This study aims to identify the transcriptional elements that regulate Mmp13 gene expression. Identification of novel Mmp13 enhancers was conducted in silico using the Encyclopaedia of DNA Elements (ENCODE), based on histone modifications (Limb H3K4me1 and Limb H3K27Ac), fibroblast coverage, chondrocyte, and embryonic limb regulatory elements from public ChIP-Seq data, and evolutionarily conserved sequences, in addition to transcription factor profiles of Runx2 and vitamin D. All prospective Mmp13 enhancer sequences were analysed using three different software tools: CIIIDER, TRANSFAC, and JASPAR, for the prediction of transcription factor binding sites. Each enhancer sequence was cloned upstream of the Hsp68 silenced promoter and lacZ gene to create a β-galactosidase reporter construct, which was then used to generate transgenic mice. Embryos were collected at E15.5 and tested for lacZ gene expression and tissue expression analysis. Constructs were also transfected in vitro into pre-osteoblasts (MC3T3- E1), NIH3T3 mouse embryo fibroblasts, human chondrocytes (SW1353), and primary chondrocytes extracted from OA patients. Among the seven tested Mmp13 enhancer regions, strong skeletal element expression was detected in the region from -21.9 to -21.1 kb, which overlaps with Runx2 and Sox9 binding sites. Other enhancers revealed some skeletal element activity but were not as prominent. Expression in various tissues and organs, including skin, was observed in multiple regions. In contrast, sequences aligning with the highest peaks of Runx2 at -29 kb and -32.5 kb did not show significant expression. In vitro, Mmp13-transfected enhancer sequences demonstrated enzyme activity, with the highest responses observed in chondrocyte and human cells at -10 kb and -29 kb regions, along with -21.4 kb that indicate a potential regulatory influence. Comparisons of potential enhancers in mouse embryos highlighted the sequences in the intronic 5', -10 kb, -19.2 kb, and -21.4 kb regions as significant Mmp13 enhancers regulating gene expression but not -29 and -32.5 kb regions. Identifying these specific enhancers could lead to targeted therapeutic strategies to modulate MMP13 activity, potentially slowing or preventing cartilage degradation in degenerative diseases.18 0Item Restricted The influence of ECM on mesenchymal stromal/stem cell activity and their interactions with monocytes/macrophages(University of Cambridge, 2024-03-23) Alkhrayef, Mohammad Nasser M; Birch, Mark; McCaskie, AndrewOsteoarthritis is a prevalent degenerative condition affecting synovial joints, resulting in pain, stiffness, and difficulty with movement. Current treatments mainly alleviate symptoms without effectively regenerating the lost cartilage tissue. An early surgical intervention for focal cartilage damage, known as marrow stimulation or microfracture, aims to repair this damage by promoting endogenous healing. However, the resulting repair tissue often lacks the properties of native cartilage, raising questions about the long-term effectiveness of this approach. Therefore, to enhance the outcome of this osteochondral repair approach, it is necessary to understand the biological mechanisms involved in the healing process following microfracture. Osteochondral repair is a complex interplay of cellular interactions and changing extracellular matrix (ECM) environment. This thesis aimed to extend the current understanding of the biology underpinning this process, focusing on the interaction between mesenchymal stromal/stem cells (MSCs) and monocytes/macrophages within dynamically changing ECM proteins found at the injury site. We hypothesized that this interaction plays a pivotal role in determining the outcome of tissue repair. In our investigation, we first characterised the deposition and remodelling patterns of key ECM proteins, fibrin and collagen type 1, during osteochondral injury repair in C57BL/6 mice. Our findings revealed that fibrin was the predominant ECM protein in the initial seven days post-injury, which was subsequently gradually replaced by collagen type 1. At eight weeks post-injury, the tissue repair outcome at the articular surface was found to contain collagen type 1, perhaps representing an immature form of hyaline cartilage. Subsequent in vitro studies, using 3D ECM models constructed from fibrin and collagen type 1, demonstrated that these environments significantly modulate MSC morphology, proliferation, migration, and immunomodulatory activity. Notably, MSCs within these 3D environments exhibit differential responses to pro-inflammatory stimuli, with the ECM potentially acting as a reservoir for secreted cytokines and growth factors, orchestrating cellular activities over time. Three molecular mechanisms, the TNFα/NFkB pathway, TNFα/JNK/AP1 pathway, and the potential involvement of ROCK, were identified as specific effectors of MSC immunomodulatory activity within these environments. Further investigations into the crosstalk between MSCs and monocytes in controlled 3D ECM environments revealed a distinct M2 macrophage phenotype, characterized as CD206+ MerTK- CD163- CD209-. This subpopulation displayed enhanced expression of IL10, IL6, and IL8. Moreover, conditioned media from these co-cultures influenced chondrocyte migration and chondrogenesis, with TGFβ1 playing a pivotal role in these observations. In conclusion, this thesis underscores the profound influence of the ECM on cellular interactions during osteochondral repair. The insights gained pave the way for innovative therapeutic strategies, potentially enhancing tissue repair outcomes and offering improved treatments for conditions like osteoarthritis.15 0Item Restricted Understanding the Structural and the Mechanical Properties of Bone(Saudi Digital Library, 2023-12-12) Almoshawah, Yasser Ali H; Rehman, Ihtesham Ur; Dall’Ara, EnricoOsteoarthritis (OA) is one of the most common chronic diseases characterised by a disorder in the subchondral bone (SB), cartilage damage, and osteophyte formation. Due to an inadequate understanding of the mechanism of disease pathology, no treatment is currently available to effectively prevent the initiation or progression of OA, and severe treatment modalities, such as hip joint replacement, are currently available. A better understanding of the chemical and mechanical properties of bone will also help improve OA’s diagnosis. This study aims to investigate the chemical properties of SB from the femoral head (FH) of patients with OA through an invasive and label-free approach. Vibrational spectroscopy has shown the potential to provide diagnostic information. A combination of Raman, Fourier-Transform Infrared (FTIR) spectroscopic, and Photoacoustic Fourier Transform Infrared Spectroscopy (FTIR-PAS) methods were used for the chemical analysis of samples. Principal Component Analysis (PCA) was used to identify variations within different tissue of OA bone. Linear Discriminant Analysis (LDA) was used to predict pathogenic markers with high sensitivity (Sn) and specificity (Sp). The combination of Infrared and Raman spectroscopy with chemometrics were very helpful in identifying new spectral markers to differentiate OA bone samples. Initially, preliminary studies were conducted on bovine bones, which are almost comparable to human bones. They were applied on Raman and FTIR to study the chemical composition concerning the different cutting directions to prevent mistakes and enhance the primary study. For Raman, the PCA bovine result showed a perfect clustering, with PC-1 and PC-2 accounting for 92% of the variation, resulting in excellent Sn and Sp of 100%. The results for FTIR also exhibited perfect clustering, with PC-1 and PC-4 accounting for 80% of the variance, resulting in 100% Sn and Sp. Raman, FTIR and FTIR-PAS have identified structural and compositional changes in OA compared to tissue-specific (subregion). Significant statistical differences were detected among the bone types, including organic and inorganic composites. The results of the PCA in all vibrational spectroscopy showed that the PCA had good clustering, accounting for 74, 75, and 86% of the variation for Raman, FTIR and FTIR-PAS, respectively, leading to excellent Sn and Sp of 100%, representing the whole spectrum. Furthermore, as the aetiology and pathogenesis of OA are not fully understood, measuring the mechanical properties of bone by applying nanoindentation to FH to extract the mechanical properties is essential in order to understand the disease profile. The mechanical results show that the reduced modulus (𝐸𝑟) and the hardness (H) averaged out to be (16.07±3.05 GPa) and (0.56±0.107 GPa), respectively. The average elastic modulus (𝐸𝑏) of bone was measured to be (14.84±2.85 GPa), whereas the indentation modulus (E_ind) was (16.31±3.14 GPa). Compared to the other bone types, the osteophyte (Osteo) bone has the lowest value, while the cortical bone (Cort) has the highest value. The parameters in RS and FTIR confirm that increasing mineralisation ratios in bone types were correlated with a decreased 𝐸𝑏 and vice versa. In conclusion, vibrational spectroscopy is a highly effective method for identifying chemical changes associated with different subtypes of bone tissue disease. This study confirms its significance in evaluating both chemical and mechanical changes in cases of severe OA affecting the human FH helping to understand the reasons for the disease process and enable an improved treatment modality. Furthermore, these findings will assist the research community in identifying regions of the skeleton where the local physical and chemical properties of bone, in addition to the mechanical properties, should be characterised during the preclinical optimisation process of treatments for skeletal diseases.31 0Item Restricted Emulsion assisted nonsteroidal anti-inflammatory drug delivery system for the osteoarthritis treatment(Saudi Digital Library, 2023-07-24) Saeedi, Tahani; Prokopovich, PolinaOsteoarthritis (OA) is one of the considerable chronic health conditions worldwide. In the UK, 8.75 million people were receiving OA treatment. Currently, there are no disease-modifying drugs, and treatment relies on the management of pain associated with OA. The delivery of OA drugs is challenging because the cartilage is dense, avascular (i.e., has no blood vessels), and relies on diffusion to transport nutrients and drugs to the cells. Moreover, cartilage matrix is composed of anionic proteoglycans that can repel negatively charged drugs, making it difficult for OA drugs to penetrate through the matrix and reach the target cells. Additionally, most OA drugs are hydrophobic, so an appropriate technique is required to deliver them inside cartilage. A novel and efficient emulsion-based local delivery of OA drugs coated with poly beta-amino ester polymers (POLY-X) into cartilage was proposed. In this project, nonsteroidal anti-inflammatory drugs as a model drug for OA treatment were examined. The characterization of the developed delivery system and its efficacy in native (non treated) and glycosaminoglycan (GAG) depleted cartilage was assessed by measuring the zeta potential and size, amount of drug uptake and retention in the cartilage. In addition, the ability of the developed drug delivery system to inhibit cytokine (IL-1α) induced glycosaminoglycan and collagen degradation, as well as the loss of cell viability in cartilage explants were tested. The data showed that the developed emulsion delivery system exhibited enhanced and prolonged drug localisation not only on non-treated cartilage tissues but also on GAG depleted sample resulting in a higher amount of drug uptake and retention in cartilage compared to the control, and the difference was statistically significant (p < 0.05). Furthermore, the developed emulsion delivery system protects the viability of the chondrocyte cell and provides a significant increased (p <0.01) in glycosaminoglycan and collagen content compared to IL-1α treated cartilage and similar to untreated control. The percentage of sGAG and collagen loss was 3 to 5 times higher in IL-1α treated cartilage compared to the untreated and developed drug delivery system treated groups. This study proved that modified emulsion-based therapy could provide a substantial improvement in the treatment of OA to maintain cartilage properties and improve OA outcomes.15 0Item Restricted Optimising Contextual Factors in the Practitioner-Patient Encounter in the Management of Osteoarthritis(University of Nottingham, 2024-08-31) Ismail, Ayah; Zhang, Weiya; Doherty, Michael; Hall, MichelleAbstract Background Contextual factors (CFs) related to the patient, healthcare practitioners, and their therapeutic relationship are integral to the overall treatment effect of any given intervention. In osteoarthritis (OA), around 75% of the treatment effect is directly attributable to CFs. Identifying and understanding the role of CFs may encourage healthcare practitioners to develop and enhance the contextual aspects of care, and thus enhance the overall treatment benefit. Objectives The overall aim of this research project is to develop a contextual enhancement package (CEP) that can be used to optimise the management of OA. To achieve this aim, the studies in this thesis aimed to realise the following objectives: [1] to identify and evaluate the current evidence for modifiable CFs that can improve clinical outcomes using quantitative systematic review and meta-analysis of randomised controlled trials (RCTs); [2] to explore and understand the experience and perspectives of patients and health practitioners about contextual enhancers in consultations for OA using qualitative systematic review and meta-aggregation; and [3] to obtain views and perspectives from clinicians, researchers, and public and patients on the identified CFs using an online survey and Public and Patient Involvement and Evaluation (PPI/E) meetings. Methods Quantitative systematic review: A systematic search was carried out, up until April 18th, 2019, on the following databases: MEDLINE via Ovid, EMBASE, AMED, PsycINFO and Cochrane library. RCTs comparing contextual enhanced interventions versus non-enhanced control in adults for any health conditions were searched. The outcomes included both self-reported outcomes and objectively measured outcomes. The effect size and 95% confidence interval were calculated using the standardised mean difference. Risk of bias was evaluated using the modified Cochrane tool. The random effects model was used to pool the results. The GRADE approach was used to assess the confidence in the body of evidence for each outcome assessed. Qualitative systematic review: A systematic search was conducted between March 15 and May 18, 2020, on the following databases: MEDLINE via Ovid, EMBASE, AMED, PsycINFO and CINAHL. The search for unpublished studies included ProQuest Dissertations and Google Scholar. The search was not limited to any language or publication year. The Joanna Briggs Institute (JBI) methodology for quality assessment, study selection, data extraction and synthesis were used. Findings were assessed for credibility, categorised based on similarity in meaning and subjected to a meta-aggregation. The ConQual approach was used to assess the confidence of the synthesised findings. Stakeholders’ involvement: An online survey was conducted using Microsoft Forms software. The responses to the survey were collected between September 20 and October 15, 2021. Results were tabulated and analysed utilising Microsoft Excel. The study sought anonymous stakeholders’ ratings and opinions. The survey involved a Likert scale question to rate the importance of each of the eight contextual factors identified in this project and an optional open-ended question about additional contextual factors related to practitioner-patient interaction that need to be considered. PPI/E meetings: The PPI/E process in this research project took two forms of involvement. In the early stages of the project, in November 2019, the in-person PPI/E meeting aimed to consider the relevance of the research topic to the public and patients. The meeting communicated the research information (i.e., research question and topic) to patients with OA. Whereas, at the later stage of the research, in September 2021, patients exchanged information and participated by providing their opinion and input on the research outcomes in the online meeting. Results Quantitative systematic review: Of 3928 records generated from the systematic search, 25 trials (5632 participants) met the inclusion criteria, and 20 were included in this meta-analysis. Conditions studied included musculoskeletal [6], cardiovascular [3], asthma [2], irritable bowel syndrome [1], diabetes [1], chronic pain [1], acute pain [3], gynaecological conditions requiring day-care surgery [1], postoperative nausea [1], and in GP or hospital-based patients [6]. Three CFs were identified from these trials: empathy, patient involvement and positive communication. All were found to be effective for patient experience (i.e., satisfaction) (SMD 0.34; 95% CI 0.27, 0.42). Positive communication was also effective for symptoms (SMD 0.17, 95%CI 0.06, 0.28) but not objective outcomes (SMD 0.10, 95%CI -0.14, 0.34). According to the GRADE guidelines for assessing confidence in the findings of systematic reviews of interventions, the certainty of the evidence was rated low for symptoms and objective outcomes and very low for patient experience outcomes. Qualitative systematic review: Of 1808 records generated from the systematic search of databases and grey literature, eight studies were included in the meta-aggregation. All included papers were moderate to high quality based on the JBI qualitative critical appraisal tool. Meta-aggregation generated three synthesised findings. According to the ConQual criteria for assessing confidence in qualitative review findings, all the synthesised findings' level of evidence was rated as moderate. The key, potentially modifiable, factors identified were empathy and positive communication; clear and relevant information provided by the health practitioner; patient expectation concerning their outcome and the consultation experience; active involvement of the patient in the consultation; sufficient consultation time; easy access to consultations; and health providers confidence. Stakeholder’s involvement: Fifty healthcare providers from various professions and four patients with OA responded to the online survey. The healthcare providers’ professions included physicians, physiotherapists, health researchers, and podiatrist. All the respondents answered the Likert scale question, and 39 answered the optional open-ended question. The stakeholders’ importance rating for each of the eight contextual factors identified from the quantitative and qualitative systematic reviews was high. Healthcare providers and patients with OA considered all factors essential and expanded their responses about how important these factors are in the open-ended question. PPI/E meetings: The first meeting confirmed the importance and relevance of the research topic to a group of OA patients. Also, the PPI suggested some CFs (i.e., regular follow-up and referral) that were considered later in developing the search strategy for the qualitative systematic review. The second meeting obtained the PPI inputs on the CFs identified from previous reviews in the research project. The PPI/E supported the delivery of all the factors and suggested tailoring the factors to patient needs. Conclusion Eight contextual factors have been identified according to their therapeutic effects, clinical importance and stakeholders’ perspectives. They are ready to be integrated to form a CEP. Further studies will be undertaken to develop an educational programme, test the feasibility of delivering CEP, and assess the clinical effectiveness and cost-effectiveness of CEP in people with osteoarthritis at the first instance.12 0