Charting our sustainability journey within the Division of Surgery and Interventional Science at University College London

This article describes a case study of our journey to running more sustainable labs within the Division of Surgery and Interventional Science at University College London (UCL), London, United Kingdom. Through the setting up of a self-assessment team within the division, we asked the key question, ‘what does sustainability mean in science and how will we apply this to our academic Division?’ Our division’s sustainability team took on the challenges to tackle unsustainable practise, primarily within our laboratories. By considering and implementing simple steps within our research department, we have reduced lab waste as well as decreased our overall carbon emission. We have clarified our hallmarks of sustainability and seek to share our changed practices to provide clear and easy guidance for how to make medical research divisions sustainable based on the actions taken in our labs. This study provides guideline on how to make academic research more sustainable by describing simple steps to implement in the laboratories. These steps were described using Division of Surgery and Interventional Science at UCL as a case study. The division’s sustainability team develops sustainable lab practices, which has led to reduction in lab waste and carbon emissions

Enhanced Biomimetics of Three-Dimensional Osteosarcoma Models: A Scoping Review

This scoping review evaluated 3D osteosarcoma (OS) models’ biomimicry, examining their ability to mimic the tumour microenvironment (TME) and their drug sensitivity. Adhering to PRISMA-ScR guidelines, the systematic search revealed 293 studies, with 70 selected for final analysis. Overall, 64% of 3D OS models were scaffold-based, compared to self-generated spheroid models. Scaffolds generated using native matrix were most common (42%) with collagen I/hydroxyapatite predominating. Both scaffold-based and scaffold-free models were used equally for drug screening. The sensitivity of cancer cells in 3D was reported to be lower than that of cells in 2D in ~90% of the drug screening studies. This correlates with the observed upregulation of drug resistance. OS cells cultured in extracellular matrix (ECM)-mimetic scaffolds and native biomaterials were more resistant than cells in 2D. Co-cultures of OS and stromal cells in 3D models enhanced osteogenic differentiation, ECM remodelling, mineralisation, and angiogenesis, suggesting that tumour–stroma crosstalk promotes disease progression. Seven studies demonstrated selective toxicity of chemotherapeutics towards OS cells while sparing stromal cells, providing useful evidence for developing biomimetic tumour–stroma models to test selective drug toxicity. In conclusion, this review highlights the need to enhance biomimicry in 3D OS models for TME recapitulation, especially in testing novel therapeutics. Future research should explore innovative 3D biomimetic models, biomaterials, and advancements in personalised medicine

Associated changes in stiffness of collagen scaffolds during osteoblast mineralisation and bone formation

OBJECTIVE: Engineering bone in 3D is important for both regenerative medicine purposes and for the development of accurate in vitro models of bone tissue. The changing material stiffness of bone tissue had not yet been monitored throughout the process of mineralisation and bone nodule formation by osteoblasts either during in vitro engineering or in development perspective. RESULTS: Within this short research note, stiffness changes (Young's modulus) during in vitro bone formation by primary osteoblasts in dense collagen scaffolds were monitored using atomic force microscopy. Data analysis revealed significant stiffening of 3D bone cultures at day 5 and 8 that was correlated with the onset of mineral deposition (p < 0.00005)

RANKL neutralisation prevents osteoclast activation in a human in vitro ameloblastoma-bone model

Ameloblastoma is a benign, locally invasive epithelial odontogenic neoplasm of the jaw. Treatment of choice is jaw resection, often resulting in significant morbidity. The aim of this study was to recapitulate ameloblastoma in a completely humanised 3D disease model containing ameloblastoma cells, osteoblasts and activated osteoclasts to investigate the RANKL pathway within the ameloblastoma stromal environment and its response to the RANKL antibody denosumab. In vitro bone was engineered by culturing human osteoblasts (hOB) in a biomimetic, dense collagen type I matrix, resulting in extensive mineral deposits by day 21 forming alizarin red positive bone like nodules throughout the 3D model. Activated TRAP + human osteoclasts were confirmed through the differentiation of human CD14+ monocytes after 10 days within the model. Lastly, the ameloblastoma cell lines AM-1 and AM-3 were incorporated into the 3D model. RANKL release was validated through TACE/ADAM17 activation chemically or through hOB co-culture. Denosumab treatment resulted in decreased osteoclast activation in the presence of hOB and ameloblastoma cells. These findings stress the importance of accurately modelling tumour and stromal populations as a preclinical testing platform

Modelling stromal compartments to recapitulate the ameloblastoma tumour microenvironment

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling

Mapping human serum induced gene networks as a basis for the creation of biomimetic periosteum for bone repair

The periosteum is a highly vascularised, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions, and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation. As such, this study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment. The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum. Master regulators of transcriptional networks were identified and an optimised periosteal derived-growth factor cocktail (PD-GFC; containing β-Estradiol, FGF2, TNFα, TGFβ, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regards to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a 3D collagen-type-1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a downregulated Wnt and TGFβ signature and an upregulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature. This study highlights the first stage in the development of a biomimetic periosteum which may have applications in bone repair

Modelling stromal compartments to recapitulate the ameloblastoma tumour microenvironment

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling

Renal tumouroids: challenges of manufacturing 3D cultures from patient derived primary cells.

Recent advancements in 3D in vitro culture have allowed for the development of cancer tissue models which accurately recapitulate the tumour microenvironment. Consequently, there has been increased innovation in therapeutic drug screening. While organoid cultures show great potential, they are limited by the time scale of their growth in vitro and the dependence upon commercial matrices, such as Matrigel, which do not allow for manipulations of their composition or mechanical properties. Here, we show a straightforward approach for the isolation and culture of primary human renal carcinoma cells and matched non-affected kidney. This approach does not require any specific selection for cancer cells, and allows for their direct culture in amenable 3D collagen-based matrices, with the preservation of cancer cells as confirmed by NGS sequencing. This method allows for culture of patient-derived cancer cells in 3D microenvironment, which can be used for downstream experimentation such as investigation of cell-matrix interaction or drug screening. [Abstract copyright: © 2022. Crown.

Stromal cells regulate mechanics of tumour spheroid

The remarkable contractility and force generation ability exhibited by cancer cells empower them to overcome the resistance and steric hindrance presented by a three-dimensional, interconnected matrix. Cancer cells disseminate by actively remodelling and deforming their extracellular matrix (ECM). The process of tumour growth and its ECM remodelling have been extensively studied, but the effect of the cellular tumour microenvironment (TME) has been ignored in most studies that investigated tumour-cell-mediated ECM deformations and realignment. This study reports the integration of stromal cells in spheroid contractility assays that impacts the ECM remodelling and invasion abilities of cancer spheroids. To investigate this, we developed a novel multilayer in vitro assay that incorporates stromal cells and quantifies the contractile deformations that tumour spheroids exert on the ECM. We observed a negative correlation between the spheroid invasion potential and the levels of collagen deformation. The presence of stromal cells significantly increased cancer cell invasiveness and altered the cancer cells' ability to deform and realign collagen gel, due to upregulation of proinflammatory cytokines. Interestingly, this was observed consistently in both metastatic and non-metastatic cancer cells. Our findings contribute to a better understanding of the vital role played by the cellular TME in regulating the invasive outgrowth of cancer cells and underscore the potential of utilising matrix deformation measurements as a biophysical marker for evaluating invasiveness and informing targeted therapeutic opportunities