A Visual, In-Expensive, and Wireless Capillary Rheometer for Characterizing Wholly-Cellular Bioinks.

Rheological measurements with in situ visualization can elucidate the microstructural origin of complex flow behaviors of an ink. However, existing commercial rheometers suffer from high costs, the need for dedicated facilities for microfabrication, a lack of design flexibility, and cabling that complicates operation in sterile or enclosed environments. To address these limitations, a low-cost ($300) visual, in-expensive and wireless rheometer (VIEWR) using 3D-printed and off-the-shelf components is presented. VIEWR measurements are validated by steady-state and transient flow responses for different complex fluids, and microstructural flow profiles and evolution of yield-planes are revealed via particle image velocimetry. Using the VIEWR, a wholly-cellular bioink system comprised of compacted cell aggregates is characterized, and complex yield-stress and viscoelastic responses are captured via concomitantly visualizing the spatiotemporal evolution of aggregate morphology. A symmetric hyperbolic extensional-flow geometry is further constructed inside a capillary tube using digital light processing. Such geometries allow for measuring the extensional viscosity at varying deformation rates and further visualizing the alignment and stretching of aggregates under external flow. Synchronized but asymmetric evolution of aggregate orientation and strain through the neck is visualized. Using varying geometries, the jamming and viscoelastic deformation of aggregates are shown to contribute to the extensional viscosity of the wholly-cellular bioinks.

Rotational thromboelastometry values across age groups in all trauma patients presenting to a level 1 trauma centre: An observational study.

OBJECTIVES: To describe rotational thromboelastometry (ROTEM) values (FIBTEM A5, EXTEM A5 and EXTEM CT) across age groups and assess for a statistical trend; and to determine whether any trend in ROTEM values is affected by severity of injury and packed red blood cells (PRBC) requirement. METHODS: Retrospective observational study at a level 1 trauma centre in Queensland, Australia. A total of 1601 consecutive trauma patients presenting to the ED. ROTEM data described included FIBTEM A5, EXTEM A5 and EXTEM CT. These values are described by age group (≤30 years, 31-45 years, 46-60 years, 61-75 years and >75 years), Injury Severity Score (ISS) category (<12, ≥12, <25 and ≥25) and number of PRBCs transfused in the first 24 h of admission (0 units, 1-4 units, 5-9 units and ≥10 units). RESULTS: The median age of participants was 37 years (interquartile range [IQR] 25-54 years), with 48.2% of patients had severe trauma (ISS >12) and 13.2% receiving at least one unit of PRBC in the first 24 h of admission. Median (IQR) values for FIBTEM A5, EXTEM A5 and EXTEM CT were 13 mm (10-16 mm), 45 mm (40-49 mm) and 62 s (56-71 s), respectively. A test for trend over progressive age groups showed an increase in FIBTEM A5 (P < 0.001) and EXTEM A5 values (P < 0.001) and a decrease in EXTEM CT values (P < 0.001). CONCLUSION: The present study demonstrated a pattern of increasing coagulability, as defined by ROTEM, with increasing age group in trauma patients, even among the severely injured. Further investigation is required to determine the clinical impact of these findings on both the ROTEM-guided management and longitudinal outcomes of these patients and whether an age-specific approach is beneficial.

Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.

Uptake of breast reconstruction following mastectomy: a Gold Coast experience.

BACKGROUND: The breast reconstruction (BR) rate for women undergoing mastectomy for breast cancer management is 18% in Australia. The Australian Access to Breast Reconstruction Collaborative Group recommends that all women should have access to BR. This study presents BR uptake and outcomes from a breast surgical unit. METHODS: A retrospective observational study identified women who had curative mastectomy for breast cancer between 1 January 2016 and 31 December 2021. Patient factors and surgical complications were compared between BR and no BR (NBR) patients. RESULTS: Out of 929 women who had a curative mastectomy, 34% underwent reconstruction. Of this, 89% were immediate, and 11% were delayed. Reconstruction increased from 27% (2016) to 35% (2021). During this time, 588 women had a discussion for BR documented at their initial consultation, 58 after initial surgery and 283 were not documented. The rate of discussion prior to mastectomy increased from 38% to 74%. Women who had BR were more likely to be younger, premenopausal and less likely to be diabetic. Complications requiring return to theatre were higher in reconstructed women (13% vs. 7%). Overall, infected seroma, cellulitis requiring intravenous antibiotics and haematoma requiring drainage were comparable between both groups. CONCLUSION: Our unit achieved a reconstruction rate of 34%, which is higher than national and international averages. Open discussion of reconstruction is crucial for women to make an informed decision. Further prospective studies exploring barriers to timely reconstruction will improve uptake of BR surgery and allow prioritization of BR services in Australia.

Clinical outcomes, Kadish-INSICA staging and therapeutic targeting of somatostatin receptor 2 in olfactory neuroblastoma.

INTRODUCTION: Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal region. We provide a comprehensive analysis of this malignancy with molecular and clinical trial data on a subset of our cohort to report on the potential efficacy of somatostatin receptor 2 (SSTR2)-targeting imaging and therapy. METHODS: We conducted a retrospective analysis of 404 primary, locally recurrent, and metastatic olfactory neuroblastoma (ONB) patients from 12 institutions in the United States of America, United Kingdom and Europe. Clinicopathological characteristics and treatment approach were evaluated. SSTR2 expression, SSTR2-targeted imaging and the efficacy of peptide receptor radionuclide therapy [PRRT](177Lu-DOTATATE) were reported in a subset of our cohort (LUTHREE trial; NCT03454763). RESULTS: Dural infiltration at presentation was a significant predictor of overall survival (OS) and disease-free survival (DFS) in primary cases (n = 278). Kadish-Morita staging and Dulguerov T-stage both had limitations regarding their prognostic value. Multivariable survival analysis demonstrated improved outcomes with lower stage and receipt of adjuvant radiotherapy. Prophylactic neck irradiation significantly reduces the rate of nodal recurrence. 82.4% of the cohort were positive for SSTR2; treatment of three metastatic cases with SSTR2-targeted peptide-radionuclide receptor therapy (PRRT) in the LUTHREE trial was well-tolerated and resulted in stable disease (SD). CONCLUSIONS: This study presents pertinent clinical data from the largest dataset, to date, on ONB. We identify key prognostic markers and integrate these into an updated staging system, highlight the importance of adjuvant radiotherapy across all disease stages, the utility of prophylactic neck irradiation and the potential efficacy of targeting SSTR2 to manage disease.

Substitution of ROTEM FIBTEM A5 for A10 in trauma: an observational study building a case for more rapid analysis of coagulopathy.

PURPOSE: Rotational thromboelastometry (ROTEM®) allows guided blood product resuscitation to correct trauma-induced coagulopathy in bleeding trauma patients. FIBTEM amplitude at 10 min (A10) has been widely used to identify hypofibrinogenaemia; locally a threshold of < 11 mm has guided fibrinogen replacement. Amplitude at 5 min (A5) carries an inherent time advantage. The primary aim was to explore the relationship between FIBTEM A5 and A10 in a trauma. Secondary aim was to investigate the use of A5 as a surrogate for A10 within a fibrinogen-replacement algorithm. METHODS: Retrospective observational cohort study of arrival ROTEM results from 1539 consecutive trauma patients at a Level 1 trauma centre in Australia. Consistency of agreement between FIBTEM A5 and A10 was assessed. A new fibrinogen replacement threshold was developed for A5 using the A5-A10 bias; this was clinically compared to the existing A10 threshold. RESULTS: FIBTEM A5 displayed excellent consistency of agreement with A10. Intraclass correlation coefficient = 0.972 (95% confidence interval [CI] 0.969-0.974). Bias of A5 to A10 was - 1.49 (95% CI 1.43-1.56) mm. 19.34% patients met the original local threshold of A10 < 11 mm; 19.28% patients met the new, bias-adjusted threshold of A5 < 10 mm. CONCLUSION: ROTEM FIBTEM A5 reliably predicts A10 in trauma. This further validates use of the A5 result over A10 allowing faster decision-making in time-critical resuscitation of trauma patients. A modification of -1 to the A10 threshold might be appropriate for use with the A5 value in trauma patients.

Implementation of point-of-care ROTEM® into a trauma major haemorrhage protocol: A before and after study.

OBJECTIVE: The aim of the present study was to assess transfusion practices with the implementation of a targeted viscoelastic haemostatic assay (VHA) (ROTEM®) guided coagulation management programme into a major haemorrhage protocol for trauma patients requiring ICU admission, starting from time of arrival in the ED. METHODS: This retrospective observational study was conducted in a major trauma centre in Australia. One hundred and sixty-two trauma patients admitted to the ICU between January 2013 and December 2015 with an Injury Severity Score ≥12 and who received blood products were included: 37 in the pre-group, 48 during implementation and 77 in post-group. The primary outcome was blood and blood product administration amounts. RESULTS: Packed red blood cell transfusion amounts did not significantly change post introduction of the ROTEM®. There was a significant increase in fibrinogen replacement between the pre- and post-groups (P < 0.001), accompanied by a reduction in the use of fresh frozen plasma (P < 0.001) and prothrombinex (P < 0.001). Platelet usage in the post-group was higher but not reaching statistical significance (P = 0.051). Post-implementation point-of-care ROTEM® testing was able to be performed in the ED in 94.8% of cases. CONCLUSION: Although there was no overall reduction of packed red blood cell usage, a change in the pattern of administration of other blood products was observed with the implementation of a targeted VHA (ROTEM®) guided coagulation management programme. Larger studies are needed to further define the role of early VHA testing to guide correction of trauma-induced coagulopathy and the effect on clinical outcomes.

Targeted cryoprecipitate transfusion in severe traumatic haemorrhage.

BACKGROUND: Severe traumatic haemorrhage is the leading cause of death in young adults. Trauma Induced Coagulopathy is a complex and multifactorial phenomenon associated with severe traumatic haemorrhage. Fibrinogen is one of the first coagulation factors to become depleted in TIC and evidence suggests that severely injured trauma patients with hypofibrinogenaemia have poor outcomes. It is postulated that early fibrinogen replacement can improve clinical outcomes. This study investigated cryoprecipitate transfusion in hyopfibrinogeneamic trauma patients. METHODS: This retrospective, single center, observational study investigated the use of cryoprecipitate in severely injured trauma patients admitted to an Australian Level I Trauma Centre. The primary outcome was time to administration of cryoprecipitate after identification of hypofibrinogenaemia using ROTEM (FIBTEM A5). Data collected included demographics, ISS, laboratory values of coagulation and blood product usage. RESULTS: 71 patients received cryoprecipitate with a median time of 61 minutes [IQR 37-93] from FIBTEM A5 result to initial cryoprecipitate administration. At 24 hours following admission to ED, Clauss Fibrinogen levels increased by 1.30g/L [IQR 0.45-1.85] and FIBTEM A5 assay increased by 8mm [IQR 3.0-11.3]. Changes in both variables were highly significant (p<0.001) and Clauss Fibrinogen versus FIBTEM A5 values showed moderate to strong correlation (R=0.75-0.80). CONCLUSION: This study demonstrated that early administration of cryoprecipitate was both feasible and efficacious in fibrinogen replacement in severe traumatic haemorrhage. High-level evidence supporting cryoprecipitate or fibrinogen concentrate replacement with regards to efficacy and feasibility is required to guide future clinical practice. This study provided baseline data to inform the design of further clinical trials investigating fibrinogen replacement in traumatic haemorrhage.

Investigating women with postmenopausal bleeding: The utility of endometrial thickness in transvaginal ultrasound.

BACKGROUND: Australian clinical guidelines recommend further investigation in females with postmenopausal bleeding (PMB) and endometrial thickness (ET) of ≥4 mm on transvaginal ultrasound (TVUS). However, the literature indicates that an ET of ≥3 mm as an upper limit is a more sensitive predictor of endometrial malignancy (EM) in females with PMB. AIMS: To assess whether Australian guidelines for PMB with an upper limit of 4 mm ET on ultrasound investigation, is sensitive enough for malignancy detection. MATERIAL AND METHODS: A retrospective study was performed on tissue results in PMB presentations to the Gold Coast Hospital and Health Service between 2011 and 2015. RESULTS: Twenty point nine percent of women with PMB had a malignancy. With an upper limit of 4 mm in ET on ultrasound, malignancy was present in 22% of participants. [Correction added on 10 July 2020, after first online publication: percentage of women with PMB that had a malignancy has been amended to twenty point nine percent.] CONCLUSIONS: A limit of 3 mm for ET in PMB, along with office endometrial biopsy, should be considered to ensure timely diagnoses.

Targeted fibrinogen concentrate use in severe traumatic haemorrhage.

OBJECTIVE: Fibrinogen is one of the first coagulation factors to be depleted during traumatic haemorrhage, and evidence suggests hypofibrinogenaemia leads to poor outcomes. A number of fibrinogen replacement products are currently available, with no clear consensus on the ideal product to use in severe traumatic haemorrhage. We hypothesised that it will be possible to rapidly administer fibrinogen concentrate (FC) guided by rotational thromboelastometry (ROTEM) FIBTEM A5 in patients presenting with trauma haemorrhage. METHODS: We examined 36 consecutive patients with trauma admitted to a level 1 trauma centre in Australia who received FC as part of their initial resuscitation. ROTEM analysis was conducted at various time points from emergency department (ED) admission to 48 hours after admission. The primary outcome was time to administration of FC after identification of hypofibrinogenaemia using ROTEM FIBTEM A5. Data were collected on quantity and timing of product transfusion, demographics, Injury Severity Score and laboratory values of coagulation. Spearman rank order correlation was used to determine the correlation between FIBTEM A5 and Clauss fibrinogen (FibC). RESULTS: Thirty-six patients received FC as their initial form of fibrinogen replacement during the study. Patients were hypofibrinogenaemic by both FIBTEM A5 (6 mm) and FibC (1.7 g/L) on presentation to the ED. It took a median of 22 minutes (IQR, 17-30 minutes) from time of a FIBTEM A5 analysis to FC administration. Both parameters increased significantly (P < 0.05) by 24 hours after admission. CONCLUSION: This study suggests that administration of FC represents a rapid and feasible method to replace fibrinogen in severe traumatic haemorrhage. However, the optimal method for replacing fibrinogen in traumatic haemorrhage is controversial and large multicentre randomised controlled trials are needed to provide further evidence. This study provided baseline data to inform the design of further clinical trials investigating fibrinogen replacement in traumatic haemorrhage.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.