Long-Term Characteristics of Human-Derived Biliary Organoids under a Single Continuous Culture Condition.

Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The goal of this study was to determine the feasibility of establishing organoids from various pediatric liver diseases and characterize the long-term evolution of cholangiocyte organoids (chol-orgs) under a single continuous culture condition. We established chol-orgs from 10 different liver conditions and characterized their multicellular organization into complex epithelial structures through budding, merging, and lumen formation. Immunofluorescent staining, electron microscopy, and single-nucleus RNA (snRNA-seq) sequencing confirmed the cholangiocytic nature of the chol-orgs. There were significant cell population differences in the transcript profiles of two-dimensional and organoid cultures based on snRNA-seq. Our study provides an approach for the generation and long-term maintenance of chol-orgs from various pediatric liver diseases under a single continuous culture condition.

DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling.

Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.

Barriers to recruitment to an orthopaedic randomized controlled trial comparing two surgical procedures for ankle arthritis : a qualitative study.

AIMS: A multicentre, randomized, clinician-led, pragmatic, parallel-group orthopaedic trial of two surgical procedures was set up to obtain high-quality evidence of effectiveness. However, the trial faced recruitment challenges and struggled to maintain recruitment rates over 30%, although this is not unusual for surgical trials. We conducted a qualitative study with the aim of gathering information about recruitment practices to identify barriers to patient consent and participation to an orthopaedic trial. METHODS: We collected 11 audio recordings of recruitment appointments and interviews of research team members (principal investigators and research nurses) from five hospitals involved in recruitment to an orthopaedic trial. We analyzed the qualitative data sets thematically with the aim of identifying aspects of informed consent and information provision that was either unclear, disrupted, or hindered trial recruitment. RESULTS: Recruiters faced four common obstacles when recruiting to a surgical orthopaedic trial: patient preferences for an intervention; a complex recruitment pathway; various logistical issues; and conflicting views on equipoise. Clinicians expressed concerns that the trial may not show significant differences in the treatments, validating their equipoise. However, they experienced role conflicts due to their own preference and perceived patient preference for an intervention arm. CONCLUSION: This study provided initial information about barriers to recruitment to an orthopaedic randomized controlled trial. We shared these findings in an all-site investigators' meeting and encouraged researchers to find solutions to identified barriers; this led to the successful completion of recruitment. Complex trials may benefit for using of a mixed-methods approach to mitigate against recruitment failure, and to improve patient participation and informed consent. Cite this article: Bone Jt Open 2021;2(8):631-637.

Clinical and patient-reported outcomes following Low Intensity Pulsed Ultrasound (LIPUS, Exogen) for established post-traumatic and post-surgical nonunion in the foot and ankle.

BACKGROUND: Biophysical methods including Low Intensity Pulsed Ultrasound (LIPUS) are emerging as potential alternatives to revision surgery for treating established nonunions. We aim to prospectively review the clinical and patient-reported outcomes of patients treated with LIPUS following post-traumatic and post-surgical nonunions in the foot and ankle. METHODS: Forty-seven consecutive patients underwent Exogen treatment. Patient-reported outcome scores included MOXFQ, EQ-5D and VAS. Patients were divided in to 3 groups: fractures (A), hindfoot procedures (B) and midfoot/forefoot procedures (C). RESULTS: Thirty-seven patients (78.7%) clinically united, 4 patients (8.5%) noticed no significant improvement but did not want further intervention and 6 patients (12.8%) underwent revision surgery. The mean duration of Exogen treatment was 6 months. Union rates of 93%, 67% and 78% were noted in the three groups. Significant improvement in functional outcomes and potential cost savings were observed. CONCLUSIONS: Exogen for established nonunion in the foot and ankle is a safe, valuable and economically viable clinical option as an alternative to revision surgery. We observed better results in the fracture and midfoot/forefoot groups and relatively poorer results in the hindfoot fusion group.

3-D computer modelling of malunited posterior malleolar fractures: effect of fragment size and offset on ankle stability, contact pressure and pattern.

BACKGROUND: The positioning of the fracture fragment of a posterior malleolus fracture is critical to healing and a successful outcome as malunion of a posterior malleolar fracture, a condition seen in clinical practice, can affect the dynamics of the ankle joint, cause posterolateral rotational subluxation of the talus and ultimately lead to destruction of the joint. Current consensus is to employ anatomic reduction with internal fixation when the fragment size is larger than 25 to 33% of the tibial plafond. METHODS: A 3-dimensional finite element (FE) model of ankle was developed in order to investigate the effect of fragment size (6-15 mm) and offset (1-4 mm) of a malunited posterior malleolus on tibiotalar joint contact area, pressure, motion of joint and ligament forces. Three positions of the joint were simulated; neutral position, 20° dorsiflexion and 30° plantarflexion. RESULTS: Compared to the intact joint our model predicted that contact area was greater in all malunion scenarios considered. In general, the joint contact area was affected more by section length than section offset. In addition fibula contact area played a role in all the malunion cases. CONCLUSIONS: We found no evidence to support the current consensus of fixing posterior malleolus fractures of greater than 25% of the tibial plafond. Our model predicted joint instability only with the highest level of fracture in a loaded limb at an extreme position of dorsiflexion. No increase of peak contact pressure as a result of malunion was predicted but contact pattern was modified. The results of our study support the view that in cases of posterior malleolar fracture, posttraumatic osteoarthritis occurs as a result of load on areas of cartilage not used to loading rather than an increase in contact pressure. Ankle repositioning resulted in increased force in two ankle ligaments. Our finding could explain commonly reported clinical observations.

Total ankle replacement versus arthrodesis (TARVA): protocol for a multicentre randomised controlled trial.

INTRODUCTION: Total ankle replacement (TAR) or ankle arthrodesis (fusion) is the main surgical treatments for end-stage ankle osteoarthritis (OA). The popularity of ankle replacement is increasing while ankle fusion rates remain static. Both treatments have efficacy but to date all studies comparing the 2 have been observational without randomisation, and there are no published guidelines as to the most appropriate management. The TAR versus arthrodesis (TARVA) trial aims to compare the clinical and cost-effectiveness of TAR against ankle arthrodesis in the treatment of end-stage ankle OA in patients aged 50-85 years. METHODS AND ANALYSIS: TARVA is a multicentre randomised controlled trial that will randomise 328 patients aged 50-85 years with end-stage ankle arthritis. The 2 arms of the study will be TAR or ankle arthrodesis with 164 patients in each group. Up to 16 UK centres will participate. Patients will have clinical assessments and complete questionnaires before their operation and at 6, 12, 26 and 52 weeks after surgery. The primary clinical outcome of the study is a validated patient-reported outcome measure, the Manchester Oxford foot questionnaire, captured preoperatively and 12 months after surgery. Secondary outcomes include quality-of-life scores, complications, revision, reoperation and a health economic analysis. ETHICS AND DISSEMINATION: The protocol has been approved by the National Research Ethics Service Committee (London, Bloomsbury 14/LO/0807). This manuscript is based on V.5.0 of the protocol. The trial findings will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT02128555.

Functional outcome following bone transport reconstruction of distal tibial defects.

BACKGROUND: Little has been written about the functional outcome of patients treated with bone transport to reconstruct a distal tibial defect. The aim of this study was to investigate the functional capabilities of patients who had undergone reconstruction with distraction osteogenesis for the treatment of a distal tibial defect in one lower limb. METHODS: At least eighteen months after completion of treatment, eight patients who had no pain and were able to walk and climb stairs without difficulty performed isometric ankle plantar flexion maximum voluntary contractions while the electromyographic activity of the tibialis anterior and triceps surae muscles was simultaneously recorded. Seven of the patients also underwent gait analysis. Data for the involved limb were compared with those collected for the contralateral limb. RESULTS: During gait, stance time (p = 0.01), the plantar flexion angular displacement and peak moment developed during the second half of stance (p < 0.046), and the amount of ankle power generated (p = 0.02) were significantly decreased in the involved limb compared with the contralateral limb. Similar decreases were observed in the plantar flexion (p = 0.01) and dorsiflexion (p = 0.01) maximum voluntary contractions and the corresponding electromyographic activity (p = 0.01). CONCLUSIONS: These results suggest that adaptive changes had occurred at the level of the transported muscles, which affected both routine and maximal effort capabilities. These findings contribute to our understanding of the functional limitations of patients who have undergone bone transport with its obligatory shortening of muscle length.