Mitochondrial contact site and cristae organizing system (MICOS) machinery supports heme biosynthesis by enabling optimal performance of ferrochelatase

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents. Nevertheless, understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Therefore, we investigated the molecular organization as well as the physical and genetic interactions of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses revealed dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity, affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic tetrapyrrole precursors that may cause oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. These data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis

Virtual Structural Analysis of Tibial Fracture Healing from Low-Dose Clinical CT Scans

Quantitative assessment of bone fracture healing remains a significant challenge in orthopaedic trauma research. Accordingly, we developed a new technique for assessing bone healing using virtual mechano-structural analysis of computed tomography (CT) scans. CT scans from 19 fractured human tibiae at 12 weeks after surgery were segmented and prepared for finite element analysis (FEA). Boundary conditions were applied to the models to simulate a torsion test that is commonly used to access the structural integrity of long bones in animal models of fracture healing. The output of each model was the virtual torsional rigidity (VTR) of the healing zone, normalized to the torsional rigidity of each patient’s virtually reconstructed tibia. This provided a structural measure to track the percentage of healing each patient had undergone. Callus morphometric measurements were also collected from the CT scans. Results showed that at 12 weeks post-op, more than 75% of patients achieved a normalized VTR (torsional rigidity relative to uninjured bone) of 85% or above. The predicted intact torsional rigidities compared well with published cadaveric data. Across all patients, callus volume and density were weakly and non-significantly correlated with normalized VTR and time to clinical union. Conversely, normalized VTR was significantly correlated with time to union (R2 = 0.383, p = 0.005). This suggests that fracture scoring methods based on the visual appearance of callus may not accurately predict mechanical integrity. The image-based structural analysis presented here may be a useful technique for assessment of bone healing in orthopaedic trauma research

Virtual Mechanical Testing from Low-Dose CT Scans Predicts Tibial Fracture Time to Union and Outperforms Subjective Outcomes Scoring

Background: Quantitative outcomes assessment remains a persistent challenge in orthopaedic trauma. Although patient-reported outcomes measures (PROMs) and radiographic assessments such as RUST scores are frequently used, very little evidence has been presented to support their validity for measuring structural bone formation or biomechanical integrity. Methods: A sequential cohort of tibial shaft fracture patients was prospectively recruited for observation following standard reamed intramedullary nailing in a Level I trauma center. Follow-ups at 6, 12, 18, and 24 weeks included X-rays and completion of PROMs (EQ-5D and pain scores). Low-dose computed-tomography (CT) scans were also completed at 12 weeks. Scans were reconstructed in 3D and subjected to virtual mechanical testing via the finite element method to assess fracture limb torsional rigidity relative to intact bone. Results: Patients reported progressive longitudinal improvement in mobility, self-care, activity, and health over time, but the PROMs were not correlated with structural bone healing. RUST scoring showed moderate intra-rater agreement (ICC = 0.727), but the scores at 12 weeks were not correlated with time to union (R2 = 0.103, p = 0.193) and were only moderately correlated with callus structural integrity (R2 = 0.346, p = 0.010). In contrast, patient-specific virtual torsional rigidity (VTR) was significantly correlated with time to union (R2 = 0.383, p = 0.005) and clearly differentiated one case of delayed union (VTR = 10%, union at 8 months) from the rest of the normally healing cohort (VTR > 60%, median union time 19 weeks) using CT data alone. Conclusions: PROMs provide insight into the natural history of the patient experience after tibial fracture, but have limited utility as a measure of structural bone healing. RUST scoring, although repeatable, is not a valid longitudinal predictor of time to union. In contrast, virtual mechanical testing from low-dose CT scans provides a quantitative and objective structural callus assessment that reliably predicts time to union and may enable early diagnosis of compromised healing. Level of Evidence: Diagnostic Level II

Role of the fibula in the stability of diaphyseal tibial fractures fixed by intramedullary nailing

Background: For tibial fractures, the decision to fix a concomitant fibular fracture is undertaken on a case-by-case basis. To aid in this clinical decision-making process, we investigated whether loss of integrity of the fibula significantly destabilises midshaft tibial fractures, whether fixation of the fibula restores stability to the tibia, and whether removal of the fibula and interosseous membrane for expediency in biomechanical testing significantly influences tibial interfragmentary mechanics. Methods: Tibia/fibula pairs were harvested from six cadaveric donors with the interosseous membrane intact. A tibial osteotomy fracture was fixed by reamed intramedullary (IM) nailing. Axial, torsion, bending, and shear tests were completed for four models of fibular involvement: intact fibula, osteotomy fracture, fibular plating, and resected fibula and interosseous membrane. Findings: Overall construct stiffness decreased slightly with fibular osteotomy compared to intact bone, but this change was not statistically significant. Under low loads, the influence of the fibula on construct stability was only statistically significant in torsion (large effect size). Fibular plating stiffened the construct slightly, but this change was not statistically significant compared to the fibular osteotomy case. Complete resection of the fibula and interosseous membrane significantly decreased construct torsional stiffness only (large effect size). Interpretation: These results suggest that fixation of the fibula may not contribute significantly to the stability of diaphyseal tibial fractures and should not be undertaken unless otherwise clinically indicated. For testing purposes, load-sharing through the interosseous membrane contributes significantly to overall construct mechanics, especially in torsion, and we recommend preservation of these structures when possible

Imaging Modalities to Assess Fracture Healing

Purpose of review: This review discusses imaging modalities for fracture repair assessment, with an emphasis on pragmatic clinical and translational use, best practices for implementation, and challenges and opportunities for continuing research. Recent findings: Semiquantitative radiographic union scoring remains the clinical gold standard, but has questionable reliability as a surrogate indicator of structural bone healing, particularly in early-stage, complex, or compromised healing scenarios. Alternatively, computed tomography (CT) scanning enables quantitative assessment of callus morphometry and mechanics through the use of patient-specific finite-element models. Dual-energy X-ray absorptiometry (DXA) scanning and radiostereometric analysis (RSA) are also quantitative, but technically challenging. Nonionizing magnetic resonance (MR) and ultrasound imaging are of high interest, but require development to enable quantification of 3D mineralized structures. Emerging image-based methods for quantitative assessment of bone healing may transform clinical research design by displacing binary outcomes classification (union/nonunion) and ultimately enhance clinical care by enabling early nonunion detection. Keywords: Computed tomography (CT); Finite element analysis (FEA); Fracture callus; Nonunion; Radiograph

Virtual mechanical tests out‐perform morphometric measures for assessment of mechanical stability of fracture healing in vivo

Finite element analysis with models derived from computed tomography (CT) scans is potentially powerful as a translational research tool because it can achieve what animal studies and cadaver biomechanics cannot—low‐risk, noninvasive, objective assessment of outcomes in living humans who have actually experienced the injury, or treatment being studied. The purpose of this study was to assess the validity of CT‐based virtual mechanical testing with respect to physical biomechanical tests in a large animal model. Three different tibial osteotomy models were performed on 44 sheep. Data from 33 operated limbs and 20 intact limbs was retrospectively analyzed. Radiographic union scoring was performed on the operated limbs and physical torsional tests were performed on all limbs. Morphometric measures and finite element models were developed from CT scans and virtual torsional tests were performed to assess healing with four material assignment techniques. In correlation analysis, morphometric measures and radiographic scores were unreliable predictors of biomechanical rigidity, while the virtual torsion test results were strongly and significantly correlated with measured biomechanical test data, with high absolute agreement. Overall, the results validated the use of virtual mechanical testing as a reliable in vivo assessment of structural bone healing. This method is readily translatable to clinical evaluation for noninvasive assessment of the healing progress of fractures with minimal risk. Clinical significance: virtual mechanical testing can be used to reliably and noninvasively assess the rigidity of a healing fracture using clinical‐resolution CT scans and that this measure is superior to morphometric and radiographic measures

Embracing ethical research: implementing the 3R principles into fracture healing research for sustainable scientific progress

As scientific advancements continue to reshape the world, it becomes increasingly crucial to uphold ethical standards and minimize the potentially adverse impact of research activities. In this context, the implementation of the 3R principles-Replacement, Reduction, and Refinement-has emerged as a prominent framework for promoting ethical research practices in the use of animals. This article aims to explore recent advances in integrating the 3R principles into fracture healing research, highlighting their potential to enhance animal welfare, scientific validity, and societal trust. The review focuses on in vitro, in silico, ex vivo, and refined in vivo methods, which have the potential to replace, reduce, and refine animal experiments in musculoskeletal, bone, and fracture healing research. Here, we review material that was presented at the workshop "Implementing 3R Principles into Fracture Healing Research" at the 2023 Orthopedic Research Society (ORS) Annual Meeting in Dallas, Texas