Faculty Bibliography

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain¹. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes², their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Na_v1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Na_v1.7 channels, with a density of 0.1 to 0.15 channels per µm² and 350 to 525 channels per cell. Serial genetic ablation of Na_v1.7 in multiple mouse models demonstrates that Na_v1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Na_v1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Na_v1.7 with selective or clinically used pan-Na_v channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Na_v1.7 blockers regulate intracellular Ca²⁺ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Na_v1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

OBJECTIVE:To compare the outcomes of patients with segmental bone loss who underwent repair with the induced membrane technique (IMT) with a matched cohort of nonunion fractures without bone loss. DESIGN/METHODS:Retrospective analysis on prospectively collected data. SETTING/METHODS:Academic medical center. PATIENTS/METHODS:Two cohorts of patients, those with upper and lower extremity diaphyseal large segmental bone loss and those with ununited fractures, were enrolled prospectively between 2013 and 2020. Sixteen patients who underwent repair of 17 extremities with segmental diaphyseal or meta-diaphyseal bone defects treated with the induced membrane technique were identified, and matched with 17 patients who were treated for 17 fracture nonunions treated without an induced membrane. Sixteen of the bone defects treated with the induced membrane technique were due to acute bone loss, and the other was a chronic aseptic nonunion. MAIN OUTCOME MEASUREMENTS/METHODS:Healing rate, time to union, functional outcome scores using the Short Musculoskeletal Functional Assessment (SMFA) and pain assessed by the Visual Analog Scale (VAS). RESULTS:The initial average defect size for patients treated with the induced membrane technique was 8.85 cm. Mean follow-up times were similar with 17.06 ± 10.13 months for patients treated with the IMT, and 20.35 ± 16.68. months for patients treated without the technique. Complete union was achieved in 15/17 (88.2%) of segmental bone loss cases treated with the IMT and 17/17 (100%) of cases repaired without the technique at the latest follow up visit. The average time to union for patients treated with the induced membrane technique was 13.0 ± 8.4 months and 9.64 ± 4.7 months for the matched cohort. There were no significant differences in reported outcomes measured by the SMFA or VAS. Patients treated with the induced membrane technique required more revision surgeries than those not treated with an induced membrane. CONCLUSION/CONCLUSIONS:Outcomes following treatment of acute bone loss from the diaphysis of long bones with the induced membrane technique produces clinical and radiographic outcomes similar to those of long bone fracture nonunions without bone loss that go on to heal. LEVEL OF EVIDENCE/METHODS:III.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.

The oocyte, a long-lived, postmitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout reproductive life. Mechanisms of oocyte aging include the accumulation of oxidative damage, mitochondrial dysfunction, and disruption of proteins, including cohesion. Nobel Laureate Bob Edwards also discovered a "production line" during oogonial replication in the mouse, wherein the last oocytes to ovulate in the adult-derived from the last oogonia to exit mitotic replication in the fetus. On the basis of this, we proposed a two-hit "telomere theory of reproductive aging" to integrate the myriad features of oocyte aging. The first hit was that oocytes remaining in older women traversed more cell cycles during fetal oogenesis. The second hit was that oocytes accumulated more environmental and endogenous oxidative damage throughout the life of the woman. Telomeres (Ts) could mediate both of these aspects of oocyte aging. Telomeres provide a "mitotic clock," with T attrition an inevitable consequence of cell division because of the end replication problem. Telomere's guanine-rich sequence renders them especially sensitive to oxidative damage, even in postmitotic cells. Telomerase, the reverse transcriptase that restores Ts, is better at maintaining than elongating T. Moreover, telomerase remains inactive during much of oogenesis and early development. Oocytes are left with short Ts, on the brink of viability. In support of this theory, mice with induced T attrition and women with naturally occurring telomeropathy suffer diminished ovarian reserve, abnormal embryo development, and infertility. In contrast, sperm are produced throughout the life of the male by a telomerase-active progenitor, spermatogonia, resulting in the longest Ts in the body. In mice, cleavage-stage embryos elongate Ts via "alternative lengthening of telomeres," a recombination-based mechanism rarely encountered outside of telomerase-deficient cancers. Many questions about Ts and reproduction are raised by these findings: does the "normal" T attrition observed in human oocytes contribute to their extraordinarily high rate of meiotic nondisjunction? Does recombination-based T elongation render embryos susceptible to mitotic nondisjunction (and mosaicism)? Can some features of Ts serve as markers of oocyte quality?

The improvements accomplished in assisted reproductive technology have emphasized more than ever the role played by chronological age, notably for predicting oocyte quality. Studies in cellular aging have directed research on telomere length measurements as possible markers of functional aging and, notably, female reproductive outcomes. Although further research is still needed, encouraging results are already available on the possibility that leucocyte telomere length may be a useful parameter for assessing reproductive potential in aging women.

We sought to determine what effect the size of a displaced coronoid fracture fragment in Monteggia injuries has on clinical outcome. Sixty-seven patients presented to an academic medical center for operative fixation of a Monteggia fracture. Radiographs were assessed for length and height of the displaced coronoid fragment using measuring tools in our center's imaging archive system. Data were analyzed using binary logistic or linear regression, as appropriate, controlling for sex, age, and Charlson Comorbidity Index. Outcome measurements included radiographic healing, range of motion, postoperative complications, and reoperation. The cohort had a mean follow-up of 16.7 months. Mean coronoid fragment area was 362.4±155.9 mm². Elbow range of motion decreased by 3.8° of elbow flexion (P<.001), 3.3° of elbow extension (P<.001), and 3.8° of forearm supination (P=.007) for every 1-cm² increase in coronoid fragment area. Complications (P=.012) and reoperation (P=.036) were associated with increasing coronoid fragment area. Nonunion rate, nerve injury, and pronation range of motion were not correlated to increasing coronoid fracture fragment area (P=.777, P=.123, and P=.351, respectively). As displaced coronoid fragment size increases in Monteggia fracture patterns, elbow range of motion decreases linearly. Coronoid displacement was also associated with increased rates of postoperative complication and need for reoperation. [Orthopedics. 2024;47(1):15-21.].

There has been a paradigm shift towards fixing the posterior malleolus in trimalleolar ankle fractures. This study evaluated whether a surgeon's preference to intraoperatively flip or not flip patients from prone to supine for medial malleolar fixation following repair of fibular and posterior malleoli impacted surgical outcomes. A retrospective patient cohort treated at a large urban academic center and level 1 trauma center was reviewed to identify all operative trimalleolar ankle fractures initially positioned prone. One hundred and forty-seven patients with mean 12-month follow up were included and divided based on positioning for medial malleolar fixation, prone or supine (following closure, flip and re-prep and drape). Data was collected on patient demographics, injury mechanism, perioperative variables, and complication rates. Postoperative reduction films were reviewed by orthopedic traumatologists to grade the accuracy of anatomic fracture reduction. Overall, 74 (50.3%) had the medial malleolus fixed prone, while 73 (49.7%) were flipped and fixed supine. No differences in demographics, injury details, and fracture type existed between the groups. The supine group had a higher rate of initial external fixation (p=0.047), longer operative time in minutes (p<0.001), and a higher use of plate and screw constructs for medial malleolar fixation (p=0.019). There were no differences in clinical and radiographic outcomes and complication rates. This study demonstrated that intraoperative change in positioning for improved medial malleolar visualization in trimalleolar ankle fractures results in longer operative times but similar radiographic and clinical results. The decision of operative position should be based on surgeon comfort.

Peptides that increase pro-reparative responses to injury and disease by modulating the functional organization of hyaluronan (HA) with its cell surface binding proteins (e.g., soluble receptor for hyaluronan-mediated motility [RHAMM] and integral membrane CD44) have potential therapeutic value. The binding of RHAMM to HA is an attractive target, since RHAMM is normally absent or expressed at low levels in homeostatic conditions, but its expression is significantly elevated in the extracellular matrix during tissue stress, response-to-injury, and in cancers and inflammation-based diseases. The HA-binding site in RHAMM contains two closely spaced sequences of clustered basic amino acids, in an alpha-helical conformation. In the present communication, we test whether an alpha-helical conformation is required for effective peptide binding to HA, and competitive disruption of HA-RHAMM interaction. The HA-binding RHAMM-competitive peptide P15-1, identified using the unbiased approach of phage display, was examined using circular dichroism spectroscopy and the conformation-predictive AI-based AlphaFold2 algorithm. Unlike the HA-binding site in RHAMM, peptide P15-1 was found to adopt irregular conformations in solution rather than alpha helices. Instead, our structural analysis suggests that the primary determinant of peptide-HA binding is associated with a specific clustering and spacing pattern of basic amino acids, allowing favorable electrostatic interaction with carboxylate groups on HA.

BACKGROUND/UNASSIGNED:Dying in a preferred place is important for a good death. Currently, no study has evaluated the extent to which the preferences for the place of death (PoD) are met among terminal cancer patients in China. This study examined the congruence between the preferred and actual PoD and its predictors among terminal cancer patients in China. METHODS/UNASSIGNED:Between 2015 and 2023, 845 terminal cancer patients from four tertiary hospitals in Wuhan, China, were enrolled and followed till death. Face-to-face surveys at baseline and telephone-based interviews in the last month of patients' lives were combined to learn patients' preferred PoD. Data on patients' actual PoD were collected from families within 1 month after patients' death. RESULTS/UNASSIGNED: = 0.011), patients were less likely to die in their preferred places. CONCLUSION/UNASSIGNED:The congruence between patients' preferred and actual PoD was fair. Advance care planning (ACP) needs to be popularized in China, and the quality of care in hospice facilities and nursing homes should be improved. The necessary policy support for hospice care should be made to respect cancer patients' end-of-life (EoL) care preferences in China.

Volume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focused ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted to in vitro tissue culture samples. Here we describe step-by-step protocols for two sample embedding methods for in vitro tissue culture cells intended to be studied using SBF-SEM. The first focuses on cell pellet embedding and the second on en face embedding. En face embedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events by light microscopy, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables.