The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain.

Most pathogenic bacteria require ferrous iron (Fe2+) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly conserved prokaryotic transporter of Fe2+, but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein, and FeoB, a membrane protein that translocates Fe2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that selected FeoC proteins bind O2-sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O2 to regulate Fe2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera (Vibrio cholerae) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of VcFeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of VcFeoC reveals a truncated winged ß-sheet in which the cluster-binding domain is notably absent. Using homology modeling, we predicted the structure of VcNFeoB and used docking to identify an interaction site with VcFeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of VcFeoC and contribute to a better understanding of its role vis-à-vis FeoB.

Ins and Outs: Recent Advancements in Membrane Protein-Mediated Prokaryotic Ferrous Iron Transport.

Iron is an essential nutrient for virtually every living organism, especially pathogenic prokaryotes. Despite its importance, however, both the acquisition and the export of this element require dedicated pathways that are dependent on oxidation state. Due to its solubility and kinetic lability, reduced ferrous iron (Fe2+) is useful to bacteria for import, chaperoning, and efflux. Once imported, ferrous iron may be loaded into apo and nascent enzymes and even sequestered into storage proteins under certain conditions. However, excess labile ferrous iron can impart toxicity as it may spuriously catalyze Fenton chemistry, thereby generating reactive oxygen species and leading to cellular damage. In response, it is becoming increasingly evident that bacteria have evolved Fe2+ efflux pumps to deal with conditions of ferrous iron excess and to prevent intracellular oxidative stress. In this work, we highlight recent structural and mechanistic advancements in our understanding of prokaryotic ferrous iron import and export systems, with a focus on the connection of these essential transport systems to pathogenesis. Given the connection of these pathways to the virulence of many increasingly antibiotic resistant bacterial strains, a greater understanding of the mechanistic details of ferrous iron cycling in pathogens could illuminate new pathways for future therapeutic developments.

Relationships between resource availability and elevation vary between metrics creating gradients of nutritional complexity.

Plant and animal community composition changes at higher elevations on mountains. Plant and animal species richness generally declines with elevation, but the shape of the relationship differs between taxa. There are several proposed mechanisms, including the productivity hypotheses; that declines in available plant biomass confers fewer resources to consumers, thus supporting fewer species. We investigated resource availability as we ascended three aspects of Helvellyn mountain, UK, measuring several plant nutritive metrics, plant species richness and biomass. We observed a linear decline in plant species richness as we ascended the mountain but there was a unimodal relationship between plant biomass and elevation. Generally, the highest biomass values at mid-elevations were associated with the lowest nutritive values, except mineral contents which declined with elevation. Intra-specific and inter-specific increases in nutritive values nearer the top and bottom of the mountain indicated that physiological, phenological and compositional mechanisms may have played a role. The shape of the relationship between resource availability and elevation was different depending on the metric. Many consumers actively select or avoid plants based on their nutritive values and the abundances of consumer taxa vary in their relationships with elevation. Consideration of multiple nutritive metrics and of the nutritional requirements of the consumer may provide a greater understanding of changes to plant and animal communities at higher elevations. We propose a novel hypothesis for explaining elevational diversity gradients, which warrants further study; the 'nutritional complexity hypothesis', where consumer species coexist due to greater variation in the nutritional chemistry of plants.

Hypoxic Preconditioning of Mesenchymal Stem Cells with Subsequent Spheroid Formation Accelerates Repair of Segmental Bone Defects.

Cell-based approaches for musculoskeletal tissue repair are limited by poor cell survival and engraftment. Short-term hypoxic preconditioning of mesenchymal stem cells (MSCs) can prolong cell viability in vivo, while the aggregation of MSCs into spheroids increases cell survival, trophic factor secretion, and tissue formation in vivo. We hypothesized that preconditioning MSCs in hypoxic culture before spheroid formation would increase cell viability, proangiogenic potential, and resultant bone repair compared with that of individual MSCs. Human MSCs were preconditioned in 1% O2 in monolayer culture for 3 days (PC3) or kept in ambient air (PC0), formed into spheroids of increasing cell density, and then entrapped in alginate hydrogels. Hypoxia-preconditioned MSC spheroids were more resistant to apoptosis than ambient air controls and this response correlated with duration of hypoxia exposure. Spheroids of the highest cell density exhibited the greatest osteogenic potential in vitro and vascular endothelial growth factor (VEGF) secretion was greatest in PC3 spheroids. PC3 spheroids were then transplanted into rat critical-sized femoral segmental defects to evaluate their potential for bone healing. Spheroid-containing gels induced significantly more bone healing compared with gels containing preconditioned individual MSCs or acellular gels. These data demonstrate that hypoxic preconditioning represents a simple approach for enhancing the therapeutic potential of MSC spheroids when used for bone healing. Stem Cells 2018;36:1393-1403.

A global comparison of the nutritive values of forage plants grown in contrasting environments.

Forage plants are valuable because they maintain wild and domesticated herbivores, and sustain the delivery of meat, milk and other commodities. Forage plants contain different quantities of fibre, lignin, minerals and protein, and vary in the proportion of their tissue that can be digested by herbivores. These nutritive components are important determinants of consumer growth rates, reproductive success and behaviour. A dataset was compiled to quantify variation in forage plant nutritive values within- and between-plant species, and to assess variation between plant functional groups and bioclimatic zones. 1255 geo-located records containing 3774 measurements of nutritive values for 136 forage plant species grown in 30 countries were obtained from published articles. Spatial variability in forage nutritive values indicated that climate modified plant nutritive values. Forage plants grown in arid and equatorial regions generally contained less digestible material than those grown in temperate and tundra regions; containing more fibre and lignin, and less protein. These patterns may reveal why herbivore body sizes, digestion and migration strategies are different in warmer and drier regions. This dataset also revealed the capacity for variation in the nutrition provided by forage plants, which may drive consumer species coexistence. The proportion of the plant tissue that was digestible ranged between species from 2 to 91%. The amount of fibre contained within plant material ranged by 23-90%, protein by 2-36%, lignin by 1-21% and minerals by 2-22%. On average, grasses and tree foliage contained the most fibre, whilst herbaceous legumes contained the most protein and tree foliage contained the most lignin. However, there were individual species within each functional group that were highly nutritious. This dataset may be used to identify forage plant species or mixtures of species from different functional groups with useful nutritional traits which can be cultivated to enhance livestock productivity and inform wild herbivore conservation strategies.

Femoral Neck Fractures in Young Patients.

Femoral neck fractures in patients 55 years or younger, although relatively uncommon, may cause considerable surgeon stress because they may be thought to be surgical emergencies and are difficult to manage, resulting in serious complications. Orthopaedic surgeons should understand the optimal timing for, the reduction options and techniques for, the fixation options for, and the results of surgical management of femoral neck fractures in patients 55 years or younger. The optimal timing of the surgical management of femoral neck fractures in these patients is a subject of debate. Anatomic reduction, which correlates with patient outcomes, is the goal in the management of femoral neck fractures whether it is attained via open or closed means. Multiple surgical approaches, including the Watson-Jones, Smith-Petersen, and Hueter approaches, may be used for the open reduction of femoral neck fractures. Multiple options are available for fixation, with cannulated screws and the compression hip screw most used in the literature. These implants should provide torsional stability, minimal bone loss, and a length-stable construct. Currently, no ideal implant exists. The outcomes of young patients with a femoral neck fracture who undergo surgical treatment depend more on fracture type, fracture reduction, and stable fixation than early surgical management; however, surgical management should not be excessively delayed.

The arterial supply of the nipple areola complex (NAC) and its relations: an analysis of angiographic CT imaging for breast pedicle design.

PURPOSE: To investigate the blood supply to the nipple areola complex (NAC) on thoracic CT angiograms (CTA) to improve breast pedicle design in reduction mammoplasty. METHODS: In a single centre, CT scans of the thorax were retrospectively reviewed for suitability by a cardiothoracic radiologist. Suitable scans had one or both breasts visible in extended fields, with contrast enhancement of breast vasculature in a female patient. The arterial sources, intercostal space perforated, glandular/subcutaneous course, vessel entry point, and the presence of periareolar anastomoses were recorded for the NAC of each breast. RESULTS: From 69 patients, 132 breasts were suitable for inclusion. The most reproducible arterial contribution to the NAC was perforating branches arising from the internal thoracic artery (ITA) (n = 108, 81.8%), followed by the long thoracic artery (LTA) (n = 31, 23.5%) and anterior intercostal arteries (AI) (n = 21, 15.9%). Blood supply was superficial versus deep in (n = 86, 79.6%) of ITA sources, (n = 28, 90.3%) of LTA sources, and 10 (47.6%) of AI sources. The most vascularly reliable breast pedicle would be asymmetrical in 7.9% as a conservative estimate. CONCLUSION: We suggest that breast CT angiography can provide valuable information about NAC blood supply to aid customised pedicle design, especially in high-risk, large-volume breast reductions where the risk of vascular-dependent complications is the greatest and asymmetrical dominant vasculature may be present. Superficial ITA perforator supplies are predominant in a majority of women, followed by LTA- and AIA-based sources, respectively.

Extracellular Matrix-Coated Composite Scaffolds Promote Mesenchymal Stem Cell Persistence and Osteogenesis.

Composite scaffolds of bioactive glass and poly(lactide-co-glycolide) provide advantages over homogeneous scaffolds, yet their therapeutic potential can be improved by strategies that promote adhesion and present instructive cues to associated cells. Mesenchymal stem cell (MSC)-secreted extracellular matrix (ECM) enhances survival and function of associated cells. To synergize the benefits of an instructive ECM with composite scaffolds, we tested the capacity of ECM-coated composite scaffolds to promote cell persistence and resultant osteogenesis. Human MSCs cultured on ECM-coated scaffolds exhibited increased metabolic activity and decreased apoptosis compared to uncoated scaffolds. Additionally, MSCs on ECM-coated substrates in short-term culture secreted more proangiogenic factors while maintaining markers of osteogenic differentiation. Upon implantation, we detected improved survival of MSCs on ECM-coated scaffolds over 3 weeks. Histological evaluation revealed enhanced cellularization and osteogenic differentiation in ECM-coated scaffolds compared to controls. These findings demonstrate the promise of blending synthetic and natural ECMs and their potential in tissue regeneration.

Internal fixation of osteoporotic fractures.

Osteoporosis leads to bone fragility and increased risk of fracture. Despite advances in diagnosis and treatment, the prevalence continues to rise. Osteoporotic fracture treatment has a unique set of difficulties related to poor bone quality and traditional approaches, and implants may not perform well. Fixation failure and repeat surgery are poorly tolerated and highly undesirable in this patient population. This review illustrates the most recent updates in internal fixation, implant design, and surgical theory regarding treatment of patients with osteoporotic fractures.

Plant and arthropod community sensitivity to rainfall manipulation but not nitrogen enrichment in a successional grassland ecosystem.

Grasslands provide many ecosystem services including carbon storage, biodiversity preservation and livestock forage production. These ecosystem services will change in the future in response to multiple global environmental changes, including climate change and increased nitrogen inputs. We conducted an experimental study over 3 years in a mesotrophic grassland ecosystem in southern England. We aimed to expose plots to rainfall manipulation that simulated IPCC 4th Assessment projections for 2100 (+15% winter rainfall and -30% summer rainfall) or ambient climate, achieving +15% winter rainfall and -39% summer rainfall in rainfall-manipulated plots. Nitrogen (40 kg ha(-1) year(-1)) was also added to half of the experimental plots in factorial combination. Plant species composition and above ground biomass were not affected by rainfall in the first 2 years and the plant community did not respond to nitrogen enrichment throughout the experiment. In the third year, above-ground plant biomass declined in rainfall-manipulated plots, driven by a decline in the abundances of grass species characteristic of moist soils. Declining plant biomass was also associated with changes to arthropod communities, with lower abundances of plant-feeding Auchenorrhyncha and carnivorous Araneae indicating multi-trophic responses to rainfall manipulation. Plant and arthropod community composition and plant biomass responses to rainfall manipulation were not modified by nitrogen enrichment, which was not expected, but may have resulted from prior nitrogen saturation and/or phosphorus limitation. Overall, our study demonstrates that climate change may in future influence plant productivity and induce multi-trophic responses in grasslands.

The critical impact of traumatic muscle loss on fracture healing: Basic science and clinical aspects.

Musculoskeletal trauma, specifically fractures, is a leading cause of patient morbidity and disability worldwide. In approximately 20% of cases with fracture and related traumatic muscle loss, bone healing is impaired leading to fracture nonunion. Over the past few years, several studies have demonstrated that bone and the surrounding muscle tissue interact not only anatomically and mechanically but also through biochemical pathways and mediators. Severe damage to the surrounding musculature at the fracture site causes an insufficiency in muscle-derived osteoprogenitor cells that are crucial for fracture healing. As an endocrine tissue, skeletal muscle produces many myokines that act on different bone cells, such as osteoblasts, osteoclasts, osteocytes, and mesenchymal stem cells. Investigating how muscle influences fracture healing at cellular, molecular, and hormonal levels provides translational therapeutic solutions to this clinical challenge. This review provides an overview about the contributions of surrounding muscle tissue in directing fracture healing. The focus of the review is on describing the interactions between bone and muscle in both healthy and fractured environments. We discuss current progress in identifying the bone-muscle molecular pathways and strategies to harness these pathways as cues for accelerating fracture healing. In addition, we review the existing challenges and research opportunities in the field.

Trifocal femur fracture with intracapsular femoral neck, open diaphyseal, and distal complete articular fractures.

We describe a trifocal femur injury with intracapsular femoral neck fracture, diaphyseal fracture with bone loss, and distal complete articular (AO/OTA C type) fracture, an injury rarely described in the literature. Surgical management utilized a not-yet-reported implant combination: screw-side plate device for the intracapsular femoral neck, retrograde nail for the diaphysis, and lag screws plus mini fragment buttress plating for the distal fracture. The patient had uneventful fracture union with no changes in alignment. Given the rarity and complexity of this injury, there is little consensus on surgical technique and implant choice. This case demonstrates a modernized approach that may be useful for surgeons who encounter similar fracture patterns in their practice.

Comparing Multipin Clamps With Outriggers With Standard Clamps for Lower Extremity Periarticular External Fixation: Similar Radiographic and Clinical Outcomes.

INTRODUCTION: Staged treatment of high-energy periarticular tibia fractures involves temporization with closed reduction and external fixation, aiming to provide early reduction and stabilization while mitigating soft-tissue complications. Various external fixator configurations exist, including those that use a "multipin" clamp capable of holding multiple pins but limiting pin placement to a single plane. The purpose of this study was to compare clinical and radiographic outcomes and associated costs of standard and multipin outrigger clamp constructs in tibial plateau and pilon fractures treated with temporary external fixation. We hypothesized that use of the multipin clamp may be associated with poorly aligned reductions and increased complication rates. METHODS: A retrospective review of 100 patients with periarticular tibial plateau (AO/OTA: 41B/C) or pilon (43B/C) fracture at a Level 1 trauma center from 2014 to 2023 was conducted. Patient, injury, and complication characteristics were collected. Patients were categorized based on the external fixator clamp used: multipin (MP) or standard (S). Clinical outcomes and complication rates were assessed. Radiographic alignment was evaluated by the change in anterior and lateral distal tibial angles, and sagittal plane translation for pilon fractures, and medial and posterior proximal tibial angles for plateau fractures. RESULTS: 70 patients underwent standard (25 pilon, 45 plateau) and 30 multipin (10 pilon, 20 plateau) external fixation. MP and S groups showed no notable differences in demographics or injury characteristics. Both groups demonstrated comparable complication rates and radiological alignment outcomes, with no notable differences observed. MP constructs were more costly than standard systems. CONCLUSION: In this retrospective study of 100 patients, there was no difference in radiographic or clinical outcomes between the standard frame and multipin frame groups. Typical costs for the multipin frame constructs were $635 to $1249 more than the standard frame constructs.

Temporary Stabilization of Tibia Fractures: Does External Fixation or Temporary Plate Fixation Result in Better Outcomes?

Background: Provisional stabilization of high-energy tibia fractures using temporary plate fixation (TPF) or external fixation (ex-fix) prior to definitive medullary nailing (MN) is a strategy common in damage control orthopaedics. There is a lack of comprehensive data evaluating outcomes between these methods. This study compares outcomes of patients stabilized with either TPF or ex-fix, and with early definitive MN only, assessing complications including nonunion and deep infection. Methods: A retrospective review was performed on adult patients with tibia fractures treated with MN followed until fracture union (≥3 months) at a single level-1 trauma center from 2014 to 2022. Medical records were evaluated for nonunion and deep infection. Demographics, injury characteristics, and fixation methods were recorded. Significance between patients who underwent TPF and ex-fix was compared with a matched cohort of early MN using Pearson's exact tests, independent t-tests, and one-way ANOVA, depending on the appropriate variable. Results: 81 patients were included; 27 were temporized with TPF (n = 12) or ex-fix (n = 15). 54 early MN cases defined the matched cohort. All groups had similar patient and fracture characteristics. The difference in rates of nonunion between groups was significant, with TPF, ex-fix, and early MN groups at 17, 40, and 11% respectively (p = 0.027). Early MN had lower rates of nonunion (11% vs. 40%, p = 0.017) and deep infection (13% vs. 40%, p = 0.028) compared to ex-fix. Conclusion: Temporary ex-fix followed by staged MN was associated with higher rates of nonunion and deep infection. There was no difference in complication rates between TPF and early definitive MN. These data suggest that ex-fix followed by MN of tibia fractures should be avoided in favor of early definitive MN when possible. If temporization is needed, TPF may be a better option than ex-fix. Level of Evidence: IV.

Surgical timing of treating injured extremities: an evolving concept of urgency.

The management of some orthopaedic extremity injuries has changed over the past decade because of changing resource availability and the risks of complications. It is helpful to review the current literature regarding orthopaedic extremity emergencies and urgencies. The effects of the techniques of damage control orthopaedic techniques and the concept of the orthopaedic trauma room have also affected the management of these injuries. The available literature indicates that the remaining true orthopaedic extremity emergencies include compartment syndrome and vascular injuries associated with fractures and dislocations. Orthopaedic urgencies include open fracture management, femoral neck fractures in young patients treated with open reduction and internal fixation, and talus fractures that are open or those with impending skin compromise. Deciding when the definitive management of orthopaedic extremity injuries will occur has evolved as the concept of damage control orthopaedics has become more commonly accepted. Patient survival rates have improved with current resuscitative protocols. Definitive fixation of extremity injuries should be delayed until the patient's physiologic and extremity soft-tissue status allows for appropriate definitive management while minimizing the risks of complications. In patients with semiurgent orthopaedic injuries, the use of an orthopaedic trauma room has led to more efficient care of patients, fewer complications, and better time management for surgeons who perform on-call service for patients with traumatic orthopaedic injuries.

Surgical treatment of trochanteric and cervical hip fractures in the United States: 2000-2009.

The objective of this study was to evaluate the most common treatments performed for hip fractures over the last decade in the United States. The leading treatment for trochanteric fractures was internal fixation, accounting for 96%-98% of surgical treatments each year. For cervical fractures, hemiarthroplasty (HA), total hip arthroplasty (THA), and internal fixation were performed nearly 61%, 5%, and 33% of the time, respectively, each year without any sign of change during the period assessed. The surgical choice for cervical fractures varied greatly by patient age. In 2009, two-thirds of patients younger than 60 years underwent internal fixation while two-thirds of patients 60 years or older underwent HA. Regardless of patient age, HA was performed more often than THA for cervical hip fractures.

Regional variation in the antibacterial activity of a wild plant, wild garlic (Allium ursinum L.).

Antibacterial activity is a common and highly studied property of plant secondary metabolites. Despite the extensive literature focusing on identifying novel antibacterial metabolites, little work has been undertaken to examine variation in levels of antibacterial activity in any plant species. Here, we used large-scale sampling of leaves of the antibacterial plant, wild garlic (Allium ursinum L.), assembling a set of tissue extracts from 168 plants, with 504 leaves collected and analysed. We assayed extracts for antibacterial activity against Bacillus subtilis and used LC-MS to carry out a chemometric analysis examining variation in individual metabolites, comparing them with several ecological parameters. We found that allicin was the only metabolite which was positively related to antibacterial activity. Soil temperature was a key determinant of variability in the concentrations of many foliar metabolites, however, neither allicin concentrations nor antibacterial activity was related to any of our measured ecological parameters, other than roadside proximity. We suggest that the synthesis of allicin precursors may be largely independent of growing conditions. This may be to ensure that allicin is synthesised rapidly and in sufficiently high concentrations to effectively prevent herbivory and pest damage. This finding contrasts with flavonoids which were found to vary greatly between plants and across sites. Our findings suggest that key biologically active metabolites are constrained in their concentration range compared to other compounds in the metabolome. This has important implications for the development of wild garlic as a health supplement or animal feed additive.

Irreducible posterior hip dislocation in the setting of a multifocal displaced pelvic ring injury: A case report.

Case report: Traumatic hip dislocations require prompt diagnosis and reduction to preserve the native joint. The classic irreducible posterior hip fracture-dislocation has been described as an immobile, slightly flexed, and internally rotated hip on physical exam. Classically, this irreducible pattern is associated with an ipsilateral femoral head fracture. The purpose of our report is to present an irreducible posterior hip dislocation with preserved motion in the setting of an unstable pelvic ring injury without associated femoral head pathology. Despite lacking clinical features of an irreducible hip, closed reduction in the emergency and operating rooms was unsuccessful, even after frame application for pelvis stability. Persistent irreducibility necessitated open reduction, where the femoral head was found to be buttonholed through the posterior hip capsule and blocking reduction. Conclusion: A posteriorly dislocated hip with preserved motion in the setting of a concomitant unstable pelvic ring injury may belie the true locked nature of the femoroacetabular dislocation and high suspicion for femoral head incarceration is required. The description of this unique irreducible fracture pattern and the stepwise approach used for reduction may be useful for other surgeons who may encounter similar patterns of injury.

Intraoperative Interpectoral and Subserratus Nerve Blocks in Breast Augmentation Surgery.

An essential component in ambulatory breast augmentation surgery is good analgesia. The demographic undergoing this operation is usually fit, low risk with few comorbidities. These patients do not require long-term hospitalization and do not want to spend excessive time in the hospital for financial reasons. Opiate analgesia can have significant side effects such as nausea, vomiting, and sedation. Reducing volumes of postoperative opiates allows faster ambulation and discharge from day surgery. We have developed two targeted nerve blocks that the operating surgeon can apply in minutes under direct vision, not requiring imaging. Anecdotally, we found that these targeted nerve blocks reduced opiate requirements and allowed accelerated discharge and faster return to normal activities. We conducted a prospective randomized, double-blind trial to test this theory. Methods: Twenty patients were randomized into saline (n = 10) or ropivacaine adrenaline solution (n = 10). The operating surgeons and anesthetists were blinded to the solution. All patients were closely followed up, and morphine equivalents were accurately recorded. Follow-up pain scores were recorded using the Overall Benefit of Analgesia pain questionnaire. Results: The ropivacaine nerve blocks significantly reduced opiate requirements postoperatively (P < 0.05). Pain scores were significantly decreased in the study group (P < 0.05). There were no side effects attributable to the nerve blocks. Conclusion: Intraoperative targeted nerve blocks significantly reduce postoperative opiate requirements in breast augmentation surgery resulting in faster recovery and higher patient satisfaction.

Characterization of Induction and Targeting of Senescent Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) from older donors have limited potential for bone tissue formation compared with cells from younger donors, and cellular senescence has been postulated as an underlying cause. There is a critical need for methods to induce premature senescence to study this phenomenon efficiently and reproducibly. However, the field lacks consensus on the appropriate method to induce and characterize senescence. Moreover, we have a limited understanding of the effects of commonly used induction methods on senescent phenotype. To address this significant challenge, we assessed the effect of replicative, hydrogen peroxide, etoposide, and irradiation-induced senescence on human MSCs using a battery of senescent cell characteristics. All methods arrested proliferation and resulted in increased cell spreading compared with low passage controls. Etoposide and irradiation increased expression of senescence-related genes in MSCs at early time points, proinflammatory cytokine secretion, DNA damage, and production of senescence-associated ß-galactosidase. We then evaluated the effect of fisetin, a flavonoid and candidate senolytic agent, to clear senescent cells and promote osteogenic differentiation of MSCs entrapped in gelatin methacryloyl (GelMA) hydrogels in vitro. When studying a mixture of nonsenescent and senescent MSCs, we did not observe decreases in senescent markers or increases in osteogenesis with fisetin treatment. However, the application of the same treatment toward a heterogeneous population of human bone marrow-derived cells entrapped in GelMA decreased senescent markers and increased osteogenesis after 14 days in culture. These results identify best practices for inducing prematurely senescent MSCs and motivate the need for further study of fisetin as a senolytic agent. Impact Statement The accumulation of senescent cells within the body has detrimental effects on tissue homeostasis. To study the role of senescent cells on tissue repair and regeneration, there is a need for effective means to induce premature cell senescence. Herein, we characterized the influence of common stressors to induce premature senescence in human mesenchymal stromal cells (MSCs). Irradiation of MSCs resulted in a phenotype most similar to quiescent, high-passage cells. These studies establish key biomarkers for evaluation when studying senescent cells in vitro.