The Intersection of Race and Sex: A New Perspective Into Diversity Trends in Orthopaedic Surgery.

INTRODUCTION: Studies on diversity in orthopaedic surgery have exclusively examined challenges from a race or sex perspective. This study examines trends in the diversity of entering orthopaedic surgery residents from the intersection of race and sex. METHODS: The American Association of Medical Colleges was queried for individuals entering orthopaedic surgery residencies in the United States from 2001 to 2020. Deidentified data on self-reported sex and race were collected. Proportions by the intersection of sex and race were calculated for 5-year intervals. RESULTS: From 2001 to 2020, most of the new female residents identified as White (mean, 71.0%). The average proportion of White female residents was lower in 2016 to 2020 than in 2001 to 2005 (71.0% vs. 73.2%) but higher than that in 2011 to 2015 (66.8%). The 2016 to 2020 average was lower than that of 2001 to 2005 for those who identified as Asian (11.7% vs. 14.9%), Black (4.1% vs. 4.8%), Hispanic (3.0% vs. 4.4%), and American Indian/Alaska Native (0.0% vs. 1.5%). Most of the new male orthopaedic surgery residents from 2001 to 2020 identified as White (mean, 74.1%), but the average decreased across every 5-year interval from 2001 to 2005 (76.1%) to 2016 to 2020 (71.1%). The 2016 to 2020 average was lower than that of 2001 to 2005 for those who identified as Asian (12.2% vs. 13.6%), Black (3.5% vs. 4.2%), Hispanic (3.0% vs. 3.4%), American Indian/Alaska Native (0.0% vs. 0.6%), and Native Hawaiian/Other Pacific Islander (0.1% vs. 0.3%). In 2020, White male residents made up to 54.2% of new residents. White female residents were the second highest group represented (12.1%). CONCLUSION: Increases in representation were observed for some subgroups of new orthopaedic surgery residents from 2001 to 2020. Although the proportion of both White female and male residents decreased by 11.5% during the 20-year study period, these individuals still made up most of the trainees in 2020. These results underscore the need for conversations and recruitment practices to take into consideration the intersectionality of identities.

Biodistribution of lentiviral transduced adipose-derived stem cells for "ex-vivo" regional gene therapy for bone repair.

Ex-vivo gene therapy has been shown to be an effective method for treating bone defects in pre-clinical models. As gene therapy is explored as a potential treatment option in humans, an assessment of the safety profile becomes an important next step. The purpose of this study was to evaluate the biodistribution of viral particles at the defect site and various internal organs in a rat femoral defect model after implantation of human ASCs transduced with lentivirus (LV) with two-step transcriptional activation (TSTA) of bone morphogenetic protein-2 (LV-TSTA-BMP-2). Animals were sacrificed at 4-, 14-, 56-, and 84-days post implantation. The defects were treated with either a standard dose (SD) of 5 million cells or a high dose (HD) of 15 million cells to simulate a supratherapeutic dose. Treatment groups included (1) SD LV-TSTA-BMP-2 (2) HD LV-TSTA-BMP-2, (3) SD LV-TSTA-GFP (4) HD LV-TSTA-GFP and (5) SD nontransduced cells. The viral load at the defect site and ten organs was assessed at each timepoint. Histology of all organs, ipsilateral tibia, and femur were evaluated at each timepoint. There were nearly undetectable levels of LV-TSTA-BMP-2 transduced cells at the defect site at 84-days and no pathologic changes in any organ at all timepoints. In conclusion, human ASCs transduced with a lentiviral vector were both safe and effective in treating critical size bone defects in a pre-clinical model. These results suggest that regional gene therapy using lentiviral vector to treat bone defects has the potential to be a safe and effective treatment in humans.

Trends in Race and Sex Representation Among Entering Orthopaedic Surgery Residents: A Continued Call for Active Diversification Efforts.

INTRODUCTION: Multiple studies have analyzed the diversity of surgical subspecialties, in which orthopaedic surgery consistently lags behind in female and minority representation. This study aims to examine contemporary data on trends in sex and racial representation among entering orthopaedic surgery residents. METHODS: The American Association of Medical Colleges' Graduate Medical Education Track data set was queried for all individuals entering surgical residencies in the United States from 2001 to 2020. Deidentified data on self-reported sex and race (American Indian or Alaska Native [AIAN]; Asian; Black or African American, Hispanic, Latino, or of Spanish Origin; Native Hawaiian or Other Pacific Islander [NHOPI]; White; and Other) for individuals across all surgical subspecialties were collected. Sex and race proportions for newly matriculating surgical residents were analyzed and aggregated across the study period. RESULTS: From 2001 to 2020, there was a 9.2% increase in the proportion of new female orthopaedic surgery residents, with approximately one in five identifying as such in 2020. By contrast, surgical specialties in aggregate saw a 16.3% increase. A 11.7% decrease was observed in entering orthopaedic residents who identified as White with a corresponding increase in representation by multiracial (9.2%) individuals and those identifying as "Other" (1.9%). The proportion of Asian (range: 10.4 to 15.4%), Black (2.5 to 6.2%), Hispanic (0.3 to 4.4%), AIAN (0.0 to 1.2%), and NHOPI (0.0 to 0.5%) new trainees has largely remained unchanged throughout the study period. A similar trend was observed among surgical specialties in aggregate. Of the identities most represented by the multiracial cohort, the most common were Asian (range: 7.0 to 50.0%), Hispanic (0.0 to 53.5%), and White (30.2 to 50.0%). CONCLUSION: Although orthopaedic surgery has improved in sex diversity in its entering class of residents, measures to increase racial diversity have been less successful. Efforts at improving the recruitment of a diverse class of trainees are necessary and will require acknowledging the importance of both racial and sex representation diversity metrics.

Spatial characterization of redox processes and speciation of Ru(III) anticancer complexes by 19F magnetic resonance imaging.

The application of CF3-labeled Ru(III) anticancer complexes to magnetic resonance (MR) imaging of tumour tissues is demonstrated. By combining anatomical chemical-shift selective (CHESS) imaging with 19F chemical-shift imaging (CSI) MR methods, we show that oxidation states and ligand-exchange processes of the complexes can be spatially encoded. Measurements on different tissue models, including a human breast adenocarcinoma tumour, validate the application of these complexes as MR theranostics for the sensing and targeting of hypoxia.

Influence of donor age and comorbidities on transduced human adipose-derived stem cell in vitro osteogenic potential.

Human adipose-derived mesenchymal stem cells (ASCs) transduced with a lentiviral vector system to express bone morphogenetic protein 2 (LV-BMP-2) have been shown to reliably heal bone defects in animal models. However, the influence of donor characteristics such as age, sex, race, and medical co-morbidities on ASC yield, growth and bone regenerative capacity, while critical to the successful clinical translation of stem cell-based therapies, are not well understood. Human ASCs isolated from the infrapatellar fat pads in 122 ASC donors were evaluated for cell growth characteristics; 44 underwent additional analyses to evaluate in vitro osteogenic potential, with and without LV-BMP-2 transduction. We found that while female donors demonstrated significantly higher cell yield and ASC growth rates, age, race, and the presence of co-morbid conditions were not associated with differences in proliferation. Donor demographics or the presence of comorbidities were not associated with differences in in vitro osteogenic potential or stem cell differentiation, except that transduced ASCs from healthy donors produced more BMP-2 at day 2. Overall, donor age, sex, race, and the presence of co-morbid conditions had a limited influence on cell yield, proliferation, self-renewal capacity, and osteogenic potential for non-transduced and transduced (LV-BMP-2) ASCs. These results suggest that ASCs are a promising resource for both autologous and allogeneic cell-based gene therapy applications.

Effects of cell seeding technique and cell density on BMP-2 production in transduced human mesenchymal stem cells.

Small animal models have demonstrated the efficacy of ex vivo regional gene therapy using scaffolds loaded with BMP-2-expressing mesenchymal stem cells (MSCs). Prior to clinical translation, optimization of seeding techniques of the transduced cells will be important to minimize time and resource expenditure, while maximizing cell delivery and BMP-2 production. No prior studies have investigated cell-seeding techniques in the setting of transduced cells for gene therapy applications. Using BMP-2-expressing transduced adipose-derived MSCs and a porous ceramic scaffold, this study compared previously described static and dynamic seeding techniques with respect to cell seeding efficiency, uniformity of cell distribution, and in vitro BMP-2 production. Static and negative pressure seeding techniques demonstrated the highest seeding efficiency, while orbital shaking was associated with the greatest increases in BMP-2 production per cell. Low density cell suspensions were associated with the highest seeding efficiency and uniformity of cell distribution, and the greatest increases in BMP-2 production from 2 to 7 days after seeding. Our results highlight the potential for development of an optimized cell density and seeding technique that could greatly reduce the number of MSCs needed to produce therapeutic BMP-2 levels in clinical situations. Further studies are needed to investigate in vivo effects of cell seeding techniques on bone healing.

Improving Lentiviral Transduction of Human Adipose-Derived Mesenchymal Stem Cells.

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34+ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.

Use of optical fluorescence agents during surgery for pituitary adenomas: current state of the field.

INTRODUCTION: Differentiation of normal pituitary from abnormal tumor tissue remains a surgical challenge despite improvements in optical visualization technology for pituitary adenoma (PA) surgery. During neurosurgical procedures for other tumor types, 5-aminolevulinic acid (5-ALA) has become a focus of investigation based on its high specificity in differentiating tumor tissue. However, the role of 5-ALA and other optical fluorescent agents in PA surgery remains less clear. OBJECTIVE: To perform a systematic review on the use of various optical fluorescent agents in PA surgery. METHOD: Using PRISMA guidelines, a systematic literature review to identify reports describing 5-ALA and other optical agents for fluorescence-guided surgery for PA was performed. Eleven research studies met inclusion criteria and were reviewed. RESULTS: In two studies, 5-ALA was not shown to be effective in aiding PA resection using standard neurosurgical endoscopic/microscopic approaches. 5-ALA photodynamic therapy was evaluated in two in-vitro models with inconsistent results. Intraoperative use of indocyanine green (ICG) concluded with varying results, but showed a tendency towards improved differentiation of functional PA. OTL38 showed potential for intraoperative identification of nonfunctioning PA, particularly in tumors with high folate receptor expression. One study reported clinically useful fluorescence following sodium fluorescein administration. CONCLUSION: We conclude that selected optical fluorescent agents, including ICG and folate receptors, are most likely to hold promise for clinical use in differentiating PA from normal tissue.

Anticancer copper pyridine benzimidazole complexes: ROS generation, biomolecule interactions, and cytotoxicity.

The Cu(II) complex CuCl2(pbzH), pbzH=2-(2-pyridyl)benzimidazole, and derivatives modified at the non-coordinated nitrogen of the benzimidazole fragment, have been studied as anticancer agents. These compounds show promising cytotoxicity against A549 adenocarcinomic alveolar basal epithelial cells with IC50 values in the range of 5-10µM. Importantly, this activity is higher than either CuCl2·2H2O or the individual ligands, demonstrating that ligand coordination to the Cu(II) centres of the complexes is required for full activity. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopies were used to characterize the solution behaviour of the complexes. These studies demonstrate: (i) two types of solvated species in buffer, (ii) both coordinate and non-coordinate interactions with albumin, and (iii) weak interactions with DNA. Further DNA studies using agarose gel electrophoresis demonstrate strand cleavage by the complexes in the presence of ascorbate, which is mediated by reactive oxygen species (ROS). Through a fluorescence-based in vitro assay, intracellular ROS generation in the A549 cell line was observed; indicating that damage by ROS is responsible for the observed activity of the complexes.

Combining a Ru(ii)-arene complex with a NO-releasing nitrate-ester ligand generates cytotoxic activity.

A Ru(ii) arene complex with a NO-releasing 4-nitrooxymethyl-pyridine ligand shows increased cytotoxicity against the non-small cell lung cancer cell line A549 as compared to either the free ligand or the unfunctionalized complex. EPR spin-trapping studies show that NO release is selective, being limited in phosphate buffered saline or human serum, but promoted by glutathione.

CF3 Derivatives of the Anticancer Ru(III) Complexes KP1019, NKP-1339, and Their Imidazole and Pyridine Analogues Show Enhanced Lipophilicity, Albumin Interactions, and Cytotoxicity.

The Ru(III) complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (NKP-1339) are leading candidates for the next generation of metal-based chemotherapeutics. Trifluoromethyl derivatives of these compounds and their imidazole and pyridine analogues were synthesized to probe the effect of ligand lipophilicity on the pharmacological properties of these types of complexes. Addition of CF3 groups also provided a spectroscopic handle for (19)F NMR studies of ligand exchange processes and protein interactions. The lipophilicities of the CF3-functionalized compounds and their unsubstituted parent complexes were quantified by the shake-flask method to give the distribution coefficient D at pH 7.4 (log D7.4). The solution behavior of the CF3-functionalized complexes was characterized in phosphate-buffered saline (PBS) using (19)F NMR, electron paramagnetic resonance (EPR), and UV-vis spectroscopies. These techniques, along with fluorescence competition experiments, were also used to characterize interactions with human serum albumin (HSA). From these studies it was determined that increased lipophilicity correlates with reduced solubility in PBS but enhancement of noncoordinate interactions with hydrophobic domains of HSA. These protein interactions improve the solubility of the complexes and inhibit the formation of oligomeric species. EPR measurements also demonstrated the formation of HSA-coordinated species with longer incubation. (19)F NMR spectra show that the trifluoromethyl complexes release axial ligands in PBS and in the presence of HSA. In vitro testing showed that the most lipophilic complexes had the greatest cytotoxic activity. Addition of CF3 groups enhances the activity of the indazole complex against A549 nonsmall cell lung carcinoma cells. Furthermore, in the case of the pyridine complexes, the parent compound was inactive against the HT-29 human colon carcinoma cell line but showed strong cytotoxicity with CF3 functionalization. Overall, these studies demonstrate that lipophilicity may be a determining factor in the anticancer activity and pharmacological behavior of these types of Ru(III) complexes.

Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMI-A by Ferrocene Functionalization.

A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the anti-metastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV-vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic (1)H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.

Pseudomonas aeruginosa-induced bleb-niche formation in epithelial cells is independent of actinomyosin contraction and enhanced by loss of cystic fibrosis transmembrane-conductance regulator osmoregulatory function.

UNLABELLED: The opportunistic pathogen Pseudomonas aeruginosa can infect almost any site in the body but most often targets epithelial cell-lined tissues such as the airways, skin, and the cornea of the eye. A common predisposing factor is cystic fibrosis (CF), caused by defects in the cystic fibrosis transmembrane-conductance regulator (CFTR). Previously, we showed that when P. aeruginosa enters epithelial cells it replicates intracellularly and occupies plasma membrane blebs. This phenotype is dependent on the type 3 secretion system (T3SS) effector ExoS, shown by others to induce host cell apoptosis. Here, we examined mechanisms for P. aeruginosa-induced bleb formation, focusing on its relationship to apoptosis and the CFTR. The data showed that P. aeruginosa-induced blebbing in epithelial cells is independent of actin contraction and is inhibited by hyperosmotic media (400 to 600 mOsM), distinguishing bacterially induced blebs from apoptotic blebs. Cells with defective CFTR displayed enhanced bleb formation upon infection, as demonstrated using bronchial epithelial cells from a patient with cystic fibrosis and a CFTR inhibitor, CFTR(Inh)-172. The defect was found to be correctable either by incubation in hyperosmotic media or by complementation with CFTR (pGFP-CFTR), suggesting that the osmoregulatory function of CFTR counters P. aeruginosa-induced bleb-niche formation. Accordingly, and despite their reduced capacity for bacterial internalization, CFTR-deficient cells showed greater bacterial occupation of blebs and enhanced intracellular replication. Together, these data suggest that P. aeruginosa bleb niches are distinct from apoptotic blebs, are driven by osmotic forces countered by CFTR, and could provide a novel mechanism for bacterial persistence in the host. IMPORTANCE: Pseudomonas aeruginosa is an opportunistic pathogen problematic in hospitalized patients and those with cystic fibrosis (CF). Previously, we showed that P. aeruginosa can enter epithelial cells and replicate within them and traffics to the membrane blebs that it induces. This "bleb-niche" formation requires ExoS, previously shown to cause apoptosis. Here, we show that the driving force for bleb-niche formation is osmotic pressure, differentiating P. aeruginosa-induced blebs from apoptotic blebs. Either CFTR inhibition or CFTR mutation (as seen in people with CF) causes P. aeruginosa to make more bleb niches and provides an osmotic driving force for blebbing. CFTR inhibition also enhances bacterial occupation of blebs and intracellular replication. Since CFTR is targeted for removal from the plasma membrane when P. aeruginosa invades a healthy cell, these findings could relate to pathogenesis in both CF and healthy patient populations.