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A 57-year-old Hispanic man with diabetes presented with dyspnoea. He had a positive SARS-CoV-2 PCR. He was intubated for severe hypoxia and treated with intermittent pressors, methylprednisolone and supportive care. He was extubated on hospital day (HD) 9 and discharged to a skilled nursing facility (SNF) on HD 18. Approximately 1 month later, he presented with melena. Endoscopy revealed two large 1.5-2 cm wide-based distal oesophageal ulcers without active bleeding. Histology showed ulcerated squamous mucosa with extensive necrosis extending to the muscularis propria and coccoid bacterial colonies with rare fungal forms suggestive of Candida He was treated with fluconazole and pantoprazole and was discharged to a SNF. Approximately 3 weeks later, he was readmitted for complications. Repeat endoscopy demonstrated improvement and histology revealed chronic inflammation with reactive epithelial changes. Incidentally, SARS-CoV-2 PCR was positive during this visit without any respiratory symptoms., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Importance: This is the first large-scale randomized clinical trial evaluating the effectiveness and safety of overminus spectacle therapy for treatment of intermittent exotropia (IXT)., Objective: To evaluate the effectiveness of overminus spectacles to improve distance IXT control., Design, Setting, and Participants: This randomized clinical trial conducted at 56 clinical sites between January 2017 and January 2019 associated with the Pediatric Eye Disease Investigator Group enrolled 386 children aged 3 to 10 years with IXT, a mean distance control score of 2 or worse, and a refractive error between 1.00 and -6.00 diopters (D). Data analysis was performed from February to December 2020., Interventions: Participants were randomly assigned to overminus spectacle therapy (-2.50 D for 12 months, then -1.25 D for 3 months, followed by nonoverminus spectacles for 3 months) or to nonoverminus spectacle use., Main Outcomes and Measures: Primary and secondary outcomes were the mean distance IXT control scores of participants examined after 12 months of treatment (primary outcome) and at 18 months (3 months after treatment ended) assessed by an examiner masked to treatment group. Change in refractive error from baseline to 12 months was compared between groups. Analyses were performed using the intention-to-treat population., Results: The mean (SD) age of 196 participants randomized to overminus therapy and 190 participants randomized to nonoverminus treatment was 6.3 (2.1) years, and 226 (59%) were female. Mean distance control at 12 months was better in participants treated with overminus spectacles than with nonoverminus spectacles (1.8 vs 2.8 points; adjusted difference, -0.8; 95% CI, -1.0 to -0.5; P < .001). At 18 months, there was little or no difference in mean distance control between overminus and nonoverminus groups (2.4 vs 2.7 points; adjusted difference, -0.2; 95% CI, -0.5 to 0.04; P = .09). Myopic shift from baseline to 12 months was greater in the overminus than the nonoverminus group (-0.42 D vs -0.04 D; adjusted difference, -0.37 D; 95% CI, -0.49 to -0.26 D; P < .001), with 33 of 189 children (17%) in the overminus group vs 2 of 169 (1%) in the nonoverminus group having a shift higher than 1.00 D., Conclusions and Relevance: Children 3 to 10 years of age had improved distance exotropia control when assessed wearing overminus spectacles after 12 months of overminus treatment; however, this treatment was associated with increased myopic shift. The beneficial effect of overminus lens therapy on distance exotropia control was not maintained after treatment was tapered off for 3 months and children were examined 3 months later., Trial Registration: ClinicalTrials.gov Identifier: NCT02807350.

Purpose: To use simulated arthroscopic knot tying to assess (1) whether epithelial cells from the surgeon's hands were transmitted to the suture and (2) whether the number of knots tied or the presence of glove tears would correlate with the number of cells transmitted., Methods: Knots were tied in a simulated arthroscopic environment using a nonabsorbable No. 2 suture over a metal hook. The surgeon was double gloved for each knot tied. For each "anchor," a surgeon's knot was tied, followed by 3 reversed half-hitches on alternating posts. Multiple skin lacerations were sustained by the surgeon during each knot-tying session. Gloves were collected after tying 2, 4, or 6 anchors. Gloves were tested for perforation by (1) electroconductivity and (2) saline solution load testing. Cytopathologic ThinPrep analysis was applied and allowed for the number of epithelial cells found on each suture (within 10 high-powered fields) to be counted. Statistical analysis included analysis of variance and logistic regression., Results: There was no significant difference in the number of epithelial cells identified in any of the groups compared with the negative control groups (P > .05) or with each other (P > .05). Glove tears were present in 3.3% of gloves (50% in inner and 50% in outer gloves) and 1.7% of gloves (50% in inner and 50% in outer gloves) by electroconductivity and saline solution load testing, respectively. There was no significant association between glove tears and the number of epithelial cells found on the suture (P > .05)., Conclusions: Epithelial cells were transmitted to the suture during simulated arthroscopic knot tying. However, despite multiple skin lacerations produced during knot-tying sessions, the number of cells transmitted was not significantly different when compared with the negative controls. The number of cells transmitted did not correlate with the number of knots tied and/or the presence of glove tears., Clinical Relevance: Skin lacerations on the surgeon's fingers are often noted after arthroscopic knot tying. However, despite these skin lacerations, no skin tissue is transferred across the surgical gloves to the suture itself., (Copyright © 2017 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(epsilon-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities-- 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds, in conjunction with fluid flow bioreactor culture, minimize diffusion constraints and provide mechanical stimulation to the marrow stromal cells, leading to enhancement of differentiation toward development of bone-like mineralized tissue. These results also demonstrate that the scaffold structure, namely, the porosity, influences the sequential development of osteoblastic cells and, in combination with the culture conditions, may affect the functionality of tissues formed in vitro.

In summary, many factors can influence the osteoblastic differentiation of marrow stromal cells when cultivated on three-dimensional tissue engineering scaffolds. In creating ideal bone tissue engineering constructs consisting of a combination of a scaffold, cells, and bioactive factors; a flow perfusion bioreactor is a much more suitable culture environment than static culture in well plates. The bioreactor eliminates mass transport limitations to the scaffold interior and provides mechanical stimulation to the seeded cells through fluid shear. Scaffold properties such as pore size impact cell differentiation, especially in flow perfusion culture. In addition, the bone-like extracellular matrix created by the in vitro culture of marrow stromal cells on porous scaffolds creates an osteoinductive environment for the differentiation of other marrow stromal cell populations. Therefore, bone tissue engineering constructs created by in vitro culture have excellent potential for bone regeneration applications in the clinic. However, more work is required to optimize this tissue engineering strategy. A biodegradable material with mechanical integrity similar to native bone and degradation properties similar to the rate of bone formation would be a more ideal scaffold material. It is also yet unclear what the optimum scaffold pore size and amount of in vitro generated extracellular matrix are to maximize bone formation. Finally, better characterization of the flow patterns within the flow perfusion bioreactor is needed to better understand the relationship between fluid shear and cell differentiation for creation of the ideal scaffold/culture combination.

Titanium fiber mesh scaffolds have been shown to be a suitable material for culture of primary marrow stromal cells in an effort to create tissue engineered constructs for bone tissue replacement. In native bone tissue, these cells are known to attach to extracellular matrix molecules via integrin receptors for specific peptide sequences, and these attachments can be a source of cell signaling, affecting cell behaviors such as differentiation. In this study, we examined the ability of primary rat marrow stromal cells at two different stages of osteoblastic differentiation to further differentiate into osteoblasts both in vitro and in vivo when seeded on titanium fiber mesh scaffolds either with or without RGD peptide tethered to the surface. In vitro, the tethered RGD peptide resulted in reduced initial cell proliferation. In vivo, there was no effect of tethered RGD peptide on ectopic bone formation in a rat subcutaneous implant model. Scaffold/cell constructs exposed to dexamethasone for 4 days prior to implantation (+dex constructs) resulted in significant bone formation whereas no bone formation was observed in--dex constructs. These results show that the osteoblastic differentiation of marrow stromal cells was not dependent on surface tethered RGD peptide, and that the initial differentiation stage of implanted cells plays an important role in bone formation in titanium fiber mesh bone tissue engineering constructs.

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% beta-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.

In this study, we cultured marrow stromal cells on titanium fiber meshes in a flow perfusion bioreactor and examined the effect of altering scaffold mesh size on cell behavior in an effort to develop a bone tissue construct composed of a scaffold, osteogenic cells, and extracellular matrix. Scaffolds of differing mesh size, that is, distance between fibers, were created by altering the diameter of the mesh fibers (20 or 40 microm) while maintaining a constant porosity. These scaffolds had a porosity of 80% and mesh sizes of 65 microm (20-microm fibers) or 119 microm (40-microm fibers). Cell/scaffold constructs were grown in static culture or under flow for up to 16 days and assayed for osteoblastic differentiation. Cellularity was higher at early time points and Ca2+ deposition was higher at later time points for flow constructs over static controls. The 20-microm mesh had reduced cellularity in static culture. Under flow conditions, mass transport limitations are mitigated allowing uniform cell growth throughout the scaffold, and there was no difference in cellularity between mesh types. There was greater alkaline phosphatase (ALP) activity, osteopontin levels, and calcium under flow at 8 days for the 40-microm mesh compared to the 20-microm mesh. However, by day 16, the trend was reversed, suggesting the time course of differentiation was dependent on scaffold mesh size under flow conditions. However, this dependence was not linear with respect to time; larger mesh size was conducive to early osteoblast differentiation while smaller mesh size was conducive to later differentiation and matrix deposition., ((c) 2005 Wiley Periodicals, Inc.)

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze-thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5 x 10(5) cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3 x 10(6) cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (+/- 13.6) microg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.

In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells. Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media of different viscosities while maintaining the fluid flow rate constant. This methodology allowed exposure of the cultured cells to increasing levels of mechanical stimulation, in the form of fluid shear stress, whereas chemotransport conditions for nutrient delivery and waste removal remained essentially constant. Under similar chemotransport for the cultured cells in the 3D porous scaffolds, increasing fluid shear forces led to increased mineral deposition, suggesting that the mechanical stimulation provided by fluid shear forces in 3D flow perfusion culture can indeed enhance the expression of the osteoblastic phenotype. Increased fluid shear forces also resulted in the generation of a better spatially distributed extracellular matrix inside the porosity of the 3D titanium fiber mesh scaffolds. The combined effect of fluid shear forces on the mineralized extracellular matrix production and distribution emphasizes the importance of mechanosensation on osteoblastic cell function in a 3D environment.