Zonal Patterning of Extracellular Matrix and Stromal Cell Populations Along a Perfusable Cellular Microchannel.

The spatial organization of biophysical and biochemical cues in the extracellular matrix (ECM) in concert with reciprocal cell-cell signaling is vital to tissue patterning during development. However, elucidating the role an individual microenvironmental factor plays using existing in vivo models is difficult due to their inherent complexity. In this work, we have developed a microphysiological system to spatially pattern the biochemical, biophysical, and stromal cell composition of the ECM along an epithelialized 3D microchannel. This technique is adaptable to multiple hydrogel compositions and scalable to the number of zones patterned. We confirmed that the methodology to create distinct zones resulted in a continuous, annealed hydrogel with regional interfaces that did not hinder the transport of soluble molecules. Further, the interface between hydrogel regions did not disrupt microchannel structure, epithelial lumen formation, or media perfusion through an acellular or cellularized microchannel. Finally, we demonstrated spatially patterned tubulogenic sprouting of a continuous epithelial tube into the surrounding hydrogel confined to local regions with stromal cell populations, illustrating spatial control of cell-cell interactions and signaling gradients. This easy-to-use system has wide utility for modeling three-dimensional epithelial and endothelial tissue interactions with heterogeneous hydrogel compositions and/or stromal cell populations to investigate their mechanistic roles during development, homeostasis, or disease.

A straightforward cell culture insert model to incorporate biochemical and biophysical stromal properties into transplacental transport studies.

The placental extracellular matrix (ECM) dynamically remodels over pregnancy and in disease. How these changes impact placental barrier function is poorly understood as there are limited in vitro models of the placenta with a modifiable stromal compartment to mechanistically investigate these extracellular factors. We developed a straightforward method to incorporate uniform hydrogels into standard cell culture inserts for transplacental transport studies. Uniform polyacrylamide (PAA) gels were polymerized within cell culture inserts by (re)using the insert packaging to create a closed, controllable environmental chamber. PAA pre-polymer solution was added dropwise via a syringe to the cell culture insert and the atmosphere was purged with an inert gas. Transport and cell culture studies were conducted to validate the model. We successfully incorporated ECM-functionalized uniform PAA gels into cell culture inserts, enabling cell adhesion and monolayer formation. Imaging and analyte transport studies validated gel formation and expected mass transport results, and successful cell studies confirmed cell viability, stiffness-mediated YAP translocation, and that the model could be used in transplacental transport studies. Detailed methods and validation protocols are included. The incorporation of a PAA gel within a cell culture insert enables independent study of placental ECM biophysical and biochemical properties in the context of transplacental transport. These straightforward and low-cost methods to build three-dimensional cellular models are readily adoptable by the wider scientific community.

A straightforward cell culture insert model to incorporate biochemical and biophysical stromal properties into transplacental transport studies.

Introduction: The placental extracellular matrix (ECM) dynamically remodels over pregnancy and in disease. How these changes impact placental barrier function is poorly understood as there are limited in vitro models of the placenta with a modifiable stromal compartment to mechanistically investigate these extracellular factors. We developed a straightforward method to incorporate uniform hydrogels into standard cell culture inserts for transplacental transport studies. Methods: Uniform polyacrylamide (PAA) gels were polymerized within cell culture inserts by (re)using the insert packaging to create a closed, controllable environmental chamber. PAA pre-polymer solution was added dropwise via a syringe to the cell culture insert and the atmosphere was purged with an inert gas. Transport and cell culture studies were conducted to validate the model. Results: We successfully incorporated and ECM functionalized uniform PAA gels to cell culture inserts enable cell adhesion and monolayer formation. Imaging and analyte transport studies validated gel formation and expected mass transport results and successful cell studies confirmed cell viability, monolayer formation, and that the model could be used transplacental transport studies. Detailed methods and validation protocols are included. Discussion: It is well appreciated that ECM biophysical and biochemical properties impact cell phenotype and cell signaling in many tissues including the placenta. The incorporation of a PAA gel within a cell culture insert enables independent study of placental ECM biophysical and biochemical properties in the context of transplacental transport. These straightforward and low-cost methods to build three dimensional cellular models are readily adoptable by the wider scientific community.

A Dual-Screw Technique for Vertebral Compression Fractures via Robotic Navigation in the Osteopenic Lumbar Spine: An In-Vitro Biomechanical Analysis.

STUDY DESIGN: Biomechanical cadaveric study. OBJECTIVES: Multi-rod constructs maximize posterior fixation, but most use a single pedicle screw (PS) anchor point to support multiple rods. Robotic navigation allows for insertion of PS and cortical screw (CS) within the same pedicle, providing 4 points of bony fixation per vertebra. Recent studies demonstrated radiographic feasibility for dual-screw constructs for posterior lumbar spinal fixation; however, biomechanical characterization of this technique is lacking. METHODS: Fourteen cadaveric lumbar specimens (L1-L5) were divided into 2 groups (n = 7): PS, and PS + CS. VCF was simulated at L3. Bilateral posterior screws were placed from L2-L4. Load control (±7.5Nm) testing performed in flexion-extension (FE), lateral bending (LB), axial rotation (AR) to measure ROM of: (1) intact; (2) 2-rod construct; (3) 4-rod construct. Static compression testing of 4-rod construct performed at 5 mm/min to measure failure load, axial stiffness. RESULTS: Four-rod construct was more rigid than 2-rod in FE (P < .001), LB (P < .001), AR (P < .001). Screw technique had no significant effect on FE (P = .516), LB (P = .477), or AR (P = .452). PS + CS 4-rod construct was significantly more stable than PS group (P = .032). Stiffness of PS + CS group (445.8 ± 79.3 N/mm) was significantly greater (P = .019) than PS (317.8 ± 79.8 N/mm). Similarly, failure load of PS + CS group (1824.9 ± 352.2 N) was significantly greater (P = .001) than PS (913.4 ± 309.8 N). CONCLUSIONS: Dual-screw, 4-rod construct may be more stable than traditional rod-to-rod connectors, especially in axial rotation. Axial stiffness and ultimate strength of 4-rod, dual-screw construct were significantly greater than rod-to-rod. In this study, 4-rod construct was found to have potential biomechanical benefits of increased strength, stiffness, stability.

The Effect of Sacroiliac Fusion and Pelvic Fixation on Rod Strain in Thoracolumbar Fusion Constructs: A Biomechanical Investigation.

STUDY DESIGN: In vitro biomechanical study. OBJECTIVE: Investigate effects of sacroiliac joint (SIJ) fusion and iliac fixation on distal rod strain in thoracolumbar fusions. SUMMARY OF BACKGROUND DATA: Instrument failure is a multifactorial, challenging problem frequently encountered by spinal surgeons. Increased rod strain may lead to instrumentation failure and rod fracture. METHODS: Seven fresh frozen human cadaveric specimens (T9-pelvis) used. Six operative constructs tested to investigate changes in rod strain at L5-S1 and S1-Ilium rods, posterior pedicle screws/rods from T10-S1 (PS), PS + bilateral iliac screw fixation, PS + unilateral iliac screw fixation (UIS), PS+UIS+3 unilateral SIJ screws, PS + 3 unilateral SIJ screws, and PS +6 bilateral SIJ screws. Uniaxial strain gauges were used to measure surface strain of rods during flexion-extension. RESULTS: In flexion-extension, bilateral iliac screws added significant strain to L5-S1 compared with long fusion constructs ending at S1 (PS) (P < 0.05). Unilateral iliac fixation exhibited highest strain to L5-S1 ipsilateral rod, was significantly higher compared with bilateral iliac fixation and PS construct. Unilateral and bilateral SIJ fusion did not significantly change L5-S1 rod strain compared with PS. When measuring S1-Ilium rod strain, unilateral pelvic fixation had highest reported rod strain, approached significance compared with bilateral iliac screws (P = 0.054). Addition of contralateral SIJ fusion did not affect rod strain at S1-ilium on side with unilateral fixation. CONCLUSION: Results showed additional fixation below S1 to pelvis added significant rod strain. Unilateral pelvic screws had highest rod strain; SIJ fusion did not affect rod strain. Findings can help guide surgeons when associated risk of rod failure is a consideration.Level of Evidence: N/A.

A Cross University-Led COVID-19 Rapid-Response Effort: Design, Build, and Distribute Drexel AJFlex Face Shields.

The purpose of this article is to demonstrate how a new cross-community leadership team came together, collaborated, coordinated across academic units with external community partners, and executed a joint mission to address the unmet clinical need for medical face shields during these unprecedented times. Key aspects of this success include the ability to forge and leverage new opportunities, overcome challenges, adapt to changing constraints, and serve the significant need across the Philadelphia region and healthcare systems. We teamed to design-build durable face shields (AJFlex Shields). This was accomplished by high-volume manufacturing via injection molding and by 3-D printing the key headband component that supports the protective shield. Partnering with industry collaborators and civic-minded community allies proved to be essential to bolster production and deliver approximately 33,000 face shields to more than 100 organizations in the region. Our interdisciplinary team of engineers, clinicians, product designers, manufacturers, distributors, and dedicated volunteers is committed to continuing the design-build effort and providing Drexel AJFlex Shields to our communities.

Tunable Blood Shunt for Neonates With Complex Congenital Heart Defects.

Despite advancements in procedures and patient care, mortality rates for neonatal recipients of the Norwood procedure, a palliation for single ventricle congenital malformations, remain high due to the use of a fixed-diameter blood shunt. In this study, a new geometrically tunable blood shunt was investigated to address limitations of the current treatment paradigm (e.g., Modified Blalock-Taussig Shunt) by allowing for controlled modulation of blood flow through the shunt to accommodate physiological changes due to the patient's growth. First, mathematical and computational cardiovascular models were established to investigate the hemodynamic requirements of growing neonatal patients with shunts and to inform design criteria for shunt diameter changes. Then, two stages of prototyping were performed to design, build and test responsive hydrogel systems that facilitate tuning of the shunt diameter by adjusting the hydrogel's degree of crosslinking. We examined two mechanisms to drive crosslinking: infusion of chemical crosslinking agents and near-UV photoinitiation. The growth model showed that 15-18% increases in shunt diameter were required to accommodate growing patients' increasing blood flow; similarly, the computational models demonstrated that blood flow magnitudes were in agreement with previous reports. These target levels of diameter increases were achieved experimentally with model hydrogel systems. We also verified that the photocrosslinkable hydrogel, composed of methacrylated dextran, was contact-nonhemolytic. These results demonstrate proof-of-concept feasibility and reflect the first steps in the development of this novel blood shunt. A tunable shunt design offers a new methodology to rebalance blood flow in this vulnerable patient population during growth and development.

Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward "band-shift" in occludin Western immunoblots. The presence of RA partially reversed TNF-α's effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a "cytokine storm" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model.

The human bronchial epithelial cell line, 16HBE14o- (16HBE), is widely used as a model for respiratory epithelial diseases and barrier function. During differentiation, transepithelial electrical resistance (TER) increased to approximately 800 Ohms × cm2, while 14C-d-mannitol flux rates (Jm) simultaneously decreased. Tight junctions (TJs) were shown by diffusion potential studies to be anion-selective with PC1/PNa = 1.9. Transepithelial leakiness could be induced by the phorbol ester, protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). Basal barrier function could not be improved by the micronutrients, zinc, or quercetin. Of methodological significance, TER was observed to be more variable and to spontaneously, significantly decrease after initial barrier formation, whereas Jm did not significantly fluctuate or increase. Unlike the strong inverse relationship between TER and Jm during differentiation, differentiated cell layers manifested no relationship between TER and Jm. There was also much greater variability for TER values compared with Jm. Investigating the dependence of 16HBE TER on transcellular ion conductance, inhibition of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel with GlyH-101 produced a large decrease in short-circuit current (Isc) and a slight increase in TER, but no significant change in Jm. A strong temperature dependence was observed not only for Isc, but also for TER. In summary, research utilizing 16HBE as a model in airway barrier function studies needs to be aware of the complexity of TER as a parameter of barrier function given the influence of CFTR-dependent transcellular conductance on TER.

A biomechanical investigation of the sacroiliac joint in the setting of lumbosacral fusion: impact of pelvic fixation versus sacroiliac joint fixation.

OBJECTIVE: The sacroiliac joint (SIJ) is a known source of low-back pain. Randomized clinical trials support sacroiliac fusion over conservative management for SIJ dysfunction. Clinical studies suggest that SIJ degeneration occurs in the setting of lumbosacral fusions. However, there are few biomechanical studies to provide a good understanding of the effect of lumbosacral fusion on the SIJ. In the present study, researchers performed a biomechanical investigation to discern the effect of pelvic versus SIJ fixation on the SIJ in lumbosacral fusion. METHODS: Seven fresh-frozen human cadaveric specimens were used. There was one intact specimen and six operative constructs: 1) posterior pedicle screws and rods from T10 to S1 (PS); 2) PS + bilateral iliac screw fixation (BIS); 3) PS + unilateral iliac screw fixation (UIS); 4) PS + UIS + 3 contralateral unilateral SIJ screws (UIS + 3SIJ); 5) PS + 3 unilateral SIJ screws (3SIJ); and 6) PS + 6 bilateral SIJ screws (6SIJ). A custom-built 6 degrees-of-freedom apparatus was used to simulate three bending modes: flexion-extension (FE), lateral bending (LB), and axial rotation (AR). Range of motion (ROM) was recorded at L5-S1 and the SIJ. RESULTS: All six operative constructs had significantly reduced ROM at L5-S1 in all three bending modes compared to that of the intact specimen (p < 0.05). In the FE mode, the BIS construct had a significant reduction in L5-S1 ROM as compared to the other five constructs (p < 0.05). SIJ ROM was greatest in the FE mode compared to LB and AR. Although the FE mode did not show any statistically significant differences in SIJ ROM across the constructs, there were appreciable differences. The PS construct had the highest SIJ ROM. The BIS construct reduced bilateral SIJ ROM by 44% in comparison to the PS construct. The BIS and 6SIJ constructs showed reductions in SIJ ROM nearly equal to those of the PS construct. UIS and 3SIJ showed an appreciable reduction in unfused SIJ ROM compared to PS. CONCLUSIONS: This investigation demonstrated the effects of various fusion constructs using pelvic and sacroiliac fixation in lumbosacral fusion. This study adds biomechanical evidence of adjacent segment stress in the SIJ in fusion constructs extending to S1. Unilateral pelvic fixation, or SIJ fusion, led to an appreciable but nonsignificant reduction in the ROM of the unfused contralateral SIJ. Bilateral pelvic fixation showed the greatest significant reduction of movement at L5-S1 and was equivalent to bilateral sacroiliac fusion in reducing SIJ motion.

Stabilizing effect of the rib cage on adjacent segment motion following thoracolumbar posterior fixation of the human thoracic cadaveric spine: A biomechanical study.

BACKGROUND: Although the rib cage provides substantial stability to the thoracic spine, few biomechanical studies have incorporated it into their testing model, and no studies have determined the influence of the rib cage on adjacent segment motion of long fusion constructs. The present biomechanical study aimed to determine the mechanical contribution of the intact rib cage during the testing of instrumented specimens. METHODS: A cyclic loading (CL) protocol with instrumentation (T4-L2 pedicle screw-rod fixation) was conducted on five thoracic spines (C7-L2) with intact rib cages. Range of motion (±5 Nm pure moment) in flexion-extension, lateral bending, and axial rotation was captured for intact ribs, partial ribs, and no ribs conditions. Comparisons at the supra-adjacent (T2-T3), adjacent (T3-T4), first instrumented (T4-T5), and second instrumented (T5-T6) levels were made between conditions (P ≤ 0.05). FINDINGS: A trend of increased motion at the adjacent level was seen for partial ribs and no ribs in all 3 bending modes. This trend was also observed at the supra-adjacent level for both conditions. No significant changes in motion compared to the intact ribs condition were seen at the first and second instrumented levels (P > 0.05). INTERPRETATION: The segment adjacent to long fusion constructs, which may appear more grossly unstable when tested in the disarticulated spine, is reinforced by the rib cage. In order to avoid overestimating adjacent level motion, when testing the effectiveness of surgical techniques of the thoracic spine, inclusion of the rib cage may be warranted to better reflect clinical circumstances.