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.

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 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.