Nanofiber-microwell cell culture system for spatially patterned differentiation of pluripotent stem cells in 3D.

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.

Development and Utilization of Multifunctional Polymeric Scaffolds for the Regulation of Physical Cellular Microenvironments.

Polymeric biomaterials exhibit excellent physicochemical characteristics as a scaffold for cell and tissue engineering applications. Chemical modification of the polymers has been the primary mode of functionalization to enhance biocompatibility and regulate cellular behaviors such as cell adhesion, proliferation, differentiation, and maturation. Due to the complexity of the in vivo cellular microenvironments, however, chemical functionalization alone is usually insufficient to develop functionally mature cells/tissues. Therefore, the multifunctional polymeric scaffolds that enable electrical, mechanical, and/or magnetic stimulation to the cells, have gained research interest in the past decade. Such multifunctional scaffolds are often combined with exogenous stimuli to further enhance the tissue and cell behaviors by dynamically controlling the microenvironments of the cells. Significantly improved cell proliferation and differentiation, as well as tissue functionalities, are frequently observed by applying extrinsic physical stimuli on functional polymeric scaffold systems. In this regard, the present paper discusses the current state-of-the-art functionalized polymeric scaffolds, with an emphasis on electrospun fibers, that modulate the physical cell niche to direct cellular behaviors and subsequent functional tissue development. We will also highlight the incorporation of the extrinsic stimuli to augment or activate the functionalized polymeric scaffold system to dynamically stimulate the cells.

Cumulative sum: An individualized proficiency metric for laparoscopic fundamentals.

BACKGROUND: A reliable metric of technical proficiency is indispensable to the training of fellows and residents. The purpose of this study was to determine whether cumulative sum (Cusum) has predictive validity in laparoscopic training. We hypothesized that Cusum would be a better predictor of technical ability in fundamentals of laparoscopic surgery (FLS) than traditional practice volume metrics. METHODS: Twenty medical students were recruited to practice three FLS tasks: peg transfer, circle cut, and intracorporeal knot tie. Up to 7hours of self-directed practice was allotted to each participant. Practice attempts were scored by standard FLS criteria and monitored via Cusum. Each participant's terminal Cusum performance was analyzed retrospectively. Posttests were conducted by faculty blinded to practice performance. RESULTS: Eighteen participants completed the study (90%). Median adjusted posttest scores were 102.3, 84.1, and 78.6 for peg transfer, circle cut, and knot tie, respectively. For the knot tie task, participants who exceeded the Cusum decision interval during their final practice attempts performed significantly higher on posttesting (81.2 vs 71.5, p=0.015). Knot tie terminal Cusum score was positively associated with posttest performance after adjusting for practice volume (p=0.031). Total practice volume and practice time were not significantly associated with posttest performance for any FLS task. CONCLUSION: Cusum score is a more valid representation of FLS proficiency than practice volume or practice time. Incorporating Cusum in a clinical setting may promote more efficient allocation of time resources and operative volume.

Vessel ligation training via an adaptive simulation curriculum.

BACKGROUND: A cost-effective model for open vessel ligation is currently lacking. We hypothesized that a novel, inexpensive vessel ligation simulator can efficiently impart transferrable surgical skills to novice trainees. MATERIALS AND METHODS: VesselBox was designed to simulate vessel ligation using surgical gloves as surrogate vessels. Fourth-year medical students performed ligations using VesselBox and were evaluated by surgical faculty using the Objective Structured Assessments of Technical Skills global rating scale and a task-specific checklist. Subsequently, each student was trained using VesselBox in an adaptive practice session guided by cumulative sum. Posttesting was performed on fresh human cadavers by evaluators blinded to pretest results. RESULTS: Sixteen students completed the study. VesselBox practice sessions averaged 21.8 min per participant (interquartile range 19.5-27.7). Blinded posttests demonstrated increased proficiency, as measured by both Objective Structured Assessments of Technical Skills (3.23 versus 2.29, P < 0.001) and checklist metrics (7.33 versus 4.83, P < 0.001). Median speed improved from 128.2 s to 97.5 s per vessel ligated (P = 0.001). After this adaptive training protocol, practice volume was not associated with posttest performance. CONCLUSIONS: VesselBox is a cost-effective, low-fidelity vessel ligation model suitable for graduating medical students and junior residents. Cumulative sum can facilitate an adaptive, individualized curriculum for simulation training.

Construct validation of a cost-effective vessel ligation benchtop simulator.

OBJECTIVE: Many benchtop surgical simulators assess laparoscopic proficiency, yet few address core open surgical skills. The purpose of this study is to describe a cost-effective benchtop vessel ligation simulator and provide construct validation. DESIGN: A prospective comparison of blinded proficiency assessments among participants performing a benchtop vessel ligation simulation task. Evaluations were performed using Objective Structured Assessments of Technical Skills. SETTING: This study took place at the University of Virginia, School of Medicine: a large academic medical institution. PARTICIPANTS: The participants included fourth-year medical students participating in a focused surgical elective course (n = 16), postgraduate year 2 to 3 surgery residents (n = 6), and surgical faculty (n = 5). RESULTS: The total fixed costs of the vessel ligation simulator was $30. Flexible costs of operation were less than $0.20 per attempt. The median task-specific checklist scores among the medical students, residents, and faculty were 4.83, 7.33, and 7.67, respectively. Median global rating scores across the 3 groups were 2.29, 4.43, and 4.76, respectively. Significant proficiency differences were noted between the students and the residents/faculty for both the metrics (p < 0.001). CONCLUSIONS: A cost-effective benchtop simulator can effectively measure proficiency with basic open surgical techniques such as vessel ligation. Among the junior surgical trainees, this tool can identify learning gaps and improve operative skills in a preclinical setting.