An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells.

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal stem cells.

Background and Aims: The intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs. Methods: 3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results. Results: Chamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells. Conclusions: We present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.

Return-to-Sport Time and Postoperative Performance in MLB Players Undergoing Wrist Arthroscopy.

BACKGROUND: Wrist injuries are common in sports and can result in prolonged time missed from playing. This study aimed to determine in Major League Baseball-players after arthroscopic wrist surgery the return-to-sport (RTS) rate, postoperative career length, and changes in performance compared with preoperative statistics and matched controls. METHODS: Major League Baseball players who underwent arthroscopic wrist surgery from 1990 to 2019 were identified. Demographic and performance data were collected for each player, and matched controls were identified. Comparisons were made via paired samples Student t tests. RESULTS: Twenty-six players (27 surgeries) were identified. The average age of included players was 28.9 ± 2.9 years with an average professional experience of 5.2 ± 3.4 years. Eighty-four percent of players returned to sport, with an average RTS time of 5.0 ± 2.7 months. A statistically significant (P < .05) decrease was seen in preoperative and postoperative runs scored per season (95.6 ± 91.3 vs 41.0 ± 29.5), batting average (BA) (0.270 ± 0.024 vs 0.240 ± 0.036), and average wins above replacement (WAR) (1.5 ± 1.1 vs 0.8 ± 0.9). CONCLUSION: Major League Baseball players who underwent arthroscopic wrist surgery had an RTS rate of 84% at a mean time of 5.0 months. There was no significant difference in performance statistics between cases postoperatively and matched controls overall, with some differences in performance found when categorized by position. However, a significant decrease in performance among case players was observed between preoperative and postoperative performance, including runs per season, BA, and WAR.

Percutaneous Needle Fasciotomy versus Collagenase Injection for Dupuytren's Contracture: A Systematic Review of Comparative Studies.

Introduction The purpose of this study was to review and compare clinical outcomes between percutaneous needle fasciotomy (PNF) and collagenase Clostridium histolyticum (CCH) injection for the treatment of Dupuytren's contracture. Materials and Methods A systematic review was performed including all level I-III evidence studies investigating the clinical outcomes of PNF and CCH injection in the treatment of Dupuytren's contracture. Results Five studies (278 CCH patients, 225 PNF patients; 285 CCH fingers, 246 PNF fingers, 405 males, and 98 females) were analyzed. Two randomized studies were level I evidence, one randomized study was level II, and two nonrandomized studies were level III. Two studies analyzed a total of 205 patients, each demonstrating statistically superior outcomes in one outcome measure (contracture improvement and Michigan Hand Questionnaire (MHQ) satisfaction subscore) with PNF, while the remaining three studies demonstrated no significant differences in outcomes between the two techniques. Three studies reported a statistically higher rate of minor complications (local pain, edema, ecchymosis, lymphadenopathy, pruritis) with CCH, while the remaining two studies demonstrated no significant difference in complication rates. Conclusion For the treatment of Dupuytren's contracture, there is some evidence that suggests superior clinical outcomes of PNF compared with CCH and a higher minor complication rate with CCH.

Performance and Return to Sport After Open Fracture in National Football League Players.

BACKGROUND: Open fractures are debilitating injuries for athletes. No prior studies have investigated open fractures in National Football League (NFL) players. PURPOSE: To compare outcomes after open fracture in NFL players in terms of (1) time to return to sport (RTS), (2) postinjury career length and games played per season, (3) postinjury performance, and (4) postinjury performance compared with matched controls. STUDY DESIGN: Retrospective comparative series; Level of evidence, 3. METHODS: Publicly available records were used to identify NFL players who had sustained an open fracture between 1970 and 2018. Controls were matched to injured players by age, experience, position, and preinjury performance. RTS was defined as playing in at least 1 NFL game after open fracture. Comparisons between injured and control players were made using the paired-samples Student t test. RESULTS: Injuries in 37 players were analyzed (age, 27.2 ± 3.6 years; experience, 4.4 ± 3.6 seasons).  The 3 most common locations for open fracture were the tibia/fibula (n = 16), hand/finger (n = 12), and forearm/wrist (n = 3). A total of 30 (81%) players had a mean time of RTS of 9.3 ± 8.2 months after open fracture; of these players, 4 (13.3%) who sustained hand/finger open fracture did not undergo surgical treatment. There was no difference in postinjury career length or games played per season between control and injured players. Postinjury performance was similar to preinjury performance in injured players, and postinjury performance scores were similar between injured and control players. There were significant differences between players who sustained upper extremity and lower extremity open fractures in RTS time (4.0 ± 4.8 vs 14.6 ± 7.4 months, respectively; P = .00007) and postinjury performance (6.4 ± 4.3 vs 3.3 ± 2.1, respectively; P = .03). CONCLUSION: RTS after open fracture among NFL players was high. Players who sustained an open fracture had similar games played per season, career length, and performance compared with matched controls. Players who sustained an upper extremity open fracture had a faster RTS time, higher RTS rate, and improved postinjury performance compared with players who sustained a lower extremity open fracture.