Factors Associated With High-Risk and Low-Risk Bone Stress Injury in Female Runners: Implications for Risk Factor Stratification and Management.

Background: Bone stress injury (BSI) is a common overuse injury in active women. BSIs can be classified as high-risk (pelvis, sacrum, and femoral neck) or low-risk (tibia, fibula, and metatarsals). Risk factors for BSI include low energy availability, menstrual dysfunction, and poor bone health. Higher vertical load rates during running have been observed in women with a history of BSI. Purpose/Hypothesis: The purpose of this study was to characterize factors associated with BSI in a population of premenopausal women, comparing those with a history of high-risk or low-risk BSI with those with no history of BSI. It was hypothesized that women with a history of high-risk BSI would be more likely to exhibit lower bone mineral density (BMD) and related factors and less favorable bone microarchitecture compared with women with a history of low-risk BSI. In contrast, women with a history of low-risk BSI would have higher load rates. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Enrolled were 15 women with a history of high-risk BSI, 15 with a history of low-risk BSI, and 15 with no history of BSI. BMD for the whole body, hip, and spine was standardized using z scores on dual-energy x-ray absorptiometry. High-resolution peripheral quantitative computed tomography was used to quantify bone microarchitecture at the radius and distal tibia. Participants completed surveys characterizing factors that influence bone health-including sleep, menstrual history, and eating behaviors-utilizing the Eating Disorder Examination Questionnaire (EDE-Q). Each participant completed a biomechanical assessment using an instrumented treadmill to measure load rates before and after a run to exertion. Results: Women with a history of high-risk BSI had lower spine z scores than those with low-risk BSI (-1.04 ± 0.76 vs -0.01 ± 1.15; P < .05). Women with a history of high-risk BSI, compared with low-risk BSI and no BSI, had the highest EDE-Q subscores for Shape Concern (1.46 ± 1.28 vs 0.76 ± 0.78 and 0.43 ± 0.43) and Eating Concern (0.55 ± 0.75 vs 0.16 ± 0.38 and 0.11 ± 0.21), as well as the greatest difference between minimum and maximum weight at current height (11.3 ± 5.4 vs 7.7 ± 2.9 and 7.6 ± 3.3 kg) (P < .05 for all). Women with a history of high-risk BSI were more likely than those with no history of BSI to sleep <7 hours on average per night during the week (80% vs 33.3%; P < .05). The mean and instantaneous vertical load rates were not different between groups. Conclusion: Women with a history of high-risk BSI were more likely to exhibit risk factors for poor bone health, including lower BMD, while load rates did not distinguish women with a history of BSI.

Impact of Micro- and Nano-Plastics on Human Intestinal Organoid-Derived Epithelium.

The development of patient-derived intestinal organoids represents an invaluable model for simulating the native human intestinal epithelium. These stem cell-rich cultures outperform commonly used cell lines like Caco-2 and HT29-MTX in reflecting the cellular diversity of the native intestinal epithelium after differentiation. In our recent study examining the effects of polystyrene (PS), microplastics (MPs), and nanoplastics (NPs), widespread pollutants in our environment and food chain, on the human intestinal epithelium, these organoids have been instrumental in elucidating the absorption mechanisms and potential biological impacts of plastic particles. Building on previously established protocols in human intestinal organoid culture, we herein detail a streamlined protocol for the cultivation, differentiation, and generation of organoid-derived monolayers. This protocol is tailored to generate monolayers incorporating microfold cells (M cells), key for intestinal particle uptake but often absent in current in vitro models. We provide validated protocols for the characterization of MPs/NPs via scanning electron microscopy (SEM) for detailed imaging and their introduction to intestinal epithelial monolayer cells via confocal immunostaining. Additionally, protocols to test the impacts of MP/NP exposure on the functions of the intestinal barrier using transendothelial electrical resistance (TEER) measurements and assessing inflammatory responses using cytokine profiling are detailed. Overall, our protocols enable the generation of human intestinal organoid monolayers, complete with the option of including or excluding M cells, offering crucial techniques for observing particle uptake and identifying inflammatory responses in intestinal epithelial cells to advance our knowledge of the potential effects of plastic pollution on human gut health. These approaches are also amendable to the study of other gut-related chemical and biological exposures and physiological responses due to the robust nature of the systems. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Human intestinal organoid culture and generation of monolayers with and without M cells Support Protocol 1: Culture of L-WRN and production of WRN-conditioned medium Support Protocol 2: Neuronal cell culture and integration into intestinal epithelium Support Protocol 3: Immune cell culture and integration into intestinal epithelium Basic Protocol 2: Scanning electron microscopy: sample preparation and imaging Basic Protocol 3: Immunostaining and confocal imaging of MP/NP uptake in organoid-derived monolayers Basic Protocol 4: Assessment of intestinal barrier function via TEER measurements Basic Protocol 5: Cytokine profiling using ELISA post-MP/NP exposure.

Biological effects of polystyrene micro- and nano-plastics on human intestinal organoid-derived epithelial tissue models without and with M cells.

Micro- and nano-plastics (MPs and NPs) released from plastics in the environment can enter the food chain and target the human intestine. However, knowledge about the effects of these particles on the human intestine is still limited due to the lack of relevant human intestinal models to validate data obtained from animal studies or tissue models employing cancer cells. In this study, human intestinal organoids were used to develop epithelia to mimic the cell complexity and functions of native tissue. Microfold cells (M cells) were induced to distinguish their role when exposure to MPs and NPs. During the exposure, the M cells acted as sensors, capturers and transporters of larger sized particles. The epithelial cells internalized the particles in a size-, concentration-, and time-dependent manner. Importantly, high concentrations of particles significantly triggered the secretion of a panel of inflammatory cytokines linked to human inflammatory bowel disease (IBD).

Crypt-Villus Scaffold Architecture for Bioengineering Functional Human Intestinal Epithelium.

Crypt-villus architecture in the small intestine is crucial for the structural integrity of the intestinal epithelium and maintenance of gut homeostasis. We utilized three-dimensional (3D) printing and inverse molding techniques to form three-dimensional (3D) spongy scaffold systems that resemble the intestinal crypt-villus microarchitecture. The scaffolds consist of silk fibroin protein with curved lumens with rows of protruding villi with invaginating crypts to generate the architecture. Intestinal cell (Caco-2, HT29-MTX) attachment and growth, as well as long-term culture support were demonstrated with cell polarization and tissue barrier properties compared to two-dimensional (2D) Transwell culture controls. Further, physiologically relevant oxygen gradients were generated in the 3D system. The various advantages of this system may be ascribed to the more physiologically relevant 3D environment, offering a system for the exploration of disease pathogenesis, host-microbiome interactions, and therapeutic discovery.

Bioengineered 3D Tissue Model of Intestine Epithelium with Oxygen Gradients to Sustain Human Gut Microbiome.

The human gut microbiome is crucial to hosting physiology and health. Therefore, stable in vitro coculture of primary human intestinal cells with a microbiome community is essential for understanding intestinal disease progression and revealing novel therapeutic targets. Here, a three-dimensional scaffold system is presented to regenerate an in vitro human intestinal epithelium that recapitulates many functional characteristics of the native small intestines. The epithelium, derived from human intestinal enteroids, contains mature intestinal epithelial cells and possesses selectively permeable barrier functions. Importantly, by properly positioning the scaffolds cultured under normal atmospheric conditions, two physiologically relevant oxygen gradients, a proximal-to-distal oxygen gradient along the gastrointestinal (GI) tract, and a radial oxygen gradient across the epithelium, are distinguished in the tissues when the lumens are faced up and down in cultures, respectively. Furthermore, the presence of the low oxygen gradients supported the coculture of intestinal epithelium along with a complex living commensal gut microbiome (including obligate anaerobes) to simulate temporal microbiome dynamics in the native human gut. This unique silk scaffold platform may enable the exploration of microbiota-related mechanisms of disease pathogenesis and host-pathogen dynamics in infectious diseases including the potential to explore the human microbiome-gut-brain axis and potential novel microbiome-based therapeutics.

Serum 25-Hydroxyvitamin D is Associated With Bone Microarchitecture and Strength in a Multiracial Cohort of Young Adults.

PURPOSE: To determine whether 25-hydroxyvitamin D (25-OH D) levels are associated with bone outcomes in a multiracial cohort of young adults. METHODS: This cross-sectional study included 165 participants (83 men, 82 women, 18-30 years of age) who self-identified as Asian, Black, or White. We measured bone microarchitecture and strength of the distal radius and tibia using high-resolution peripheral quantitative computed tomography. We used linear regression to estimate the association between 25-OH D (ng/mL) and bone measurements, adjusting for race, sex, age, weight, height, calcium intake, physical activity, and season. RESULTS: A total of 43.6% of participants were 25-OH D deficient (<20 ng/mL) with greater prevalence in Asian (38.9%) and Black (43.1%) compared with White (18.0%) participants (P < 0.001). At the distal radius, 25-OH D was positively associated with cortical area, trabecular density, cortical thickness, cortical porosity, and failure load (P < 0.05 for all). At the distal tibia, higher 25-OH D was associated with higher cortical area, trabecular density, trabecular number, failure load, and lower trabecular separation and cortical density (P < 0.05 for all). After multivariable adjustment, those with 25-OH D deficiency had generally worse bone microarchitecture than those with 25-OH D sufficiency. Black individuals had largely more favorable bone outcomes than Asian and White individuals, despite higher prevalence of 25-OH D deficiency. CONCLUSIONS: We found a high prevalence of 25-OH D deficiency in a multiracial cohort of young adults. Lower 25-OH D was associated with worse bone outcomes at the distal radius and tibia at the time of peak bone mass, warranting further attention to vitamin D status in young adults.

Restrictive Eating and Prior Low-Energy Fractures Are Associated With History of Multiple Bone Stress Injuries.

Bone stress injuries (BSIs) are common among athletes and have high rates of recurrence. However, risk factors for multiple or recurrent BSIs remain understudied. Thus, we aimed to explore whether energy availability, menstrual function, measures of bone health, and a modified Female Athlete Triad Cumulative Risk Assessment (CRA) tool are associated with a history of multiple BSIs. We enrolled 51 female runners (ages 18-36 years) with history of ≤1 BSI (controls; n = 31) or ≥3 BSIs (multiBSI; n = 20) in this cross-sectional study. We measured lumbar spine, total hip, and femoral neck areal bone mineral density by dual-energy X-ray absorptiometry, bone material strength index using impact microindentation, and volumetric bone mineral density, microarchitecture, and estimated strength by high-resolution peripheral quantitative computed tomography. Participants completed questionnaires regarding medical history, low-energy fracture history, and disordered eating attitudes. Compared with controls, multiBSI had greater incidence of prior low-energy fractures (55% vs. 16%, p = .005) and higher modified Triad CRA scores (2.90 ± 2.05 vs. 1.84 ± 1.59, p = .04). Those with multiBSI had higher Eating Disorder Examination Questionnaire (0.92 ± 1.03 vs. 0.46 ± 0.49, p = .04) scores and a greater percentage difference between lowest and highest body mass at their current height (15.5% ± 6.5% vs. 11.5% ± 4.9% p = .02). These preliminary findings indicate that women with a history of multiple BSIs suffered more prior low-energy fractures and have greater historical and current estimates of energy deficit compared with controls. Our results provide strong rationale for future studies to examine whether subclinical indicators of energy deficit contribute to risk for multiple BSIs in female runners.

The Short-Chain Fatty Acids Propionate and Butyrate Augment Adherent-Invasive Escherichia coli Virulence but Repress Inflammation in a Human Intestinal Enteroid Model of Infection.

Short-chain fatty acids (SCFAs), which consist of six or fewer carbons, are fermentation products of the bacterial community that inhabits the intestine. Due to an immunosuppressive effect on intestinal tissue, they have been touted as a therapeutic for inflammatory conditions of the bowel. Here, we study the impact of acetate, propionate, and butyrate, the three most abundant SCFAs in the intestine, on gene expression in the intestinal pathobiont adherent-invasive Escherichia coli. We pair this with adherence, invasion, and inflammation in Caco-2 and human intestinal enteroid (HIE)-derived monolayer models of the intestinal epithelium. We report that propionate and butyrate upregulate transcription of adherent-invasive Escherichia coli (AIEC) flagellar synthesis genes and decrease expression of capsule assembly and transport genes. These changes are predicted to augment AIEC invasiveness. In fact, SCFA supplementation increases AIEC adherence to and invasion of the Caco-2 monolayer but has no effect on these parameters in the HIE model. We attribute this to the anti-inflammatory effect of propionate and butyrate on HIEs but not on Caco-2 cells. We conclude that the potential of SCFAs to increase the virulence of intestinal pathogens should be considered in their use as anti-inflammatory agents. IMPORTANCE The human terminal ileum and colon are colonized by a community of microbes known as the microbiota. Short-chain fatty acids (SCFAs) excreted by bacterial members of the microbiota define the intestinal environment. These constitute an important line of communication within the microbiota and between the microbiota and the host epithelium. In inflammatory conditions of the bowel, SCFAs are often low and there is a preponderance of a conditionally virulent bacterium termed adherent-invasive Escherichia coli (AIEC). A connection between SCFA abundance and AIEC has been suggested. Here, we study AIEC in monoculture and in coculture with human intestinal enteroid-derived monolayers and show that the SCFAs propionate and butyrate increase expression of AIEC virulence genes while concurrently bolstering the intestinal epithelial barrier and reducing intestinal inflammation. While these SCFAs have been promoted as a therapy for inflammatory bowel conditions, our findings demonstrate that their effect on bacterial virulence must be considered.

Physical Activity, Menstrual History, and Bone Microarchitecture in Female Athletes with Multiple Bone Stress Injuries.

PURPOSE: To determine differences in health and physical activity history, bone density, microarchitecture, and strength among female athletes with a history of multiple BSI, athletes with ≤1 BSI, and nonathletes. METHODS: We enrolled 101 women (age, 18-32 yr) for this cross-sectional study: nonathlete controls (n = 17) and athletes with a history of ≥3 BSIs (n = 21) or ≤1 BSI (n = 63). We collected subjects' health and training history and measured bone microarchitecture of the distal tibia via high-resolution peripheral quantitative computed tomography (HR-pQCT) and areal bone mineral density of the hip and spine by dual-energy X-ray absorptiometry. RESULTS: Groups did not differ according to age, body mass index, age at menarche, areal bone mineral density, or tibial bone microarchitecture. Women with multiple BSI had a higher prevalence of primary and secondary amenorrhea (P < 0.01) compared with other groups. Total hours of physical activity in middle school were similar across groups; however, women with multiple BSI performed more total hours of physical activity in high school (P = 0.05), more hours of uniaxial loading in both middle school and high school (P = 0.004, P = 0.02), and a smaller proportion of multiaxial loading activity compared with other groups. CONCLUSIONS: These observations suggest that participation in sports with multiaxial loading and maintaining normal menstrual status during adolescence and young adulthood may reduce the risk of multiple bone stress injuries.

Changes in Volumetric Bone Mineral Density Over 12 Months After a Tibial Bone Stress Injury Diagnosis: Implications for Return to Sports and Military Duty.

BACKGROUND: Bone stress injuries (BSIs) occur in up to 20% of runners and military personnel. Typically, after a period of unloading and gradual return to weightbearing activities, athletes return to unrestricted sports participation or military duty approximately 4 to 14 weeks after a BSI diagnosis, depending on the injury location and severity. However, the time course of the recovery of the bone's mechanical competence is not well-characterized, and reinjury rates are high. PURPOSE: To assess the bone microarchitecture and volumetric bone mineral density (vBMD) over 12 months after a tibial BSI diagnosis. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: We enrolled 30 female athletes from the local community (aged 18-35 years) with a tibial BSI (grade ≥2 of 4 on magnetic resonance imaging) for this prospective observational study. Participants completed a baseline visit within 3 weeks of the diagnosis. At baseline and 6, 12, 24, and 52 weeks after the BSI diagnosis, we collected high-resolution peripheral quantitative computed tomography scans of the ultradistal tibia (4% of tibial length) of the injured and uninjured legs as well as pain and physical activity assessment findings. RESULTS: From baseline to 12 weeks after the diagnosis, total, trabecular, and cortical vBMD declined by 0.58% to 0.94% (P < .05 for all) in the injured leg. Total and trabecular vBMD also declined by 0.61% and 0.67%, respectively, in the uninjured leg (P < .05 for both). At 24 weeks, mean values for all bone parameters were nearly equivalent to baseline values, and by 52 weeks, several mean values had surpassed baseline values. Of the 30 participants, 10 incurred a subsequent BSI during the course of the study, and 1 of these 10 incurred 2 subsequent BSIs. Participants who suffered an additional BSI were younger and had a later age of menarche, a greater incidence of previous fractures, and lower serum parathyroid hormone levels (P < .05 for all). CONCLUSION: Bone density declined in both the injured and the uninjured legs and, on average, did not return to baseline for 3 to 6 months after a tibial BSI diagnosis. The observed time to the recovery of baseline vBMD, coupled with the high rate of recurrent BSIs, suggests that improved return-to-sports and military duty guidelines may be in order.

Influence of soft tissue on bone density and microarchitecture measurements by high-resolution peripheral quantitative computed tomography.

High-resolution peripheral quantitative computed tomography (HR-pQCT) is a non-invasive method of measuring volumetric bone mineral density (vBMD) and microarchitecture at the distal radius and tibia. With increasing use of this technology, it is crucial to understand the potential impact of overlying soft tissue on the accuracy of HR-pQCT measures. Thus, we examined the effects of a simulated increase in adiposity (via 6- and 12-mm thick layers of overlying circumferential fat) on HR-pQCT measures of a hydroxyapatite (HA) phantom and in women (n = 20, aged 18-75 years). In the phantom, increasing the amount of overlying fat tissue led to a corresponding decrease in the mean measured density for each HA rod. In women, fat-layering led to a decrease in total vBMD (-2.9 to -3.7%, p < 0.001), cortical vBMD (-1.4% to -5.5%, p < 0.001), and estimated failure load (-1.4 to -5.7%, p = 0.002) at the radius, with similar changes in the tibia. Trabecular microarchitectural measurements were also impacted by simulated adiposity, with fat-layering leading to decreased trabecular thickness and separation and increased trabecular number at the radius (Δ's = 5 to 12%) with more pronounced differences at the tibia (Δ's = 14 to 40%). At the tibia, fat-layering also led to decreased cortical thickness and increased cortical porosity. Altogether, these results demonstrate that overlying adipose tissue can lead to artifacts in bone measurements by HR-pQCT, resulting in an underestimation of vBMD and generally, an overestimation of bone microarchitecture impairment. Therefore, soft tissue artifact should be considered when interpreting HR-pQCT results, particularly in those with high BMI and/or marked changes in adiposity.