Tunable hydrogel viscoelasticity modulates human neural maturation.

Human-induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biomechanical signaling cues imparted by the extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in neural maturation gene expression. Together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation.

Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

Measurement and assessment of grief in a large international sample.

BACKGROUND: In 2022, the International Classification of Diseases (ICD-11) and an update of the Diagnostic Statistical Manual of Mental Disorders (DSM 5 TR) were released for implementation worldwide and now include the new Prolonged Grief Disorder (PGD). The newest definition of PGD is based on robust clinical research from the Global North yet until now has not been tested for global applicability. METHODS: The current study assesses the new PGD ICD-11 criteria in a large international sample of 1393 bereaved adults. The majority of the sample was included from the USΑ. Additionally, we conduct a sub-sample analysis to evaluate the psychometric properties, probable caseness of PGD, and differences in network structure across three regions of residency (USA, Greece-Cyprus, Turkey-Iran). RESULTS: The psychometric validity and reliability of the 33-item International Prolonged Grief Disorder Scale (IPGDS) were confirmed across the whole sample and for each regional group. Using the strict diagnostic algorithm, the probable caseness for PGD for the whole sample was 3.6 %. Probable caseness was highest for the Greece-Cyprus group (6.9 %) followed by Turkey-Iran (3.2 %) and the USA (2.8 %). Finally, the network structure of the IPGDS standard items and cultural supplement items (total of 33 items) confirmed the strong connection between central items of PGD, and revealed unique network connections within the regional groups. LIMITATIONS: Future research is encouraged to include larger sample sizes and a more systematic assessment of culture. CONCLUSION: Overall, our findings confirm the global applicability of the new ICD-11 PGD disorder definition as evaluated through the newly developed IPGDS. This scale includes culturally sensitive grief symptoms that may improve clinical precision and decision-making.

Associations Between Primary Residence and Mental Health in Global Marginalized Populations.

Scholars suggest that marginalized people in non-urban areas experience higher distress levels and fewer psychosocial resources than in urban areas. Researchers have yet to test whether precise proximity to urban centers is associated with mental health for marginalized populations. We recruited 1733 people who reported living in 45 different countries. Participants entered their home locations and completed measures of anxiety, depression, social support, and resilience. Regression and thematic analyses were used to determine what role distance from legislative and urban centers may play in mental health when marginalized people were disaggregated. Greater distance from legislative center predicted higher anxiety and resilience. Greater distance from urban center also predicted more resilience. Thematic analyses yielded five categories (e.g., safety, connection) that further illustrated the impact of geographic location on health. Implications for community mental health are discussed including the need to better understand and further expand resilience in rural areas.

Safe drilling zones for anteriorly, central, and posteriorly angulated syndesmotic stabilization devices.

PURPOSE: The purposes of the study were to (1) analyze the shape of the distal fibula at the location of syndesmotic stabilization and to (2) define safe zones at the distal-lateral fibula for three different drilling tunnel orientations: anteriorly-, posteriorly angulated and center-center. METHODS: Postoperative, bilateral CT images of adult patients that underwent syndesmotic stabilization (suture-button system) for an acute, unilateral ankle injury were analyzed. Manual axial CT reconstructions of the uninjured side were generated. First, the axial shape of the distal fibula was classified. The aspect ratio between the anterio-lateral and the posterior-lateral surfaces of the fibula was calculated to assess symmetry. Second, the same axial planes were used to define the safe zones. Each drilling-tunnel orientation (anterior, central, posterior) comprised a fixed medial tibial anchor point and a safe zone on the lateral fibula. For each of the three orientations, the most anteriorly and posteriorly drilling tunnel location was simulated. Next to a cumulative visual analysis, a quantitative analysis of the most anterior and posterior point on the anterio- and posterior-lateral surfaces was calculated. RESULTS: A total of 96 CT datasets were analyzed. (1) 81% of fibulae revealed a triangular convex-, 10% an irregular-, and 8% a quadrilateral shape. The lateral surface ratio was 1.0 ± 0.2 (range: 0.7-1.5), not differing between the fibula types (n.s.). (2) The safe corridor on the lateral surface of the fibula for an anteriorly angulated drilling tunnel was - 8% to - 41%, for a posteriorly angulated drilling tunnel was 0% to 46%, and for a center-center alignment - 7 ± 11% (range: - 28 to 18%). CONCLUSION: The meta-diaphyseal region of the distal fibula revealed a homogeneous crosssectional shape. The lateral apex of the fibula can serve as a landmark defining safe zones to place the drilling tunnels correctly. Applying these safe zones in clinical practice could help to avoid the misplacement of the syndesmotic fixation device. LEVEL OF EVIDENCE: Level III, retrospective radiographic study.

Influence of kinematic alignment on femorotibial kinematics in medial stabilized TKA design compared to mechanical alignment.

INTRODUCTION: Worldwide more and more primary knee replacements are being performed. Kinematic alignment (KA) as one of many methods of surgical alignment has been shown to have a significant impact on kinematics and function. The aim of the present study was to compare KA and mechanical alignment (MA) with regard to femorotibial kinematics. MATERIALS AND METHODS: Eight fresh frozen human specimens were tested on a knee rig during active knee flexion from 30 to 130°. Within the same specimen a medial stabilized (MS) implant design was used first with KA and then with MA. RESULTS: The femorotibial kinematics showed more internal rotation of the tibia in KA compared to MA. At the same time, there was a larger medial rotation point in KA. Both alignment methods showed femoral rollback over the knee bend. CONCLUSION: Relating to an increased internal rotation and a more precise medial pivot point, it can be concluded that KA combined with a MS implant design may partially support the reproduction of physiological knee joint mechanics.

Tuning pro-survival effects of human induced pluripotent stem cell-derived exosomes using elastin-like polypeptides.

Exosome-based regenerative therapies are potentially easier to manufacture and safer to apply compared to cell-based therapies. However, many questions remain about how to bio-manufacture reproducible and potent exosomes using animal-free reagents. Here we evaluate the hypothesis that designer biomaterial substrates can be used to alter the potency of exosomes secreted by human induced pluripotent stem cells (iPSCs). Two animal-free designer matrices were fabricated based on recombinant elastin-like polypeptides (ELPs): one including a cell-adhesive RGD ligand and a second with a non-adhesive RDG peptide. While iPSCs cultured on these two substrates and Matrigel-coated controls had similar levels of proliferation, the RDG-ELP substrate significantly increased protein expression of stemness markers OCT4 and SOX2 and suppressed spontaneous differentiation compared to those on RGD-ELP. The pro-survival potency of iPSC-derived exosomes was evaluated using three distinct stress tests: serum starvation in murine fibroblasts, hypoxia in human endothelial cells, and hyperosmolarity in canine kidney cells. In all three cases, exosomes produced by iPSCs grown on RDG-ELP substrates had similar pro-survival effects to those produced using iPSCs grown on Matrigel, while use of RGD-ELP substrates led to significantly reduced exosome potency. These data demonstrate that recombinant substrates can be designed for the robust bio-manufacturing of iPSC-derived, pro-survival exosomes.

A multicentre, randomised, double-blind, phase II study to evaluate the tolerability of an induction dose escalation of everolimus in patients with metastatic breast cancer (DESIREE).

BACKGROUND: Stomatitis is one of the main reasons to discontinue everolimus in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC). To decrease stomatitis and subsequently early treatment discontinuations or dose reductions, the DESIREE trial investigated the use of a stepwise dose-escalation schedule of everolimus (EVE esc). PATIENTS AND METHODS: DESIREE is a phase II, multicentre, randomised, double-blind, placebo-controlled trial in patients with HR+/HER2- mBC and progression/relapse after nonsteroidal aromatase inhibitor treatment. Patients were randomised to EVE esc (2.5 mg/day, week 1; 5 mg/day, week 2; 7.5 mg/day, week 3; 10 mg/day, weeks 4-24) or everolimus 10 mg/day (EVE 10mg) for 24 weeks plus exemestane. The primary endpoint was the incidence of stomatitis episodes grade ≥2 within 12 weeks of treatment. The secondary endpoints included toxicity, relative total dose intensity (RTDI) and quality of life (QoL). RESULTS: A total of 160 patients were randomised and 156 started treatment (EVE esc: 80; EVE 10mg: 76). The median age of patients was 64 years (range 33-85), 56.3% patients in the EVE esc arm versus 42.1% in the EVE 10mg arm had liver metastasis (P = 0.081) and 62.5% versus 51.3% received over one metastatic therapy line (P = 0.196). Within 12 weeks, the incidence of stomatitis episodes grade ≥2 was significantly lower in the EVE esc arm compared with the EVE 10mg arm (28.8% versus 46.1%; odds ratio 0.47, 95% confidence interval 0.24-0.92; P = 0.026). Toxicity was in line with the known safety profile without new safety concerns. The median RTDI was 91.1% in the EVE esc arm versus 80.0% in the EVE 10mg arm (P = 0.329). Discontinuation rate in the first 3 weeks was 6.3% versus 15.8%, respectively (P = 0.073). QoL was comparable between the two treatment arms. CONCLUSIONS: A dose-escalation schema of everolimus over 3 weeks can be successfully used to reduce the incidence of high-grade stomatitis in the first 12 weeks of treatment in patients with HR+/HER2- mBC. TRIAL REGISTRATION: ClinicalTrials.govNCT02387099; https://clinicaltrials.gov/ct2/show/NCT02387099.

In Vitro Performance of Different Universal Adhesive Systems on Several CAD/CAM Restorative Materials After Thermal Aging.

OBJECTIVE: To evaluate the microshear bond strength (mSBS) of 10 universal adhesive systems applied on five different CAD/CAM restorative materials, immediately and after thermal aging. METHODS AND MATERIALS: Five CAD/CAM materials were selected: 1) feldspathic glass ceramic (FeCe); 2) pre-polymerized reinforced resin composite (ReRC); 3) leucite-reinforced glass ceramic (LeGC); 4) lithium disilicate (LiDi); and 5) yttrium-stabilized zirconium dioxide (ZiDi). For each material, 15 blocks were cut into four rectangular sections (6 × 6 × 6 mm; n=60 per group) and processed as recommended by the respective manufacturer. For each indirect material, the following adhesive systems were applied according to the respective manufacturer's instructions: 1) AdheSE Universal [ADU]; 2) All-Bond Universal [ABU]; 3) Ambar Universal [AMB]; 4) Clearfil Universal Bond [CFU]; 5) Futurabond U [FBU]; 6) One Coat 7 Universal [OCU]; 7) Peak Universal Bond [PUB]; 8) Prime&Bond Elect [PBE]; 9) Scotchbond Universal Adhesive [SBU]; 10) Xeno Select [XEN, negative control]. After the application of the adhesive system, cylinder-shaped transparent matrices were filled with a dual-curing resin cement (NX3) and light cured. Specimens were tested in shear mode at 1.0 mm/min (mSBS), after 24 hours and 10,000 thermal cycles (TC). All data were submitted to statistical analysis (α=0.05). RESULTS: For FeCe, there was no significant decrease in mean mSBS for AMB, FBU, and SBU after TC when compared at 24 hours. For ReRC, AMB and SBU showed higher mean mSBS when compared to CFU and XEN, after 24 hours and TC. For LiDi, FBU and OCU showed higher mean mSBS when compared to CFU and XEN, after 24 hours and TC. For LeGC, AMB and PUB showed higher mean mSBS when compared to XEN, after 24 hours and TC. For ZiDi, OCU and SBU showed higher mean mSBS when compared to XEN, after 24 hours and TC. In addition, PBE and XEN showed the lowest mean mSBS after TC with higher percentage of bond strength reduction. CONCLUSIONS: The mean mSBS among the different universal adhesives varied widely for each CAD/CAM material used. In addition, most universal adhesives underwent a statistically significant bond strength reduction after TC.

Next-generation cancer organoids.

Organotypic models of patient-specific tumours are revolutionizing our understanding of cancer heterogeneity and its implications for personalized medicine. These advancements are, in part, attributed to the ability of organoid models to stably preserve genetic, proteomic, morphological and pharmacotypic features of the parent tumour in vitro, while also offering unprecedented genomic and environmental manipulation. Despite recent innovations in organoid protocols, current techniques for cancer organoid culture are inherently uncontrolled and irreproducible, owing to several non-standardized facets including cancer tissue sources and subsequent processing, medium formulations, and animal-derived three-dimensional matrices. Given the potential for cancer organoids to accurately recapitulate the intra- and intertumoral biological heterogeneity associated with patient-specific cancers, eliminating the undesirable technical variability accompanying cancer organoid culture is necessary to establish reproducible platforms that accelerate translatable insights into patient care. Here we describe the current challenges and recent multidisciplinary advancements and opportunities for standardizing next-generation cancer organoid systems.

Feeding guanidinoacetic acid to broiler chickens can compensate for low dietary metabolisable energy formulation.

1. This study compared the responses of broilers to diets supplemented with the same level of guanidinoacetic acid (GAA) but formulated to have different N-corrected apparent metabolisable energy (AMEn) contents. The study involved 1280, one-day-old Ross 308 broilers, in 64 pens comprising 32 pens of males and 32 pens females, (20 birds in each) aged from 0 to 42 d.2. Commercial AME levels of 12.55 MJ/kg, 12.97 MJ/kg and 13.18 MJ/kg in the starter, grower and finisher diets, respectively, were set for the positive control (PC) feed. Four dietary treatments were prepared: PC (as above); negative control 1 (NC; PC - 0.21 MJ ME /kg); NC1 + 0.06% GAA; NC2 (PC - 0.42 MJ ME/kg + 0.06% GAA). Each diet was provided in 16 pens (eight male and eight female), following randomisation.3. Overall, birds fed NC1 had lower feed intakes (FI) compared to birds fed the PC and NC2+ GAA, lower weight gain (WG) compared to all the other diets and lower final body weight than birds fed the GAA diets (P < 0.05). There was a diet x sex interaction (P = 0.039), whereby feeding NC+GAA to female birds improved feed efficiency compared to being fed NC2 and NC1+ GAA, but not in males. Birds fed diets with GAA had a higher poultry efficiency factor (P < 0.001) than those fed NC1.4. There were no effects of treatment or sex on litter moisture, footpad score, white striping, wooden breast, AMEn, dry matter and fat retention (P > 0.05). However, the diet NC1+ GAA had 11.2% higher nitrogen retention coefficient compared to the NC1 diet (P = 0.038).5. Overall, the results implied that lower performance induced by a reduction of dietary AMEn in the range of 0.21 to 0.42 MJ/kg was more than compensated by supplementing 600 g/t GAA to the feed.

Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling.

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-ß signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.

Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids.

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

Microrheology reveals simultaneous cell-mediated matrix stiffening and fluidization that underlie breast cancer invasion.

Living tissues embody a unique class of hybrid materials in which active and thermal forces are inextricably linked. Mechanical characterization of tissues demands descriptors that respect this hybrid nature. In this work, we develop a microrheology-based force spectrum analysis (FSA) technique to dissect the active and passive fluctuations of the extracellular matrix (ECM) in three-dimensional (3D) cell culture models. In two different stromal models and a 3D breast cancer spheroid model, our FSA reveals emergent hybrid dynamics that involve both high-frequency stress stiffening and low-frequency fluidization of the ECM. We show that this is a general consequence of nonlinear coupling between active forces and the frequency-dependent viscoelasticity of stress-stiffening networks. In 3D breast cancer spheroids, this dual active stiffening and fluidization is tightly connected with invasion. Our results suggest a mechanism whereby breast cancer cells reconcile the seemingly contradictory requirements for both tension and malleability in the ECM during invasion.

Effects of methylsulfonylmethane and neutralizing anti-IL-10 antibody supplementation during a mild Eimeria challenge infection in broiler chickens.

A 28-day experiment was conducted in broilers to study the effects of feeding methylsulfonylmethane (MSM) and IL-10-neutralizing antibody from dried egg product (DEP) on the growth performance, immune responsivity, oxidative stress parameters, and gut health outcomes during a mild infection with mixed species of Eimeria. A total of 500 male Ross 308 chicks were allocated to five treatments: sham-inoculated (uninfected) chickens fed control diet (UCON), Eimeria-infected chickens fed control diet (ICON), and Eimeria-infected chickens fed control diet supplemented with 287 U/tonne of DEP (I-DEP), 0.4% MSM, or their combination (I-DEP-MSM), with 10 replicate cages of 10 birds per treatment. All infected groups received 1 mL of an oral inoculum containing Eimeria acervulina (10,000 oocysts), Eimeria maxima (5,000 oocysts), and Eimeria tenella (5,000 oocysts) on study days 7 and 14. Data were analyzed as a two-way ANOVA for all treatments including Eimeria-infected groups, in addition to a single degree of freedom contrast to compare uninfected and infected groups receiving the control diet. Mild Eimeria infection did not influence the growth performance in ICON compared with UCON at any time points. Overall (day 0-28) growth performance parameters were not influenced by either infection or dietary supplementation of MSM or DEP. However, birds in I-DEP-MSM showed improved ADG during study day 7 to 14 (i.e., 7 d after primary inoculation) indicating a beneficial effect immediately after Eimeria infection. Although MSM supplementation reduced thiobarbituric acid reactive substances (day 21 and 28), both MSM and DEP improved the total antioxidant capacity (day 21) in the plasma of infected birds. Histopathological outcomes were not influenced by treatments, and fecal oocyst output was higher in MSM- and DEP-supplemented groups than with ICON, indicating no beneficial effects. Similarly, expression of cecal inflammatory cytokines (IL-10, IL-1ß, and interferon-γ) was not affected by MSM, DEP, or their combination. Overall, the current results suggest that both MSM and DEP supplementation may benefit birds during a mild Eimeria infection as indicated by improvements in ADG and oxidative stress outcomes.