Fibroblast and myofibroblast activation in normal tissue repair and fibrosis.

The term 'fibroblast' often serves as a catch-all for a diverse array of mesenchymal cells, including perivascular cells, stromal progenitor cells and bona fide fibroblasts. Although phenotypically similar, these subpopulations are functionally distinct, maintaining tissue integrity and serving as local progenitor reservoirs. In response to tissue injury, these cells undergo a dynamic fibroblast-myofibroblast transition, marked by extracellular matrix secretion and contraction of actomyosin-based stress fibres. Importantly, whereas transient activation into myofibroblasts aids in tissue repair, persistent activation triggers pathological fibrosis. In this Review, we discuss the roles of mechanical cues, such as tissue stiffness and strain, alongside cell signalling pathways and extracellular matrix ligands in modulating myofibroblast activation and survival. We also highlight the role of epigenetic modifications and myofibroblast memory in physiological and pathological processes. Finally, we discuss potential strategies for therapeutically interfering with these factors and the associated signal transduction pathways to improve the outcome of dysregulated healing.

The Clinical Impact of Paravalvular Leaks With Transcutaneous Aortic Valve Implantation (TAVI) Versus Surgical Aortic Valve Replacement (SAVR): A Systematic Review and Meta-Analysis.

BACKGROUND: Aortic valve stenosis is a common cardiac condition that requires intervention for symptomatic and/or prognostic reasons. The two most common interventions are surgical aortic valve replacement (SAVR) and transcatheter aortic valve implantation (TAVI). The ratio of TAVI:SAVR has increased twofold over the past few years and is now being considered in intermediate-risk patients as well. One of the significant benefits of TAVI is that it is less invasive; however, one of the drawbacks is a high paravalvular leaks (PVLs) rate compared to SAVR. To assess the impact of PVLs on survival, progression of heart failure, and the need for re-intervention. METHOD: We conducted a comprehensive systematic literature search from the conception of TAVI 2002 until December 2022 through Embase (Ovid), MEDLINE (Ovid), Science Direct, and CENTRAL (Wiley). We followed PRISMA guidelines and checklists. Review protocol registration ID in PROSPERO: CRD42023393742. RESULTS: We identified 28 studies that met our eligibility criteria, and only 24 studies were suitable for pooling in a meta-analysis (including their hazard ratio with a confidence interval of 95%) assessing our primary outcome (all-cause mortality). The remaining four studies were narratively synthesised. RevMan V5.4 (Version 5.4. Cochrane Collaboration, 2020) was utilised to pool meta-analysis data to assess effect estimates of PVLs in both intervention arms, using a random effect model for calculation (hazard ratio 1.14 confidence interval 95% 1.08-1.21 [p<0.0001]), with a follow-up duration between 30 days to 5 years. CONCLUSION: Patients with mild or higher degrees of PVLs in both intervention arms incurred unfavourable outcomes. The incidence of PVLs was significantly higher with TAVI; even a mild degree led to poor quality of life and increased all-cause mortality on long-term follow-up.

The interplay of fibroblasts, the extracellular matrix, and inflammation in scar formation.

Various forms of fibrosis, comprising tissue thickening and scarring, are involved in 40% of deaths across the world. Since the discovery of scarless functional healing in fetuses prior to a certain stage of development, scientists have attempted to replicate scarless wound healing in adults with little success. While the extracellular matrix (ECM), fibroblasts, and inflammatory mediators have been historically investigated as separate branches of biology, it has become increasingly necessary to consider them as parts of a complex and tightly regulated system that becomes dysregulated in fibrosis. With this new paradigm, revisiting fetal scarless wound healing provides a unique opportunity to better understand how this highly regulated system operates mechanistically. In the following review, we navigate the four stages of wound healing (hemostasis, inflammation, repair, and remodeling) against the backdrop of adult versus fetal wound healing, while also exploring the relationships between the ECM, effector cells, and signaling molecules. We conclude by singling out recent findings that offer promising leads to alter the dynamics between the ECM, fibroblasts, and inflammation to promote scarless healing. One factor that promises to be significant is fibroblast heterogeneity and how certain fibroblast subpopulations might be predisposed to scarless healing. Altogether, reconsidering fetal wound healing by examining the interplay of the various factors contributing to fibrosis provides new research directions that will hopefully help us better understand and address fibroproliferative diseases, such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis.

The use of the independent sector in providing NHS services during the Covid-19 outbreak; two hospitals experience.

This paper reviews the activity undertaken between a teaching hospital and its adjacent Independent Hospital and its implementation under the Independent Sector Provider Contract between NHSE and the Independent Sector. RESULTS: From the instigation of the NHSE contract with the Independent Sector up until 28th June 2020 The Norfolk and Norwich University NHS Trust (NNUH) delivered 9016 episodes of care including 576 surgical episodes at its nearby Independent Hospital. During the time that a seven day household isolation period was required, no patients from the 31 tested postoperatively were recorded as testing positive for Covid-19. In the month after moving to a mandatory 14 day period of household isolation, 29 patients had their surgery postponed as they were unable to comply with the required period of isolation. CONCLUSION: Working cooperatively with the independent sector can deliver significant additional capacity for the NHS. Fourteen days household isolation may impact on a patient's decision to have surgery, despite, in some cases, that surgery being time-sensitive. The recommendation from NICE reducing the length of isolation largely reversed this impact.

Myocardial fibrosis in asymptomatic and symptomatic chronic severe primary mitral regurgitation and relationship to tissue characterisation and left ventricular function on cardiovascular magnetic resonance.

BACKGROUND: Myocardial fibrosis occurs in end-stage heart failure secondary to mitral regurgitation (MR), but it is not known whether this is present before onset of symptoms or myocardial dysfunction. This study aimed to characterise myocardial fibrosis in chronic severe primary MR on histology, compare this to tissue characterisation on cardiovascular magnetic resonance (CMR) imaging, and investigate associations with symptoms, left ventricular (LV) function, and exercise capacity. METHODS: Patients with class I or IIa indications for surgery underwent CMR and cardiopulmonary exercise testing. LV biopsies were taken at surgery and the extent of fibrosis was quantified on histology using collagen volume fraction (CVFmean) compared to autopsy controls without cardiac pathology. RESULTS: 120 consecutive patients (64 ± 13 years; 71% male) were recruited; 105 patients underwent MV repair while 15 chose conservative management. LV biopsies were obtained in 86 patients (234 biopsy samples in total). MR patients had more fibrosis compared to 8 autopsy controls (median: 14.6% [interquartile range 7.4-20.3] vs. 3.3% [2.6-6.1], P < 0.001); this difference persisted in the asymptomatic patients (CVFmean 13.6% [6.3-18.8], P < 0.001), but severity of fibrosis was not significantly higher in NYHA II-III symptomatic MR (CVFmean 15.7% [9.9-23.1] (P = 0.083). Fibrosis was patchy across biopsy sites (intraclass correlation 0.23, 95% CI 0.08-0.39, P = 0.001). No significant relationships were identified between CVFmean and CMR tissue characterisation [native T1, extracellular volume (ECV) or late gadolinium enhancement] or measures of LV function [LV ejection fraction (LVEF), global longitudinal strain (GLS)]. Although the range of ECV was small (27.3 ± 3.2%), ECV correlated with multiple measures of LV function (LVEF: Rho = - 0.22, P = 0.029, GLS: Rho = 0.29, P = 0.003), as well as NTproBNP (Rho = 0.54, P < 0.001) and exercise capacity (%PredVO2max: R = - 0.22, P = 0.030). CONCLUSIONS: Patients with chronic primary MR have increased fibrosis before the onset of symptoms. Due to the patchy nature of fibrosis, CMR derived ECV may be a better marker of global myocardial status. Clinical trial registration Mitral FINDER study; Clinical Trials NCT02355418, Registered 4 February 2015, https://clinicaltrials.gov/ct2/show/NCT02355418.

Dynamic assembly of ultrasoft colloidal networks enables cell invasion within restrictive fibrillar polymers.

In regenerative medicine, natural protein-based polymers offer enhanced endogenous bioactivity and potential for seamless integration with tissue, yet form weak hydrogels that lack the physical robustness required for surgical manipulation, making them difficult to apply in practice. The use of higher concentrations of protein, exogenous cross-linkers, and blending synthetic polymers has all been applied to form more mechanically robust networks. Each relies on generating a smaller network mesh size, which increases the elastic modulus and robustness, but critically inhibits cell spreading and migration, hampering tissue regeneration. Here we report two unique observations; first, that colloidal suspensions, at sufficiently high volume fraction (ϕ), dynamically assemble into a fully percolated 3D network within high-concentration protein polymers. Second, cells appear capable of leveraging these unique domains for highly efficient cell migration throughout the composite construct. In contrast to porogens, the particles in our system remain embedded within the bulk polymer, creating a network of particle-filled tunnels. Whereas this would normally physically restrict cell motility, when the particulate network is created using ultralow cross-linked microgels, the colloidal suspension displays viscous behavior on the same timescale as cell spreading and migration and thus enables efficient cell infiltration of the construct through the colloidal-filled tunnels.

LEM domain-containing protein 3 antagonizes TGFß-SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol.

Transforming growth factor-ß (TGFß) signaling through SMAD2/3 is an important driver of pathological fibrosis in multiple organ systems. TGFß signaling and extracellular matrix (ECM) stiffness form an unvirtuous pathological circuit in which matrix stiffness drives activation of latent TGFß, and TGFß signaling then drives cellular stress and ECM synthesis. Moreover, ECM stiffness also appears to sensitize cells to exogenously activated TGFß through unknown mechanisms. Here, using human fibroblasts, we explored the effect of ECM stiffness on a putative inner nuclear membrane protein, LEM domain-containing protein 3 (LEMD3), which is physically connected to the cell's actin cytoskeleton and inhibits TGFß signaling. We showed that LEMD3-SMAD2/3 interactions are inversely correlated with ECM stiffness and TGFß-driven luciferase activity and that LEMD3 expression is correlated with the mechanical response of the TGFß-driven luciferase reporter. We found that actin polymerization but not cellular stress or LEMD3-nuclear-cytoplasmic couplings were necessary for LEMD3-SMAD2/3 interactions. Intriguingly, LEMD3 and SMAD2/3 frequently interacted in the cytosol, and we discovered LEMD3 was proteolytically cleaved into protein fragments. We confirmed that a consensus C-terminal LEMD3 fragment binds SMAD2/3 in a stiffness-dependent manner throughout the cell and is sufficient for antagonizing SMAD2/3 signaling. Using human lung biopsies, we observed that these nuclear and cytosolic interactions are also present in tissue and found that fibrotic tissues exhibit locally diminished and cytoplasmically shifted LEMD3-SMAD2/3 interactions, as noted in vitro Our work reveals novel LEMD3 biology and stiffness-dependent regulation of TGFß by LEMD3, providing a novel target to antagonize pathological TGFß signaling.

Vendor-specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4+ Foxp3+ regulatory T cell levels.

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4+ Foxp3+ regulatory T cells (Tregs ) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad-spectrum antibiotics decreases Tregs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3-driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of Tregs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through Treg -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period.

Suppression of TGFß-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition.

BACKGROUND: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy. METHODS: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo. RESULTS: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFß-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo. CONCLUSIONS: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.

Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability.

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5ß1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvß3 integrin binding. In vitro, α3/α5ß1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5ß1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvß3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.

Minimising the risk of Mycobacterium chimaera infection during cardiopulmonary bypass by the removal of heater-cooler units from the operating room.

INTRODUCTION: Mycobacterium chimaera ( M. chimaera) is a recently characterised bacterium that can cause life-threatening infections in small numbers of patients who undergo cardiopulmonary bypass during cardiac surgery. The likely mode of transmission is thought to occur through aerosolisation from contaminated water reservoirs. The airborne bacteria then contaminate the surgical field, leading to an infection months or even years later. The preferred practical solution to disrupt the transmission of these airborne bacteria to the patient is to remove the heater-cooler units (HCUs) from the operating room (OR). We describe a process of achieving this in order to provide information to guide other institutions who wish to do a similar thing. METHODS: A multidisciplinary team was assembled to work on the project. The planning phase involved trialling different OR layouts and simulating the alterations in the HCU circuit function. The changes to the OR were made over a weekend to minimise disruption to the operating schedule. RESULTS: The HCU was moved to the dirty utility room adjacent to the OR. Standard operating procedures (SOP) and risk assessments were made to enable this to be used for a dual purpose. One of the ORs was reconfigured to allow the cardiopulmonary bypass machine to be located close to the HCU in the dirty utility room. The total cost of the alterations was £6,158. Although we have provided a physical barrier to interrupt patient exposure to aerosolised M. chimaera from HCUs, we continue to perform cultures and decontamination as per the national recommendations. The SOP was designed to be auditable to ensure compliance with the protocols. CONCLUSIONS: We show a method by which the HCU can be removed from the OR in a relatively low-cost, straightforward and practical manner.

A prospective study examining the role of myocardial Fibrosis in outcome following mitral valve repair IN DEgenerative mitral Regurgitation: rationale and design of the mitral FINDER study.

BACKGROUND: The optimal management of chronic severe primary degenerative mitral regurgitation (MR) is to repair the valve but identification of the optimal timing of surgery remains challenging. Current guidelines suggest 'watchful waiting' until the onset of symptoms or left ventricular (LV) dysfunction but these have been challenged as promoting 'rescue surgery'. Better predictors are required to inform decision-making in relation to the necessity and timing of surgery. Chronic volume overload is a stimulus for adverse adaptive LV remodelling. Subclinical reduction in LV strain before mitral repair predicts a fall in LV ejection fraction following surgery and is thought to reflect the development of myocardial fibrosis in response to chronic volume overload. Myocardial fibrosis can be detected non-invasively using cardiac magnetic resonance (CMR) imaging techniques as an expansion of the extracellular volume (ECV). METHODS/DESIGN: This study investigates whether: 1) patients with above median ECV will have smaller reduction in end-systolic volume index (as a measure of the degree of reverse LV remodelling) on CMR following mitral valve repair, compared to those with below median ECV; and 2) higher ECV on CMR, validated through histology, adversely impacts upon post-operative complications and symptomatic improvement following surgery. This is a multi-centre, prospective, cross-sectional comparison of patients prior to and 9 months following surgery for chronic severe primary degenerative MR. To establish the natural history of ECV in MR, an additional cohort of patients with asymptomatic MR who do not wish to consider early repair will be followed. Investigations include CMR, cardiopulmonary exercise test, stress echocardiography, signal-averaged electrocardiogram, 24-h electrocardiogram monitoring, laboratory tests and patient-reported outcome measures. Patients undergoing surgery will have cardiac biopsies performed at the time of mitral valve repair for histological quantification of fibrosis. DISCUSSION: This study will advance our understanding of ventricular remodelling in MR, its impact on patient symptoms and ventricular response following surgery. Establishing the link between myocardial fibrosis (measured on CMR and validated through histology), with early ventricular dysfunction, will offer physicians a novel non-invasive biomarker that can further inform the timing of surgery. TRIAL REGISTRATION: This trial was registered at ClinicalTrials.gov ( NCT02355418 ) on 30th November 2015.

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation.

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbß3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.

Sprint interval and moderate-intensity continuous training have equal benefits on aerobic capacity, insulin sensitivity, muscle capillarisation and endothelial eNOS/NAD(P)Hoxidase protein ratio in obese men.

KEY POINTS: Skeletal muscle capillary density and vasoreactivity are reduced in obesity, due to reduced nitric oxide bioavailability. Sprint interval training (SIT) has been proposed as a time efficient alternative to moderate-intensity continuous training (MICT), but its effect on the skeletal muscle microvasculature has not been studied in obese individuals. We observed that SIT and MICT led to equal increases in capillarisation and endothelial eNOS content, while reducing endothelial NOX2 content in microvessels of young obese men. We conclude that SIT is equally effective at improving skeletal muscle capillarisation and endothelial enzyme balance, while being a time efficient alternative to traditional MICT. ABSTRACT: Sprint interval training (SIT) has been proposed as a time efficient alternative to moderate-intensity continuous training (MICT), leading to similar improvements in skeletal muscle capillary density and microvascular function in young healthy humans. In this study we made the first comparisons of the muscle microvascular response to SIT and MICT in an obese population. Sixteen young obese men (age 25 ± 1 years, BMI 34.8 ± 0.9 kg m(-2) ) were randomly assigned to 4 weeks of MICT (40-60 min cycling at ∼65% V̇O2 peak , 5 times per week) or constant load SIT (4-7 constant workload intervals of 200% Wmax 3 times per week). Muscle biopsies were taken before and after training from the m. vastus lateralis to measure muscle microvascular endothelial eNOS content, eNOS serine(1177) phosphorylation, NOX2 content and capillarisation using quantitative immunofluorescence microscopy. Maximal aerobic capacity (V̇O2 peak ), whole body insulin sensitivity and arterial stiffness were also assessed. SIT and MICT increased skeletal muscle microvascular eNOS content and eNOS ser(1177) phosphorylation in terminal arterioles and capillaries (P < 0.05), but the latter effect was eliminated when normalised to eNOS content (P = 0.217). SIT and MICT also reduced microvascular endothelial NOX2 content (P < 0.05) and both increased capillary density and capillary-fibre perimeter exchange index (P < 0.05). In parallel, SIT and MICT increased V̇O2 peak (P < 0.05) and whole body insulin sensitivity (P < 0.05), and reduced central artery stiffness (P < 0.05). As no significant differences were observed between SIT and MICT it is concluded that SIT is a time efficient alternative to MICT to improve aerobic capacity, insulin sensitivity and muscle capillarisation and endothelial eNOS/NAD(P)Hoxidase protein ratio in young obese men.

Integrin α3ß1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat.

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbß3 and α5ß1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3ß1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9(R)→(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3ß1 to the Fn fragments. Integrin α3ß1 had the highest affinity for FnIII9'10 and FnIII9(2G)10. Mutation within the synergy site decreased integrin α3ß1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that α3ß1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3ß1 binding to Fn.

Fibrin Network Changes in Neonates after Cardiopulmonary Bypass.

BACKGROUND: Quantitative and qualitative differences in the hemostatic systems exist between neonates and adults, including the presence of "fetal" fibrinogen, a qualitatively dysfunctional form of fibrinogen that exists until 1 yr of age. The consequences of "fetal" fibrinogen on clot structure in neonates, particularly in the context of surgery-associated bleeding, have not been well characterized. Here, the authors examine the sequential changes in clotting components and resultant clot structure in a small sample of neonates undergoing cardiac surgery and cardiopulmonary bypass (CPB). METHODS: Blood samples were collected from neonates (n = 10) before surgery, immediately after CPB, and after the transfusion of cryoprecipitate (i.e., adult fibrinogen component). Clots were formed from patient samples or purified neonatal and adult fibrinogen. Clot structure was analyzed using confocal microscopy. RESULTS: Clots formed from plasma obtained after CPB and after transfusion were more porous than baseline clots. Analysis of clots formed from purified neonatal and adult fibrinogen demonstrated that at equivalent fibrinogen concentrations, neonatal clots lack three-dimensional structure, whereas adult clots were denser with significant three-dimensional structure. Clots formed from a combination of purified neonatal and adult fibrinogen were less homogenous than those formed from either purified adult or neonatal fibrinogen. CONCLUSIONS: The results of this study confirm that significant differences exist in clot structure between neonates and adults and that neonatal and adult fibrinogen may not integrate well. These findings suggest that differential treatment strategies for neonates should be pursued to reduce the demonstrated morbidity of blood product transfusion.

Ultrasoft microgels displaying emergent platelet-like behaviours.

Efforts to create platelet-like structures for the augmentation of haemostasis have focused solely on recapitulating aspects of platelet adhesion; more complex platelet behaviours such as clot contraction are assumed to be inaccessible to synthetic systems. Here, we report the creation of fully synthetic platelet-like particles (PLPs) that augment clotting in vitro under physiological flow conditions and achieve wound-triggered haemostasis and decreased bleeding times in vivo in a traumatic injury model. PLPs were synthesized by combining highly deformable microgel particles with molecular-recognition motifs identified through directed evolution. In vitro and in silico analyses demonstrate that PLPs actively collapse fibrin networks, an emergent behaviour that mimics in vivo clot contraction. Mechanistically, clot collapse is intimately linked to the unique deformability and affinity of PLPs for fibrin fibres, as evidenced by dissipative particle dynamics simulations. Our findings should inform the future design of a broader class of dynamic, biosynthetic composite materials.

Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells.

Neural stem cells (NSCs) possess great potential for neural tissue repair after traumatic injuries to the central nervous system (CNS). However, poor survival and self-renewal of NSCs after injury severely limits its therapeutic potential. Sulfated chondroitin sulfate glycosaminoglycans (CS-GAGs) linked to CS proteoglycans (CSPGs) in the brain extracellular matrix (ECM) have the ability to bind and potentiate trophic factor efficacy, and promote NSC self-renewal in vivo. In this study, we investigated the potential of CS-GAG hydrogels composed of monosulfated CS-4 (CS-A), CS-6 (CS-C), and disulfated CS-4,6 (CS-E) CS-GAGs as NSC carriers, and their ability to create endogenous niches by enriching specific trophic factors to support NSC self-renewal. We demonstrate that CS-GAG hydrogel scaffolds showed minimal swelling and degradation over a period of 15 days in vitro, absorbing only 6.5 ± 0.019% of their initial weight, and showing no significant loss of mass during this period. Trophic factors FGF-2, BDNF, and IL10 bound with high affinity to CS-GAGs, and were significantly (p < 0.05) enriched in CS-GAG hydrogels when compared to unsulfated hyaluronic acid (HA) hydrogels. Dissociated rat subventricular zone (SVZ) NSCs when encapsulated in CS-GAG hydrogels demonstrated ∼88.5 ± 6.1% cell viability in vitro. Finally, rat neurospheres in CS-GAG hydrogels conditioned with the mitogen FGF-2 demonstrated significantly (p < 0.05) higher self-renewal when compared to neurospheres cultured in unconditioned hydrogels. Taken together, these findings demonstrate the ability of CS-GAG based hydrogels to regulate NSC self-renewal, and facilitate growth factor enrichment locally.

Phage-based molecular probes that discriminate force-induced structural states of fibronectin in vivo.

Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins and these forces are presumed to activate integrin "switches." The mechanism of such switches is thought to be the partial unfolding of integrin-binding domains within fibronectin (Fn). However, integrin switches remain largely hypothetical due to a dearth of evidence for their existence, and relevance, in vivo. By using phage display in combination with the controlled deposition and extension of Fn fibers, we report the discovery of peptide-based molecular probes capable of selectively discriminating Fn fibers under different strain states. Importantly, we show that the probes are functional in both in vitro and ex vivo tissue contexts. The development of such tools represents a critical step in establishing the relevance of theoretical mechanotransduction events within the cellular microenvironment.