Low Concentrations Of Collagen Research Articles

Improved Composite Hydrogel for Bioengineered Tracheal Graft Demonstrates Effective Early Angiogenesis.

Background/Objectives: Collagen-agarose hydrogel blends currently used in tracheal graft bioengineering contain relatively high concentrations of collagen to withstand mechanical stresses associated with native trachea function (e.g., breathing). Unfortunately, the high collagen content restricts effective cell infiltration into the hydrogel. In this study, we created an improved hydrogel blend with lower concentrations of collagen (<5 mg/mL) and characterized its capacity for fibroblast invasion and angiogenesis. Methods: Four collagen-agarose hydrogel blends were created: 1 mg/mL type 1 collagen (T1C) and 0.25% agarose, 1 mg/mL T1C and 0.125% agarose, 2 mg/mL T1C and 0.25% agarose, and 2 mg/mL T1C and 0.125% agarose. The hydrogel surface was seeded with fibroblasts, while both endothelial cells and fibroblasts (3:1 ratio) were mixed within the hydrogel matrix. We assessed early angiogenesis by observing fibroblast migration and endothelial cell morphology (elongation and branching) at 7 days. In addition, we performed immunostaining for alpha-smooth muscle actin (aSMA) and explored the gene expression of various angiogenic markers (including vascular endothelial growth factor; VEGF). Results: Gels with lower agarose concentrations (0.125%) with 1 or 2 mg/mL T1C were more effective in allowing early attachment and migration of surface-applied fibroblasts compared to gels with higher (0.25%) agarose concentrations. The low-agarose gels also allowed cells to quickly adopt a spread morphology and self-assemble into elongated structures indicative of early angiogenesis, while demonstrating positive immunostaining for aSMA and increased gene expression of VEGF by day 7. Conclusions: Hydrogel blends with collagen and low agarose concentrations may be effective in allowing early cellular infiltration and angiogenesis, making such gels a suitable cell substrate for use in the development of composite bioengineered tracheal grafts. The collagen-agarose hydrogel blend is meant to be cast around a three-dimensional (3D) printed polycaprolactone support structure and wrapped in porcine small intestine submucosa ECM to create an off-the-shelf bioengineered tracheal implant.

Microscale strain concentrations in tissue-engineered osteochondral implants are dictated by local compositional thresholds and architecture

Tissue-engineered osteochondral implants manufactured from condensed mesenchymal stem cell bodies have shown promise for treating focal cartilage defects. Notably, such manufacturing techniques have shown to successfully recapture the bulk mechanical properties of native cartilage. However, the relationships among the architectural features, local composition, and micromechanical environment within tissue-engineered cartilage from cell-based aggregates remain unclear. Understanding such relationships is crucial for identifying critical parameters that can predict in vivo performance. Therefore, this study investigated the relationship among architectural features, composition, and micromechanical behavior of tissue-engineered osteochondral implants. We utilized fast-confocal microscopy combined with a strain mapping technique to analyze the micromechanical behavior under quasi-static loading, as well as Fourier Transform Infrared Spectroscopy to analyze the local compositions. More specifically, we investigated the architectural features and compositional distributions generated from tissue maturation, along with macro- and micro-level strain distributions. Our results showed that under compression, cell-based aggregates underwent deformation followed by body movement, generating high local strain around the boundaries, where local aggrecan concentration was low and local collagen concentration was high. By analyzing the micromechanics and composition at the single aggregate length scale, we identified a strong threshold relationship between local strain and compositions. Namely at the aggrecan concentration below 0.015 arbitrary unit (A.U.) and the collagen concentration above 0.15 A.U., the constructs experienced greater than threefold increase in compressive strain. Overall, this study suggests that local compositional features are the primary driver of the local mechanical environment in tissue-engineered cartilage constructs, providing insight into potential quality control parameters for manufacturing tissue-engineered constructs.

3D Printing of Collagen Nerve Scaffold with Multichannel Characteristics

Given the difficulty in preparing multichannel nerve scaffolds for peripheral nerve repair, a method of manufacturing multichannel collagen nerve scaffolds by multi-nozzle 3D printing technology was proposed. Gelatin material is used as the core melting material because of its temperature-sensitive characteristics. A collagen nerve scaffold with a multichannel structure was manufactured by temperature-rising removal after printing, and the scaffold with different collagen concentrations was analyzed and evaluated in terms of microtubule diameter and mechanical properties. The research results show that the mechanical properties of the scaffold are gradually enhanced with an increase in collagen concentration. When the concentration of collagen is between 5% and 6%, the quality of the microtubule is significantly improved compared with the 4% collagen scaffold. Combined with the distribution of microtubule diameter, a lower concentration of collagen will lead to the collapse and deformation of the print structure, while a higher concentration of collagen will lead to the existence of voids in the print structure. Considering the manufacturing process, microtubule diameter, and mechanical properties of collagen nerve scaffold, 5% collagen nerve scaffold is the best and is expected to meet the requirements of peripheral nerve regeneration.

Effect of UV Irradiation of Pre-Gel Solutions on the Formation of Collagen Gel Tubes

Hollow collagen gels are promising materials for drug/cell delivery systems to promote tissue regeneration because they may be able to function as carriers for these types of loads. Controlling the cavity size and swelling suppression is essential to expand the applications and improve the usability of such gel-like systems. We investigated the effects of UV-treated collagen solutions as a pre-gel aqueous mixture on the formation and properties of the hollow collagen gels in terms of their preparation range limits, morphology, and swelling ratio. The UV treatment thickened the pre-gel solutions, which allowed hollowing at lower collagen concentrations. This treatment also prevents the over-swelling of the hollow collagen rods in PBS buffer solutions. The UV-treated collagen solutions provided a large lumen space in the prepared collagen hollow fiber rods with a limited swelling ratio, allowing vascular endothelial cells and ectodermal cells to be cultured separately in the outer and inner lumen.

In vitro hair follicle growth model for drug testing

In vitro models of human hair follicle-like tissue could be fundamental tools to better understand hair follicle morphogenesis and hair drug screening. During prenatal development and postnatal cyclic hair regeneration, hair follicle morphogenesis is triggered by reciprocal interactions and the organization of the epithelial and mesenchymal cell populations. Given this mechanism, we developed an approach to induce hair peg-like sprouting in organoid cultures composed of epithelial and mesenchymal cells. Human fetal/adult epithelial and mesenchymal cells were cultured in a medium supplemented with a low concentration of either Matrigel or collagen I. These extracellular matrices significantly enhanced the self-organization capabilities of the epithelial and mesenchymal cells, resulting in spherical aggregation and subsequent hair peg-like sprouting. The length of the hair peg sprouting and associated gene expression significantly increased in the presence of a well-known hair drug, minoxidil. This approach may be beneficial for testing hair growth-promoting drug candidates.

Pre-glycation impairs gelation of high concentration collagen solutions.

There remains a need for stiffer collagen hydrogels for tissue engineering and disease modeling applications. Pre-glycation, or glycation of collagen in solution prior to gelation, has been shown to increase the mechanics of collagen hydrogels while maintaining high viability of encapsulated cells. The stiffness of glycated collagen gels can be increased by increasing the collagen concentration, sugar concentration, and glycation time. However, previous studies on pre-glycation of collagen have used low collagen concentrations and/or low sugar concentrations and have not investigated the effect of glycation time. Therefore, the objective of this study was to determine the effects of pre-glycation with high sugar concentrations (up to 500 mM) and extended glycation times (up to 21 days) on high concentration collagen (8mg/ml). The addition of sugar to the collagen and the formation of advanced glycation end products (AGEs) were quantified. The ability to gel successfully and rheological properties were determined and correlated with biochemical characterizations. Successful collagen gelation and rheological properties of pre-glycated collagen were found to be strongly dependent on the ratio of added sugars to added AGEs with high ratios impairing gelation and low ratios resulting in optimal storage moduli. There is likely a competing effect during pre-glycation of the formation of AGEs resulting in crosslinking of collagen and the formation of Amadori intermediates acting to increase collagen solubility. Overall, this study shows that collagen glycation can be optimized by increasing the formation of AGEs while maintaining a low ratio of added sugar to added AGEs.

Efficient deformation mechanisms enable invasive cancer cells to migrate faster in 3D collagen networks

Cancer cell migration is a widely studied topic but has been very often limited to two dimensional motion on various substrates. Indeed, less is known about cancer cell migration in 3D fibrous-extracellular matrix (ECM) including variations of the microenvironment. Here we used 3D time lapse imaging on a confocal microscope and a phase correlation method to follow fiber deformations, as well as cell morphology and live actin distribution during the migration of cancer cells. Different collagen concentrations together with three bladder cancer cell lines were used to investigate the role of the metastatic potential on 3D cell migration characteristics. We found that grade-3 cells (T24 and J82) are characterized by a great diversity of shapes in comparison with grade-2 cells (RT112). Moreover, grade-3 cells with the highest metastatic potential (J82) showed the highest values of migration speeds and diffusivities at low collagen concentration and the greatest sensitivity to collagen concentration. Our results also suggested that the small shape fluctuations of J82 cells are the signature of larger migration velocities. Moreover, the displacement fields generated by J82 cells showed significantly higher fiber displacements as compared to T24 and RT112 cells, regardless of collagen concentration. The analysis of cell movements enhanced the fact that bladder cancer cells were able to exhibit different phenotypes (mesenchymal, amoeboid). Furthermore, the analysis of spatio-temporal migration mechanisms showed that cancer cells are able to push or pull on collagen fibers, therefore producing efficient local collagen deformations in the vicinity of cells. Our results also revealed that dense actin regions are correlated with the largest displacement fields, and this correlation is enhanced for the most invasive J82 cancer cells. Therefore this work opens up new routes to understand cancer cell migration in soft biological networks.

Multi-pin contact drawing enables production of anisotropic collagen fiber substrates for alignment of fibroblasts and monocytes

Type I collagen is the most abundant protein in the human body and is known to play important roles in numerous biological processes including tissue morphogenesis and wound healing. As such, it is one of the most frequently used substrates for cell culture, and there have been considerable efforts to develop collagen-based cell culture substrates that mimic the structural organization of collagen as it is found in native tissues, i.e., collagen fibers. However, producing collagen fibers from extracted collagen has been notoriously difficult, with existing methods providing only low throughput production of collagen fibers. In this study, we prepared collagen fibers using a highly efficient, bio-friendly, and cost-effective approach termed contact drawing, which uses an entangled polymer fluid to aid in fiber formation. Contact drawing technology has been demonstrated previously for collagen using highly concentrated dextran solutions with low concentrations of collagen. Here, we show that by replacing dextran with polyethylene oxide (PEO), high collagen content fibers may be readily formed from mixtures of soluble collagen and PEO, a polymer that readily forms fibers by contact drawing at concentrations as low as 0.5%wt. The presence of collagen and the formation of well-ordered collagen structures in the resulting fibers were characterized by attenuated total reflectance Fourier-transform infrared spectromicroscopy, Raman spectromicroscopy, and fluorescence microscopy. Corresponding to well-ordered collagen, the mechanical properties of the PEO-collagen fibers approximated those observed for native collagen fibers. Growth of cells on aligned PEO-collagen fibers attached to a polydimethyl siloxane support was examined for human dermal fibroblast (WS1) and human peripheral leukemia blood monocyte (THP-1) cell lines. WS1 and THP-1 cells readily attached, displayed alignment through migration and spreading, and proliferated on the collagen fiber substrate over the course of several days. We also demonstrated the retrieval of viable cells from the PEO-collagen fiber substrates through enzymatic digestion of the collagen substrate with collagenase IV.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

Addition of collagen type I in agarose created a dose-dependent effect on matrix production in engineered cartilage.

Extracellular-matrix composition impacts mechanical performance in native and engineered tissues. Previous studies showed collagen type I-agarose blends increased cell-matrix interactions and extracellular matrix production. However, long-term impacts on protein production and mechanical properties of engineered cartilage are unknown. Our objective was to characterize the effect of collagen type I on the matrix production of chondrocytes embedded in agarose hydrogels. We hypothesized that the addition of collagen would improve long-term mechanical properties and matrix production (e.g. collagen and glycosaminoglycans) through increased bioactivity. Agarose hydrogels (2% w/v) were mixed with varying concentrations of collagen type I (0, 2 and 5 mg/ml). Juvenile bovine chondrocytes were added to the hydrogels to assess matrix production over 4 weeks through biochemical assays, and mechanical properties were assessed through unconfined compression. We observed a dose-dependent effect on cell bioactivity, where 2 mg/ml of collagen improved bioactivity, but 5 mg/ml had a negative impact on bioactivity. This resulted in a higher modulus for scaffolds supplemented with lower collagen concentration as compared to the higher collagen concentration, but not when compared to the control. In conclusion, the addition of collagen to agarose constructs provided a dose-dependent impact on improving glycosaminoglycan production but did not improve collagen production or compressive mechanics.

Vibrational circular dichroism spectroscopy as a probe for the detection of collagen fibril and fibrillation in solution

The detection of collagen fibrils and fibrillation is crucial for tissue regeneration, biomedical applications of collagen, and for studying the interaction of small molecules with the collagen fibrils in the development of therapeutics for collagen-based diseases. At present, collagen fibrillation is probed by high-value microscopic techniques, which limits real-time monitoring of collagen fibril growth and measurement only in dry form. In this context, a quick sensing method is necessary for detecting these fibrils, especially monitoring fibril growth in solution. Herein, we show that vibrational circular dichroism (VCD), a growing spectroscopic technique, can probe collagen fibrils in aqueous solutions by generating an enhanced amide I VCD signal (intensity) compared to monomer. Unlike regular protein VCD measurements, VCD of collagen fibrils require a very low concentration (5 mg/mL) of collagen and can be probed in a very short time (∼10 min). Moreover, VCD responds to the diameter and height of collagen fibrils as evidenced by an increased VCD intensity (close to 103) with pH and NaCl, making VCD a sensitive tool for monitoring the growth of the fibril diameter and height. VCD was also found to be suitable for detecting the time-dependent increase in fibril diameter under fibrillation conditions. Because no other probe is presently available for rapid detection of collagen fibrils in solution form, the ability of VCD to rapidly probe collagen fibrillation in solution and in real-time opens a new way of studying collagen fibrillation in different fields and applications.

Novel Digital Light Processing Printing Strategy Using a Collagen-Based Bioink with Prospective Cross-Linker Procyanidins.

Three-dimensional (3D) bioink plays a vital role in the construction of tissues and organs by 3D bioprinting. Collagen has outstanding biocompatibility and is widely used in the field of tissue engineering. However, due to poor mechanical properties and slow self-assembly, it is challenging to manufacture high-precision 3D bioprinted collagen scaffolds. Herein, a novel digital light processing (DLP) bioink which can satisfy the printing of complex structures has been developed. This photocurable bioink is based on collagen and supplemented with a small amount of procyanidins (PA) as a cross-linking agent. The low concentration of collagen gives the bioink good fluidity and excellent biocompatibility, and a small amount of PA increases the cross-linking density of the system to obtain better mechanical properties. Using commercial digital light processing (DLP) printers, this collagen-based ink can effectively print structures with micrometer resolution, and the fidelity of the 3D structures can reach above 90%. Cells were able to be loaded in the bioink and distributed uniformly in the collagen scaffold in an unscathed way. This photocurable collagen bioink has broad application potential in DLP 3D bioprinting.

Effects of the Btk-Inhibitors Remibrutinib (LOU064) and Rilzabrutinib (PRN1008) With Varying Btk Selectivity Over Tec on Platelet Aggregation and in vitro Bleeding Time.

Background: Bruton tyrosine kinase inhibitors (BTKi) are used in B-cell malignancies and in development against various autoimmune diseases. Since Btk is also involved in specific pathways of platelet activation, BTKi might be considered to target platelet GPVI/GPIb-mediated atherothrombosis and platelet FcγRIIA-dependent immune disorders. However, BTKi treatment of patients with B-cell malignancies is frequently associated with mild bleeding events caused possibly by off-target inhibition of Tec. Here, we compared the platelet effects of two novel BTKi that exhibit a high (remibrutinib) or low (rilzabrutinib) selectivity for Btk over Tec.Methods and Results: Remibrutinib and rilzabrutinib were pre-incubated with anticoagulated blood. Platelet aggregation and in vitro bleeding time (closure time) were studied by multiple electrode aggregometry (MEA) and platelet-function analyzer-200 (PFA-200), respectively. Both BTKi inhibited atherosclerotic plaque-stimulated GPVI-mediated platelet aggregation, remibrutinib being more potent (IC50 = 0.03 μM) than rilzabrutinib (IC50 = 0.16 μM). Concentrations of remibrutinib (0.1 μM) and rilzabrutinib (0.5 μM), >80% inhibitory for plaque-induced aggregation, also significantly suppressed (>90%) the Btk-dependent pathways of platelet aggregation upon GPVI, von Willebrand factor/GPIb and FcγRIIA activation stimulated by low collagen concentrations, ristocetin and antibody cross-linking, respectively. Both BTKi did not inhibit aggregation stimulated by ADP, TRAP-6 or arachidonic acid. Remibrutinib (0.1 μM) only slightly prolonged closure time and significantly less than rilzabrutinib (0.5 μM).Conclusion: Remibrutinib and rilzabrutinib inhibit Btk-dependent pathways of platelet aggregation upon GPVI, VWF/GPIb, and FcγRIIA activation. Remibrutinib being more potent and showing a better profile of inhibition of Btk-dependent platelet activation vs. hemostatic impairment than rilzabrutinib may be considered for further development as an antiplatelet drug.

The brain-derived neurotrophic factor prompts platelet aggregation and secretion

AbstractBrain-derived neurotrophic factor (BDNF) has both autocrine and paracrine roles in neurons, and its release and signaling mechanisms have been extensively studied in the central nervous system. Large quantities of BDNF have been reported in circulation, essentially stored in platelets with concentrations reaching 100- to 1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. At low concentrations, BDNF primed platelets, acting synergistically with classical agonists. At high concentrations, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. Neurotrophin-4, but not nerve growth factor, and an activating antibody against the canonical BDNF receptor tropomyosin-related kinase B (TrkB) induced similar platelet responses to BDNF, suggesting TrkB could be the mediator. Platelets expressed, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking its tyrosine kinase domain. BDNF-induced platelet aggregation was prevented by inhibitors of Ras-related C3 botulinum toxin substrate 1 (Rac1), protein kinase C, and phosphoinositide 3-kinase. BDNF-stimulated platelets secreted a panel of angiogenic and inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Two families with autism spectrum disorder were found to carry rare missense variants in the BDNF gene. Platelet studies revealed defects in platelet aggregation to low concentrations of collagen, as well as reduced adenosine triphosphate secretion in response to adenosine diphosphate. In summary, circulating BDNF levels appear to regulate platelet activation, aggregation, and secretion through activation of a truncated TrkB receptor and downstream kinase-dependent signaling.

Platelet Adhesion and Thrombus Formation in Microchannels: The Effect of Assay-Dependent Variables.

Microfluidic flow chambers (MFCs) allow the study of platelet adhesion and thrombus formation under flow, which may be influenced by several variables. We developed a new MFC, with which we tested the effects of different variables on the results of platelet deposition and thrombus formation on a collagen-coated surface. Methods: Whole blood was perfused in the MFC over collagen Type I for 4 min at different wall shear rates (WSR) and different concentrations of collagen-coating solutions, keeping blood samples at room temperature or 37 °C before starting the experiments. In addition, we tested the effects of the antiplatelet agent acetylsalicylic acid (ASA) (antagonist of cyclooxygenase-1, 100 µM) and cangrelor (antagonist of P2Y12, 1 µM). Results: Platelet deposition on collagen (I) was not affected by the storage temperature of the blood before perfusion (room temperature vs. 37 °C); (II) was dependent on a shear rate in the range between 300/s and 1700/s; and (III) was influenced by the collagen concentration used to coat the microchannels up to a value of 10 µg/mL. ASA and cangrelor did not cause statistically significant inhibition of platelet accumulation, except for ASA at low collagen concentrations. Conclusions: Platelet deposition on collagen-coated surfaces is a shear-dependent process, not influenced by the collagen concentration beyond a value of 10 µg/mL. However, the inhibitory effect of antiplatelet drugs is better observed using low concentrations of collagen.

Biochemical and functional quality assessment of platelet concentrates

Background/Aim. Preparation of platelet concentrate (PC) from a unit of whole blood is a method dependent on a number of factors, which alone or together, can have a significant impact on the quality of the final product. Quality of PCs is determined with criteria defined in the Guide to the preparation, use and quality assurance of the Council of European Committee for Blood Transfusion. The aim of the study was to analyze the quality of PCs prepared from buffy coat (BC) and to allocate the factors of improvement and standardization of the quality of PCs. Methods. The study included a total of 80 PCs prepared from BC according to the standard procedure in the Blood Transfusion Institute Nis. The quality of PCs was determined according to the product volume and laboratory testing on the first (PC1), the third (PC3) and the fifth day (PC5) of the PC storage. The following parameters were determined: platelets, residual erythrocytes and leukocytes count, gas analysis [partial pressure of oxygen, carbon dioxide (pO2, pCO2)], pH, sterility and platelet aggregation triggered by using 3.2, 6.4 and 9.6 ?g collagen/mL (impedance aggregometry). Results. There were significantly lower platelet count, pO2, pCO2 and pH in the PC3 and PC5 samples (p &lt; 0.001). Except for the fulfillment of the criteria for platelet count, all the other quality parameters were in accordance with recommended criteria. Platelet aggregation for all the concentrations of collagen showed a decrease during the storage period, with statistically significant differences for PC3 and PC5 as compared to PC1 (p &lt; 0.01). There was statistically significant decrease in activity of PCs triggered with higher concentrations of collagen (6.4 and 9.6 ?g collagen/mL) in comparison with lower concentration of collagen (3.2 ?g collagen/mL). Conclusion. Platelet count, evaluated biochemical parameters and the platelet function were significantly changed during the storage period. In order to improve the quality of PCs it is important to store the products under proper conditions, change the type of plastic bag for PC storage and use platelet additive solutions (PAS) instead of plasma.

Platelet CD34 Expression and a Congenital Collar Bone Malformation Associated with a Partial CBFB Deletion in a Case with a Bleeding Disorder

Mutations in the transcription factor Growth Factor Independence 1B (GFI1B) and Runt-related transcription factor 1 (RUNX1) are causal to familial bleeding disorders. Mutant RUNX1 and GFI1B affect megakarocyte development, resulting in decreased numbers of platelets with significantly reduced α-granules and platelets that express the hematopoietic stem and progenitor cell marker CD34. A case that presented in our clinic with a bleeding disorder (Tosetto bleeding score of 11) was diagnosed with a δ-granule secretion defect. Platelet light transmission aggregometry revealed an aggregation defect upon stimulation with epinephrine and low concentrations of collagen and ADP. ATP release from δ-granules was normal after stimulation with a high concentration of collagen, however, no ATP was released upon stimulation with epinephrine and low levels were measured after stimulation with the thromboxane analogue U46619. Whole mount electron microscopy showed increased numbers of δ-granules in platelets derived from the index case compared to controls (average 5.84 δ-granules vs 3.66, per platelet respectively). To identify a genetic cause for the disease, a bleeding disorder gene panel was screened following whole exome sequencing. No disease-causing variants were called. Open exome analysis detected a ~100Kb heterozygous chromosome 16q22.1 deletion that was confirmed by SNP array. The observed deletion covers nine genes, including the last exon of CBFB. To date, none of the deleted genes have been implicated in inherited bleeding or platelet disorders. However, conditional knockout of Cbfb in mice results in a plethora of hematopoietic abnormalities including disturbed megakaryopoiesis and thrombocytopenia. CBFB is part of a heterodimeric transcription factor complex with either RUNX1, -2 or -3. Because of the CBFB-RUNX1 interaction and the fact that bleeding disorders caused by RUNX1 mutations result in platelet CD34 expression, we tested the platelets of the case reported here. Using flow cytometry, we clearly detected platelet CD34 expression compared to healthy controls, albeit to a lower level compared to RUNX1 and GFI1B affected cases. Mutations in RUNX2 may cause cleidocranial dysplasia and entire CBFB deletions have been reported in rare cases with congenital bone abnormalities. The medical record of the case presented here indicated that she had undergone surgery at the age of 13 for a congenital malformed collar bone. These data suggest a relation between the 16q/CBFB deletion and the observed abnormalities. To address this, we determined whether the abnormalities segregated with the 16q deletion. The mother and son of the index case had no history of clinical bleedings, no signs of bone malformations and did not have the 16q deletion. The father who was deceased was reported by the family not to have a bleeding propensity nor bone abnormalities. Platelets from the son did not express CD34. Remarkably, platelets from the son showed an aggregation defect upon stimulation with epinephrine and ADP, similar to the index patient. No ATP was released from δ-granules following stimulation with epinephrine. Thus, the 16q deletion associates with platelet CD34 expression and a congenital bone abnormality but is not responsible for the δ -granule secretion defect as the latter was observed in the son who did not have the 16q deletion, nor bleedings. Defective downmodulation of CD34 during platelet formation in the index case reported here suggests abnormal megakaryocyte development that might contribute in a multifactorial manner to bleedings. Recently, we reported CD34 expression on platelets from patients with rare GFI1B variants that may predispose to a bleeding propensity, but do not cause bleedings on their own. Thus, it will be important to determine whether the 16q deletion (and C-terminal truncation of CBFB) reported here affects megakaryocyte development and if so, contributes to the bleeding disorder in the index case. In contrast to Cbfb null mice, Cbfb heterozygous mice do not exhibit discernable hematopoietic phenotypes. Thus, if the C-terminal CBFB truncation contributes to the disease characteristics, it is likely that this occurs through a dominant-negative mechanism rather than haploinsufficiency. The findings reported here warrant further studies into the role of 16q (and CBFB) deletions in megakaryocyte development. Disclosures No relevant conflicts of interest to declare.

Varying solvent type modulates collagen coating and stem cell mechanotransduction on hydrogel substrates.

Type I collagen is the most abundant extracellular matrix protein in the human body and is commonly used as a biochemical ligand for hydrogel substrates to support cell adhesion in mechanotransduction studies. Previous protocols for conjugating collagen I have used different solvents; yet, how varying solvent pH and composition impacts the efficiency and distribution of these collagen I coatings remains unknown. Here, we examine the effect of varying solvent pH and type on the efficiency and distribution of collagen I coatings on polyacrylamide hydrogels. We further evaluate the effects of varying solvent on mechanotransduction of human mesenchymal stem cells (MSCs) by characterizing cell spreading and localization of Yes-Associated Protein (YAP), a key transcriptional regulator of mechanotransduction. Increasing solvent pH to 5.2 and above increased the heterogeneity of coating with collagen bundle formation. Collagen I coating highly depends on the solvent type, with acetic acid leading to the highest conjugation efficiency and most homogeneous coating. Compared to HEPES or phosphate-buffered saline buffer, acetic acid-dissolved collagen I coatings substantially enhance MSC adhesion and spreading on both glass and polyacrylamide hydrogel substrates. When acetic acid was used for collagen coatings, even the low collagen concentration (1 μg/ml) induced robust MSC spreading and nuclear YAP localization on both soft (3 kPa) and stiff (38 kPa) substrates. Depending on the solvent type, stiffness-dependent nuclear YAP translocation occurs at a different collagen concentration. Together, the results from this study validate the solvent type as an important parameter to consider when using collagen I as the biochemical ligand to support cell adhesion.

The shear mechanics of enzymatically degraded articular cartilage are predicted by a rigidity percolation model

Purpose: An accurate model for the mechanics of cartilage would allow for the prediction of future damage to diseased tissue and would direct the necessary advancements for artificial construct development. Recently it has been shown that orders of magnitude of variation of the shear modulus of articular cartilage are captured by describing the disordered collagen network with a rigidity percolation model. This model suggests that low concentrations of collagen, found near the surface of cartilage, are not sufficiently connected to support a shear load, leading to the mechanical properties in this low concentration regime to be dictated by the background aggrecan gel. This result contrasts with the deeper regions of the tissue where there is a higher concentration of collagen. In this region, the collagen rigidity percolates and the shear modulus of the tissue rapidly increases by several orders of magnitude. This model provides a framework for understanding the effect compositional changes cause to the mechanics of the tissue. The model predicts that the removal of the reinforcing background modulus shifts the rigidity percolation threshold of the collagen concentration, and results in a large decrease in shear modulus for the same collagen concentration. In the surface region of the tissue, as the mechanics are dominated by the aggrecan gel, then the shear modulus of the tissue will greatly decrease when the gel is removed - unlike deep into the tissue where the removal of the aggrecan gel will result in only a small decrease of shear modulus as the collagen network is still sufficiently connected. To test these predictions, we degrade the proteoglycan network of cartilage using the enzyme trypsin. Methods: Cartilage explants (N=24) were harvested from femoral condyles of neonatal calves (Fig 1.A.). The plugs (N=12) were coated by an epoxy surface which protected everywhere except the surface. The samples were then submerged in trypsin-EDTA (0.25%) at 37°C for 300 minutes, which removed proteoglycans as a front from the surface towards the deep zone. The samples were then rinsed with protease inhibitors. These plugs were bisected, and one half was used for measurement of the depth dependent shear modulus and biochemical analysis. The other half was fixed and used for histology and FTIR measurement, then biochemical essays were used to measure proteoglycan and collagen content. We were able to stain the tissue to validate proteoglycan loss, and the FTIR measurement provided a relative concentration of the aggrecan and collagen concentration throughout the tissue. We performed our simulations in Fortran, treating collagen as a random Kagome network with a reinforcing uniform background. We were able to calculate a shear modulus of this network, and then add in the reinforcing background afterwards. Results: The histology staining shows that we have a consistent front progressing with depth (Fig 1.B.). This verifies our FTIR measurements which shows a region of significantly reduced aggrecan concentration, with an unchanged collagen content (Fig 1.D.1.). Our shear measurements showed that the loss of proteoglycans caused an uneven decrease of shear modulus by up to a factor of 5 (Fig 1.D.2.). We then fitted the measurements of both the healthy articular cartilage and the degraded tissue (Fig 1.D.3.), and by scaling the reinforcing background modulus in the mechanical simulations, we can make predictions about the mechanical properties of other tissues for a range of proteoglycan concentrations. Conclusions: The trypsin degradation was able to mostly remove the aggrecan gel but deeper in the tissue there was still a non-zero concentration of aggrecan. Despite the removal of most of the proteoglycan background, the shear modulus of the deep zone was still an order of magnitude larger than that of the healthy surface tissue. This backs the prediction of the model which suggests that at high concentrations of collagen, it is the collagen network which provides most of the tissue’s structural support. This rigidity percolation model captures the mechanics of both healthy and deteriorated articular cartilage. Using this model, full mechanical descriptions of articular cartilage can be described using simple constituent scans of the collagen and proteoglycan content of the tissue. The model can also be used for the diagnosis of early proteoglycan loss through mechanical measurement. Finally, this model provides the understanding to direct the necessary advancements in artificial construct development.

Optimizing Platelet GPVI Inhibition versus Haemostatic Impairment by the Btk Inhibitors Ibrutinib, Acalabrutinib, ONO/GS-4059, BGB-3111 and Evobrutinib.

Ibrutinib and acalabrutinib are approved for B cell malignancies and novel Bruton's tyrosine kinase (Btk) inhibitors undergo clinical testing also in B cell-driven autoimmune disorders. Btk in platelets mediates platelet activation via glycoprotein (GP) VI, which is crucial for atherosclerotic plaque-induced platelet thrombus formation. This can be selectively inhibited by Btk inhibitors. Since patients on second-generation Btk inhibitors apparently show less bleeding than patients on ibrutinib, we compared the effects of ibrutinib and four novel irreversible Btk inhibitors on GPVI-dependent platelet aggregation in blood and in vitro bleeding time. Low concentrations of collagen which induced the same low degree of GPVI-mediated platelet aggregation as atherosclerotic plaque material were applied. IC50 values for collagen (0.2-0.5 µg/mL)-induced platelet aggregation after 15-minute pre-incubation were: ibrutinib 0.12 µM, BGB-3111 0.51 µM, acalabrutinib 1.21 µM, ONO/GS-4059 1.20 µM and evobrutinib 5.84 µM. Peak venous plasma concentrations of ibrutinib (0.5 µM), acalabrutinib (2 µM) and ONO/GS-4059 (2 µM) measured after anti-proliferative dosage inhibited collagen-induced platelet aggregation, but did not increase PFA-200 closure time on collagen/epinephrine. Closure times were moderately increased by 2- to 2.5-fold higher concentrations of these inhibitors, but not by BGB-3111 (1 µM) and evobrutinib (10 µM). Prolonging platelet drug exposure to 60 minutes lowered IC50 values of any Btk inhibitor for GPVI-mediated aggregation by several fold, and 5- to 10-fold below anti-proliferative therapeutic drug plasma levels. In conclusion, low blood concentrations of ibrutinib and the novel Btk inhibitors suffice for GPVI selective platelet inhibition relevant for atherothrombosis but do not impair primary haemostasis.