Collagen Surface Research Articles

Static magnetic field effects on the secondary structure and elasticity of collagen molecules; a possible biophysical approach to treat keratoconus

Type I collagen is among the major extracellular proteins that play a significant role in the maintenance of the cornea's structural integrity and is essential in cell adhesion, differentiation, growth, and integrity. Here, we investigated the effect of 300 mT Static Magnetic Field (300 mT SMF) on the structure and molecular properties of acid-solubilized collagens (ASC) isolated from the rat tail tendon. The SMF effects at molecular and atomic levels were investigated by various biophysical approaches like Circular Dichroism Spectropolarimetery (CD), Fourier Transform Infrared Spectroscopy (FTIR), Zetasizer light Scattering, and Rheological assay. Exposure of isolated type I collagen to 300 mT SMF retained its triple helix. The elasticity of collagen molecules and the keratoconus (KCN) cornea treated with SMF decreased significantly after 5 min and slightly after 10, 15, and 20 min of treatments. The exposure to 300 mT SMF shifted the Amid I bond random coil to antiparallel wave number from 1647 to 1631 cm−1. The pH of the 300 mT SMF treated collagen solution increased by about 25 %. The treatment of the KCN corneas with 300 mT SMF decreased their elasticity significantly. The promising results of the effects of 300 mT SMF on the collagen molecules and KCN cornea propose a novel biophysical approach capable of manipulating the collagen's elasticity, surface charges, electrostatic interactions, cross binding, network formation and fine structure. Therefore, SMF treatment may be considered as a novel non-invasive, direct, non-chemical and fast therapeutic and manipulative means to treat KCN cornea where the deviated physico-chemical status of collagen molecules cause deformation.

Platelet-specific Rictor knockout inhibits platelet production and activation and reduces thrombosis in mice

To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice. PF4-Cre and Rictorfl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout (Rictor-KO) mice and wild-type mice (n=65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbβ3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy. Compared with the wild-type mice, Rictor-KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbβ3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice. Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.

Methylglyoxal alters collagen fibril nanostiffness and surface potential

Collagen fibrils are fundamental to the mechanical strength and function of biological tissues. However, they are susceptible to changes from non-enzymatic glycation, resulting in the formation of advanced glycation end-products (AGEs) that are not reversible. AGEs accumulate with aging and disease and can adversely impact tissue mechanics and cell-ECM interactions. AGE-crosslinks have been related, on the one hand, to dysregulation of collagen fibril stiffness and damage and, on the other hand, to altered collagen net surface charge as well as impaired cell recognition sites. While prior studies using Kelvin probe force microscopy (KPFM) have shown the effect glycation has on collagen fibril surface potential (i.e., net charge), the combined effect on individual and isolated collagen fibril mechanics, hydration, and surface potential has not been documented. Here, we explore how methylglyoxal (MGO) treatment affects the mechanics and surface potential of individual and isolated collagen fibrils by utilizing atomic force microscopy (AFM) nanoindentation and KPFM. Our results reveal that MGO treatment significantly increases nanostiffness, alters surface potential, and modifies hydration characteristics at the collagen fibril level. These findings underscore the critical impact of AGEs on collagen fibril physicochemical properties, offering insights into pathophysiological mechanical and biochemical alterations with implications for cell mechanotransduction during aging and in diabetes. Statement of SignificanceCollagen fibrils are susceptible to glycation, the irreversible reaction of amino acids with sugars. Glycation affects the mechanical properties and surface chemistry of collagen fibrils with adverse alterations in biological tissue mechanics and cell-ECM interactions. Current research on glycation, at the level of individual collagen fibrils, is sparse and has focused either on collagen fibril mechanics, with contradicting evidence, or surface potential. Here, we utilized a multimodal approach combining Kelvin probe force (KPFM) and atomic force microscopy (AFM) to examine how methylglyoxal glycation induces structural, mechanical, and surface potential changes on the same individual and isolated collagen fibrils. This approach helps inform structure-function relationships at the level of individual collagen fibrils.

Fabrication and Characterization of Collagen–Magnetic Particle Composite Microbeads for Targeted Cell Adhesion and Proliferation

The integration of the biocompatibility of collagen and the remote-control ability of magnetic elements serves as both a cell scaffold and an actuator. We studied the preparation, characterization, and potential applications of collagen–magnetic particle composite microbeads (CMPMBs). The interplay among collagen concentration, particle size, and surface roughness was found to influence cell adhesion and proliferation. Adsorption and desorption tests showed the reversible attachment of the particles to magnetic sheets, enabling precise spatial control and targeted cell delivery. The particles demonstrated their utility as cell carriers, supporting cell migration and proliferation. These findings showcase the potential of CMPMBs as a promising platform for advanced cell delivery and tissue regeneration applications. The ability to fine-tune particle properties and manipulate them using magnetic fields offers new possibilities for creating complex tissue constructs and controlling cellular behavior, which could contribute to the development of more effective regenerative therapies and tissue engineering approaches.

Affimer reagents as tool molecules to modulate platelet GPVI-ligand interactions and specifically bind GPVI dimer

AbstractGlycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5′-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.

Preliminarily revealing rheological behaviors of mesoscale collagen fibrils in suspension

Using natural mesoscopic collagen as the structural unit has gradually emerged as a promising processing strategy for the preparation of high-performance collagen materials. These mesoscopic collagen fibrils (CAs) are exfoliated from biological tissues and dispersed in liquid media to prepare subsequent materials. The present work explores the effects of solid content, size, and temperature on the rheological behavior of collagen fibril suspensions under shearing through steady-state and dynamic rheological studies. Suspensions tend to exhibit shear-thinning and gel-like behavior, attributed to mesoscopic collagen forming a rigid network. Suspensions with large-size collagen fibers (aspect ratio > 100) exhibit high resistance to shear deformation at low solid contents (0.5 wt%) due to the intensive physical entanglement. The viscosity of the suspension was temperature-dependent and decreased rapidly near the hydrated thermal denaturation temperature of collagen. This work reveals the interactions between mesoscale collagen's surface interfaces and provides the necessary theoretical basis for collagen processing.

Fish‐Derived Collagen: A Promising Biomaterial for Regulating the Migration Behavior of MCF‐7 Cells

The uncontrollable migration of cancer cells is one of the reasons for the poor prognosis. The interaction between collagen and cellular processes is usually dominated by the full triple‐helix structure of collagen. However, it remains to be explored how the collagen configuration affects the migration behaviors of cancer cells. Michigan cancer foundation‐7 cells are chosen to study this relationship. Meanwhile, the grass carp is one of the representative fish species in Hubei and is virtually unaffected by zoonotic diseases. Therefore, grass carp‐derived collagen is prepared and used to modulate the migration behaviors. The collagen sample is pretreated via several procedures including the hydrothermal process and EDC/NHS, respectively. The underlying mechanisms affecting migration behavior are explored by tuning the degree of denaturation, assembly states, and cross‐linking conditions of collagen. Results demonstrate that moderate cross‐linking and assembly contribute to the migration processes, while denaturation significantly suppresses migration. Of particular note is the positive correlation of F‐actin expression levels with these phenomena. More importantly, the hydrophilic and hydrophobic properties of the collagen surface also affect the migration process. In summary, the study may provide insights to flexibly tune the migration behavior of cancer cells, which may be promising in inhibiting tumor metastasis.

Plasma-treated collagen functionalized with chondroitin sulfate as bioactive and nanostructured extracellular matrix mimics.

Aim: Cell microenvironment contains a plethora of information that influences cell modulation. Indeed, the extracellular matrix plays a central role in tissue development. Reproducing the cell-extracellular matrix crosstalk able to recapitulate both physical and biochemical signals is crucial to obtain functional tissue models or regenerative strategies. Materials & methods: Here, a combined method is proposed to easily functionalize collagen surface films, tailoring morphological properties. Oxygen nonthermal plasma treatment and glyco-conjugation with chondroitin sulfate are used to modify surface properties. Results: It resultsin higher adhesion, proliferationand morphological organization of U87 glioblastoma cells. Conclusion: Our finding suggests new promising strategies for the development of collagen-based biomaterials, which can be employed for advanced in vitro models.

Mechanical properties change of immobilized skeletal muscle in short position measured by shear wave elastography and pure shearing test

The purpose of this study was to evaluate the effects of immobilization on mechanical properties of skeletal muscle over the time. An in vivo rat model was used to investigate the shear modulus change of the flexor carpi ulnaris (FCU) in a short position. Measurements were performed by shear wave elastography (SWE) to compare contralateral and immobilized cases. The results showed a significant increase of 18.1% (p = 3.86. 10−7) in the shear modulus of immobilized skeletal muscle after two weeks (D14) when compared with the contralateral case. For the purposes of comparison, in vitro mechanical pure shearing tests were performed on samples collected from the skeletal muscles of the same rats. Although the difference between contralateral and immobilized cases was 17.6% (p = 0.32) at D14, the shear modulus difference was 35.7% (p = 0.0126 and p = 1.57.10−5 for immobilization and contralateral respectively) between in vivo and in vitro approaches. The mechanical properties changes were then correlated with the density of collagen from histological analysis, and it was shown that the contralateral collagen surface density was 25.4% higher than the immobilized density at D14 (p = 0.001). Thus, the results showed the feasibility of the comparison between the two approaches, which can surely be improved by optimizing the experimental protocols.

Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow

BackgroundDuring clotting, thrombin generates fibrin monomers and activates plasma-derived transglutaminase factor (F) XIIIa; collagen and thrombin-activated platelets offer thrombin-independent cellular FXIIIa (cFXIIIa) for clotting. Detecting fibrin on collagen and tissue factor surfaces in whole blood clotting typically uses complex reagents like fluorescent fibrinogen or antifibrin antibody. ObjectivesWe want to test whether the peptide using the α2- antiplasmin crosslinking mechanism by FXIIIa is a useful tool in both monitoring FXIIIa activity, and visualize and monitor fibrin formation, deposition, and extent of crosslinking within fibrin structures in whole blood clots formed under flow. MethodsWe tested a fluorescent peptide derived from α2-antiplasmin sequence (Ac-GNQEQVSPLTLLKWC-fluorescein) to monitor the location of transglutaminase activity and fibrin during whole blood clotting under microfluidic flow (wall shear rate, 100 s−1). ResultsThe peptide rapidly colocated with accumulating fibrin due to transglutaminase activity, confirmed by Phe-Pro-Arg-chloromethylketone inhibiting fibrin and peptide labeling. The FXIIIa inhibitor T101 had no effect on fibrin generation but ablated the labeling of fibrin by the peptide. Similarly, Gly-Pro-Arg-Pro abated fibrin formation and thus strongly attenuated the peptide signal. At arterial wall shear rate (1000 s−1), less fibrin was formed, and consequently, less peptide labeling of fibrin was detected compared with venous conditions. The addition of tissue plasminogen activator caused a reduction of both fibrin and peptide signals. Also, the peptide strongly colocalized with fibrin (but not platelets) in clots from laser-injured mouse cremaster arterioles. For clotting under flow, FXIIIa activity was most likely plasma-derived since a RhoA inhibitor did not block α2-antiplasmin fragment cross-linking to fibrin. ConclusionUnder flow, the majority of FXIIIa-dependent fibrin labeling with peptide during clotting was distal of thrombin activity. The synthetic peptide provided a strong and sustained labeling of fibrin as it formed under flow.

Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) and Cryoprecipitated AHF Contain Von Willebrand Factor with Comparable Binding to Collagen and Support Shear-Induced Platelet Thrombus Formation

Traumatic hemorrhage is a leading cause of morbidity and mortality and is a potentially preventable cause of death. Trauma-induced coagulopathy (TIC) exhibits complex pathophysiology. Fibrinogen (FIB) deficiency, a major mediator of TIC, is an independent predictor of mortality; and one of the first factors to reach critically low levels during hemorrhage. Early supplementation of FIB using cryoprecipitated AHF (Cryo) administration in resuscitation has been shown to improve outcomes in the treatment of TIC. Unlike FIB concentrates, Cryo prepared from conventional plasma is enriched in von Willebrand factor (VWF), factor VIII, factor XIII, and fibronectin that are impacted by TIC in addition to FIB. However, Cryo has a limited post thaw shelf-life of 4-6 hours at room temperature. This causes logistical impediments for early clinical application in trauma often resulting in use late in resuscitation with significant wastage. Pooling of individual Cryo units to achieve therapeutic doses increases donor exposure with risk of bacterial and viral transfusion transmitted infections (TTI). FDA approved Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) manufactured from Amotosalen/UVA treated plasma (PR plasma) is an alternative FIB source that has a 5-day post-thaw shelf-life at RT with reduced TTI risk. Due to its longer post-thaw shelf-life, it can be thawed in advance so that it is readily available and can be administered early in massive transfusion protocols with significantly lower wastage. VWF is a key component of Cryo and plays a major role in hemostasis during resuscitation with TIC. Here we have compared VWF contained in Cryo and IFC in various functional assays. At equimolar concentrations as assessed by measured VWF antigen, IFC and Cryo supported similar ristocetin-induced platelet aggregation. Accordingly, VWF multimeric composition, particularly presence of the largest molecular species, was similar in IFC and Cryo. We then evaluated the ability of VWF in IFC and Cryo compared to native blood to support platelet adhesion and platelet-rich thrombus formation onto a fibrillar type I collagen surface exposed to a linearly increasing shear rate gradient (0 t0 20,000 s -1) in the direction of flow from inlet to outlet of a Hele-Shaw flow chamber. Briefly, human blood, collected in EDTA, Lepirudin and supplemented with PGE 1 was centrifuged to separate blood cells from plasma. Red cells and platelets were washed to remove all plasma VWF and reconstituted to original hematocrit with Tyrode buffer at pH 7.4 spiked with either Cryo or IFC up to 1 U/ml of VWF. Platelets in native blood and in the different mixtures were fluorescently labeled with 3,3'-dihexyloxacarbocyanine iodide (DiOC 6) for visualization, and image sequences were acquired at different shear rates along the gradient. Images were analyzed to quantify platelet adhesion (the proportion of the field of view covered by platelets) and aggregation (i.e., the number and area of aggregates) at any given shear rate. As a negative control, blood reconstituted with buffer instead of a VWF source was used to ascertain that plasma VWF was effectively removed. As positive control, normal donor blood was used without washing out VWF (native blood). At all tested shear rates, the total surface coverage (1 pixel = 0.099 µm 2) by platelets was comparable with VWF in AHF or IFC added to washed blood and native blood (Figure). Of note, the individual size of platelet aggregates tended to increase with increasing shear rates and the number of individual aggregates tended to decrease. Specific monoclonal antibodies against VWF A1 and A3 domains - blocking interaction with GPIbα and collagen, respectively - markedly reduced the number and size of platelet aggregates over the surface. These results indicate that VWF derived from IFC, comparable to Cryo and native blood, retains functions strictly required to support platelet adhesion and aggregation at extremely elevated levels of shear rate/stress. The figure shows the size where 1 pixel = 0.099 µm 2 of each individual platelet thrombus on the surface.

Abstract 16373: Pyridoxamine (Vitamin B6) Mitigation of Protein Glycation and Deleterious Platelet-Material Interactions in Bioprosthetic Heart Valve Leaflets

Introduction: Though bioprosthetic heart valves (BHV) most often do not require anticoagulation, it has been reported that 12% of BHVs have significant leaflet thrombosis. We identified Vitamin B6 (pyridoxamine, abbr. PYR) as a pretreatment agent to reduce advanced glycation end-product (AGE) accumulation within bovine pericardial (BP) BHVs. AGEs contribute to valvular degeneration through collagen crosslinking and immune signaling. PYR is also known to inhibit platelet aggregation. Hypothesis: PYR pretreatment and supplementation mitigate BHV thrombosis. Methods: Glutaraldehyde fixed collagen coated (GFC) PVC tubing was used as a leaflet surface model and methylglyoxal (MGO) pretreatment to simulate glycation. PYR pretreated collagen surfaces and perfusate PYR supplementation were compared to untreated controls. These studies were repeated with glycated GFC. Fresh citrated human whole blood from healthy donors was perfused over the surfaces for 4 hours at 37 °C and arterial shear using the Chandler loop apparatus. Post perfusion platelet activation (CD62P expression via flow cytometry) and nitric oxide synthase (NOS) activity were measured. Platelets adhered to the tubing sections were visualized and quantitated via scanning electron microscopy. Results: PYR pretreatment of GFC surfaces reduced platelet activation after perfusion compared to the control (Fig1a). Surface glycation with MGO resulted in increased platelet activation, but PYR pretreatment of the glycated surface mitigated the effect of glycation on platelet activation (Fig1b). NOS activity was impaired by surface glycation and PYR treatment appears to rescue function in glycated environments (Fig1c). Platelet adhesion to GFC was increased by glycation (Fig1d-f). Conclusions: Glycated surfaces, even in euglycemia, are prothrombotic when compared to non-glycated surfaces. Vitamin B6 reduces platelet activation and adhesion in both glycated and non-glycated environments.

Antagonistic Roles of Human Platelet Integrin αIIbβ3 and Chemokines in Regulating Neutrophil Activation and Fate on Arterial Thrombi Under Flow.

Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches. Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbβ3. We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca2+]i rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide. Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.

An in vitro culture platform to study the extracellular matrix remodeling potential of human mesenchymal stem cells

The ability of mesenchymal stem cells (MSCs) to synthesize and degrade extracellular matrix (ECM) is important for MSC-based therapies. However, the therapeutic effects associated with ECM remodeling in cultured MSCs have been limited by the lack of a method to assess the ability of cultured cells to degrade ECM in vitro. Here, we describe a simple in vitro culture platform for studying the ECM remodeling potential of cultured MSCs using a high-density collagen (CL) surface. Cells on the CL surface have remarkable ability to degrade collagen fibrils by secreting matrix metalloproteinase (MMP); to study this, the marker collagen hybridizing peptide (CHP) was used. Confirming the ECM remodeling potential of MSCs with different population doublings (PDs), young and healthy γ-H2AX-negative cells, a marker of DNA damage and senescence, showed more extensive collagen degradation on the CL surface, whereas damaged cells of γ-H2AX-positive cells showed no collagen degradation. The frequency of γ-H2AX-/CHP + cells at PD = 0 was 49%, which was 4.9-fold higher than that at PD=13.07, whereas the frequency of γ-H2AX+/CHP- at PD=13.07 was 50%, which was 6.4-folds higher than that at PD=0. Further experimentation examining the in vitro priming effect of MSCs with the pro-inflammatory cytokine interferon-γ treatment showed increased frequency of cells with ECM remodeling potential with higher MMP secretion. Thus, this culture surface can be used for studying the ECM remodeling capacity of ex vivo–expanded MSCs in vitro and may serve as a platform for prediction in vivo ECM remodeling effect. Statement of significanceThe extracellular matrix (ECM) remodeling potential of cultured mesenchymal stem cells (MSCs) is important for assessing the effectiveness of MSC-based therapy. However, methods to assess the ability of cultured cells to degrade ECM in vitro are still lacking. Here, we developed a simple in vitro culture platform to study the ECM remodeling potential of cultured MSCs using high-density collagen surfaces. This platform was used to evaluate the ECM remodeling potential of long-term ex vivo–expanded MSCs in vitro.

Wound Coating Collagen-Based Composites with Ag Nanoparticles: Synthesis, Structure and Biological Activity

The search for materials for a new generation of wound coatings is important due to the increase in antibiotic-resistant microorganisms and the number of patients with untreatable chronic purulent wounds. Metal nanoparticles, specifically silver nanoparticles, have antimicrobial activity and do not induce known bacterial resistance. To obtain new Ag-containing nanocomposites, type I collagen was extracted by an enzyme–acid method from cattle tendons. Silver nanoparticles were obtained by an environmentally safe method, metal-vapor synthesis (MVS), which enables obtaining metal nanoparticles without impurities. For this, metal vapors were cocondensed in a vacuum of 10−2 Pa on the walls of a quartz reactor cooled to 77 K using acetone as an organic dispersion medium. The composition of the collagen surface was determined by XPS using the spectra of C1s, N1s, and O1s. The presence of a peak with a binding energy of approximately 368.57 eV in the Ag 3d5/2 spectrum indicates the state of Ag0 silver atoms in the nanocomposite. SEM images showed that collagen contributes to the effective stabilization of Ag nanoparticles with an average size of 13.0 ± 3.5 nm. It was found that collagen is non-toxic and biocompatible with skin cells and fibroblasts. The collagen–Ag nanoparticle nanocomposites exhibited antimicrobial activity against bacteria Bacillus subtilis, Escherichia coli, and fungi Aspergillus niger.

Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based chip.

The development of cell-based microfluidic assays offers exciting new opportunities in toxicity testing, allowing for integration of new functionalities, automation, and high throughput in comparison to traditional well-plate assays. As endocrine disruption caused by environmental chemicals and pharmaceuticals represents a growing global health burden, the purpose of the current study was to contribute towards the miniaturization of the H295R steroidogenesis assay, from the well-plate to the microfluidic format. Microfluidic chip fabrication with the established well-plate material polystyrene (PS) is expensive and complicated; PDMS and thiol-ene were therefore tested as potential chip materials for microfluidic H295R cell culture, and evaluated in terms of cell attachment, cell viability, and steroid synthesis in the absence and presence of collagen surface modification. Additionally, spike-recovery experiments were performed, to investigate potential steroid adsorption to chip materials. Cell aggregation with poor steroid recoveries was observed for PDMS, while cells formed monolayer cultures on the thiol-ene chip material, with cell viability and steroid synthesis comparable to cells grown on a PS surface. As thiol-ene overall displayed more favorable properties for H295R cell culture, a microfluidic chip design and corresponding cell seeding procedure were successfully developed, achieving repeatable and uniform cell distribution in microfluidic channels. Finally, H295R perfusion culture on thiol-ene chips was investigated at different flow rates (20, 10, and 2.5 µL/min), and 13 steroids were detected in eluting cell medium over 48h at the lowest flow rate. The presented work and results pave the way for a time-resolved microfluidic H295R steroidogenesis assay.

Hyaluronic acid-mediated collagen intrafibrillar mineralization and enhancement of dentin remineralization

Non-collagenous proteins (NCPs) in the extracellular matrix (ECM) of bone and dentin are known to play a critical regulatory role in the induction of collagen fibril mineralization and are embedded in hyaluronic acid (HA), which acts as a water-retaining glycosaminoglycan and provides necessary biochemical and biomechanical cues. Our previous study demonstrated that HA could regulate the mineralization degree and mechanical properties of collagen fibrils, yet its kinetics dynamic mechanism on mineralization is under debate. Here, we further investigated the role of HA on collagen fibril mineralization and the possible mechanism. The HA modification can significantly promote intrafibrillar collagen mineralization by reducing the electronegativity of the collagen surface to enhance calcium ions (Ca2+) binding capacity to create a local higher supersaturation. In addition, the HA also provides additional nucleation sites and shortens the induction time of amorphous calcium phosphate (ACP)-mediated hydroxyapatite (HAP) crystallization, which benefits mineralization. The acceleration effect of HA on intrafibrillar collagen mineralization is also confirmed in collagen hydrogel and in vitro dentin remineralization. These findings offer a physicochemical view of the regulation effect of carbohydrate polymers in the body on biomineralization, the fine prospect for an ideal biomaterial to repair collagen-mineralized tissues.

Role of SHP2 (PTPN11) in glycoprotein VI-dependent thrombus formation: Improved platelet responsiveness by the allosteric drug SHP099 in Noonan syndrome patients

IntroductionThe protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. Materials and methodsWashed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. ResultsPharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbβ3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. ConclusionPharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.

Nanoscale Dynamics of Streptococcal Adhesion to AGE-Modified Collagen

The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes in collagen—including dentinal collagen—is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)–derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type I collagen in the presence and absence of MGO-derived AGEs by employing bacterial cell force spectroscopy with atomic force microscopy (AFM). Type I collagen gels were treated with 10 mM MGO to induce AGE formation, which was characterized with microscopy and enzyme-linked immunosorbent assay. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or Streptococcus sanguinis SK 36 cells and probed against collagen surfaces to obtain force curves displaying bacterial attachment in real time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in silico computer simulation docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in silico simulations suggest that this effect is due to increased specific and nonspecific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to aging and glycation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, among others.

Platelet glycoprotein VI cluster size is related to thrombus formation and phosphatidylserine exposure in collagen-adherent platelets under arterial shear

BackgroundCollagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. ObjectivesTo determine the importance of GPVI clusters for thrombus formation in whole blood under shear. MethodsWe utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. ResultsAutomated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. ConclusionLabeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.