Collagen Surface Research Articles

Immobilization of Growth Factors to Collagen Surfaces Using Pulsed Visible Light.

In the treatment of traumatic injuries, burns, and ulcers of the eye, inadequate epithelial tissue healing remains a major challenge. Wound healing is a complex process involving the temporal and spatial interplay between cells and their extracellular milieu. It can be impaired by a variety of causes including infection, poor circulation, loss of critical cells, and/or proteins, and a deficiency in normal neural signaling (e.g., neurotrophic ulcers). Ocular anatomy is particularly vulnerable to lasting morbidity from delayed healing, whether it be scarring or perforation of the cornea, destruction of the conjunctival mucous membrane, or cicatricial changes to the eyelids and surrounding skin. Therefore, there is a major clinical need for new modalities for controlling and accelerating wound healing, particularly in the eye. Collagen matrices have long been explored as scaffolds to support cell growth as both two-dimensional coatings and substrates, as well as three-dimensional matrices. Meanwhile, the immobilization of growth factors to various substrates has also been extensively studied as a way to promote enhanced cellular adhesion and proliferation. Herein we present a new strategy for photochemically immobilizing growth factors to collagen using riboflavin as a photosensitizer and exposure to visible light (∼458 nm). Epidermal growth factor (EGF) was successfully bound to collagen-coated surfaces as well as directly to endogenous collagen from porcine corneas. The initial concentration of riboflavin and EGF as well as the blue light exposure time were keys to the successful binding of growth factors to these surfaces. The photocrosslinking reaction increased EGF residence time on collagen surfaces over 7 days. EGF activity was maintained after the photocrosslinking reaction with a short duration of pulsed blue light exposure. Bound EGF accelerated in vitro corneal epithelial cell proliferation and migration and maintained normal cell phenotype. Additionally, the treated surfaces were cytocompatible, and the photocrosslinking reaction was proven to be safe, preserving nearly 100% cell viability. These results suggest that this general approach is safe and versatile may be used for targeting and immobilizing bioactive factors onto collagen matrices in a variety of applications, including in the presence of live, seeded cells or in vivo onto endogenous extracellular matrix collagen.

Bioimaterials: “Tissue Papers” from Organ‐Specific Decellularized Extracellular Matrices (Adv. Funct. Mater. 34/2017)

In article number 1700992, Ramille N. Shah and co-workers investigate “Tissue Papers” (TPs) created directly from decellularized bovine and porcine tissues and organs. The researchers create a series of topographical maps illustrating the collagen distribution and surface profiles for six different tissues. Top row from left to right: Cardiac TP, Kidney TP, Liver TP. Bottom row from left to right: Muscle TP, Ovary TP, Uterus TP.

Antimicrobial activity of collagen material with thymol addition for potential application as wound dressing

Recently, special attention has been paid to the development of active wound dressing materials based on biopolymers. Collagen is a natural polymer, which meets the requirements of modern materials for medical applications. However, despite its unique properties, collagen has no antimicrobial activity. In this work thymol was incorporated into collagen films to meet antimicrobial properties of the material. Thymol is a naturally occurring phenolic compound recognized as an antimicrobial agent. Collagen/thymol thin films were obtained through solvent evaporation using collagen solutions containing different amounts of thymol. The structure of the obtained materials was studied using FTIR-ATR spectroscopy. The inhibition ability on the growth of several strains of microorganisms was tested. The standard ISO 22196:2007 was used to define the bactericidal properties of the material. The growth of the following bacteria on the collagen/thymol films was studied: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Enterobacter aerogenes, Candida albicans. The results showed that the growth of Staphylococcus aureus was the most inhibited compared to the other tested strains. Collagen/thymol material is more efficient against pathogens through direct contact compared to the diffusion of thymol from the material. In general, the thymol addition inhibits biofilm formation on the collagen surface.

Collagen density gradient on three-dimensional printed poly(ε-caprolactone) scaffolds for interface tissue engineering.

The ability to engineer scaffolds that resemble the transition between tissues would be beneficial to improve repair of complex organs, but has yet to be achieved. In order to mimic tissue organization, such constructs should present continuous gradients of geometry, stiffness and biochemical composition. Although the introduction of rapid prototyping or additive manufacturing techniques allows deposition of heterogeneous layers and shape control, the creation of surface chemical gradients has not been explored on three-dimensional (3D) scaffolds obtained through fused deposition modelling technique. Thus, the goal of this study was to introduce a gradient functionalization method in which a poly(ε-caprolactone) surface was first aminolysed and subsequently covered with collagen via carbodiimide reaction. The 2D constructs were characterized for their amine and collagen contents, wettability, surface topography and biofunctionality. Finally, chemical gradients were created in 3D printed scaffolds with controlled geometry and porosity. The combination of additive manufacturing and surface modification is a viable tool for the fabrication of 3D constructs with controlled structural and chemical gradients. These constructs can be employed for mimicking continuous tissue gradients for interface tissue engineering.

Tethering Growth Factors to Collagen Surfaces Using Copper-Free Click Chemistry: Surface Characterization and in Vitro Biological Response.

Surface modifications with tethered growth factors have mainly been applied to synthetic polymeric biomaterials in well-controlled, acellular settings, followed by seeding with cells. The known bio-orthogonality of copper-free click chemistry provides an opportunity to not only use it in vitro to create scaffolds or pro-migratory tracks in the presence of living cells, but also potentially apply it to living tissues directly as a coupling modality in situ. In this study, we studied the chemical coupling of growth factors to collagen using biocompatible copper-free click chemistry and its effect on the enhancement of growth factor activity in vitro. We verified the characteristics of modified epidermal growth factor (EGF) using mass spectrometry and an EGF/EGF receptor binding assay, and evaluated the chemical immobilization of EGF on collagen by copper-free click chemistry using surface X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR) spectroscopy, and enzyme-linked immunosorbent assay (ELISA). We found that the anchoring was noncytotoxic, biocompatible, and rapid. Moreover, the surface-immobilized EGF had significant effects on epithelial cell attachment and proliferation. Our results demonstrate the possibility of copper-free click chemistry as a tool for covalent bonding of growth factors to collagen in the presence of living cells. This approach is a novel and potentially clinically useful application of copper-free click chemistry as a way of anchoring growth factors to collagen and foster epithelial wound healing.

Abstract 342: Coagulation Factor XII Promotes Platelet Consumption in the Presence of Microbial Polyphosphate Under Shear Flow

Background: Terminal complications of bacterial sepsis include development of consumptive coagulopathy referred to as disseminated intravascular coagulation. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway is capable of promoting thrombin generation and platelet activation and consumption in whole blood distal to thrombus formation under shear ex vivo and in vivo . Aim: Test the hypothesis that the presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods and Results: Presence of long-chain polyP in whole blood promoted platelet aggregation on immobilized collagen surfaces under shear flow. Long-chain polyP enhanced fibrin formation and shortened clotting times of plasma and whole blood. The addition of long-chain polyP promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow. Distal to the sites of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of the coagulation pathway reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could exaggerate sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

Chondrocyte-Specific Loss of the Branched Actin Mediator ARP2/3 Results in Growth Plate Fusion and Proteoglycan Loss in Articular Cartilage

Purpose: The actin cytoskeleton regulates cell shape changes that are associated with chondrogenic differentiation in the developing growth plate and with phenotypic changes to articular chondrocytes during monolayer culture. However, it has been challenging to determine the role of the actin cytoskeleton during cartilage homeostasis in vivo. The Arp2/3 complex generates branched actin and is involved in many cellular processes including cell motility in response to extra-cellular matrix cues. We hypothesized that chondrocyte-specific deletion of Arp2/3 in vivo would alter cell-matrix interactions and result in both growth plate disorganization and premature osteoarthritis (OA). Methods: C57BL/6 mice harboring flanking loxP sites around a critical exon of an Arp2/3 subunit (p34) were crossed to two chondrocyte-specific Cre driver strains. Type II collagen (Col2)-Cre resulted in constitutive loss of Arp2/3, whereas Aggrecan-CreERT2 was used to induce Arp2/3 loss by intraperitoneal injection of tamoxifen. The cellular organization of the tibial growth plate was analyzed using hematoxylin and eosin stained histological sections. Mid-coronal sections stained with Safranin-O were evaluated for disrupted articular cartilage structure and proteoglycan loss using established semiquantitative scales of 0–12 for medial and lateral sides of the tibial and femoral cartilage. Statistical analysis was performed by ANOVA and Tukey's post-hoc test with greater than 6 mice per group. Live cell imaging was used to quantify cell spreading in isolated cells after induction of Arp2/3 loss in vivo. Pure populations of growth plate and articular chondrocytes were sorted based on fluorescence in cells successfully recombined by Aggrecan-CreERT2 using a lox-stop-lox zsgreen reporter allele. Results: Mice with constitutive loss of Arp2/3 in chondrocytes showed reduced viability (5/90 pups; expected ratio 1/4). Mutant mice that did survive were smaller than littermate controls and showed disorganization of the growth plate with secondary ossification failure (Fig. 1). When Arp2/3 loss was induced at 4 months, growth plates showed partial closure at 6 months and complete fusion at 12 months (Fig. 2). The articular cartilage structure at 6 and 12 months was not affected by Arp2/3 loss (Fig. 3a). However, the proteoglycan content was significantly reduced at 12 months in mice with Arp2/3 loss as compared to other groups (Fig. 3b). Osteophytes were noted in 4 out of 8 mice with Arp2/3 loss at 12 months as compared to 1 out of 6 controls. Using an in vitro assay of cell-matrix interaction, growth plate and articular chondrocytes directly isolated from 3 week old mice with induced Arp2/3 loss showed less spread cell area on fibronectin and type I collagen coated dishes as compared to heterozygous controls (all combinations p<0.01). Conclusions: The ability of chondrocytes to sense and respond to extra-cellular matrix cues plays important roles during both development and homeostasis. Arp2/3 appears to mediate chondrocyte-matrix interactions, as cells with Arp2/3 loss were unable to spread normally on collagen and fibronectin surfaces. Growth plate chondrocytes with constitutive Arp2/3 loss failed to organize the highly ordered columns that result in longitudinal bone growth. When Arp2/3 loss was induced after skeletal maturity, growth plates progressively fused in a manner that does not typically occur in mice. This suggests that cell-matrix interactions or other Arp2/3 functions contribute to the active maintenance of the growth plate throughout adulthood. Loss of Arp2/3 in articular chondrocytes did not result in extensive erosion of cartilage matrix at 12 months, but did cause reduced staining for proteoglycans and more osteophytes. These observations suggest a role for the actin cytoskeleton in maintaining cartilage homeostasis over the long-term, although longer follow-up may be necessary to determine whether loss of Arp2/3 would result in more severe OA. This work has implications for understanding developmental phenomena such as chondrodysplasias and catch-up growth, as well as providing insight into the mechanisms that govern how cell-matrix interactions in articular cartilage may alter the risk for OA progression.Figure 2: H&E stained sections of the tibia of mice at 6 months (top) and 12 months (bottom) of age. All mice had Aggrecan-CreERT2 and received tamoxifen at 4 months of age, but controls retained one copy of functional Arp2/3. Scale bars are 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3. Safranin-O stained sections were graded for articular cartilage structure (A) and proteoglycan loss (B). A semiquantitative score of 0–12 was summed across four quadrants of the joint (high scores consistent with OA, max = 48). # indicates p<0.05 to all other groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Investigation of Human Dental Pulp Cells on a Potential Injectable Poly(lactic-co-glycolic acid) Microsphere Scaffold

IntroductionPoly(lactic-co-glycolic acid) (PLGA) has been extensively explored in the tissue engineering field with good biocompatibility and biodegradability. PLGA microspheres' injectable potency makes it highly desirable in dentin-pulp complex regeneration. Therefore, we investigated the cell adhesion, proliferation, odontogenic differentiation, and matrix mineralization of human dental pulp cells (HDPCs) on a PLGA microsphere scaffold. We hypothesized that this scaffold might be suitable for dentin-pulp complex regeneration. MethodsPLGA microsphere scaffolds were fabricated using the double-emulsion solvent extraction technique with or without type I collagen surface modification. HDPCs were isolated from freshly extracted premolar or third molar teeth with patients' informed consent and ethical approval. Fourth-passage HDPCs (1 × 105 cells/ml) were seeded onto surface-modified or -unmodified PLGA microspheres and cultured in vitro. Cell adhesion, proliferation, and alkaline phosphatase activity were evaluated at different time points. Odontogenic-related gene expression (DMP1, DSPP, COL1, OPN, and OCN) were analyzed using quantitative real-time polymerase chain reaction. After 8 weeks of culture, samples were observed under scanning electron microscopy. ResultsSurface modification using type I collagen significantly enhanced HDPC attachment to the PLGA microspheres and promoted cell spreading. Alkaline phosphatase activity and odontogenic-related gene expression of HDPCs cultured with PLGA microsphere scaffolds were enhanced significantly compared with HDPCs cultured without PLGA microsphere scaffolds. After 8 weeks of culture, HDPCs combined with PLGA microspheres formed 3-dimensional structures. Partial degradation of the scaffolds and matrix mineralization were also observed. ConclusionsHDPCs can adhere to the PLGA microspheres, proliferate and differentiate into odontoblastlike cells, and form a 3-dimensional complex with matrix mineralization. This study may provide insight into the clinical dentin-pulp complex restoration with HDPCs and PLGA microsphere constructs.

Adsorption of Water to Collagen as Studied Using Infrared (IR) Microspectroscopy Combined with Relative Humidity Control System and Quartz Crystal Microbalance.

Infrared (IR) microspectroscopy combined with a quartz crystal microbalance (QCM) together with an original relative humidity (RH) control system has been developed for studying water adsorption on a collagen film. The adsorbed water weights measured by QCM are almost similar for wetting and drying processes at 28 ℃, indicating that the collagen film is close to the water adsorption/desorption equilibria. A broad OH + NH stretching band area (3000-3700 cm-1) in the IR spectra of the collagen film increased linearly with the adsorbed weight until about 1.2 μg/8.0 μg dry collagen film at relative humidity (RH) = 40%, while at higher RH (60%, 80%), the band area deviates from the linear trend to the lower side, due to viscoelasticity and others. The OH + NH band can be simulated by four Gaussian components at 3440, 3330, 3210, and 3070 cm-1 with the relatively constant band areas of 3330 and 3070 cm-1 components due to amide A and B (NH) for increasing and decreasing RH. Bound water (3210 cm-1 component: short H bond) constitutes around 70% of total water (3440 + 3210 cm-1 band areas) at RH = 4.9% but decreases to 23% at RH = 80.3%, where free water (3440 cm-1 component: long H bond) becomes dominant over 70%. The peak shifts of C=O stretching (Amide I) and N-H bending (Amide II) can be understood by increasing hydrogen bonding of water molecules (bound water) bound to peptides at lower RH. The higher wavenumber shifts of CH stretching can be due to the loose binding of water molecules (free water) to aliphatic chains on the collagen surface, especially at higher RH. The present combined QCM-IR method is useful for studying amounts and natures of water adsorbing on biomolecules.

The effects of inner vertical outer spiral complex scaffold in repairing long segment of urethral defect

Objective To explore the possibility of the inner vertical outer spiral complex tubular urethra scaffold vascularization in repairing long segment of urethral defect. Methods From August 2014 to October 2015, 27 clean male New Zealand white rabbits were divided into 3 groups, S1 group was transfected recombinant vascular endothelial growth factor(VEGF) gene lentiviral vector group. S2 group was vascular pedicle transfer tube group. C group was simple stent group. A 3.0 cm inner vertical outer spiral complex scaffold was constructed by using the small intestine acellular matrix (SIS) and polylactic acid copolymer (PLGA) modified by type Ⅰ collagen surface, and adipose-derived mesenchymal stem cells (ADSC) and smooth muscle cells after transformation from New Zealand white rabbits. In S1 group, the seed cells were transfected by recombinant vascular endothelial growth factor (VEGF) gene lentivirus, which express VEGF protein.The complex scaffold was used to repair 3.0 cm rabbit urethral defect In S2 group, the un-transfected cells were seeded into the scaffold and embedded in the skin near the groin artery 3 weeks for repairing urethral defect with vascular pedicle transfer tube. In group C, the unseeded scaffold was used to repair the urethral defect alone.Postoperative observation and urethrography were followed 4, 8 and 24 weeks after implantation. The HE staining, fluorescence tracing, immunohistochemical and scanning electron microscopy were evaluated at the same phase. Results In S1 group, there were one urinary fistula and one urethral stricture-related death, respectively. The urethra was smooth and patent, histological examination showed active hyperplasia of urethral capillary. In S2 group, there were one urinary fistula and two urethral stricture-related deaths, respectively. The urethral was rough, local thinning or dilated. Fat accumulation and mucosal contraction were found in the urethral submucosal, respctively. In C group, there were one urinary fistula, three hypospadias, and three urethral stricture-related deaths. The thickness of the urethra was uneven and stricture bending. The urethral mucosa was poorly repaired and the scar was narrow. HE and CD31 staining showed that S1 and S2 groups were active in the proliferation of urethral capillaries, and the angiogenesis was abundant. VEGF staining showed that the cytoplasm of endothelial cell layer, smooth muscle layer of vascular wall and the urothelial epithelial cell layer were fully expressed at 24 weeks, especially in epithelial cell layer. CKpan staining showed that the epithelium of S1 and S2 group developed to stratified epithelium, and the morphology of urethra was similar to normal urethra at 24 weeks. The urethral epithelial in C group of grew poor as single-level, irregular arrangement, 24 weeks is still a lack of effective stratified epithelium. HE and α-SMA staining showed that the smooth muscle and actin gradually increased in group S1 and S2, α-SMA staining in group C was scarce and increased at 24 weeks. PLGA was encapsulated by the surrounding tissue and the structure of electrospinning was clear after 4 weeks, absorbed and degraded after 8 weeks and absorbed after 24 weeks. Conclusions The inner vertical outer spiral complex tubular urethra scaffold maybe a reasonable method in repairing long segment urethral defects, and the methods of tubular urethra scaffold vascularization by transfected VEGF gene recombinant lentiviral vector and vascular flap deserve more research. Key words: Tissue engineering; Scaffold; Urethra defect; Vascularization

Structure and properties of nitrides on the surface of collagen

A study of the structure and composition of titanium and hafnium nitride coatings, deposited by condensation from plasma phase, on the leather by methods of scanning electron microscopy and x-ray fluorescence analysis. Nanostructured nitride coatings don’t degrade the properties of the leather and also inhibits the growth of pathogenic microflora and slow the migration of chromium ions.

Platelet-Delivered Recombinant ADAMTS13 Inhibits Platelet Adhesion and Aggregation on Collagen Surface Under Flow in the Presence of Autoantibodies from Acquired Thrombotic Thrombocytopenic Purpura

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal syndrome. It is primarily caused by severe deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF), thereby reducing the platelet adhesion and aggregation under flow. Plasma exchange is the only effective initial therapy for acquired autoimmune TTP. However, in hospital mortality rate remains as high as 10-20% in addition to the concern about the complications associated with the placement of central catheter and the use of plasma products. Therefore, a better and more effective therapy is urgently needed.Objectives: To develop a more effective therapy, we determined the uptake and package of recombinant ADAMTS13 (rA13) in platelets and assessed the efficacy of platelets-delivered rA13 in inhibiting platelet adhesion and aggregation and thrombus formation under arterial shear.Methods: Human platelets were isolated from whole blood, washed, and incubated with various concentrations of rA13 (1-100 microgram/ml) at 25 and 37 degree Celsius for various times. The amount of rA13 uptake by platelets was determined in cell lysate by Western blotting and FRETS-VWF73 assays and in fixed cells by immunofluoresent microscopy and flow cytometry. The function of platelet delivered rA13 was also determined by shear-induced platelet adhesion and aggregation on collagen surface using a microfluidic system.Results: Freshly isolated and blood bank stored platelets were able to update rA13 in a temperature, concentration, and time-dependent manner. When rA13 (5 microgram/ml) was incubated with platelets, nearly all platelets were positive for rA13, assessed by immunofluoresent microscopy and flow cytometry. The rA13 inside platelets remained intact and was proteolytically active in cleaving FRETS-VWF73. Confocal imaging revealed that rA13 was partially co-localized with VWF in alpha granules of platelets. Microfluidic assay demonstrated that platelet-delivered rA13 was able to dramatically inhibit platelet adhesion and aggregation on collagen-coated surface under arterial shear (100 dyne/cm2) in the absence and presence of a human monoclonal antibody against ADAMTS13 (scFv4-20) that was isolated from a patient with acquired autoimmune TTP. These results were consistent with the inhibitory effects observed with mouse transgenic platelets expressing rA13 under the same conditions. When 1/3 of whole blood Adamts13-/- platelets were replaced with transgenic platelets (containing rA13), the coverage of all fluorescent platelets onto a VWF-collagen surface was dramatically reduced (data not shown).Conclusion: Our results demonstrate that platelets uptake and deliver rA13 to the site of thrombus formation under flow and the platelet-delivered rA13 may be efficacious for treating acquired TTP with inhibitors. DisclosuresZheng:Ablynx: Consultancy; Alexion: Research Funding.

Therapeutic efficacy of the platelet glycoprotein Ib antagonist anfibatide in murine models of thrombotic thrombocytopenic purpura

AbstractThrombotic thrombocytopenic purpura (TTP), a potentially fatal blood clot disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from acquired autoantibodies. Plasma exchange is the only effective initial therapy. However, the high mortality rate and the complications associated with plasma exchange therapy remain a major concern. To address unmet clinical needs, therapeutic efficacies of anfibatide, a snake venom-derived platelet glycoprotein Ib antagonist, in murine models of spontaneous thrombocytopenia and shigatoxin-induced TTP were determined. A light scattering platelet aggregometry, microfluidic shear-based assay, and murine models of TTP were used in the study. We showed that purified anfibatide inhibits ristocetin- or botrocetin-induced human or murine platelet agglutination in the presence of von Willebrand factor in a concentration-dependent manner. Anfibatide could also dramatically inhibit the adhesion and aggregation of murine and human platelets on a collagen surface under arterial shear stress, in the presence or absence of plasma ADAMTS13 activity. Most importantly, we demonstrated that an intraperitoneal administration of anfibatide at the dose of 60 ng/g body weight twice daily mitigated spontaneous thrombocytopenia and prevented shigatoxin-induced TTP in Adamts13−/− and disease-susceptible mice (CAST/Ei strain). Thus, we conclude that anfibatide, when administered at the optimal dosage, route, and interval, is efficacious in treating spontaneous and bacterial shigatoxin-induced TTP in the murine models. Our findings may provide the basis for further development of anfibatide for the treatment of acute TTP in humans.

Collagen-binding nanoparticles for extracellular anti-inflammatory peptide delivery decrease platelet activation, promote endothelial migration, and suppress inflammation.

In this manuscript, we present our work on the development and characterization of a novel temperature sensitive collagen-binding nanoparticle system. We demonstrate that when bound to a collagenous matrix, the nanoparticles are able to promote endothelial migration while avoiding cellular uptake. We also show that the nanoparticles are able to reduce inflammation via the release of anti-inflammatory peptides which, when combined with its ability to inhibit platelet binding, could lead to reduced intimal hyperplasia following balloon angioplasty. The drug delivery platform presented represents a unique dual therapy biomaterial wherein the nanoparticle itself plays a crucial role in the system's overall therapeutic potential while simultaneously releasing anti-inflammatory peptides.

Collagen intrafibrillar mineralization as a result of the balance between osmotic equilibrium and electroneutrality.

Mineralisation of fibrillar collagen with biomimetic process-directing agents has enabled scientists to gain insight into the potential mechanisms involved in intrafibrillar mineralisation. Here, by using polycation- and polyanion-directed intrafibrillar mineralisation, we challenge the popular paradigm that electrostatic attraction is solely responsible for polyelectrolyte-directed intrafibrillar mineralisation. Because there is no difference when a polycationic or a polyanionic electrolyte is used to direct collagen mineralisation, we argue that additional types of long-range non-electrostatic interactions are responsible for intrafibrillar mineralisation. Molecular dynamics simulations of collagen structures in the presence of extrafibrillar polyelectrolytes show that the outward movement of ions and intrafibrillar water through the collagen surface occurs irrespective of the charges of polyelectrolytes, resulting in the experimentally verifiable contraction of the collagen structures. The need to balance electroneutrality and osmotic equilibrium simultaneously to establish Gibbs-Donnan equilibrium in a polyelectrolyte-directed mineralisation system establishes a new model for collagen intrafibrillar mineralisation that supplements existing collagen mineralisation mechanisms.

Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Protease-activated receptor 4 is more important than protease-activated receptor 1 for the thrombin-induced procoagulant effect on platelets

Collagen Substrate Stiffness Anisotropy Affects Cellular Elongation, Nuclear Shape, and Stem Cell Fate toward Anisotropic Tissue Lineage.

Rigidity of substrates plays an important role in stem cell fate. Studies are commonly carried out on isotropically stiff substrate or substrates with unidirectional stiffness gradients. However, many native tissues are anisotropically stiff and it is unknown whether controlled presentation of stiff and compliant material axes on the same substrate governs cytoskeletal and nuclear morphology, as well as stem cell differentiation. In this study, electrocompacted collagen sheets are stretched to varying degrees to tune the stiffness anisotropy (SA) in the range of 1 to 8, resulting in stiff and compliant material axes orthogonal to each other. The cytoskeletal aspect ratio increased with increasing SA by about fourfold. Such elongation was absent on cellulose acetate replicas of aligned collagen surfaces indicating that the elongation was not driven by surface topography. Mesenchymal stem cells (MSCs) seeded on varying anisotropy sheets displayed a dose-dependent upregulation of tendon-related markers such as Mohawk and Scleraxis. After 21 d of culture, highly anisotropic sheets induced greater levels of production of type-I, type-III collagen, and thrombospondin-4. Therefore, SA has direct effects on MSC differentiation. These findings may also have ramifications of stem cell fate on other anisotropically stiff tissues, such as skeletal/cardiac muscles, ligaments, and bone.

Molecular basis of retinol anti-ageing properties in naturally aged human skin in vivo.

Retinoic acid has been shown to improve the aged-appearing skin. However, less is known about the anti-ageing effects of retinol (ROL, vitamin A), a precursor of retinoic acid, in aged human skin in vivo. This study aimed to investigate the molecular basis of ROL anti-ageing properties in naturally aged human skin in vivo. Sun-protected buttock skin (76 ± 6 years old, n = 12) was topically treated with 0.4% ROL and its vehicle for 7 days. The effects of topical ROL on skin epidermis and dermis were evaluated by immunohistochemistry, in situ hybridization, Northern analysis, real-time RT-PCR and Western analysis. Collagen fibrils nanoscale structure and surface topology were analysed by atomic force microscopy. Topical ROL shows remarkable anti-ageing effects through three major types of skin cells: epidermal keratinocytes, dermal endothelial cells and fibroblasts. Topical ROL significantly increased epidermal thickness by stimulating keratinocytes proliferation and upregulation of c-Jun transcription factor. In addition to epidermal changes, topical ROL significantly improved dermal extracellular matrix (ECM) microenvironment; increasing dermal vascularity by stimulating endothelial cells proliferation and ECM production (type I collagen, fibronectin and elastin) by activating dermal fibroblasts. Topical ROL also stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, and thus enriched the deposition of ECM in aged human skin in vivo. 0.4% topical ROL achieved similar results as seen with topical retinoic acid, the biologically active form of ROL, without causing noticeable signs of retinoid side effects. 0.4% topical ROL shows remarkable anti-ageing effects through improvement of the homeostasis of epidermis and dermis by stimulating the proliferation of keratinocytes and endothelial cells, and activating dermal fibroblasts. These data provide evidence that 0.4% topical ROL is a promising and safe treatment to improve the naturally aged human skin.

Magneto-responsive liquid crystalline elastomer nanocomposites as potential candidates for dynamic cell culture substrates

Recently, liquid crystalline elastomers (LCEs) have been proposed as active substrates for cell culture due to their potential to attach and orient cells, and impose dynamic mechanical signals through the application of external stimuli. In this report, the preparation of anisotropic and oriented nematic magnetic-sensitized LCEs with iron oxide nanoparticles, and the evaluation of the effect of particle addition at low concentrations on the resultant structural, thermal, thermo-mechanical, and mechanical properties is presented. Phase transformations produced by heating in alternating magnetic fields were investigated in LCEs in contact with air, water, and a common liquid cell culture medium was also evaluated. The inclusion of nanoparticles into the elastomers displaced the nematic-to-isotropic phase transition, without affecting the nematic structure as evidenced by similar values of the order parameter, while reducing the maximum thermomechanical deformations. Remote and reversible deformations of the magnetic LCEs were achieved through the application of alternating magnetic fields, which induces the nematic–isotropic phase transition through nanoparticle heat generation. Formulation parameters can be modified to allow for remote actuation at values closer to the human physiological temperature range and within the range of deformations that can affect the cellular behavior of fibroblasts. Finally, a collagen surface treatment was performed to improve compatibility with NIH-3T3 fibroblast cultures, which enabled the attachment and proliferation of fibroblasts on substrates with and without magnetic particles under quiescent conditions. The LCEs developed in this work, which are able to deform and experience stress changes by remote contact-less magnetic stimulation, may allow for further studies on the effect of substrate morphology changes and dynamic mechanical properties during in vitro cell culture.

Effects of acid concentration and the UHP pretreatment on the gelatinisation of collagen and the properties of extracted gelatins

SummaryAn ultra‐high‐pressure (UHP) transmission medium with HCl was applied as a pretreatment to extract gelatin. The effects of the acid concentration (0–1% (w/v)) on the gelatinisation of collagen and the properties of gelatin were investigated. An increase in the acid concentration decreased the thermostability of collagen and increased the yield of gelatin (from 64% increased to 80%). The content of the subunit components in the pretreated collagen and the gelatin declined slightly as the acid concentration was increased, resulted in slight decrease (P &gt; 0.05) in the gel strength (from 435 g decreased to 408 g). The analysis of Fourier transform infrared spectroscopy (FTIR) spectra showed that the triple‐helical structure, secondary structures of the collagen and covalent cross‐linking in the collagenous fibres were damaged gradually as the acid concentration was increased. The scanning electron microscopy (SEM) images showed that the smooth surface of collagen was partly disrupted with microvoids after UHP/1% HCl pretreatment, which may be related to the damage on the covalent cross‐linking.