Microscale mechanical properties of single elastic fibers: The role of fibrillin–microfibrils

Micromechanical properties of single elastic fibers and fibrillin–microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin–microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3–1.5 MPa were determined, values not significantly affected by the presence of fibrillin–microfibrils. To further understand the role of fibrillin–microfibrils in vertebrate elastic fibers, layers of fibrillin–microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74 MPa were calculated. The results suggest that fibrillin–microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.     

Controlled biomineralization of electrospun poly(ε-caprolactone) fibers to enhance their mechanical properties

Electrospun polymeric fibers have been investigated as scaffolding materials for bone tissue engineering. However, their mechanical properties, and in particular stiffness and ultimate tensile strength, cannot match those of natural bones. The objective of the study was to develop novel composite nanofiber scaffolds by attaching minerals to polymeric fibers using an adhesive material – the mussel-inspired protein polydopamine – as a “superglue”. Herein, we report for the first time the use of dopamine to regulate mineralization of electrospun poly(ε-caprolactone) (PCL) fibers to enhance their mechanical properties. We examined the mineralization of the PCL fibers by adjusting the concentration of HCO₃^? and dopamine in the mineralized solution, the reaction time and the surface composition of the fibers. We also examined mineralization on the surface of polydopamine-coated PCL fibers. We demonstrated the control of morphology, grain size and thickness of minerals deposited on the surface of electrospun fibers. The obtained mineral coatings render electrospun fibers with much higher stiffness, ultimate tensile strength and toughness, which could be closer to the mechanical properties of natural bone. Such great enhancement of mechanical properties for electrospun fibers through mussel protein-mediated mineralization has not been seen previously. This study could also be extended to the fabrication of other composite materials to better bridge the interfaces between organic and inorganic phases.     

Effects of crystalline phase on the biological properties of collagen–hydroxyapatite composites

The objective of this study was to investigate the effects of spatial structure and crystalline phase on the biological performance of collagen–hydroxyapatite (Col–HA) composite prepared by biomineralization crystallization. Two types of Col–HA composites were prepared using mineralization crystallization (MC composites) and pre-crystallization (PC composites), respectively. Structural characteristics were analyzed by scanning electron microscopy and transmission electron microscopy. Surface elemental compositions were measured by electron spectroscopy for chemical analysis (ESCA). These composites were used in in vivo repair of bone defects. The effects of the crystalline phase on the biological performance of Col–HA composites were investigated using radionuclide bone scan, histopathology and morphological observation. It was observed that in MC composites, HA was located on the surface of the collagen fibers and aggregated into crystal balls, whereas HA in PC composites was scattered among the collagen fibers. ESCA showed that phosphorus and calcium were 8.99% and 17.56% on MC composite surface, compared with 4.39% and 5.86% on the PC composite surface. In vivo bone defect repair experiments revealed that radionuclide uptake was significantly higher in the area implanted with the PC composite than in the contralateral area implanted with the MC composite. Throughout the whole repair process, the PC composite proved to be superior to the MC composite with regard to capillary-forming capacity and the amount of newly formed bone tissue. So it could be concluded that HA placement on collagen fibers affected the biological performance of Col–HA composites. Pre-crystallization made HA scattered among collagen fibers, creating a better structure for bone defect repair in comparison with MC Col–HA composites.     

Bioactivity and mechanical properties of collagen composite membranes reinforced by chitosan and β-tricalcium phosphate

In this study, we analyzed the effects of varying concentrations of chitosan (CS) and β-tricalcium phosphate [β-TCP, Ca(3) (PO(4) )(2) ] on the mechanical and cell-adhesion properties of a collagen (CG) matrix for use in guided bone regeneration (GBR). Three different CS concentrations (0.5, 1, and 2%) and five different contents of β-TCP (0, 17, 29, 38, and 44%) were investigated. The composite membranes were analyzed by scanning electron microscopy and cell-adhesion, flexural-strength, and tear-strength assays. The results show that the cell-adhesion and mechanical properties of the composite membranes improved with increasing β-TCP and CS contents, yielding suitable levels of the adhesion of cells and adequate mechanical stability to ensure successful GBR. The CS adhered to the microsized β-TCP, which was distributed uniformly in the composite membranes. The β-TCP and CS have no negative effect on the cell morphology, viability, and proliferation and possess good biocompatibility. This study demonstrates that β-TCP/CS/CG composite membranes are good candidates for GBR membranes in future applications. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

The mechanical properties and bioactivity of poly(methyl methacrylate)/SiO2–CaO nanocomposite

The mechanical properties and bioactivity of poly(methyl methacrylate)/SiO₂–CaO nanocomposite were investigated using dimethyldiethoxysilane (DMDES) and tetraethoxysilane (TEOS), which could produce two and four siloxane linkages, respectively, after a sol–gel reaction. Methyl methacrylate was co-polymerized with 3-(trimethoxysilyl)propyl methacrylate and then co-condensed with DMDES (specimen D) and TEOS (specimen T), respectively, with calcium nitrate tetrahydrate under acidic conditions. The fracture toughness of specimen D was much improved compared to that of specimen T, whereas its fracture strength, hardness, and apatite-forming ability in simulated body fluid (SBF) were slightly decreased. The improved fracture toughness of specimen D without losing apatite-forming ability was explained by the decrease of siloxane linkage numbers and the introduction of alkyl groups in silica structure because covalently bonded siloxane linkages produce hard and brittle fracture behavior in the nanocomposite while the alkyl groups help to make the silica as linear chain structure. The practical implication of these results is that this new nanocomposite can be applied to the filler materials for bone cement and dental composite resin because of its good bioactivity and improved mechanical properties.     

The influence of carbon fibres on the resorption time and mechanical properties of the lactide–glycolide co-polymer

In this study the influence of short carbon fibres (CF) on mechanical properties and degradation time of the lactide–glycolide co-polymer (PGLA) and on the mechanism of bone ingrowth into the implants was determined. Mechanical properties and push-out tests were measured. The pH of solutions and the implants' weights were tested after incubation in Ringer fluid. Analysis was based upon FT-IR and SEM with EDS studies. Pathological examinations were also performed. The in vitro examination revealed that carbon fibres accelerated polymer degradation process and increased the mechanical strength of polymer. In the case of PGLA + CF under in vivo conditions, initially, the superficial polymer degradation with new tissue in-growth was observed. Next, the degradation process included also the inner part of the implant, while the bone began to grow on exposed carbon fibres. In the case of pure PGLA the growth of soft tissue can be observed at the bone–implant interface and in the implant area. Our research indicates that PGLA + CF composite can be used in bone surgery as a short-term multifunctional load-bearing implant, which initially provides a mechanical support. During the time of controlled resorption of PGLA, carbon fibres act as a scaffold for the bone growth.     

Alteration of dentin–enamel mechanical properties due to dental whitening treatments

The mechanical properties of dentin and enamel affect the reliability and wear properties of a tooth. This study investigated the influence of clinical dental treatments and procedures, such as whitening treatments or etching prior to restorative procedures. Both autoclaved and non-autoclaved teeth were studied in order to allow for both comparison with published values and improved clinical relevance. Nanoindentation analysis with the Oliver–Pharr model provided elastic modulus and hardness across the dentin–enamel junction (DEJ). Large increases were observed in the elastic modulus of enamel in teeth that had been autoclaved (52.0 GPa versus 113.4 GPa), while smaller increases were observed in the dentin (17.9 GPa versus 27.9 GPa). Likewise, there was an increase in the hardness of enamel (2.0 GPa versus 4.3 GPa) and dentin (0.5 GPa versus 0.7 GPa) with autoclaving. These changes suggested that the range of elastic modulus and hardness values previously reported in the literature may be partially due to the sterilization procedures. Treatment of the exterior of non-autoclaved teeth with Crest Whitestrips?, Opalescence? or UltraEtch? caused changes in the mechanical properties of both the enamel and dentin. Those treated with Crest Whitestrips? showed a reduction in the elastic modulus of enamel (55.3 GPa to 32.7 GPa) and increase in the elastic modulus of dentin (17.2 GPa to 24.3 GPa). Opalescence? treatments did not significantly affect the enamel properties, but did result in a decrease in the modulus of dentin (18.5 GPa to 15.1 GPa). Additionally, as expected, UltraEtch? treatment decreased the modulus and hardness of enamel (48.7 GPa to 38.0 GPa and 1.9 GPa to 1.5 GPa, respectively) and dentin (21.4 GPa to 15.0 GPa and 1.9 GPa to 1.5 GPa, respectively). Changes in the mechanical properties were linked to altered protein concentration within the tooth, as evidenced by fluorescence microscopy and Fourier transform infrared spectroscopy.     

Bioactive silica–collagen composite xerogels modified by calcium phosphate phases with adjustable mechanical properties for bone replacement

The development of composites has been recognized as a promising strategy to fulfil the complex requirements of biomaterials. The present study reports on the modification of a novel silica–collagen composite material by varying the inorganic/organic mass ratio and introducing calcium phosphate cement (CPC) as a third component. The sol–gel technique is used for processing, followed by xerogel formation under specific temperature and relative humidity conditions. Cylindrical monolithic samples up to 400 mm³ were obtained without any sintering processes. Various hierarchical phases of the organic component were applied, ranging from tropocollagen and collagen fibrils up to collagen fibers, each characterized by atomic force microscopy. Focusing on the application of fibrils, various inorganic/organic mass ratios were used: 100/0, 85/15 and 70/30; their influence on the structure of the composite material was demonstrated by scanning electron microscopy. The composition was extended by the addition of 25 wt.% CPC which led to increased bioactivity by accelerating the formation of bone apatite layers in simulated body fluid. Synchrotron microcomputed tomography demonstrated the homogeneous distribution of the cement particles in the silica–collagen matrix. Compressive strength tests showed that the mechanical properties of the brittle pure silica gel are changed significantly due to collagen addition. The highest ultimate strength of about 115 MPa at about 18% total strain was registered for the 70/30 silica–collagen composite xerogels. Incorporation of CPC lowered the gel’s strength. By demonstrating differentiation of human monocytes into osteoclast-like cells, an important feature of the composite material regarding successful bone remodeling is fulfilled.     

Microstructural and mechanical differences between digested collagen–fibrin co-gels and pure collagen and fibrin gels

Collagen and fibrin are important extracellular matrix (ECM) components in the body, providing structural integrity to various tissues. These biopolymers are also common scaffolds used in tissue engineering. This study investigated how co-gelation of collagen and fibrin affected the properties of each individual protein network. Collagen–fibrin co-gels were cast and subsequently digested using either plasmin or collagenase; the microstructure and mechanical behavior of the resulting networks were then compared with the respective pure collagen or fibrin gels of the same protein concentration. The morphologies of the collagen networks were further analyzed via three-dimensional network reconstruction from confocal image z-stacks. Both collagen and fibrin exhibited a decrease in mean fiber diameter when formed in co-gels compared with the pure gels. This microstructural change was accompanied by an increased failure strain and decreased tangent modulus for both collagen and fibrin following selective digestion of the co-gels. In addition, analysis of the reconstructed collagen networks indicated the presence of very long fibers and the clustering of fibrils, resulting in very high connectivities for collagen networks formed in co-gels.     

The role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen–silk fibroin construct

Airway tracts serve as a conduit of transport in the respiratory system. Architecturally, these are composed of cartilage rings that offer flexibility and prevent collapse during normal breathing. To this end, the successful regeneration of an airway tract requires the presence of differentiated chondrocytes and airway smooth muscle cells. This study investigated the role of physiological dynamic mechanical stimulation, in vitro, on the differentiation of mesenchymal stem cells (MSCs), three-dimensionally seeded within a tubular dense collagen matrix construct-reinforced with rings of electrospun silk fibroin mat (TDC–SFC). In particular, the role of either shear stress supplied by laminar fluid flow or cyclic shear stress in combination with circumferential strain, provided by pulsatile flow, on the chondrogenic differentiation, and contractile lineage of MSCs, and their effects on TDC–SFC morphology and mechanical properties were analysed. Chondrogenic differentiation of MSCs was observed in the presence of chondrogenic supplements under both static and laminar flow cultures. In contrast, physiological pulsatile flow resulted in preferential cellular orientation within TDC–SFC, as dictated by dynamic circumferential strain, and induced MSC contractile phenotype expression. In addition, pulsatile flow decreased MSC-mediated collagen matrix remodelling and increased construct circumferential strength. Therefore, TDC–SFC demonstrated the central role of a matrix in the delivery of mechanical stimuli over chemical factors, by providing an in vitro niche to control MSC differentiation, alignment and its capacity to remodel the matrix.     

Comparison of the properties of collagen–chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking

The property of collagen–chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen–chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen–chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen–chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.     

Deficient degradation of homotrimeric type I collagen, α1(I)3 glomerulopathy in oim mice

Col1a2-deficient (oim) mice synthesize homotrimeric type I collagen due to nonfunctional proα2(I) collagen chains. Our previous studies revealed a postnatal, progressive type I collagen glomerulopathy in this mouse model, but the mechanism of the sclerotic collagen accumulation within the renal mesangium remains unclear. The recent demonstration of the resistance of homotrimeric type I collagen to cleavage by matrix metalloproteinases (MMPs), led us to investigate the role of MMP-resistance in the glomerulosclerosis of Col1a2-deficient mice. We measured the pre- and post-translational expression of type I collagen and MMPs in glomeruli from heterozygous and homozygous animals. Both the heterotrimeric and homotrimeric isotypes of type I collagen were equally present in whole kidneys of heterozygous mice by immunohistochemistry and biochemical analysis, but the sclerotic glomerular collagen was at least 95–98% homotrimeric, suggesting homotrimeric type I collagen is the pathogenic isotype of type I collagen in glomerular disease. Although steady-state MMP and Col1a1 mRNA levels increased with the disease progression, we found these changes to be a secondary response to the deficient clearance of MMP-resistant homotrimers. Increased renal MMP expression was not sufficient to prevent homotrimeric type I collagen accumulation.     

Pathogenicity of IgG subclass autoantibodies to type VII collagen: Induction of dermal–epidermal separation

In different autoimmune diseases, tissue damage is mediated by the Fc portion of autoantibodies. These include autoimmunity to type VII collagen, a major hemidesmosomal skin constituent, where autoantibodies activate both complement and leukocytes, leading to separation within the dermal–epidermal junction. Fc-dependent effector functions differ among IgG subclasses. To elucidate the still controversial role of IgG subclasses in the pathogenesis of autoimmunity to type VII collagen, we generated a unique set of V gene-matched recombinant chimeric anti-type VII collagen autoantibodies of the four human IgG subclasses. Binding specificities and avidities of all four autoantibodies were comparable. Using ex vivo models, our results demonstrate that a monoclonal autoantibody is sufficient to activate complement and to induce dermal–epidermal separation. However, only IgG1 and IgG3, but not IgG2 and IgG4 against type VII collagen, were pathogenic in our ex vivo model systems. To our knowledge, this is the first time that a full-length recombinant disease-related human autoantibody has been investigated. Our results demonstrate the usefulness of recombinant antibody technology to dissect the contribution of F(ab′)₂ and Fc portions of autoantibodies to their biological effects. These findings may eventually contribute to novel diagnostic tools for monitoring disease and to the development of more specific therapies in autoantibody-mediated diseases, i.e. the generation of subclass-specific adsorbers, used for extracorporal immunoapheresis, or the shifting of the autoimmune response to production of non-pathogenic autoantibodies.     

Microstructure,mechanical and corrosion properties of Mg–Dy–Gd–Zr alloys for medical applications

In previous investigations, a Mg–10Dy (wt.%) alloy with a good combination of corrosion resistance and cytocompatibility showed great potential for use as a biodegradable implant material. However, the mechanical properties of Mg–10Dy alloy are not satisfactory. In order to allow the tailoring of mechanical properties required for various medical applications, four Mg–10(Dy + Gd)–0.2Zr (wt.%) alloys were investigated with respect to microstructure, mechanical and corrosion properties. With the increase in Gd content, the number of second-phase particles increased in the as-cast alloys, and the age-hardening response increased at 200 °C. The yield strength increased, while the ductility reduced, especially for peak-aged alloys with the addition of Gd. Additionally, with increasing Gd content, the corrosion rate increased in the as-cast condition owing to the galvanic effect, but all the alloys had a similar corrosion rate (～0.5 mm year^?1) in solution-treated and aged condition.     

Microstructure,mechanical properties and bio-corrosion properties of Mg–Si(–Ca,Zn) alloy for biomedical application

Mg–Si alloy was investigated for biomedical application due to the biological function of Si in the human body. However, Mg–Si alloy showed a low ductility due to the presence of coarse Mg₂Si. Ca and Zn elements were used to refine and modify the morphology of Mg₂Si in order to improve the corrosion resistance and the mechanical properties. The cell toxicity of Mg, Zn and Ca metals was assessed by an MTT test. The test results indicated that increasing the concentrations of Mg, Zn and Ca ions did not cause cell toxicity, which showed that the release of these three elements would not lead to cell toxicity. Then, microstructure, mechanical properties and bio-corrosion properties of as-cast Mg–Si(–Ca, Zn) alloys were investigated by optical microscopy, scanning electronic microscopy, mechanical properties testing and electrochemical measurement. Ca element can slightly refine the grain size and the morphology Mg₂Si phase in Mg–Si alloy. The bio-corrosion resistance of Mg–Si alloys was improved by the addition of Ca due to the reduction and refinement of Mg₂Si phase; however, no improvement was observed in the strength and elongation. The addition of 1.6% Zn to Mg–0.6Si can modify obviously the morphology of Mg₂Si phase from course eutectic structure to a small dot or short bar shape. As a result, tensile strength, elongation and bio-corrosion resistance were all improved significantly; especially, the elongation improved by 115.7%. It was concluded that Zn element was one of the best alloying elements of Mg–Si alloy for biomedical application.     

PCL–gelatin composite nanofibers electrospun using diluted acetic acid–ethyl acetate solvent system for stem cell-based bone tissue engineering

Composite nanofibrous scaffolds with various poly(ε-caprolactone) (PCL)/gelatin ratios (90:10, 80:20, 70:30, 60:40, 50:50 wt.%) were successfully electrospun using diluted acetic and ethyl acetate mixture. The effects of this solvent system on the solution properties of the composites and its electrospinning properties were investigated. Viscosity and conductivity of the solutions, with the addition of gelatin, allowed for the electrospinning of uniform nanofibers with increasing hydrophilicity and degradation. Composite nanofibers containing 30 and 40 wt.% gelatin showed an optimum combination of hydrophilicity and degradability and also maintained the structural integrity of the scaffold. Human mesenchymal stem cells (hMSCs) showed favorable interaction with and proliferation on, the composite scaffolds. hMSC proliferation was highest in the 30 and 40 wt.% gelatin containing composites. Our experimental data suggested that PCL–gelatin composite nanofibers containing 30–40 wt.% of gelatin and electrospun in diluted acetic acid–ethyl acetate mixture produced nanofiber scaffolds with optimum hydrophilicity, degradability, and bio-functionality for stem cell-based bone tissue engineering.     

Transforming growth factor β upregulates the integrin-mediated adhesion of human prostatic carcinoma cells to type I collagen

Prostate cancer frequently metastasizes to bone, and we propose that this process may be facilitated by the adhesion of metastatic cells to bone-derived type I collagen. We examined collagen receptor function and regulation in osteotropic PC-3 human prostatic carcinoma cells. PC-3 cell adhesion to immobilized human type I collagen was promoted by Mn and Mg ions and was RGD-independent. Antibodies directed against 1 or 2 integrin subunits inhibited adhesion to collagen by 90% and 53%, respectively, suggesting involvement of the 21 receptor. Anti-1 or anti-3 antibodies had no effect on adhesion. Flow cytometry and immunoprecipitation of [S]methionine-labeled cells demonstrated that 21 was the major collagen receptor expressed by PC-3 cells. The pretreatment of PC-3 cells with transforming growth factor-1 (TGF-1), a major bone-derived growth factor, caused a rapid (2 h) 2-fold increase in the de novo synthesis of 2 and 1 integrin subunits, and also increased by 2- to 3-fold the adhesion and spreading of PC-3 cells on collagen. We conclude that 21 is the major collagen receptor employed by PC-3 cells, and that 21 upregulation by TGF- is associated with an increased adhesion and spreading on collagen. The data suggest that exposure of metastatic PC-3 cells to the high levels of TGF- in bone may promote their ability to adhere to bone-derived collagen, which may thereby facilitate the localization of metastatic cells in the skeleton.     

The relation of motion sickness to the spatial–temporal properties of velocity storage

Tilting the head in roll to or from the upright while rotating at a constant velocity (roll while rotating, RWR) alters the position of the semicircular canals relative to the axis of rotation. This produces vertical and horizontal nystagmus, disorientation, vertigo, and nausea. With recurrent exposure, subjects habituate and can make more head movements before experiencing overpowering motion sickness. We questioned whether promethazine lessened the vertigo or delayed the habituation, whether habituation of the vertigo was related to the central vestibular time constant, i.e., to the time constant of velocity storage, and whether the severity of the motion sickness was related to deviation of the axis of eye velocity from gravity. Sixteen subjects received promethazine and placebo in a double-blind, crossover study in two consecutive 4-day test series 1 month apart, termed series I and II. Horizontal and vertical eye movements were recorded with video-oculography while subjects performed roll head movements of approx. 45° over 2 s to and from the upright position while being rotated at 138°/s around a vertical axis. Motion sickness was scaled from 1 (no sickness) to an endpoint of 20, at which time the subject was too sick to continue or was about to vomit. Habituation was determined by the number of head movements that subjects made before reaching the maximum motion sickness score of 20. Head movements increased steadily in each session with repeated testing, and there was no difference between the number of head movements made by the promethazine and placebo groups. Horizontal and vertical angular vestibulo-ocular reflex (aVOR) time constants declined in each test, with the declines being closely correlated to the increase in the number of head movements. The strength of vertiginous sensation was associated with the amount of deviation of the axis of eye velocity from gravity; the larger the deviation of the eye velocity axis from gravity, the more severe the motion sickness. Thus, promethazine neither reduced the nausea associated with RWR, nor retarded or hastened habituation. The inverse relationship between the aVOR time constants and number of head movements to motion sickness, and the association of the severity of motion sickness with the extent, strength, and time of deviation of eye velocity from gravity supports the postulate that the spatiotemporal properties of velocity storage, which are processed between the nodulus and uvula of the vestibulocerebellum and the vestibular nuclei, are likely to represent the source of the conflict responsible for producing motion sickness.     

The promotion of neural progenitor cells proliferation by aligned and randomly oriented collagen nanofibers through β1 integrin/MAPK signaling pathway

In regenerative medicine, accumulating evidence demonstrates that the property of substrates monitors neural stem cells behavior. However, how stem cells sense and interpret biochemical and topographical cues remains elusive. This study aimed to explore the mechanism how nanofibrous scaffold modulated stem cells behavior. Spinal cord derived neural progenitor cells (NPCs) were cultured on electrospun aligned and randomly oriented collagen nanofibrous scaffolds. A 30% increase in proliferation and an elevation of BrdU incorporation were observed in NPCs on collagen nanofibers, compared to that on collagen-coated surface. In particular, NPCs expanded faster on aligned nanofibers in comparison with that on randomly oriented nanofibers. Moreover, an alteration in cell cycle progression with a reduced percentage of cells in G0/G1 phase and increased cell proliferation index (S phase plus G2/M phase) was also detected in NPCs cultured on collagen nanofibers. Incubating NPCs with anti-β1 integrin antibody or U1026 (an inhibitor of mitogen-activated protein kinase kinase, MEK) eliminated the altered cell cycle dynamics and BrdU incorporation induced by collagen nanofibers. In addition, cyclin D1 and cyclin dependent kinase 2 (CDK2), downstream genes of β1 integrin/mitogen-activated protein kinase (MAPK) pathway that control G1/S phase transition, were correspondingly regulated by nanofibers. Collectively, these data suggested that the property of substrate modulated NPCs proliferation by promoting cell cycle through β1 integrin/MAPK pathway. Our findings provide a better understanding of the interaction between NPCs and the substrate and therefore will pave way for regenerative medicine.     

The influence of ascorbic acid,TGF-β1, and cell-mediated remodeling on the bulk mechanical properties of 3-D PEG–fibrinogen constructs

Two-dimensional cell culture studies have shown that matrix rigidity can influence cell function, but little is known about how matrix physical properties, and their changes with time, influence cell function in 3-D. Biosynthetic hydrogels based on PEGylated fibrinogen permit the initial decoupling of matrix chemical and mechanical properties, and are thus potentially attractive for addressing this question. However, the mechanical stability of these gels due to passive hydrolysis and cell-mediated remodeling has not previously been addressed. Here, we show that the bulk mechanical properties of acellular PEG–fibrinogen hydrogels significantly decrease over time in PBS regardless of matrix cross-linking density in 7 days. To compensate, smooth muscle cells (SMCs) were encapsulated and stimulated to produce their own matrix using ascorbic acid or TGF-β1. Ascorbic acid treatment improved the mechanical properties of the constructs after 14 days in less cross-linked matrices, but TGF-β1 did not. The increase in matrix modulus of the constructs was not due to an increase in type I collagen deposition, which remained low and pericellular regardless of cross-link density or the soluble factor applied. Instead, ascorbic acid, but not TGF-β1, preferentially enhanced the contractile SMC phenotype in the less cross-linked gels. Inhibition of contractility reduced cell spreading and the expression of contractile markers, and eliminated any beneficial increase in matrix modulus induced by cell-generated contraction of the gels. Together, these data show that PEG–fibrinogen hydrogels are susceptible to both hydrolysis and proteolysis, and suggest that some soluble factors may stimulate matrix remodeling by modulating SMC phenotype instead of inducing ECM synthesis in a 3-D matrix.