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Calcium (Ca2+) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca2+ concentration, and the levels of bone mineralization regulator proteins [phosphate-regulating neutral endopeptidase on chromo- some X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLO-Y4 cells was determined using the live/dead assay following treatment with various Ca2+ concentrations (1.8, 6, 12, 18, 24 and 50 mM) for different durations (15 and 60 min, and 24 h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase-3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15 min, there were more live cells than dead cells; however, after 60 min, the number of dead cells was increased following treatment with 24 and 50 mM Ca2+. After 24 h, there were more dead cells than live cells following treatment with 50 mM Ca2+. After 24 h of Ca2+ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24 mM Ca2+. In addition, as Ca2+ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24 mM Ca2+ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future. © 2022 Spandidos Publications. All rights reserved.

Osteoporotic fracture is one of the most common bone diseases in middle and old age, as the most serious consequence of osteoporosis. Sclerostin single‐chain antibody fragments (SCL‐scFv) have been proven to promote bone formation by binding to scleroprotein, a natural antagonist of the Wnt pathway, but it is difficult to rule alone due to the weak permeability and immunogenicity. Herein, we prepared poly(lactic‐co‐glycolic acid) microspheres as a sustained‐release vehicle to prolong the activity of SCL‐scFv. The morphology of microspheres were uniform and nearly sphere, loading efficiency and encapsulation efficiency of SCL‐scFv microspheres were 6.28 ± 1.04% and 48.37 ± 8.11%, respectively. Approximately 90% of the SCL‐scFvs were released from the microspheres over 28 days with initial burst releasing (38%) in the first 4 days. Sustained‐release of active SCL‐scFv from microspheres promoted bone marrow mesenchymal stem cells osteogenic differentiation in vitro and enhanced fracture healing in ovariectomized rats by improving bone mass and bone formation in the fracture region. All these findings demonstrate that the microspheres are able to simultaneously achieve localized long‐term SCL‐scFv controlled release and effectively promote bone formation, which provides a promising approach for osteoporotic fracture.

The dearth of knowledge on the diverse structures and functions in bacterial collagen-like proteins is in stark contrast to the deep grasp of structures and functions in mammalian collagen, the ubiquitous triple-helical scleroprotein that plays a central role in tissue architecture, extracellular matrix organization, and signal transduction. To fill and highlight existing gaps due to the general paucity of data on bacterial CLPs, we comprehensively reviewed the latest insight into their functional and structural diversity from multiple perspectives of biology, computational simulations, and materials engineering. The origins and discovery of bacterial CLPs were explored. Their genetic distribution and molecular architecture were analyzed, and their structural and functional diversity in various bacterial genera was examined. The principal roles of computational techniques in understanding bacterial CLPs' structural stability, mechanical properties, and biological functions were also considered. This review serves to drive further interest and development of bacterial CLPs, not only for addressing fundamental biological problems in collagen but also for engineering novel biomaterials. Hence, both biology and materials communities will greatly benefit from intensified research into the diverse structures and functions in bacterial collagen-like proteins.

Scleroproteins are an important category of proteins within the human body that adopt filamentous, elongated conformations in contrast with typical globular proteins. These include keratin, collagen, and elastin, which often serve a common mechanical function in structural support of cells and tissues. Genetic mutations alter these proteins, disrupting their functions and causing diseases. Computational characterization of these mutations has proven to be extremely valuable in identifying the intricate structure-function relationships of scleroproteins from the molecular scale up, especially if combined with multiscale experimental analysis and the synthesis of model proteins to test specific structure-function relationships. In this work, we review numerous critical diseases that are related to keratin, collagen, and elastin, and through several case studies, we propose ways of extensively utilizing multiscale modeling, from atomistic to coarse-grained molecular dynamics simulations, to uncover the molecular origins for some of these diseases and to aid in the development of novel cures and therapies. As case studies, we examine the effects of the genetic disease Epidermolytic Hyperkeratosis (EHK) on the structure and aggregation of keratins 1 and 10; we propose models to understand the diseases of Osteogenesis Imperfecta (OI) and Alport syndrome (AS) that affect the mechanical and aggregation properties of collagen; and we develop atomistic molecular dynamics and elastic network models of elastin to determine the role of mutations in diseases such as Cutis Laxa and Supravalvular Aortic Stenosis on elastin's structure and molecular conformational motions and implications for assembly.

Collagen from marine organisms has a broad prospect in biomedical field, yet the knowledge on marine-derived type II collagen is rare. Herein, a novel type II collagen was successfully isolated from squid cartilage for the first time. After being characterized, the immunogenicity of squid type II collagen (SCII) was evaluated and compared with that of bovine type II collagen (BCII). Then investigations were further conducted for the impacts of SCII on pro-inflammatory macrophages and macrophage chemotaxis. The degenerative osteoarthritis (OA) -relieving effects of SCII were explored using OA rat model in vivo. Our results demonstrated that the isolated SCII maintained triple-superhelical structure of native collagen with high purity. Different from BCII, SCII presented no immunogenicity since it neither induced abnormal proliferation of lymphocytes in vitro nor changed the basic levels of IgM, IgG, anti-type II collagen IgG and CD4+/CD8+ lymphocytes ratio in vivo. Additionally, SCII also exerted prominent anti-inflammatory effects. SCII significantly reduced the production of pro-inflammatory cytokines by enhancing the activity of TCPTP and subsequently prompting the dephosphorylation of p-STAT1 in pro-inflammatory macrophages. Besides, it indirectly prevented hypertrophic changes of chondrocytes, and markedly impeded chemotaxis of macrophages. Moreover, inflammation condition in OA rats was significantly alleviated under treatment with SCII. These data suggested that the newly developed SCII could not only avoid the immunogenic risks of collagen derived from terrestrial animals, but more importantly, provide new choice for the control and treatment of OA.

Electrospun keratin nanofiber fabricated with non-toxic solvents and crosslinking reagents are promising scaffolds for tissue engineering applications. Keratin from chicken feathers was solubilized in aqueous alkaline conditions and electrospun using aqueous poly(vinyl alcohol)(PVA)/citric acid solution as electrospinning polymer aid. Keratin/PVA nanofibers were successfully prepared containing 10, 20 and 30% keratin to PVA mass ratio. The increase in keratin concentration from 0 to 20% resulted in decreased viscosity of keratin/PVA solutions leading to a reduction in the fiber diameter from 565 nm in to 274 nm. Further decrease in viscosity with 30% keratin resulted in beads-on-fiber type of morphology. Nanofibers were thermally cross-linked through esterification reaction between carbonyl groups in citric acid and hydroxyl groups in PVA. Cross-linked nanofibers maintained their morphology and pore structure after soaking in water. The incorporation of keratin in PVA nanofiber was confirmed by FTIR and XPS results. Proliferation of fibroblasts after 14 days was higher in scaffolds containing 20% keratin which was attributed to the superior biological properties of keratin and higher surface area to volume ratio compared to 30% keratin/PVA scaffolds. Safer extraction, electrospinning and crosslinking conditions of keratin/PVA nanofiber are important for potential uses in tissue engineering. Keywords: Nanofiber, Electrospinning, Keratin, Polyvinyl alcohol, Scaffold

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1’s effects on liver by comparing wild-type controls, heterozygous igf1+/−, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage. Electronic supplementary material The online version of this article (doi:10.1007/s13105-016-0545-x) contains supplementary material, which is available to authorized users.

In the latest decade, mesenchymal stem cells (MSCs) have wildly considered as a source of seeded cells in tissue engineering, not only because of its multi-differentiation potentials, but also due to its immunoregulation ability. The main immunoregulatory features of MSCs could be divided into low self-immunogenicity and secretion of soluble factors. In this study, we explored how scaffold structures modulated the secretion of soluble immunoregulatory factors in MSCs under an allogeneic cartilage tissue engineering background. MSCs were seeded in four different collagen-based scaffolds. Their proliferation, differentiation, and secretion of various soluble factors associated with the immunosuppressive effects were evaluated. In this study, qRT-PCR, ELISA and immunoregulation results showed a great variability of the factor secretion by MSCs seeded in scaffolds with different structures. Compared with two-dimensional (2D) monolayer culture condition, three-dimensional (3D) groups (hydrogels and sponge) could effectively promote the mRNA expression and the protein production of soluble immune-related factors. Also, the supernatants collected from 3D groups obviously showed inhibition on allogeneic lymphocyte activating. These results suggested that scaffold structures might modulate MSCs' secretion of soluble immunoregulatory factors, and our study might enlighten the scaffold designs for desired tissue regeneration to control the host immune rejection through immune-regulation reaction.

Background: Biomaterials are non-drug substances used to treat, enhance or replace functions of body tissues or organs. Natural sources of biomaterials have recently become the focus of several research activities. Cowry shell constitutes one of the most promising natural sources of biomaterials because of its chemical stability, biodegradability and biocompatibility in the body. However, its applications may be limited due to immunogenic and toxic responses that may occur following implantation, hence this study.Materials and Methods: Crude fibrous protein extracted with citrate buffer from pulverised cowry shells (Cypraea moneta (L)), was resolved into two components (CSP1 and CSP2) by gel filtration. Immunological studies were performed with antisera obtained from rabbits by double immunodiffusion and immunoelectrophoresis techniques. Mice treated with the proteins were observed for signs of toxicity and their liver, kidney, lungs and spleen were processed histologically.Results: The native molecular weight of CSP1 and CSP2 determined by gel filtration were 91kDa and 33kDa respectively. CSP1 and CSP2 displayed single bands on SDS-PAGE with subunit molecular weight values of 19kDa and 19.5kDa respectively. Antisera obtained from rabbits immunised with the crude citrate buffer extracts precipitated the antigen in double immunodiffusion tests. Histopathological examinations revealed a dose-dependent damaging effect of the shell proteins on liver, kidney, lung and spleen tissues of the treated mice.Conclusion: This study showed that cowry shells contain fibrous proteins which are immunogenic and toxic in mice at relatively high concentrations, causing visible organ damage without concurrent physical manifestations.Keywords: cowry shell protein, purification, immune activation, toxicity

For cartilage repair, ideal scaffolds should mimic natural extracellular matrix (ECM) exhibiting excellent characteristics, such as biocompatibility, suitable porosity, and good cell affinity. This study aimed to prepare a collagen/silk fibroin composite scaffold incorporated with poly-lactic-co-glycolic acid (PLGA) microsphere that can be applied in repairing cartilage. To obtain optimum conditions for manufacturing a composite scaffold, a scaffold composed of different collagen-to-silk fibroin ratios was evaluated by determining porosity, water absorption, loss rate in hot water, and cell proliferation. Results suggested that the optimal ratio of collagen and silk fibroin composite scaffold was 7:3. The microstructure and morphological characteristics of the obtained scaffold were also examined through scanning electron microscopy and Fourier transform infrared spectroscopy. The results of in vitro fluorescence staining of bone marrow stromal cells revealed that collagen/silk fibroin composite scaffold enhanced cell proliferation without eliciting side effects. The prepared composite scaffold incorporated with PLGA microsphere was implanted in fully thick articular cartilage defects in rabbits. Collagen/silk fibroin composite scaffold with PLGA microspheres could enhance articular cartilage regeneration and integration between the repaired cartilage and the surrounding cartilage. Therefore, this composite will be a promising material for cartilage repair and regeneration.

Hierarchically porous structures and bioactive compositions of artificial biomaterials play a positive role in bone defect healing and new bone regeneration. Herein, cerium oxide nanoparticles-modified bioglass (Ce-BG) scaffolds were firstly constructed by the incorporation of hollow mesoporous Ce-BG microspheres in CTS via a freeze-drying technology. The interconnected macropores in Ce-BG scaffolds facilitated the in-growth of bone cells/tissues from material surfaces into the interiors, while the hollow cores and mesopore shells in Ce-BG microspheres provides more active sites for bone mineralization. The cerium oxide nanoparticles in the scaffolds rapidly promoted the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs), as confirmed by the up-regulation of osteogenesis-related markers such as OCN, ALP and COL-1. The enhanced osteoinductivity of Ce-BG scaffolds was mainly related to the activated ERK pathway, and it was blocked by adding a selective ERK1/2 inhibitor (SCH772984). In vivo rat cranial defect models revealed that Ce-BG scaffolds accelerated collagen deposition, osteoblast formation and bone regeneration as compared to BG scaffolds. The exciting results demonstrated that the synergistic effects between hierarchically porous structures and cerium oxide nanoparticles contributed to osteogenic ability, and hollow mesoporous Ce-BG scaffolds would be a novel platform for bone regeneration.

A collagen solution was added as a joint-lubricating fluid to extend the service life of UHMWPE liners. Adding this solution could effectively increase the adsorption of collagen onto zirconia surfaces, thereby reducing grinding parts (UHMWPE) wear. In this experiment, the thermal denaturation properties of collagen were used to manipulate the structural state of collagen molecules. The resulting structural alterations were observed using Fourier transform infrared spectroscopy, which indicated that the molecular structure of collagen changed from a triple-helix structure to a disordered molecular structure due to thermal effects. Collagen adsorption experiments also demonstrated that helical collagen molecules displayed higher rates of adsorption than disordered collagen molecules. Actual wear tests showed that larger quantities of adsorbed collagen led to lower friction coefficients and wear.

Mineralized collagen has been used clinically as a bone grafting material in oral applications. We have developed a mineralized collagen/Mg-Ca alloy combined scaffold to overcome the mechanical limitations of mineralized collagen. This study discusses the cytocompatibility of the mineralized collage/Mg-Ca alloy combined scaffold in vitro and the bone regeneration following tooth extraction in vivo. Using an indirect proliferation assay adapted from ISO 10993-5, it was found that mineralized collagen/Mg-Ca alloy combined scaffold enhanced cell proliferation and migration in addition to MC3T3-E1 cells not showing a cytotoxicity response in vitro. Finally, the ability of the combined scaffold to enhance osteogenesis was assessed in a canine socket preservation model. Cone beam CT, x-ray microscopes and biomechanical analysis showed the mineralized collagen/Mg-Ca alloy combined scaffold to be more effective at reducing the absorption of alveolar ridge and preserving the socket site than mineralized collagen alone. The combined scaffolds can promote bone regeneration with good biocompatibility, providing a new concept of the combined application of mineralized collagen and magnesium alloy.

To duplicate collagen's in vivo liquid crystalline (LC) phase and investigate the relationship between the morphology of LC collagen and osteoblast behavior, a self-assembly method was introduced for preparing collagen films with a stabilized LC phase. The LC texture and topological structure of the films before and after stabilization were observed with polarizing optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). The relationship between the collagen films and osteoblast behavior was studied with the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide method, proliferation index detection, alkaline phosphatase measurements, osteocalcin assay, inverted microscopy, SEM observation, AFM observation, and cytoskeleton fluorescence staining. The results showed that the LC collagen film had continuously twisting orientations in the cholesteric phase with a typical series of arced patterns. The collagen fibers assembled in a well-organized orientation in the LC film. Compared to the non-LC film, the LC collagen film can promote cell proliferation, and increase ALP and osteocalcin expression, revealing a contact guide effect on osteoblasts.

Biocompatible keratin-based hydrogels prepared by electron beam irradiation (EBI) were examined in wound healing. As the EBI dose increased to 60 kGy, the tensile strength of the hydrogels increased, while the percentage of elongation of the hydrogels decreased. After 7 days, the dehydrated wool-based hydrogels show the highest mechanical properties (the % elongation of 1341 and the tensile strength of 6030 g/cm(2) at an EBI dose of 30 kGy). Excision wound models were used to evaluate the effects of human hair-based hydrogels and wool-based hydrogels on various phases of healing. On post-wounding days 7 and 14, wounds treated with either human hair-based or wool-based hydrogels were greatly reduced in size compared to wounds that received other treatments, although the hydrocolloid wound dressing-treated wound also showed a pronounced reduction in size compared to an open wound as measured by a histological assay. On the 14th postoperative day, the cellular appearances were similar in the hydrocolloid wound dressing and wool-based hydrogel-treated wounds, and collagen fibers were substituted with fibroblasts and mixed with fibroblasts in the dermis. Furthermore, the wound treated with a human hair-based hydrogel showed almost complete epithelial regeneration, with the maturation of immature connective tissue and hair follicles and formation of a sebaceous gland.

The aim of this study was to investigate the influence of calcitriol on osteoinduction following local administration into mandibular bone defects. Calcitriol-loaded absorbable collagen membrane scaffolds were prepared using the polydopamine coating method and characterized by scanning electron microscopy. Composite scaffolds were implanted into rat mandibular bone defects in the following groups: no graft material (control), bare collagen membrane (CM group), collagen membrane bearing polydopamine coating (DOP/CM group), and collagen membrane bearing polydopamine coating absorbed with calcitriol (CAL/DOP/CM group). At 1, 2, 4 and 8 weeks post-surgery, the osteogenic potential of calcitriol was examined by histological and immunohistochemical methods. Following in vivo implantation, calcitriol-loaded composite scaffolds underwent rapid degradation with pronounced replacement by new bone and induced reunion of the bone marrow cavity. Calcitriol showed strong potential in inhibiting osteoclastogenesis and promotion of osteogenic differentiation at weeks 1, and 2. Furthermore, statistical analysis revealed that the newly formed bone volume in the CAL/DOP/CM group was significantly higher than other groups at weeks 1, and 2. At weeks 4, and 8, the CAL/DOP/CM group showed more mineralized bone and uniform collagen structure. These data suggest that local administration of calcitriol is promising in promoting osteogenesis and mineralization for restoration of mandibular bone defects.

Stem cells derived from oral tissue represent a highly attractive alternative source for clinical bone regeneration because they can be collected by non-invasive or minimally invasive procedures. Herein, we describe the human dental stem cells (DSCs) deriving from buccal fat pads (BFP), dental pulp (DP) of impacted teeth, and periodontal ligaments (PDL) to obtain BFPSCs, DPSCs, and PDLSCs, respectively. Cells were purified with selected medium and expanded through passages in stem cell culture medium. Purified cells were characterized for stemness by their growth rate, immunostaining, and multilineage differentiation ability. They showed plastic adherence, expression of stemness-specific markers, and multilineage differentiation potential. Immunocytochemistry analysis confirmed that DPSCs had more osteogenic potential than BFSCs and PDLSCs. Calcium-rich deposits, evaluated by von Kossa and Alizarin red staining, showed greater mineralization when DPSCs were cultured on collagen type I matrix than without collagen. Furthermore, DPSC-seeded collagen type I matrix maintained consistent osteogenesis and boosted mineral formation by 1-2 weeks over that in DPSCs cultured without collagen. Radiographic analysis of DPSC-seeded collagen type I matrix transplanted into rat cranial defects showed significant bone regeneration after 8 weeks. These results suggested that the redundant oral tissue can be used as a source of adult multipotent stem cells for clinical bone regeneration. Triple overlay images with biomarkers (red), nuclei (blue) and bright field morphology of DPSCs. The specifically osteo-differentiation shown by osteocalcin (left) expression and lack of sox9 (right) expressed in the images below which were cultured with collagen matrix, contrast with no collagen matrix group above.

In this study, we show a novel simple method to prepare a sponge-like porous keratin hydrogel through the extraction of wool keratin in a solution containing guanidine hydrochloride and 2-mercaptoethanol followed by dialysis for both aggregation of keratin and recrosslink. The gel had a highly porous structure and a fast-swelling property in rehydration after freeze-drying. It had also high mechanical strength both in the tensile test and the measurement of dynamic viscoelasticity. Three types of animal cells, PC12 cells, HOS cells and murine embryonic fibroblasts, well attached and grew on the surface of the porous hydrogel.

In the present study, poly(ethylene glycol) (PEG) and collagen I were sequentially immobilized on the titanium surface to simultaneously improve the anticoagulation and endothelial cell proliferation. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy analysis confirmed that PEG and collagen I were successfully immobilized on the titanium surface. Water contact angle results suggested the excellent hydrophilic surface after the immobilization. The anticoagulation experiments demonstrated that the immobilized PEG and collagen I on the titanium surface could not only obviously prevent platelet adhesion and aggregation but also prolong activated partial thromboplastin time (APTT), leading to the improved blood compatibility. Furthermore, immobilization of collagen to the end of PEG chain did not abate the anticoagulation. As compared to those on the pristine and PEG-modified titanium surfaces, endothelial cells exhibited improved proliferative profiles on the surface modified by the sequential immobilization of PEG and collagen in terms of CCK-8 assay, implying that the modified titanium may promote endothelialization without abating the blood compatibility. Our method may be used to modify the surface of blood-contacting biomaterials such as titanium to promote endothelialization and improve the anticoagulation, it may be helpful for development of the biomedical devices such as coronary stents, where endothelializaton and excellent anticoagulation are required.

3D porous scaffolds are relevant biomaterials to bone engineering as they can be used as templates to tissue reconstruction. The aim of the present study was to produce and characterize in vitro 3D magnesium-carbonate apatite/collagen (MCA/col) scaffolds. They were prepared by using biomimetic approach, followed by cross-linking with 0.25% glutaraldehyde solution (GA) and liofilization. Results obtained with Fourier-transform infrared spectroscopy (FT-IR) confirmed the type-B carbonate substitution, while by X-ray diffraction (XRD), a crystallite size of ~10nm was obtained. Optical and electron microscopy showed that the cylindrical samples exhibited an open-porous morphology, with apatite nanocrystals precipitated on collagen fibrils. The cross-linked 3D scaffolds showed integrity when immersed in culture medium up to 14 days. Also, the immersion of such samples into an acid buffer solution, to mimic the osteoclastic resorption environment, promotes the release of important ions for bone repair, such as calcium, phosphorus and magnesium. Bone cells (SaOs2) adhered, and proliferated on the 3D composite scaffolds, showing that synthesis and the cross-linking processes did not induce cytotoxicity.

Arteriovenous fistulae have been previously created by our group, through implantation of e-PTFE grafts between the carotid artery and jugular vein in healthy pigs, to gather comprehensive data on the time-course of the adapted geometry, composition, and biomechanical properties of the venous wall exposed to chronic increases in pressure and flow. The aim of this study was to mathematically assess the biomechanical adaptation of venous wall, by characterizing our previous in vitro inflation/extension testing data obtained 2, 4, and 12 weeks post-fistula, using a microstructure-based material model. Our choice for such a model considered a quadratic function for elastin with a four-fiber family term for collagen, and permitted realistic data characterization for both overloaded and control veins. As structural validation to the hemodynamically-driven differences in the material response, computerized histology was employed to quantitate the composition and orientation of collagen and elastin-fiber networks. The parameter values optimized showed marked differences among the overloaded and control veins, namely decrease in the quadratic function parameters and increase in the four-fiber family parameters. Differences among the two vein types were highlighted with respect to the underlying microstructure, namely the reduced elastin and increased collagen contents induced by pressure and flow-overload. Explicit correlations were found of the material parameters with the two basic scleroprotein contents, substantiating the material model used and the characterization findings presented. Our results are expected to improve the current understanding of the dynamics of venous adaptation under sustained pressure- and flow-overload conditions, for which data are largely unavailable and contradictory.

Objective: Patients with congenital and acquired heart diseases or arteriopathy require small-diameter vascular grafts for arterial reconstruction. Autologous veins are the most suitable graft, but when absent, an alternative is necessary. This work addresses the issue. Background: Tissue-engineering efforts to create such grafts by modifications of acellular natural scaffolds are considered a promising area. Methods: Homologous saphenous veins harvested from cadavers and organ donors were processed by decellularization with detergent and enzymatic digestion, followed by crosslinking by dye-mediated photooxidation. They were validated for acellularity, mechanical strength, and crosslink stability. In-vitro and in-vivo cytotoxicity and hemocompatibility studies were conducted. Collagen conformity was studied by Fourier transform infrared spectroscopy, and heat stability by differential scanning calorimetry. A limited large animal study was performed. Results: The processing method delivered biocompatible, hemocompatible, effectively crosslinked grafts, with high heat stability of 126 ℃, an enthalpy value of 183.5 J·g−1, and collagen conformity close to that of the native vein. The mechanical strength was 250% better than the native vein. The presence of extracellular matrix proteins allowed the acellular vein to become a triple-layered vascular structure in the sheep venous system. Conclusion: Crosslinking after decellularization by the dye-mediated photooxidation method could be reproduced in any human vein to obtain a small-diameter vascular grafts.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:01Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:29:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-01 The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd. UNICAMP-Universidade de Campinas Piracicaba School of Dentistry, Piracicaba, SP, 13414-903 UNESP-University Estadual Paulista Araraquara School of Dentistry, Araraquara, SP, 14801-903 UNESP-University Estadual Paulista Araraquara School of Dentistry, Araraquara, SP, 14801-903

Information on the biomechanical properties of aortic root aneurysms that would facilitate our understanding of their rupture modes is currently unavailable. In this study, whole-thickness wall specimens from aortic root aneurysms were studied in vitro so as to compare the biomechanical properties with gross histomorphology and composition, in relation to age, region, and direction. The stress-strain relationship was determined under uniaxial loading conditions and characterized by the Fung-type material model in terms of optimized material constants; failure properties were recorded. The connective tissue contents of the basic scleroproteins were also determined through computerized histology. Aging had a deleterious influence on the tensile strength of the aneurysmal sinus tissue, causing also stiffening and reduced extensibility that was consistent with the deficient elastin and collagen contents. Direction-dependent differences were demonstrated in the noncoronary sinus, with the circumferential being stiffer and stronger than the longitudinal direction, justified by the preferred collagen reinforcement along that direction there. In the left and right coronary sinus, the material constants and failure properties were essentially the same in the two directions, justified by the arbitrary orientation of medial (collagen and elastin fibers, and cellular) components relative to the circumferential-longitudinal directions. The material characterization results afforded, and the regional and age-related differences in the strength of the sinus wall, i.e. in its capacity to withstand hemodynamic stresses, are hoped to provide novel insight into the pathophysiological mechanisms responsible for the highest incidence of ruptured aortic root aneurysms in the right coronary and noncoronary sinus.

Circulating levels of IGF-1 may decrease under several circumstances like ageing, metabolic syndrome, and advanced cirrhosis. This reduction is associated with insulin resistance, dyslipidemia, progression to type 2 diabetes, and increased risk for cardiovascular diseases. However, underlying mechanisms between IGF-1 deficiency and cardiovascular disease remain elusive. The specific aim of the present work was to study whether the partial IGF-1 deficiency influences heart and/or coronary circulation, comparing vasoactive factors before and after of ischemia-reperfusion (I/R). In addition, histology of the heart was performed together with cardiac gene expression for proteins involved in structure and function (extracellular matrix, contractile proteins, active peptides); carried out using microarrays, followed by RT-qPCR confirmation of the three experimental groups. IGF-1 partial deficiency is associated to a reduction in contractility and angiotensin II sensitivity, interstitial fibrosis as well as altered expression pattern of genes involved in extracellular matrix proteins, calcium dynamics, and cardiac structure and function. Although this work is descriptive, it provides a clear insight of the impact that partial IGF-1 deficiency on the heart and establishes this experimental model as suitable for studying cardiac disease mechanisms and exploring therapeutic options for patients under IGF-1 deficiency conditions. © 2017 González-Guerra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

This article examines order and disorder in the extracellular matrix (ECM). The mechanical and structural properties of the tissues in the human body are largely determined by the ECM, the fibrillar network of proteins surrounding the cells. The main component of the ECM is collagen, which self-assembles in vivo into fibrils and fibers that supply rigidity and tensile strength to the ECM. The protein is often found together with elastin, another self-assembling ECM scleroprotein, whose properties of elasticity, extensibility, and elastic recoil supplement the mechanical properties of collagen. This article first provides an overview of protein synthesis and structure, with particular emphasis on tropocollagen, tropoelastin, and recombinantly-made polypeptides. It then considers the hierarchical supramolecular organization of the ECM, collagen and elastin before concluding with an assessment of structure-function relations at the matrix level.

// Vasiliki Gkretsi 1 , Andreas Stylianou 1 , Maria Louca 1 , Triantafyllos Stylianopoulos 1 1 Cancer Biophysics Laboratory, Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus Correspondence to: Triantafyllos Stylianopoulos, email: tstylian@ucy.ac.cy Keywords: Ras suppressor-1, extracellular matrix stiffness, invasion, tumor spheroids, atomic force microscopy Received: July 16, 2016 Accepted: February 20, 2017 Published: March 09, 2017 ABSTRACT Breast cancer (BC) is the most common malignant disease in women, with most patients dying from metastasis to distant organs, making discovery of novel metastasis biomarkers and therapeutic targets imperative. Extracellular matrix (ECM)-related adhesion proteins as well as tumor matrix stiffness are important determinants for metastasis. As traditional two-dimensional culture does not take into account ECM stiffness, we employed 3-dimensional collagen I gels of increasing concentration and stiffness to embed BC cells of different invasiveness (MCF-7, MDA-MB-231 and MDA-MB-231-LM2) or tumor spheroids. We tested the expression of cell-ECM adhesion proteins and found that Ras Suppressor-1 (RSU-1) is significantly upregulated in increased stiffness conditions. Interestingly, RSU-1 siRNA-mediated silencing inhibited Urokinase Plasminogen Activator, and metalloproteinase-13, whereas tumor spheroids formed from RSU-1-depleted cells lost their invasive capacity in all cell lines and stiffness conditions. Kaplan-Meier survival plot analysis corroborated our findings showing that high RSU-1 expression is associated with poor prognosis for distant metastasis-free and remission-free survival in BC patients. Taken together, our results indicate the important role of RSU-1 in BC metastasis and set the foundations for its validation as potential BC metastasis marker.

During the 1930s and 1940s the technique of X-ray diffraction was applied widely by William Astbury and his colleagues to a number of naturally-occurring fibrous materials. On the basis of the diffraction patterns obtained, he observed that the structure of each of the fibres was dominated by one of a small number of different types of molecular conformation. One group of fibres, known as the k-m-e-f group of proteins (keratin - myosin - epidermin - fibrinogen), gave rise to diffraction characteristics that became known as the α-pattern. Others, such as those from a number of silks, gave rise to a different pattern - the β-pattern, while connective tissues yielded a third unique set of diffraction characteristics. At the time of Astbury's work, the structures of these materials were unknown, though the spacings of the main X-ray reflections gave an idea of the axial repeats and the lateral packing distances. In a breakthrough in the early 1950s, the basic structures of all of these fibrous proteins were determined. It was found that the long protein chains, composed of strings of amino acids, could be folded up in a systematic manner to generate a limited number of structures that were consistent with the X-ray data. The most important of these were known as the α-helix, the β-sheet, and the collagen triple helix. These studies provided information about the basic building blocks of all proteins, both fibrous and globular. They did not, however, provide detailed information about how these molecules packed together in three-dimensions to generate the fibres found in vivo. A number of possible packing arrangements were subsequently deduced from the X-ray diffraction and other data, but it is only in the last few years, through the continued improvements of electron microscopy, that the packing details within some fibrous proteins can now be seen directly. Here we outline briefly some of the milestones in fibrous protein structure determination, the role of the amino acid sequences and how new techniques, including electron microscopy, are helping to define fibrous protein structures in three-dimensions. We also introduce the idea that, from the known sequence characteristics of different fibrous proteins, new molecules can be designed and synthesized, thereby generating new biological materials with specific structural properties. Some of these, for example, are planned for use in drug delivery systems. Along the way we also introduce the various Chapters of the book, where individual fibrous proteins are discussed in detail.

In this study, ribose was proposed as a promising, non-toxic, low-cost crosslinker to enhance the structural integrity and stiffness of type I collagen matrices. The main objective was to determine the optimal conditions of glycation by ribose to fabricate 3D porous collagen scaffolds and to verify their effectiveness for use as scaffolds for cartilage tissue engineering, by physicochemical and biological characterization. Two different crosslinking strategies were investigated including variation in the amount of ribose and the time of reaction: pre-crosslinking (PRE) and post-crosslinking (POST). All ribose-glycated collagen scaffolds demonstrated good swelling properties and interconnected porous microstructure suitable for cell growth and colonization. The POST samples were superior to PRE, in terms of porosity, degree of crosslinking, fluid uptake ability, and resistance to enzymatic digestion. Moreover, the mechanical properties of the scaffolds were significantly improved upon glycation when compared to non-crosslinked collagen, manifesting the best performance for POST matrices crosslinked for 5 d and in the highest amount of sugar. In vitro studies analyzing cell-material interactions revealed scaffold cytocompatibility with higher cell viability and cell proliferation as well as higher glycosaminoglycan secretion for POST scaffolds with respect to PRE. This report demonstrated the feasibility of developing 3D collagen scaffolds by ribose glycation and highlighted the POST-crosslinking strategy as being more favorable than the PRE-crosslinking to achieve scaffolds suitable for cartilage regeneration.

Aims: Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H2O2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. Results: Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with ß-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H2O2 and O2 .- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mitoO, the antioxidant apocynin, or the H2O2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mitoO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. Innovation: We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. Conclusion: LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. © 2017, Mary Ann Liebert, Inc.

This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering.

Endothelial cells (ECs) form a monolayer lining over the entire vascular wall and play an important role in maintaining vascular homeostasis and cancer metastasis. Loss of proper endothelial function can lead to vascular diseases. Therefore, the endothelial monolayer is particularly important in tissue regeneration and mimicking vascular tissue in vitro. Numerous studies have described the effects of ECs on nanofibers made from a variety of synthetic polymer materials designed to mimic the extracellular matrix (ECM). However, little is known about maintaining the integrity of ECs in in vitro systems. Here we describe polycaprolactone nanofibrous membranes coated with collagen gel that overcome many limitations of conventional nanofibers used for engineering endothelia. We investigated cell-cell and cell-ECM junctional complexes using collagen-coated and conventional nanofibrous membranes. Conventional nanofibrous membranes alone did not form a monolayer with ECs, whereas collagen-coated nanofibrous membranes did. Several concentrations of collagen in the gel coating promoted the formation of cell-cell junctional complexes, facilitated the deposition of laminin, and increased the focal contact organization of ECs. These results suggest the possible use of collagen-coated nanofibrous membranes for vascular tissue engineering applications and a vascular platform for organ-on-a-chip systems.

Lichen sclerosus is a progressive, inflammatory dermatosis characterized with porcelain white sclerotic plaques most commonly in the anogenital area of postmenopausal women. Children and men can also be affected. Extragenital involvement may occur. Even though the etiology is unknown; autoimmune, genetic, hormonal and infectious factors are suggested. Lichen sclerosus is associated with autoimmune diseases such as thyroid disease, alopecia areata, vitiligo, diabetes and pernicious anemia. An increased incidence of autoantibodies to the extracellular matrix protein 1 is shown in sera of patients with lichen sclerosus. Familial occurence and an association with HLA DQ7 supports the presence of genetic factors in its etiology. However, the role of estrogen and androgens could not be demonstrated as etiologic factors. Clinical similarity with acrodermatitis chronica atrophicans led researches to be held for the detection of Borrelia burgdorferi antibodies ended with conflicting results. Disease may be triggered with trauma in the presence of genetic predisposition. Pruritus is the main symptom. Pain, burning and dysesthesia may occur. Lichen sclerosus may be confused with sexual abuse in girls. Furthermore, it is more severe in the presence of sexual abuse. If not treated it leads to permanent anatomical disorders in the anogenital region. It is one of the most common cause of acquired phimosis in boys. Meatal stenosis and urinary obstruction may develop. Erectile dysfunction and dyspareunia due to introital stenosis may cause psychosexual problems in men and women. Perianal involvement causes constipation in women and girls. Long-standing anogenital lesions have the risk of developing squamous cell carcinoma. Verrucous carcinoma rarely occurs. Extragenital symptoms do not have similar risks. Ultra-potent topical corticosteroids are used as the first line therapy. In the case of unresponsiveness to corticosteroids the alternative approaches are topical immunmodulators, UVA1, calcipotriol, retinoids, and photodynamic therapy. In the presence of neoplasia and other complications surgical interventions are considered. Due to mainly preputial involvement, circumcision is an alternative procedure to topical corticosteroids in men. Often it may be curative. Lichen sclerosus is a disease with serious complications that needs life-long follow-up. Patients should be trained and encouraged for effective participation to the treatment and follow-up period.(Turk J Dermatol 2012; 6: 27-34)

The zero-stress state of the mucosa-submucosa and two muscle esophageal layers has been delineated, but their multi-axial response has not, because muscle dissection may not leave tubular specimens intact for inflation/extension testing. The histomechanical behavior of the three-layered porcine esophagus was investigated in this study, through light microscopic examination and uniaxial tension, with two-dimensional strain measurement in pairs of orthogonally oriented specimens. The two-dimensional Fung-type strain–energy function described suitably the pseudo-elastic tissue response, affording faithful simulations to our data. Differences in the scleroprotein content and configuration were identified as a function of layer, topography, and orientation, substantiating the macromechanical differences found. In view of the failure and optimized material parameters, the mucosa-submucosa was stronger and stiffer than muscle, associating it with a higher collagen content. A notable topographical distribution was apparent, with data for the abdominal region differentiated from that for the cervical region, owing to the existence of inner muscle with a circumferential arrangement and of outer muscle with a longitudinal arrangement in the former region, and of both muscle layers with oblique arrangement in the latter region, with thoracic esophagus being a transition zone. Tissue from the mucosa-submucosa was stronger and stiffer longitudinally, relating with a preferential collagen reinforcement along that axis, but more extensible in the orthogonal axis.

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen–alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.

Complex formation of scleroprotein models with an N-methylmorpholine N-oxide solvent was studied by the semiempirical PM3 method.

The impacts of glycosylation on biomineralization protein function are largely unknown. This is certainly true for the mollusk shell, where glycosylated intracrystalline proteins such as AP24 (Haliotis rufescens) exist but their functions and the role of glycosylation remain elusive. To assess the effect of glycosylation on protein function, we expressed two recombinant variants of AP24: an unglycosylated bacteria-expressed version (rAP24N) and a glycosylated insect cell-expressed version (rAP24G). Our findings indicate that rAP24G is expressed as a single polypeptide containing variations in glycosylation that create microheterogeneity in rAP24G molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic monosialylated and bisialylated, and monosulfated and bisulfated monosaccharides on the protein molecules. AFM and DLS experiments confirm that both rAP24N and rAP24G aggregate to form protein phases, with rAP24N exhibiting a higher degree of aggregation, compared to rAP24G. With regard to functionality, we observe that both recombinant proteins exhibit similar behavior within in vitro calcium carbonate mineralization assays and potentiometric titrations. However, rAP24G modifies crystal growth directions and is a stronger nucleation inhibitor, whereas rAP24N exhibits higher mineral phase stabilization and nanoparticle containment. We believe that the post-translational addition of anionic groups (via sialylation and sulfation), along with modifications to the protein surface topology, may explain the changes in glycosylated rAP24G aggregation and mineralization behavior, relative to rAP24N.

Morphological modifications of the cellular membrane of human keratinocytes treated with HgCl2 at different concentrations were investigated employing atomic force microscopy and Raman microspectroscopy techniques. Important changes in the surface structure of the keratinocytes membrane occur when this chemical treatment is performed. Mercury action gives rise to roughness and hole-like depressions, especially at cytotoxic concentration. Such surface features are mainly localized in peripheral zones of cells. Although the viability assay reveals that the exposure of keratinocytes to HgCl2 at a concentration of 10−6 M has no cytotoxic effect, morphological modifications are also evident in cellular membrane at such concentration. Raman microspectroscopy measurements suggest that such morphological modifications are related to modifications occurring in the lipidic layer. Our findings show that atomic force microscopy can be a valid and useful tool in studying the changes in morphology of cells when exposed to chemical stress.

Angiogenesis, or neovascularization, is tightly controlled by positive and negative regulators, many of which reside in the extracellular matrix. We have now identified eight novel 19- to 20-residue peptides derived from the alpha4, alpha5, and alpha6 fibrils of type IV collagen, which we have designated tetrastatins, pentastatins, and hexastatins, respectively. We have shown that these endogenous peptides suppress the proliferation and migration of HUVECs in vitro. By performing clustering analyses of the sequences using sequence similarity criteria and of the experimental results using a hierarchical algorithm, we report that the clusters identified by the experimental results coincide with the sequence-based clusters, indicating a tight relationship between peptide sequence and anti-angiogenic potency. These peptides may have potential as anti-angiogenic therapeutic agents.

Introduction: In patients with idiopathic pulmonary fibrosis (IPF) transforming growth factorbeta 1 (TGFβ1) induces excessive extracellular matrix (ECM) protein deposition leading to fibrosis. Our recent studies have shown that the glycoprotein fibulin-1 is increased in serum and lung tissue from patients with IPF and that their fibroblasts, which are the main fibrosis effector cells, produce more fibulin-1 than those from healthy people. Aim: To examine the modulation of fibroblast derived fibulin-1 from people with and without IPF. Methods: Primary parenchymal fibroblasts were derived from 5 patients with IPF and 5 without IPF (nonIPF). The production of fibulin-1 in the presence of cigarette smoke extract (CSE), foetal bovine serum (FBS) or TGFβ1 in the presence or absence of BIBF1120 (a triple kinase inhibitor the same as nintedanib) was measured by QPCR or ELISA. Proliferation was measured by MTT and CyQuant and cell attachment assessed by toluidine blue absorbance in fibroblasts from both groups. Results: CSE did not modulate fibulin-1 expression. Fibroblasts from IPF patients proliferated faster in 10% FBS and deposited more fibulin-1 in the presence of TGFβ1, compared to nonIPF fibroblasts, however attachment did not differ between the groups. BIBF1120 reduced proliferation and fibulin-1 deposition in the ECM, but did not influence attachment of fibroblasts. There were no differences in the degree of response to BIBF1120 between fibroblasts from patients with and without IPF. Conclusion: BIBF1120 inhibits fibroblastic behaviours that contribute to the pathogenesis of IPF. This new inhibitor may be effective in targeting the development of fibrosis in IPF patients.

Introduction: In patients with idiopathic pulmonary fibrosis (IPF) transforming growth factorbeta 1 (TGFβ1) induces excessive extracellular matrix (ECM) protein deposition leading to fibrosis. Our recent studies have shown that the glycoprotein fibulin-1 is increased in serum and lung tissue from patients with IPF and that their fibroblasts, which are the main fibrosis effector cells, produce more fibulin-1 than those from healthy people. Aim: To examine the modulation of fibroblast derived fibulin-1 from people with and without IPF. Methods: Primary parenchymal fibroblasts were derived from 5 patients with IPF and 5 without IPF (nonIPF). The production of fibulin-1 in the presence of cigarette smoke extract (CSE), foetal bovine serum (FBS) or TGFβ1 in the presence or absence of BIBF1120 (a triple kinase inhibitor the same as nintedanib) was measured by QPCR or ELISA. Proliferation was measured by MTT and CyQuant and cell attachment assessed by toluidine blue absorbance in fibroblasts from both groups. Results: CSE did not modulate fibulin-1 expression. Fibroblasts from IPF patients proliferated faster in 10% FBS and deposited more fibulin-1 in the presence of TGFβ1, compared to nonIPF fibroblasts, however attachment did not differ between the groups. BIBF1120 reduced proliferation and fibulin-1 deposition in the ECM, but did not influence attachment of fibroblasts. There were no differences in the degree of response to BIBF1120 between fibroblasts from patients with and without IPF. Conclusion: BIBF1120 inhibits fibroblastic behaviours that contribute to the pathogenesis of IPF. This new inhibitor may be effective in targeting the development of fibrosis in IPF patients.

Background CDP-choline (cytidine-5'-diphosphocholine) improves functional recovery, promotes nerve regeneration, and decreases perineural scarring in rat peripheral nerve injury. The aim of the present study was to investigate the mechanism of action of CDP-choline with regard to matrix metalloproteinase (MMP) activity in the rat-transected sciatic nerve injury model. Materials and methods Male Wistar rats were randomized into Sham, Saline, and CDP-choline groups. Rats in Sham group received Sham surgery, whereas rats in Saline and CDP-choline groups underwent right sciatic nerve transection followed by immediate primary saturation and injected intraperitoneally with 0.9% NaCl (1 mL/kg) and CDP-choline (600 μg/kg), respectively. Sciatic nerve samples were obtained 1, 3, and 7 d after the surgery and analyzed for levels and activities of MMP-2 and MMP-9, levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-3, and axonal regeneration. Results CDP-choline treatment decreased the levels and activities of MMP-2 and MMP-9, whereas increasing levels of TIMP-1 and TIMP-3 significantly on the third and seventh day after injury compared to Saline group. In addition, CDP-choline administration resulted in new axon formation and formation and advancement of myelination on newly formed islets (compartments) of axonal regrowth. Conclusions Our data show, for the first time, that CDP-choline modulates MMP activity and promotes the expression of TIMPs to stimulate axonal regeneration. These data help to explain one mechanism by which CDP-choline provides neuroprotection in peripheral nerve injury.

As a general rule, smooth muscle cells (SMC) are able to switch from a contractile phenotype to a less mature synthetic phenotype. This switch is accompanied by a loss of differentiation with decreased expression of contractile markers, increased proliferation as well as the synthesis and the release of several signaling molecules such as pro-inflammatory cytokines, chemotaxis-associated molecules, and growth factors. This SMC phenotypic plasticity has extensively been investigated in vascular diseases, but interest is also emerging in the field of gastroenterology. It has in fact been postulated that altered microenvironmental conditions, including the composition of microbiota, could trigger the remodeling of the enteric SMC, with phenotype changes and consequent alterations of contraction and impairment of gut motility. Several molecular actors participate in this phenotype remodeling. These include extracellular molecules such as cytokines and extracellular matrix proteins, as well as intracellular proteins, for example, transcription factors. Epigenetic control mechanisms and miRNA have also been suggested to participate. In this review key roles and actors of smooth muscle phenotypic switch, mainly in GI tissue, are described and discussed in the light of literature data available so far. J. Cell. Physiol. 231: 295-302, 2016. © 2015 Wiley Periodicals, Inc.

Poly(D,L-lactide) films were surface-modified by grafting collagen via NH(3) plasma to improve cell affinity. The modified films were characterized by IR analysis, contact angle measurement, SEM analysis and collagen quantity determination. It was demonstrated that -NH(2) and collagen were incorporated into the surface of PDLLA films. The hydrophilicity of the PDLLA film increased after NH(3) plasma treatment, but decreased with further collagen modification. More collagen was incorporated into the PDLLA films by a grating method as compared to that with an anchorage treatment. L929 fibroblast cells were used to evaluate the cell affinity of the modified films and control. It was shown that PDLLA films surface-modified by grafting collagen via NH(3) plasma more efficiently enhanced the cells attachment and proliferation than those films modified by collagen anchorage or only NH(3) plasma treatment.

Two species of alcyonarian corals, Lobophytum crassum and Sinularia polydactyla, are closely related to each other. It is reported that the calcified organic substances in the skeletons of both contain a protein-polysaccharide complex playing a key role in the regulation of biocalcification. However, information on the matrix proteins of endoskeletal sclerite has been lacking. Hence we studied the proteinaceous organic matrices of sclerites for both species, to analyze the sequences and the functional properties of the proteins present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed four bands of proteins with apparent molecular masses of 102, 67, 48, and 37 kDa for L. crassum and seven bands of 109, 83, 70, 63, 41, 30, and 22 kDa for S. polydactyla. A major protein band of about 67 kDa in L. crassum and two bands of proteins of about 70 and 63 kDa in S. polydactyla yielded N-terminal amino acid sequences. Periodic acid-Schiff staining indicated that the 67-kDa protein in L. crassum, and 83- and 63-kDa proteins in S. polydactyla were glycosylated. For detection of calcium binding proteins, a Ca(2+) overlay analysis was conducted in the extract via (45)Ca autoradiography. The 102- and 67-kDa calcium binding proteins in L. crassum, and the 109- and 63-kDa Ca(2+) binding proteins in S. polydactyla were found to be radioactive. An assay for carbonic anhydrase (CA), which is thought to play an important role in the process of calcification, revealed specific activities. Newly derived protein sequences were subjected to standard sequence analysis involving identification of similarities to other proteins in databases. The significantly different protein expressions and compositional analysis of sequences between two species were demonstrated.