Laminin‐modified and aligned poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)/polyethylene oxide nanofibrous nerve conduits promote peripheral nerve regeneration

Poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) has received much attention for its biodegradability and biocompatibility, characteristics that are required in tissue engineering. In this study, polyethylene oxide (PEO)‐incorporated PHBV nanofibres with random or aligned orientation were obtained by electrospinning. For further use in vivo, the nanofibre films were made into nerve conduits after treatment with NH₃ plasma, which could improve the hydrophilicity of inner surfaces of nerve conduits and then facilitate laminin adsorption via electrostatic interaction for promoting cell adhesion and proliferation. Morphology of the surfaces of modified PHBV/PEO nanofibrous scaffolds were examined by scanning electron microscopy. Schwann cell viability assay was conducted and the results confirmed that the functionalized nanofibres were favourable for cell growth. Morphology of Schwann cells cultured on scaffolds showed that aligned nanofibrous scaffolds provided topographical guidance for cell orientation and elongation. Furthermore, three‐dimensional PHBV/PEO nerve conduits made from aligned and random‐oriented nanofibres were implanted into 12‐mm transected sciatic nerve rat model and subsequent analysis were conducted at 1 and 2 months postsurgery. The above functionalized PHBV/PEO scaffolds provide a novel and promising platform for peripheral nerve regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Platelet‐rich fibrin‐based matrices to improve angiogenesis in an in vitro co‐culture model for bone tissue engineering

In the context of prevascularization strategies for tissue‐engineering purposes, co‐culture systems consisting of outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs) have been established as a promising in vitro tool to study regeneration mechanisms and to identify factors that might positively influence repair processes such as wound healing or angiogenesis. The development of autologous injectable platelet‐rich fibrin (PRF), which can be generated from peripheral blood in a minimal invasive procedure, fulfils several requirements for clinically applicable cell‐based tissue‐engineering strategies. During this study, the established co‐culture system of OECs and pOBs was mixed with injectable PRF and was cultivated in vitro for 24 h or 7 days. The aim of this study was to analyse whether PRF might have a positive effect on wound healing processes and angiogenic activation of OECs in the co‐culture with regard to proinflammatory factors, adhesion molecules and proangiogenic growth factor expression. Histological cell detection revealed the formation of lumina and microvessel‐like structures in the PRF/co‐culture complexes after 7 days of complex cultivation. Interestingly, the angiogenic activation of OECs was accompanied by an upregulation of wound healing‐associated factors, as well as by a higher expression of the proangiogenic factor vascular endothelial growth factor, which was evaluated both on the mRNA level as well as on the protein level. Thus, PRF might positively influence wound healing processes, in particular angiogenesis, in the in vitro co‐culture, making autologous PRF‐based matrices a beneficial therapeutic tool for tissue‐engineering purposes by simply profiting from the PRF, which contains blood plasma, platelets and leukocytes.     

Decorin mimic promotes endothelial cell health in endothelial monolayers and endothelial–smooth muscle co‐cultures

Non‐specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti‐restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC–SMC co‐cultures, we investigated the ability of DS–SILY₂₀, a decorin mimic, to promote EC health. DS–SILY₂₀ increased EC proliferation and migration by 1.5‐ and 2‐fold, respectively, which corresponded to increased phosphorylation of ERK‐1/2. Interestingly, IL‐6 secretion and the production of both E‐selectin and P‐selectin were reduced in the presence of 10 μm DS–SILY₂₀, even in the presence of the potent pro‐inflammatory cytokine platelet‐derived growth factor (PDGF). In hyperplastic and quiescent EC–SMC co‐cultures, DS–SILY₂₀ treatment reduced the secretion of IFNγ, IL‐1β, IL‐6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E‐selectin and P‐selectin expression was further reduced following DS–SILY₂₀ treatment in both co‐culture models. These results indicate that DS–SILY₂₀ promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.     

Mesenchymal stem cell expression of SDF‐1β synergizes with BMP‐2 to augment cell‐mediated healing of critical‐sized mouse calvarial defects

Bone has the potential for spontaneous healing. This process, however, often fails in patients with comorbidities. Tissue engineering combining functional cells, biomaterials and osteoinductive cues may provide alternative treatment strategies. We have recently demonstrated that stromal cell‐derived factor‐1β (SDF‐1β) works in concert with bone morphogenetic protein‐2 (BMP‐2) to potentiate osteogenic differentiation of bone marrow‐derived mesenchymal stem/stromal cells (BMSCs). Here, we test the hypothesis that SDF‐1β overexpressed in Tet‐Off‐SDF‐1β BMSCs, delivered on acellular dermal matrix (ADM), synergistically augments BMP‐2‐induced healing of critical‐sized mouse calvarial defects. BMSC therapies alone showed limited bone healing, which was increased with co‐delivery of BMP‐2. This was further enhanced in Tet‐Off‐SDF‐1β BMSCs + BMP‐2. Only limited BMSC retention on ADM constructs was observed after 4 weeks in vivo, which was increased with BMP‐2 co‐delivery. In vitro cell proliferation studies showed that supplementing BMP‐2 to Tet‐Off BMSCs significantly increased the cell number during the first 24 h. Consequently, the increased cell numbers decreased the detectable BMP‐2 levels in the medium, but increased cell‐associated BMP‐2. The data suggest that SDF‐1β provides synergistic effects supporting BMP‐2‐induced, BMSC‐mediated bone formation and appears suitable for optimization of bone augmentation in combination therapy protocols. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of a co‐produced mental health service to traditional services: A co‐produced mixed‐methods cross‐sectional study

This study investigates the differences between a co‐produced experimental mental health centre and traditional day centres. For this purpose, we used a collaborative and mixed‐method approach in two complementary studies: (i) a quantitative cross‐sectional study designed to compare users' hospitalization rates and their use of psychiatric medications and (ii) a qualitative study designed to explore and document the experienced differences between co‐produced and traditional services. In the quantitative cross‐sectional study, surveys were administered to 37 users of one co‐produced mental health service and to 40 users of traditional mental health services. A negative binomial regression analysis was performed to examine the relationships between predictors and users' hospitalization rates. After adjusting for the potential confounders, users of the co‐produced centre reported a 63.2% reduced rate of hospitalizations compared with users of traditional mental health services (P = 0.002). Furthermore, 39% of users of the co‐produced centre reported a reduction or even withdrawal from psychiatric medications against 22% of the comparison group (P = 0.036). In the qualitative study, six main differences emerged from a thematic analysis of a large user‐led focus group. In the participants' experiences, the co‐produced service focused on (i) parity and respectful relationships, (ii) people's strengths, (iii) freedom, (iv) psychological continuity, (v) social inclusion, and (vi) recovery orientation. Our research provides empirical evidence concerning the ‘preventive aspect’ of co‐produced mental health services. Additionally, new insights into how different stakeholders, particularly users of co‐produced mental health services, experience the differences between co‐produced and traditional mental health services are provided.     

Surrogate MR markers of response to chemo‐ or radiotherapy in association with co‐treatments: a retrospective analysis of multi‐modal studies

The study of magnetic resonance (MR) markers over the past decade has provided evidence that the tumor microenvironnement and hemodynamics play a major role in determining tumor response to therapy. The aim of the present work is to predict and monitor the efficacy of co‐treatments to radio‐ and chemotherapy by noninvasive MR imaging. Ten different co‐treatments were involved in this retrospective analysis of our previously published data, including NO‐mediated co‐treatments (insulin and isosorbide dinitrate), anti‐inflammatory drugs (hydrocortisone, NS‐398), anti‐angiogenic agents (thalidomide, SU5416 and ZD6474), a vasoactive agent (xanthinol nicotinate), botulinum toxin and carbogen breathing. Dynamic contrast enhanced (DCE) MRI, intrinsic susceptibility‐weighted (BOLD) MRI and electronic paramagnetic resonance (EPR) oximetry all reflect tumor microenvironment hemodynamic variables that are known to influence tumor response. Eight MR‐derived parameters (markers) were tested for their ability to predict therapeutic outcome (factor of increase in regrowth delay) in experimental tumor models (TLT and FSaII) after radiation therapy and/or chemotherapy with cyclophosphamide, namely tumor pO₂ and O₂ consumption rate (using EPR oximetry); tumor blood flow and permeability, i.e. V _p, K _trans, K _ep and percentage of perfused vessels (using DCE‐MRI); and BOLD signal intensity and R ₂* (using functional MRI). This multi‐modal comparison of co‐treatment efficacy points out the limitations of each MR marker and identifies in vivo pO₂ as a relevant endpoint for radiation therapy. DCE parameters (V _p and K _ep) were identified as a relevant endpoints for cyclophosphamide chemotherapy in our tumor models. This study helps qualify relevant imaging endpoints in the preclinical setting of cancer therapy. Copyright © 2010 John Wiley & Sons, Ltd.     

P(NIPAAM–co‐HEMA) thermoresponsive hydrogels: an alternative approach for muscle cell sheet engineering

Loss of skeletal muscle tissue caused by traumatic injury or damage due to myopathies produces a deficit of muscle function for which there is still no clinical treatment. Transplantation of myogenic cells, themselves or combined with materials, has been proposed to increase the regenerative capacity of skeletal muscle but it is hampered by many limitations, such as low cell survival and engraftment or immunological reaction and low biocompatibility of the exogenous materials. Recently, myoblast sheet engineering, obtained with thermoresponsive culture dishes, has attracted attention as a new technique for muscle damage treatment. For this purpose, a series of thermoresponsive hydrogels, constituted by poly(N‐isopropylacrylamide‐co‐2‐hydroxyethylmethacrylate) [p(NIPAAM‐co‐HEMA)] were synthesized by a simple and inexpensive free‐radical polymerization of the two co‐monomers with a redox initiator. Different ratios of N‐isopropylacrylamide (NIPAAm) and 2‐hydroxyethylmethacrylate (HEMA) have been examined to evaluate the effects on physicochemical, mechanical and optical hydrogel properties. The murine muscle cell line C₂C₁₂ has been exploited to test the cytotoxicity of the thermoresponsive hydrogels, depending on different synthesis conditions. In this study, we have identified a thermoresponsive hydrogel that allows cell adhesion and viability, together with the detachment of viable sheet of muscle cells, giving the chance to develop further applications for muscle damage and disease. Copyright © 2014 John Wiley & Sons, Ltd.     

Pressure‐activated microsyringe (PAM) fabrication of bioactive glass–poly(lactic‐co‐glycolic acid) composite scaffolds for bone tissue regeneration

The aim of this work was the fabrication and characterization of bioactive glass–poly(lactic‐co‐glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA–CEL2 composite scaffolds were innovatively produced by a pressure‐activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2–PLGA composite films (CEL2 is an experimental bioactive SiO₂–P₂O₅–CaO–MgO–Na₂O–K₂O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10–50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone‐like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast‐like cells and periosteal‐derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone‐like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT–PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd.     

Cyclosporine‐impregnated allograft bone sterilized with low‐temperature plasma

Deep‐freezing, freeze‐drying and gamma (γ)‐irradiation have deleterious effects on bone healing and mechanical properties of allograft bones. We tried preparing bone allografts using cyclosporine plus low‐temperature‐plasma sterilization. To explore the feasibility of this method of preparation, segmental defects in the right radii of rabbits were repaired with cyclosporine‐impregnated allograft bones (CABs) sterilized with low‐temperature‐plasma (in the study group) and deep‐frozen/freeze‐dried irradiated allograft bones (D/FIABs) (in the control group). X‐ray and quantitative histological analysis, peripheral blood T lymphocyte subset analysis and CD₂₅ molecule immunohistochemistry stain, the four‐point bending test and safety evaluations were respectively conducted to compare bone‐healing, immunosuppression, mechanical properties and safety between the two groups. X‐ray scores were higher in the study group than those in the control (p = 0.032). There were significant differences in new bone areas at most repairs in both groups (p < 0.05). There were no significant differences in the percentages of CD₄⁺ T, CD₈⁺ T, ratios of CD₄⁺ T:CD₈⁺ T or serum concentrations of GPT/Cr in both groups (p > 0.05). At 16 weeks postoperatively, the density of CD₂₅ molecules in the control group was higher than that in the study group. The ultimate loading in the study group was significantly higher than that in the control (p = 0.048). Bone marrow stromal cells (BMSCs) grew thickly around and on the surface of a cyclosporine‐impregnated allograft. Livers and kidneys in the study and control groups remained histologically normal at 7 days postoperatively. These results indicate that the CAB might be a better material than the D/FIAB in terms of bone healing, preservation of mechanical properties and immunosuppression without severe side‐effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas‐foamed poly(lactide‐co‐glycolide) and poly(lactide‐co‐glycolide) with bioactive glass fibres demonstrate insufficient bone repair in lapine osteochondral defects

Deep osteochondral defects may leave voids in the subchondral bone, increasing the risk of joint structure collapse. To ensure a stable foundation for the cartilage repair, bone grafts can be used for filling these defects. Poly(lactide‐co‐glycolide) (PLGA) is a biodegradable material that improves bone healing and supports bone matrix deposition. We compared the reparative capacity of two investigative macroporous PLGA‐based biomaterials with two commercially available bone graft substitutes in the bony part of an intra‐articular bone defect created in the lapine femur. New Zealand white rabbits (n = 40) were randomized into five groups. The defects, 4 mm in diameter and 8 mm deep, were filled with neat PLGA; a composite material combining PLGA and bioactive glass fibres (PLGA–BGf); commercial beta‐tricalcium phosphate (β‐TCP) granules; or commercial bioactive glass (BG) granules. The fifth group was left untreated for spontaneous repair. After three months, the repair tissue was evaluated with X‐ray microtomography and histology. Relative values comparing the operated knee with its contralateral control were calculated. The relative bone volume fraction (?BV/TV) was largest in the β‐TCP group (p ≤ 0.012), which also showed the most abundant osteoid. BG resulted in improved bone formation, whereas defects in the PLGA–BGf group were filled with fibrous tissue. Repair with PLGA did not differ from spontaneous repair. The PLGA, PLGA–BGf, and spontaneous groups showed thicker and sparser trabeculae than the commercial controls. We conclude that bone repair with β‐TCP and BG granules was satisfactory, whereas the investigational PLGA‐based materials were only as good as or worse than spontaneous repair.     

Redox control of β2‐glycoprotein I–von Willebrand factor interaction by thioredoxin‐1

Summary. Background: β₂‐Glycoprotein I (β₂GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β₂GPI in thrombus formation is unknown. We have recently shown that β₂GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β₂GPI can take place on the platelet surface. Methods: β₂GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe N^a‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β₂GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β₂GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β₂GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β₂GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β₂GPI reduced by thioredoxin‐1, in comparison with non‐reduced β₂GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β₂GPI with VWF may contribute to the redox regulation of platelet adhesion.     

Matrix formation is enhanced in co‐cultures of human meniscus cells with bone marrow stromal cells

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co‐cultured in vitro in three‐dimensional (3D) pellet culture at three different cell–cell ratios for 3 weeks under normal (21% O₂) or low (3% O₂) oxygen tension in the presence of serum‐free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT–PCR. Co‐cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3–1.7‐fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O₂. GAG matrix formation was also enhanced (1.3–1.6‐fold) under 3% O₂ culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG‐rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co‐cultured pellets. However, SOX9 and HIF‐1α mRNA expression were not significantly modulated by co‐culture. Co‐culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co‐delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects. Copyright © 2012 John Wiley & Sons, Ltd.     

The effectiveness of silver‐releasing dressings in the management of non‐healing chronic wounds: a meta‐analysis

Aim. The purpose of this study was to examine the efficacy of silver‐releasing dressings in the management of non‐healing chronic wounds. Background. Non‐healing chronic wounds often have a negative physical impact on patients and place a financial burden on healthcare systems. Silver dressings are wound products designed to control infection and provide a wound environment conducive to healing. However, validation of the clinical efficacy of these dressings is lacking. Design. Systematic review and meta‐analysis. Methods. A systematic search of the major electronic databases PubMed, CINAHL, Cochrane, MEDLINE, British Nursing Index, EBSCO, OCLC and Proquest between 1950–June 2007 was conducted. Hand searches of selected periodicals, textbooks and checking reference lists and contacting experts was also performed. Results. Eight studies were selected from a potentially relevant 1957 references screened. Analysis incorporated data from 1399 participants in the eight randomised control trials. We found that silver dressings significantly improved wound healing (CI₉₅: 0·16–0·39, p < 0·001), reduced odour (CI₉₅: 0·24–0·52, p < 0·001) and pain‐related symptoms (CI₉₅: 0·18–0·47, p < 0·001), decreased wound exudates (CI₉₅: 0·17–0·44, p < 0·001) and had a prolonged dressing wear time (CI₉₅: 0·19–0·48, p = 0·028) when compared with alternative wound management approaches. An analysis of sensitivity in these studies by subgroup analysis generally supported these associations. Furthermore, studies indicated an improvement in quality of life (CI₉₅: 0·04–0·33, p = 0·013) using silver dressings in wound management with no associated severe adverse events. Conclusion. This meta‐analysis confirms the effectiveness of silver dressings in wound healing and improving patients’ quality of life. However, it also highlights the need for additional well‐designed randomised controlled trials to evaluate the effectiveness of silver‐related dressings further. Relevance to clinical practice. The results of this study provide objective data on the effectiveness of silver‐related dressing when applied to non‐healing chronic wounds.     

Interactive endothelial phenotype maintenance and osteogenic differentiation of adipose tissue stromal vascular fraction SSEA‐4+‐derived cells

Bone formation relies on complex processes that require well‐orchestrated interactions between several cell types, such as bone‐forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co‐culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co‐culture of microvascular‐like ECs and pre‐OBs, both derived from the SSEA‐4⁺ cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA‐4⁺ hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre‐OBs and maintenance of the EC phenotype. Co‐cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre‐OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31⁺ cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre‐OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co‐culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA‐4⁺ hASCs as a single‐cell source is promising to attain 3D bone‐like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2‐antiplasmin

Summary. Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of α₂‐antiplasmin (α₂AP) incorporated within fibrin. Circulating prolyl‐specific serine proteinase, antiplasmin‐cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met‐α₂AP between ‐Pro12‐Asn13‐ to yield Asn‐α₂AP, which is crosslinked to fibrin approximately 13× more rapidly than Met‐α₂AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met‐α₂AP to Asn‐α₂AP and thereby enhance endogenous fibrinolysis. Methods and results: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl‐Arg‐(8‐amino‐3,6‐dioxaoctanoic acid)‐d ‐Ala‐l ‐boroPro selectively inhibited APCE vs. DPPIV, with an apparent K_i of 5.7 nm vs. 6.1 μm , indicating that an approximately 1000‐fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K_i of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose‐dependent decrease of APCE‐mediated Met‐α₂AP cleavage, which ultimately shortened plasminogen activator‐induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC₅₀ value in plasma remaining comparable to that in phosphate buffer. Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.     

Endothelial invasive response in a co‐culture model with physically‐induced osteodifferentiation

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co‐culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity‐induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co‐culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co‐culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen‐like morphology, suggesting that cell‐cell interaction in the co‐culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co‐culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre‐vascularized bone grafts. Copyright © 2012 John Wiley & Sons, Ltd.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

Evaluation of in vitro spermatogenesis using poly(D,L‐lactic‐co‐glycolic acid) (PLGA)‐based macroporous biodegradable scaffolds

Successful in vitro differentiation of spermatogenic cells into spermatids appears to offer extremely attractive potential for the treatment of impaired spermatogenesis and male infertility. Experimental evidence indicates that biocompatible polymers may improve in vitro reconstitution and regeneration of tissues of various origins. Here, we fabricated highly porous biodegradable poly(D,L ‐lactic‐co‐glycolic acid) or PLGA co‐polymer scaffolds by combining the gas‐foaming and salt‐leaching methods, using ammonium bicarbonate as a porogen, which allowed us to generate polymer scaffolds with a high density of interconnected pores of 400–500 µm in average diameter, concomitant with a high malleability to mould a wide range of temporal tissue scaffolds requiring a specific shape and geometry. The PLGA scaffolds were biocompatible and biodegradable, as evidenced by the fact that they survived almost 3 month long subcutaneous xenografting into immunodeficient host mice and became easily destroyable after recovery. Immature rat testicular cells that were seeded onto the surface of the scaffold exhibited about 65% seeding efficiency and up to 75% viability after 18 days in culture. Furthermore, our scaffolds enhanced the proliferation and differentiation of spermatogenic germ cells to a greater extent than conventional in vitro culture methods, such as monolayer or organ culture. Taken together, an implication of the present findings is that the PLGA‐based macroporous scaffold may provide a novel means by which spermatocytes could be induced to differentiate into presumptive spermatids. Copyright © 2010 John Wiley & Sons, Ltd.