Density gradients at hydrogel interfaces for enhanced cell penetration.

We report that stiffness gradients facilitate infiltration of cells through otherwise cell-impermeable hydrogel interfaces. By enabling the separation of hydrogel manufacturing and cell seeding, and by improving cell colonization of additively manufactured hydrogel elements, interfacial density gradients present a promising strategy to progress in the creation of 3D tissue models.

Engineered cell instructive matrices for fetal membrane healing.

Iatrogenic preterm prelabour rupture of fetal membranes (iPPROM) occurs in 6-45% of the cases after fetoscopic procedures, posing a significant threat to fetal survival and well-being. The number of diagnostic and therapeutic prenatal interventions available is increasing, thus developing treatment options for iPPROM is becoming more important than ever before. Fetal membranes exhibit very restricted regeneration and little is known about factors which might modulate their healing potential, rendering various materials and strategies to seal or heal fetal membranes pursued over the past decades relatively fruitless. Additionally, biocompatible materials with tunable in vivo stability and mechanical and biological properties have not been available. Using poly(ethylene glycol)-based biomimetic matrices, we provide evidence that, upon presentation of appropriate biological cues in three dimensions, mesenchymal progenitor cells from the amnion can be mobilized, induced to proliferate and supported in maintaining their native extracellular matrix production, thus creating a suitable environment for healing to take place. These data suggest that engineering materials with defined mechanical and biochemical properties and the ability to present migration- and proliferation-inducing factors, such as platelet-derived growth factor, basic fibroblast growth factor or epidermal growth factor, could be key in resolving the clinical problem of iPPROM and allowing the field of fetal surgery to move forward.

Locally controlling mesenchymal stem cell morphogenesis by 3D PDGF-BB gradients towards the establishment of an in vitro perivascular niche.

The perivascular niche is a complex microenvironment containing mesenchymal stem cells (MSCs), among other perivascular cells, as well as temporally organized biochemical and biophysical gradients. Due to a lack of conclusive phenotypic markers, MSCs' identity, heterogeneity and function within their native niche remain poorly understood. The in vitro reconstruction of an artificial three-dimensional (3D) perivascular niche would offer a powerful alternative to study MSC behavior under more defined conditions. To this end, we here present a poly(ethylene glycol)-based in vitro model that begins to mimic the spatiotemporally controlled presentation of biological cues within the in vivo perivascular niche, namely a stably localized platelet-derived growth factor B (PDGF-BB) gradient. We show that 3D-encapsulated MSCs respond to soluble PDGF-BB by proliferation, spreading, and migration in a dose-dependent manner. In contrast, the exposure of MSCs to 3D matrix-tethered PDGF-BB gradients resulted in locally restricted morphogenetic responses, much as would be expected in a native perivascular niche. Thus, the herein presented artificial perivascular niche model provides an important first step towards modeling the role of MSCs during tissue homeostasis and regeneration.

Mussel mimetic tissue adhesive for fetal membrane repair: initial in vivo investigation in rabbits.

OBJECTIVE: Iatrogenic preterm prelabour rupture of fetal membranes (iPPROM) remains the main complication after invasive interventions into the intrauterine cavity. The aim of this study was to evaluate the sealing capability and tissue interaction of mussel-mimetic tissue adhesive (mussel glue) in comparison to fibrin glue on punctured fetal membranes in vivo. STUDY DESIGN: A mid-gestational rabbit model was used for testing the materials. The fetal sacs of pregnant rabbits at day 23 were randomly assigned into experimental groups: unoperated (negative control), unclosed puncture (positive control), commercially available fibrin glue (FG) with decellularized amnion scaffold (DAM), mussel glue (MG) with DAM, or mussel glue alone. Evaluation was done at term (30 days' gestation) assessing fetal survival, fetal membrane integrity and histology of the membranes. RESULTS: Fetal survival was not significantly lower in any of the treatment groups compared to the negative control. All plugging materials could be found at the end of the pregnancy and no adverse effects on the fetus or the pregnant does could be observed. Sac integrity was higher in all treatment groups compared to the positive control group but significant only in the FG+DAM group. Cellular infiltration could be seen in fibrin glue and DAM in contrast to mussel glue which was only tightly adhering to the surrounding tissue. These cells were mostly of mesenchymal phenotype staining positive for vimentin. CD68 positive macrophages were found clustered around all the plugging materials, but their numbers were only significantly increased for the mussel glue alone group compared to negative controls. CONCLUSIONS: Mussel glues performance in sealing fetal membranes in the rabbit model was comparable to that of fibrin glue. Taking into account its other favorable properties, it is a noteworthy candidate for a clinically applicable fetal membrane sealant.

Second harmonic generation microscopy of fetal membranes under deformation: normal and altered morphology.

INTRODUCTION: Insight into the microstructure of fetal membrane and its response to deformation is important for understanding causes of preterm premature rupture of the membrane. However, the microstructure of fetal membranes under deformation has not been visualized yet. Second harmonic generation microscopy, combined with an in-situ stretching device, can provide this valuable information. METHODS: Eight fetal membranes were marked over the cervix with methylene blue during elective caesarean section. One sample per membrane of reflected tissue, between the placenta and the cervical region, was cyclically stretched with a custom built inflation device. Samples were mounted on an in-situ stretching device and imaged with a multiphoton microscope at different deformation levels. Microstructural parameters such as thickness and collagen orientation were determined. Image entropy was evaluated for the spongy layer. RESULTS: The spongy layer consistently shows an altered collagen structure in the cervical and cycled tissue compared with the reflected membrane, corresponding to a significantly higher image entropy. An increased thickness of collagenous layers was found in cervical and stretched samples in comparison to the reflected tissue. Significant collagen fibre alignment was found to occur already at moderate deformation in all samples. CONCLUSIONS: For the first time, second harmonic generation microscopy has been used to visualize the microstructure of fetal membranes. Repeated mechanical loading was shown to affect the integrity of the amnion-chorion interface which might indicate an increased risk of premature rupture of fetal membrane. Moreover, mechanical loading might contribute to morphological alterations of the fetal membrane over the cervical region.

Multiaxial mechanical behavior of human fetal membranes and its relationship to microstructure.

This study was directed to the measurement of the mechanical response of fetal membranes to physiologically relevant loading conditions. Characteristic mechanical parameters were determined and their relation to the microstructural constituents collagen and elastin as well as to the pyridinium cross-link concentrations analyzed. 51 samples from twelve fetal membranes were tested on a custom-built inflation device, which allows mechanical characterization within a multiaxial state of stress. Methods of nonlinear continuum mechanics were used to extract representative mechanical parameters. Established biochemical assays were applied for the determination of the collagen and elastin content. Collagen cross-link concentrations were determined by high-performance liquid chromatography measurements. The results indicate a distinct correlation between the mechanical parameters of high stretch stiffness and membrane tension at rupture and the biochemical data of collagen content and pyridinoline as well as deoxypyridinoline concentrations. No correlation was observed between the mechanical parameters and the elastin content. Moreover, the low stretch stiffness is, with a value of 105 ± 31 × 10(-3) N/ mm much higher for a biaxial state of stress compared to a uniaxial stress configuration. Determination of constitutive model equations leads to better predictive capabilities for a reduced polynomial hyperelastic model with only terms related to the second invariant, I 2, of the right Cauchy-Green deformation tensor. Relevant insights were obtained on the mechanical behavior of fetal membranes. Collagen and its cross-linking were shown to determine membrane's stiffness and strength for multiaxial stress states. Their nonlinear deformation behavior characterizes the fetal membranes as I 2 material.

Mussel-mimetic tissue adhesive for fetal membrane repair: an ex vivo evaluation.

Iatrogenic preterm prelabor rupture of membranes (iPPROM) remains the main complication after invasive interventions into the intrauterine cavity. Here, the proteolytic stability of mussel-mimetic tissue adhesive (mussel glue) and its sealing behavior on punctured fetal membranes are evaluated. The proteolytic degradation of mussel glue and fibrin glue were compared in vitro. Critical pressures of punctured and sealed fetal membranes were determined under close to physiological conditions using a custom-made inflation device. An inverse finite element procedure was applied to estimate mechanical parameters of mussel glue. Mussel glue was insensitive whereas fibrin glue was sensitive towards proteolytic degradation. Mussel glue sealed 3.7mm fetal membrane defect up to 60mbar (45mmHg) when applied under wet conditions, whereas fibrin glue needed dry membrane surfaces for reliable sealing. The mussel glue can be represented by a neo-Hookean material model with elastic coefficient C(1)=9.63kPa. Ex-vivo-tested mussel glue sealed fetal membranes and resisted pressures achieved during uterine contractions. Together with good stability in proteolytic environments, this makes mussel glue a promising sealing material for future applications.

Mussel-mimetic tissue adhesive for fetal membrane repair: a standardized ex vivo evaluation using elastomeric membranes.

OBJECTIVE: Iatrogenic preterm premature rupture of membranes (iPPROM), the main complication of invasive interventions in the prenatal period, seriously limits the benefit of diagnostic or surgical prenatal procedures. This study aimed to evaluate preventive plugging of punctured fetal membranes in an ex vivo situation using a new mussel-mimetic tissue adhesive (mussel glue) to inhibit leakage. METHODS: A novel biomechanical test device that tests the closure of injured membranes under near-physiological conditions was used. Mussel glue, a poly(ethylene glycol)-based hydrogel, was used to seal membrane defects of up to 3 mm in mechanically well-defined elastomeric membranes with three different degrees of stiffness. RESULTS: Elastomeric test membranes were successfully employed for testing mussel glue under well-defined conditions. Mussel glue plugs were distended by up to 94%, which translated to an improved sealing efficiency on elastomeric membranes with high stiffness. For the stiffest membrane tested, a critical burst pressure of 48 mbar (36 mmHg) was accomplished in this ex vivo setting. CONCLUSIONS: Mussel glue appears to efficiently seal membrane defects under well-standardized ex vivo conditions. As repaired membranes resist pressures measured in amniotic cavities, mussel glue might represent a novel sealing method for iatrogenic membrane defects.

Elucidating the role of matrix stiffness in 3D cell migration and remodeling.

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

Improved efficacy of chemotherapy by parvovirus-mediated sensitisation of human tumour cells.

Increasing resistance of tumour cells towards the cytotoxic action of chemotherapeutic drugs is a major limitation in the treatment of cancer patients. The non-pathogenic human adeno-associated viruses (AAV) have been reported to sensitise HeLa cervical cancer cells to gamma irradiation in vivo and in vitro. To test whether these parvoviruses might render other human tumour cells more sensitive towards chemotherapeutic drugs, we analysed the effects of AAV type 2 (AAV-2) infection on established cancer cell lines and freshly explanted tumour biopsies treated with chemotherapeutic agents (e.g. cisplatin). AAV-2 infection significantly increased the cytotoxic activity of chemotherapeutic drugs compared with uninfected controls. AAV-2 infection without concomitant chemotherapeutic treatment had no significant effect on viability of the cells. In nude mice, combined application of AAV-2 infection and chemotherapeutic treatment significantly increased the therapeutic activity on tumours arising from subcutaneously injected tumour cells compared with tumours treated by chemotherapeutics only. These results indicate that AAV-2 infection sensitises human cancer cells towards the cytotoxic action of chemotherapeutic drugs.

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus.

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens ("initiators") as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.