The Importance of Cell-Cell Interaction Dynamics in Bottom-Up Tissue Engineering: Concepts of Colloidal Self-Assembly in the Fabrication of Multicellular Architectures.

Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell-cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell-cell interactions by pulsed light activation results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic, compact and round multicellular clusters under thermodynamic control form, while otherwise branched and loose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction- and diffusion-limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell-cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.

Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology.

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing-thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing.

Histological processing of un-/cellularized thermosensitive electrospun scaffolds.

Histological processing of thermosensitive electrospun poly(ε-caprolactone)/poly(L-lactide) (PCL/PLA) scaffolds fails, as poly(ε-caprolactone) (PCL) is characterized by its low-melting temperature (Tm = 60 °C). Here, we present an optimized low-temperature preparation method for the histological processing of un-/cellularized thermosensitive PCL/PLA scaffolds.Our study is aimed at the establishment of an optimized dehydration and low-melting-point paraffin-embedding method of electrospun PCL/PLA scaffolds (un-/cellularized). Furthermore, we compared this method with (a) automatized dehydration and standard paraffin embedding, (b) gelatin embedding followed by automatized dehydration and standard paraffin embedding, (c) cryofixation, and (d) acrylic resin embedding methods. We investigated pepsin and proteinase K antigen retrieval for their efficiency in epitope demasking at low temperatures and evaluated protocols for immunohistochemistry and immunofluorescence for cytokeratin 7 (CK7) and in situ padlock probe technology for beta actin (ACTB). Optimized dehydration and low-melting-point paraffin embedding preserved the PCL/PLA scaffold, as the diameter and structure of its fibers were unchanged. Cells attached to the PCL/PLA scaffolds showed limited alterations in size and morphology compared to control. Epitope demasking by enzymatic pepsin digestion and immunostaining of CK7 displayed an invasion of attached cells into the scaffold. Expression of ACTB and CK7 was shown by a combination of mRNA-based in situ padlock probe technology and immunofluorescence. In contrast, gelatin stabilization followed by standard paraffin embedding led to an overall shrinkage and melting of fibers, and therefore, no further analysis was possible. Acrylic resin embedding and cyrofixation caused fiber structures that were nearly unchanged in size and diameter. However, acrylic resin-embedded scaffolds are limited to 3 µm sections, whereas cyrofixation led to a reduction of the cell size by 14% compared to low-melting paraffin embedding. The combination of low-melting-point paraffin embedding and pepsin digestion as an antigen retrieval method offers a successful opportunity for histological investigations in thermosensitive specimens.

CD8- dendritic cells and macrophages cross-present poly(D,L-lactate-co-glycolate) acid microsphere-encapsulated antigen in vivo.

The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly(D,L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8(+) and CD8(-) DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after s.c. injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8(-) DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8(+) DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8(-) DCs and MΦs.

Neuromuscular adaptations after osseointegration of a bone-anchored prosthesis in a unilateral transfemoral amputee - a case study.

PURPOSE: Many individuals with a lower limb amputation experience problems with the fitting of the socket of their prosthesis, leading to dissatisfaction or device rejection. Osseointegration (OI)- the implantation of a shaft directly interfacing with the remaining bone- is an alternative for these patients. In this observational study, we investigated how bone anchoring influences neuromuscular parameters during balance control in a patient with a unilateral transfemoral amputation. MATERIAL AND METHODS: Center of pressure (CoP) and electromyography (EMG) signals from muscles controlling the hip and the ankle of the intact leg were recorded during quiet standing six months before and one and a half years after this patient underwent an OI surgery. Results were compared to a control group of nine able-bodied individuals. RESULTS: Muscle co-activation and EMG intensity decreased after bone anchoring, approaching the levels of able-bodied individuals. Muscle co-activation controlling the ankle decreased in the high-frequency range, and the EMG intensity spectrum decreased in the lower-frequency range for all muscles when vision was allowed. With eyes closed, the ankle extensor muscle showed an increased EMG intensity in the high-frequency range post-surgery. CoP length increased in the mediolateral direction of the amputated leg. CONCLUSIONS: These findings point to shifts in the patient's neuromuscular profile towards the one of able-bodied individuals.

Development of a dual-component infection-resistant arterial replacement for small-caliber reconstructions: A proof-of-concept study.

Introduction: Synthetic vascular grafts perform poorly in small-caliber (<6mm) anastomoses, due to intimal hyperplasia and thrombosis, whereas homografts are associated with limited availability and immunogenicity, and bioprostheses are prone to aneurysmal degeneration and calcification. Infection is another important limitation with vascular grafting. This study developed a dual-component graft for small-caliber reconstructions, comprising a decellularized tibial artery scaffold and an antibiotic-releasing, electrospun polycaprolactone (PCL)/polyethylene glycol (PEG) blend sleeve. Methods: The study investigated the effect of nucleases, as part of the decellularization technique, and two sterilization methods (peracetic acid and γ-irradiation), on the scaffold's biological and biomechanical integrity. It also investigated the effect of different PCL/PEG ratios on the antimicrobial, biological and biomechanical properties of the sleeves. Tibial arteries were decellularized using Triton X-100 and sodium-dodecyl-sulfate. Results: The scaffolds retained the general native histoarchitecture and biomechanics but were depleted of glycosaminoglycans. Sterilization with peracetic acid depleted collagen IV and produced ultrastructural changes in the collagen and elastic fibers. The two PCL/PEG ratios used (150:50 and 100:50) demonstrated differences in the structural, biomechanical and antimicrobial properties of the sleeves. Differences in the antimicrobial activity were also found between sleeves fabricated with antibiotics supplemented in the electrospinning solution, and sleeves soaked in antibiotics. Discussion: The study demonstrated the feasibility of fabricating a dual-component small-caliber graft, comprising a scaffold with sufficient biological and biomechanical functionality, and an electrospun PCL/PEG sleeve with tailored biomechanics and antibiotic release.

A Cavopulmonary Assist Device for Long-Term Therapy of Fontan Patients.

Treatment of univentricular hearts remains restricted to palliative surgical corrections (Fontan pathway). The established Fontan circulation lacks a subpulmonary pressure source and is commonly accompanied by progressively declining hemodynamics. A novel cavopulmonary assist device (CPAD) may hold the potential for improved therapeutic management of Fontan patients by chronic restoration of biventricular equivalency. This study aimed at translating clinical objectives toward a functional CPAD with preclinical proof regarding hydraulic performance, hemocompatibility and electric power consumption. A prototype composed of hemocompatible titanium components, ceramic bearings, electric motors, and corresponding drive unit was manufactured for preclinical benchtop analysis: hydraulic performance in general and hemocompatibility characteristics in particular were analyzed in-silico (computational fluid dynamics) and validated in-vitro. The CPAD's power consumption was recorded across the entire operational range. The CPAD delivered pressure step-ups across a comprehensive operational range (0-10 L/min, 0-50 mm Hg) with electric power consumption below 1.5 W within the main operating range. In-vitro hemolysis experiments (N = 3) indicated a normalized index of hemolysis of 3.8 ± 1.6 mg/100 L during design point operation (2500 rpm, 4 L/min). Preclinical investigations revealed the CPAD's potential for low traumatic and thrombogenic support of a heterogeneous Fontan population (pediatric and adult) with potentially accompanying secondary disorders (e.g., elevated pulmonary vascular resistance or systemic ventricular insufficiency) at distinct physical activities. The low power consumption implied adequate settings for a small, fully implantable system with transcutaneous energy transfer. The successful preclinical proof provides the rationale for acute and chronic in-vivo trials aiming at the confirmation of laboratory findings and verification of hemodynamic benefit.

Tumor eradication by immunotherapy with biodegradable PLGA microspheres--an alternative to incomplete Freund's adjuvant.

In experimental tumor immunotherapy, incomplete Freund's adjuvant (IFA) has been considered as the "gold standard" for T-cell vaccination in mice and humans in spite of its considerable adverse effects. Recently, we succeeded in eliciting strong CTL responses in mice after vaccination with biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS). In our study, we compared the immune response to IFA and PLGA-MS containing ovalbumin (OVA) and CpG-oligodeoxynucleotide (MS-OVA/CpG) or we used a mixture of MS-OVA/CpG and MS-polyI:C. A single vaccination with MS-OVA/CpG elicited long-lasting titers of IgG1 and IgG2a, but only low IgE titers, and also the T-cell response was biased toward Th(1) differentiation. Antigen presentation to CD4(+) and CD8(+) cells and activation of a cytotoxic T-cell response in mice vaccinated with PLGA-MS and IFA lasted for over 3 weeks. Preconditioning of the injection site with TNF-α and heterologous prime-boost regimen further enhanced the cytotoxic response. PLGA-MS were as efficient or superior to IFA in eradication of preexisting tumors and suppression of lung metastases. Taken together, PLGA-MS are well-defined, biodegradable and clinically compatible antigen carrier systems that compare favorably with IFA in their efficacy of tumor immunotherapy in mouse models and hence deserve to be tested for their effectiveness against human malignant diseases.

Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell-Cell Interactions.

The regulation of cell-cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell-cell interactions with high precision are of great interest to a better understanding of their roles and building tissue-like structures. Herein, the green light-responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell-cell interactions form and lead to cell clustering in a concentration-dependent manner. Upon green light illumination, the CarH based cell-cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell-cell interactions impact cell migration, as observed in a wound-healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell-cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell-cell interactions in biological processes.

Influence of Augmented Visual Feedback on Balance Control in Unilateral Transfemoral Amputees.

Patients with a lower limb amputation rely more on visual feedback to maintain balance than able-bodied individuals. Altering this sensory modality in amputees thus results in a disrupted postural control. However, little is known about how lower limb amputees cope with augmented visual information during balance tasks. In this study, we investigated how unilateral transfemoral amputees incorporate visual feedback of their center of pressure (CoP) position during quiet standing. Ten transfemoral amputees and ten age-matched able-bodied participants were provided with real-time visual feedback of the position of their CoP while standing on a pressure platform. Their task was to keep their CoP within a small circle in the center of a computer screen placed at eye level, which could be achieved by minimizing their postural sway. The visual feedback was then delayed by 250 and 500 ms and was combined with a two- and five-fold amplification of the CoP displacements. Trials with eyes open without augmented visual feedback as well as with eyes closed were further performed. The overall performance was measured by computing the sway area. We further quantified the dynamics of the CoP adjustments using the entropic half-life (EnHL) to study possible physiological mechanisms behind postural control. Amputees showed an increased sway area compared to the control group. The EnHL values of the amputated leg were significantly higher than those of the intact leg and the dominant and non-dominant leg of controls. This indicates lower dynamics in the CoP adjustments of the amputated leg, which was compensated by increasing the dynamics of the CoP adjustments of the intact leg. Receiving real-time visual feedback of the CoP position did not significantly reduce the sway area neither in amputees nor in controls when comparing with the eyes open condition without visual feedback of the CoP position. Further, with increasing delay and amplification, both groups were able to compensate for small visual perturbations, yet their dynamics were significantly lower when additional information was not received in a physiologically relevant time frame. These findings may be used for future design of neurorehabilitation programs to restore sensory feedback in lower limb amputees.

Orthogonal Blue and Red Light Controlled Cell-Cell Adhesions Enable Sorting-out in Multicellular Structures.

The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell-cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell-cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.

Electrospun PCL/PLA Scaffolds Are More Suitable Carriers of Placental Mesenchymal Stromal Cells Than Collagen/Elastin Scaffolds and Prevent Wound Contraction in a Mouse Model of Wound Healing.

Mesenchymal stem/stromal cells (MSCs) exert beneficial effects during wound healing, and cell-seeded scaffolds are a promising method of application. Here, we compared the suitability of a clinically used collagen/elastin scaffold (Matriderm) with an electrospun Poly(ε-caprolactone)/poly(l-lactide) (PCL/PLA) scaffold as carriers for human amnion-derived MSCs (hAMSCs). We created an epidermal-like PCL/PLA scaffold and evaluated its microstructural, mechanical, and functional properties. Sequential spinning of different PCL/PLA concentrations resulted in a wide-meshed layer designed for cell-seeding and a dense-meshed layer for apical protection. The Matriderm and PCL/PLA scaffolds then were seeded with hAMSCs, with or without Matrigel coating. The quantity and quality of the adherent cells were evaluated in vitro. The results showed that hAMSCs adhered to and infiltrated both scaffold types but on day 3, more cells were observed on PCL/PLA than on Matriderm. Apoptosis and proliferation rates were similar for all carriers except the coated Matriderm, where apoptotic cells were significantly enhanced. On day 8, the number of cells decreased on all carrier types except the coated Matriderm, which had consistently low cell numbers. Uncoated Matriderm had the highest percentage of proliferative cells and lowest apoptosis rate of all carrier types. Each carrier also was topically applied to skin wound sites in a mouse model and analyzed in vivo over 14 days via optical imaging and histological methods, which showed detectable hAMSCs on all carrier types on day 8. On day 14, all wounds exhibited newly formed epidermis, and all carriers were well-integrated into the underlying dermis and showing signs of degradation. However, only wounds treated with uncoated PCL/PLA maintained a round appearance with minimal contraction. Overall, the results support a 3-day in vitro culture of scaffolds with hAMSCs before wound application. The PCL/PLA scaffold showed higher cell adherence than Matriderm, and the effect of the Matrigel coating was negligible, as all carrier types maintained sufficient numbers of transplanted cells in the wound area. The anti-contractive effects of the PCL/PLA scaffold offer potential new therapeutic approaches to wound care.

Blue Light Switchable Cell-Cell Interactions Provide Reversible and Spatiotemporal Control Towards Bottom-Up Tissue Engineering.

Controlling cell-cell interactions is central for understanding key cellular processes and bottom-up tissue assembly from single cells. The challenge is to control cell-cell interactions dynamically and reversibly with high spatiotemporal precision noninvasively and sustainably. In this study, cell-cell interactions are controlled with visible light using an optogenetic approach by expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of cells. CRY2 and CIBN expressing cells form specific heterophilic interactions under blue light providing precise control in space and time. Further, these interactions are reversible in the dark and can be repeatedly and dynamically switched on and off. Unlike previous approaches, these genetically encoded proteins allow for long-term expression of the interaction domains and respond to nontoxic low intensity blue light. In addition, these interactions are suitable to assemble cells into 3D multicellular architectures. Overall, this approach captures the dynamic and reversible nature of cell-cell interactions and controls them noninvasively and sustainably both in space and time. This provides a new way of studying cell-cell interactions and assembling cellular building blocks into tissues with unmatched flexibility.

Leptin inhibits cell growth of human vascular smooth muscle cells.

Elevated leptin levels are thought to contribute to the individual cardiovascular risk, however, the role of leptin in the pathogenesis of atherosclerosis remains unclear. The aim of our study was to elucidate the effects of leptin on growth of human vascular smooth muscle cells (VSMC) and leptin receptor expression. By establishing a new quantitative real-time PCR for leptin receptor (ObR) isoforms we showed that the short isoforms of ObR were expressed in a 10- to 27-fold excess compared to the long isoform in cultured human VSMCs. Incubation of VSMCs with 100 ng/ml leptin downregulated the short isoforms significantly, whereas the long isoform was not influenced. Increasing leptin concentrations of 50 and 100 ng/ml significantly reduced the cell number of VSMCs compared to untreated controls. Our findings suggest a role for leptin in vascular smooth muscle cell growth, associated to a downregulation of leptin receptor isoforms.

Detection of disseminated pancreatic cells by amplification of cytokeratin-19 with quantitative RT-PCR in blood, bone marrow and peritoneal lavage of pancreatic carcinoma patients.

AIM: To evaluate the diagnostic potential of cytokeratin-19 (CK-19) mRNA for the detection of disseminated tumor cells in blood, bone marrow and peritoneal lavage in patients with ductal adenocarcinoma of the pancreas. METHODS: Sixty-eight patients with pancreatic cancer (n = 37), chronic pancreatitis (n = 16), and non-pancreatic benign surgical diseases (n = 15, control group) were included in the study. Venous blood was taken preoperatively, intraoperatively and at postoperative d 1 and 10. Preoperative bone marrow aspirates and peritoneal lavage taken before mobilization of the tumor were analyzed. All samples were evaluated for disseminated tumor cells by CK-19-specific nested-PCR and quantitative fluorogenic RT-PCR. RESULTS: CK-19 mRNA expression was increased in 24 (64%) blood samples and 11 (30%) of the peritoneal lavage samples in the patients with pancreatic cancer. In 15 (40%) of the patients with pancreatic cancer, disseminated tumor cells were detected in venous blood and bone marrow and/or peritoneal lavage. In the peritoneal lavage, the detection rates were correlated with the tumor size and the tumor differentiation. CK-19 levels were increased in pT3/T4 and moderately/poorly differentiated tumors (G2/G3). Pancreatic cancer patients with at least one CK-19 mRNA-positive sample showed a trend towards shorter survival. Pancreatic cancer patients showed significantly increased detection rates of disseminated tumor cells in blood and peritoneal lavage compared to the controls and the patients with chronic pancreatitis. CONCLUSION: Disseminated tumor cells can be detected in patients with pancreatic ductal adenocarcinoma by CK-19 fluorogenic RT-PCR. In peritoneal lavage, detection rate is correlated with tumor stage and differentiation. In the clinical use, CK-19 is suitable for the distinction between malignant and benign pancreatic disease in combination with other tumor-specific markers.

Fixation devices for whole-body 18F-FDG PET/CT: patient perspectives and technical aspects.

OBJECTIVE: To evaluate the use of a fixation device in whole-body postiron emission tomography/computed tomography (PET/CT). METHODS: Two hundred and thirty patients were prospectively included over a period of 3 months. Different single-phase and multiphase contrast-enhanced PET/CT protocols were used for whole-body examination. An unforced expiration state was applied as breathing protocol for CT examination. Patients were placed on a deflating device (1.0 m x 1.5 m) with arms elevated but supported in order to prevent full extension in shoulders and elbows providing comfortable positioning. Image quality was assessed by means of alignment of the liver quantitatively on co-registered PET/CT images. After the examination, patients were asked to complete a survey on subjective sensations such as pain in different body regions (yes/no). They were asked to give a final evaluation for the whole-body PET/CT examination (comfortable/not comfortable). Additionally, a control group (n=30) was assessed without the aid of additional devices. RESULTS: Examination protocols using the device showed minor misalignment of 5 mm. Different protocols did not reveal significant differences in misalignment. When comparing the control group misalignment was significantly higher with approx. 7 mm. The majority (75%) evaluated the positioning as comfortable despite 46% of the patients in this group feeling more or less severe pain in at least one body region. For controls, misalignment was slightly higher whereas only 39% found the positioning comfortable (chi(2)=13.03; P<0.0005) and 61% reported pain (NS). CONCLUSION: Both the technical aspects and patient evaluations favour the use of the vacuum device in whole-body PET/CT examinations. In particular, in time consuming protocols using multiphase CT examination the fixation device leads to excellent co-registration quality and patient compliance.

The Osteogenic and Tenogenic Differentiation Potential of C3H10T1/2 (Mesenchymal Stem Cell Model) Cultured on PCL/PLA Electrospun Scaffolds in the Absence of Specific Differentiation Medium.

The differentiation potential of mesenchymal stem cells (MSC) has been extensively tested on electrospun scaffolds. However, this potential is often assessed with lineage-specific medium, making it difficult to interpret the real contribution of the properties of the scaffold in the cell response. In this study, we analyzed the ability of different polycaprolactone/polylactic acid PCL/PLA electrospun scaffolds (pure or blended compositions, random or aligned fibers, various fiber diameters) to drive MSC towards bone or tendon lineages in the absence of specific differentiation medium. C3H10T1/2 cells (a mesenchymal stem cell model) were cultured on scaffolds for 96 h without differentiation factors. We performed a cross-analysis of the cell-scaffold interactions (spreading, organization, and specific gene expression) with mechanical (elasticity), morphological (porosity, fibers diameter and orientation) and surface (wettability) characterizations of the electrospun fibers. We concluded that (1) osteogenic differentiation can be initiated on pure PCL-based electrospun scaffolds without specific culture conditions; (2) fiber alignment modified cell organization in the short term and (3) PLA added to PCL with an increased fiber diameter encouraged the stem cells towards the tendon lineage without additional tenogenic factors. In summary, the differentiation potential of stem cells on adapted electrospun fibers could be achieved in factor-free medium, making possible future applications in clinically relevant situations.

Rapid phenotypic stress-based microfluidic antibiotic susceptibility testing of Gram-negative clinical isolates.

Bacteremia is a life-threatening condition for which antibiotics must be prescribed within hours of clinical diagnosis. Since the current gold standard for bacteremia diagnosis is based on conventional methods developed in the mid-1800s-growth on agar or in broth-identification and susceptibility profiling for both Gram-positive and Gram-negative bacterial species requires at least 48-72 h. Recent advancements in accelerated phenotypic antibiotic susceptibility testing have centered on the microscopic growth analysis of small bacterial populations. These approaches are still inherently limited by the bacterial growth rate. Our approach is fundamentally different. By applying environmental stress to bacteria in a microfluidic platform, we can correctly assign antibiotic susceptibility profiles of clinically relevant Gram-negative bacteria within two hours of antibiotic introduction rather than 8-24 h. The substantial expansion to include a number of clinical isolates of important Gram-negative species-Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa-reported here underscores the broad utility of our approach, complementing the method's proven utility for Gram-positive bacteria. We also demonstrate that the platform is compatible with antibiotics that have varying mechanisms of action-meropenem, gentamicin, and ceftazidime-highlighting the versatility of this platform.

Optimized contrast-enhanced CT protocols for diagnostic whole-body 18F-FDG PET/CT: technical aspects of single-phase versus multiphase CT imaging.

UNLABELLED: The purpose of this study was to compare various PET/CT examination protocols that use contrast-enhanced single-phase or contrast-enhanced multiphase CT scans under different breathing conditions. METHODS: Sixty patients with different malignant tumors were randomized into 4 different PET/CT protocols. Single-phase protocols included an intravenous contrast-enhanced (Ultravist 370; iodine at 370 mg/mL) single-phase whole-body CT scan (90 mL at 1.8 mL/min; delay, 90 s) during shallow breathing (protocol A) or during normal expiration (NormExp; protocol B). Multiphase protocols included 2 separate CT scans in the arterial contrast enhancement phase (90 mL at 2.5-2.8 mL/min; bolus tracking; scan range, base of the skull to the kidneys) and the portal-venous contrast enhancement phase (delay, 90 s; scan range, base of the lungs to the proximal thighs) during shallow breathing (protocol C) or during NormExp (protocol D) followed by a low-dose CT scan during shallow breathing for attenuation correction and whole-body PET. Feasibility was assessed by comparing the misalignment of the upper abdominal organs quantitatively by means of the craniocaudal, lateral, and anterior-posterior differences on coregistered PET/CT images. For image quality, the occurrence of CT artifacts and mismatching of rigid body points were evaluated qualitatively. RESULTS: Misalignment was significantly lower for protocol B in almost all organs and represented the best coregistration quality. Surprisingly, protocol A showed significantly better alignment than the multiphase CT scans during NormExp. Misalignment values between the multiphase protocols were not significantly different, with a trend toward lower values for protocol D. The best CT image quality, with a significantly lower occurrence of artifacts, was found for protocols B and D (NormExp). The levels of mismatching of rigid body points because of patient movement in between the transmission and emission scans were similar for all protocols. CONCLUSION: Multiphase CT protocols presented a technical disadvantage represented by suboptimal image coregistration compared with single-phase protocols. Nevertheless, multiphase protocols are technically feasible and should be considered for patients who will benefit from a contrast-enhanced multiphase CT examination for diagnosis.