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The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide-polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules.
Fibrous tissue growth and loss of residual hearing after cochlear implantation can be reduced by application of the glucocorticoid dexamethasone-21-phosphate-disodium-salt (DEX). To date, sustained delivery of this agent to the cochlea using a number of pharmaceutical technologies has not been entirely successful. In this study we examine a novel way of continuous local drug application into the inner ear using a refillable hydrogel functionalized silicone reservoir. A PEG-based hydrogel made of reactive NCO-sP(EO-stat-PO) prepolymers was evaluated as a drug conveying and delivery system in vitro and in vivo. Encapsulating the free form hydrogel into a silicone tube with a small opening for the drug diffusion resulted in delayed drug release but unaffected diffusion of DEX through the gel compared to the free form hydrogel. Additionally, controlled DEX release over several weeks could be demonstrated using the hydrogel filled reservoir. Using a guinea-pig cochlear trauma model the reservoir delivery of DEX significantly protected residual hearing and reduced fibrosis. As well as being used as a device in its own right or in combination with cochlear implants, the hydrogel-filled reservoir represents a new drug delivery system that feasibly could be replenished with therapeutic agents to provide sustained treatment of the inner ear.
Myocardial infarction (MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time-span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time-span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel (FDH) for delivering Met-CCL5 over 24hrs and a slow degradable hydrogel (SDH) for a gradual release of protease-resistant CXCL12 (S4V) over 4weeks. We demonstrate that the time-controlled release using Met-CCL5-FDH and CXCL12 (S4V)-SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time-controlled, biopolymer-mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell-based therapies.
Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 mu m and 80 mu m and spacings between 5 mu m and 20 mu m that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.
Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches.
Calcium phosphate biocements based on calcium phosphate chemistry are well-established biomaterials for the repair of non-load bearing bone defects due to the brittle nature and low flexural strength of such cements. This article features reinforcement strategies of biocements based on various intrinsic or extrinsic material modifications to improve their strength and toughness. Altering particle size distribution in conjunction with using liquefiers reduces the amount of cement liquid necessary for cement paste preparation. This in turn decreases cement porosity and increases the mechanical performance, but does not change the brittle nature of the cements. The use of fibers may lead to a reinforcement of the matrix with a toughness increase of up to two orders of magnitude, but restricts at the same time cement injection for minimal invasive application techniques. A novel promising approach is the concept of dual-setting cements, in which a second hydrogel phase is simultaneously formed during setting, leading to more ductile cement-hydrogel composites with largely unaffected application properties.
Additive manufacturing of scaffolds with sub-micron filaments via melt electrospinning writing
(2015)
The aim of this study was to explore the lower resolution limits of an electrohydrodynamic process combined with direct writing technology of polymer melts. Termed melt electrospinning writing, filaments are deposited layer-by-layer to produce discrete three-dimensional scaffolds for in vitro research. Through optimization of the parameters (flow rate, spinneret diameter, voltage, collector distance) for poly-ϵ-caprolactone, we could direct-write coherent scaffolds with ultrafine filaments, the smallest being 817 ± 165 nm. These low diameter filaments were deposited to form box-structures with a periodicity of 100.6 ± 5.1 μm and a height of 80 μm (50 stacked filaments; 100 overlap at intersections). We also observed oriented crystalline regions within such ultrafine filaments after annealing at 55 °C. The scaffolds were printed upon NCO-sP(EO-stat-PO)-coated glass slide surfaces and withstood frequent liquid exchanges with negligible scaffold detachment for at least 10 days in vitro.
Background
There is a need to establish more cell lines from breast tumors in contrast to immortalized cell lines from metastatic effusions in order to represent the primary tumor and not principally metastatic biology of breast cancer. This investigation describes the simultaneous isolation, characterization, growth and function of primary mammary epithelial cells (MEC), mesenchymal cells (MES) and adipose derived stem cells (ADSC) from four normal breasts, one inflammatory and one triple-negative ductal breast tumors.
Methods
A total of 17 cell lines were established and gene expression was analyzed for MEC and MES (n = 42) and ADSC (n = 48) and MUC1, pan-KRT, CD90 and GATA-3 by immunofluorescence. DNA fingerprinting to track cell line identity was performed between original primary tissues and isolates. Functional studies included ADSC differentiation, tumor MES and MEC invasion co-cultured with ADSC-conditioned media (CM) and MES adhesion and growth on 3D-printed scaffolds.
Results
Comparative analysis showed higher gene expression of EPCAM, CD49f, CDH1 and KRTs for normal MEC lines; MES lines e.g. Vimentin, CD10, ACTA2 and MMP9; and ADSC lines e.g. CD105, CD90, CDH2 and CDH11. Compared to the mean of all four normal breast cell lines, both breast tumor cell lines demonstrated significantly lower ADSC marker gene expression, but higher expression of mesenchymal and invasion gene markers like SNAI1 and MMP2. When compared with four normal ADSC differentiated lineages, both tumor ADSC showed impaired osteogenic and chondrogenic but enhanced adipogenic differentiation and endothelial-like structures, possibly due to high PDGFRB and CD34. Addressing a functional role for overproduction of adipocytes, we initiated 3D-invasion studies including different cell types from the same patient. CM from ADSC differentiating into adipocytes induced tumor MEC 3D-invasion via EMT and amoeboid phenotypes. Normal MES breast cells adhered and proliferated on 3D-printed scaffolds containing 20 fibers, but not on 2.5D-printed scaffolds with single fiber layers, important for tissue engineering.
Conclusion
Expression analyses confirmed successful simultaneous cell isolations of three different phenotypes from normal and tumor primary breast tissues. Our cell culture studies support that breast-tumor environment differentially regulates tumor ADSC plasticity as well as cell invasion and demonstrates applications for regenerative medicine.
The development and formulation of printable inks for extrusion-based 3D bioprinting has been a major challenge in the field of biofabrication. Inks, often polymer solutions with the addition of crosslinking to form hydrogels, must not only display adequate mechanical properties for the chosen application but also show high biocompatibility as well as printability. Here we describe a reproducible two-step method for the assessment of the printability of inks for bioprinting, focussing firstly on screening ink formulations to assess fibre formation and the ability to form 3D constructs before presenting a method for the rheological evaluation of inks to characterise the yield point, shear thinning and recovery behaviour. In conjunction, a mathematical model was formulated to provide a theoretical understanding of the pressure-driven, shear thinning extrusion of inks through needles in a bioprinter. The assessment methods were trialled with a commercially available crème, poloxamer 407, alginate-based inks and an alginate-gelatine composite material. Yield stress was investigated by applying a stress ramp to a number of inks, which demonstrated the necessity of high yield for printable materials. The shear thinning behaviour of the inks was then characterised by quantifying the degree of shear thinning and using the mathematical model to predict the window of printer operating parameters in which the materials could be printed. Furthermore, the model predicted high shear conditions and high residence times for cells at the walls of the needle and effects on cytocompatibility at different printing conditions. Finally, the ability of the materials to recover to their original viscosity after extrusion was examined using rotational recovery rheological measurements. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterisation of a wide variety of inks for bioprinting.
Intercellular adhesion plays a major role in tissue development and homeostasis. Yet, technologies to measure mature cell-cell contacts are not available. We introduce a methodology based on fluidic probe force microscopy to assess cell-cell adhesion forces after formation of mature intercellular contacts in cell monolayers. With this method we quantify that L929 fibroblasts exhibit negligible cell-cell adhesion in monolayers whereas human endothelial cells from the umbilical artery (HUAECs) exert strong intercellular adhesion forces per cell. We use a new in vitro model based on the overexpression of Muscle Segment Homeobox 1 (MSX1) to induce Endothelial-to-Mesenchymal Transition (EndMT), a process involved in cardiovascular development and disease. We reveal how intercellular adhesion forces in monolayer decrease significantly at an early stage of EndMT and we show that cells undergo stiffening and flattening at this stage. This new biomechanical insight complements and expands the established standard biomolecular analyses. Our study thus introduces a novel tool for the assessment of mature intercellular adhesion forces in a physiological setting that will be of relevance to biological processes in developmental biology, tissue regeneration and diseases like cancer and fibrosis.
Dicalcium phosphate cement preparation requires the addition of setting retarders to meet clinical requirements regarding handling time and processability. Previous studies have focused on the influence of different setting modifiers on material properties such as mechanical performance or injectability, while ignoring their influence on biological cement properties as they are used in low concentrations in the cement pastes and the occurrence of most compounds in human tissues. Here, analyses of both material and biological behavior were carried out on samples with common setting retardants (citric acid, sodium pyrophosphate, sulfuric acid) and novel (phytic acid). Cytocompatibility was evaluated by in vitro tests with osteoblastic (hFOB 1.19) and osteoclastic (RAW 264.7) cells. We found cytocompatibility was better for sodium pyrophosphate and phytic acid with a three-fold cell metabolic activity by WST-1 test, whereas samples set with citric acid showed reduced cell number as well as cell activity. The compressive strength (CS) of cements formed with phytic acid (CS = 13 MPa) were nearly equal to those formed with citric acid (CS = 15 MPa) and approximately threefold higher than for other setting retardants. Due to a proven cytocompatibility and high mechanical strength, phytic acid seems to be a candidate replacement setting retardant for dicalcium phosphate cements.
Calcium phosphate cement (CPC) is a well-established bone replacement material in dentistry and orthopedics. CPC mimics the physicochemical properties of natural bone and therefore shows excellent in vivo behavior. However, due to their brittleness, the application of CPC implants is limited to non-load bearing areas. Generally, the fiber-reinforcement of ceramic materials enhances fracture resistance, but simultaneously reduces the strength of the composite. Combining strong C-fiber reinforcement with a hydroxyapatite to form a CPC with a chemical modification of the fiber surface allowed us to adjust the fiber–matrix interface and consequently the fracture behavior. Thus, we could demonstrate enhanced mechanical properties of CPC in terms of bending strength and work of fracture to a strain of 5% (WOF5). Hereby, the strength increased by a factor of four from 9.2 ± 1.7 to 38.4 ± 1.7 MPa. Simultaneously, the WOF5 increased from 0.02 ± 0.004 to 2.0 ± 0.6 kJ∙m−2, when utilizing an aqua regia/CaCl2 pretreatment. The cell proliferation and activity of MG63 osteoblast-like cells as biocompatibility markers were not affected by fiber addition nor by fiber treatment. CPC reinforced with chemically activated C-fibers is a promising bone replacement material for load-bearing applications.
In this study, we evaluate hydrogels based on oxidized hyaluronic acid, cross-linked with adipic acid dihydrazide, for their suitability as bioinks for 3D bioprinting. Aldehyde containing hyaluronic acid (AHA) is synthesized and cross-linked via Schiff Base chemistry with bifunctional adipic acid dihydrazide (ADH) to form a mechanically stable hydrogel with good printability. Mechanical and rheological properties of the printed and casted hydrogels are tunable depending on the concentrations of AHA and ADH cross-linkers.
Reinforcing hydrogels with micro-fibre scaffolds obtained by a Melt-Electrospinning Writing (MEW) process has demonstrated great promise for developing tissue engineered (TE) constructs with mechanical properties compatible to native tissues. However, the mechanical performance and reinforcement mechanism of the micro-fibre reinforced hydrogels is not yet fully understood. In this study, FE models, implementing material properties measured experimentally, were used to explore the reinforcement mechanism of fibre-hydrogel composites. First, a continuum FE model based on idealized scaffold geometry was used to capture reinforcement effects related to the suppression of lateral gel expansion by the scaffold, while a second micro-FE model based on micro-CT images of the real construct geometry during compaction captured the effects of load transfer through the scaffold interconnections. Results demonstrate that the reinforcement mechanism at higher scaffold volume fractions was dominated by the load carrying-ability of the fibre scaffold interconnections, which was much higher than expected based on testing scaffolds alone because the hydrogel provides resistance against buckling of the scaffold. We propose that the theoretical understanding presented in this work will assist the design of more effective composite constructs with potential applications in a wide range of TE conditions.
Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384–402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.
Dual setting cements composed of an in situ forming hydrogel and a reactive mineral phase combine high compressive strength of the cement with sufficient ductility and bending strength of the polymeric network. Previous studies were focused on the modification with non-degradable hydrogels based on 2-hydroxyethyl methacrylate (HEMA). Here, we describe the synthesis of suitable triblock degradable poly(ethylene glycol)-poly(lactide) (PEG-PLLA) cross-linker to improve the resorption capacity of such composites. A study with four different formulations was established. As reference, pure hydroxyapatite (HA) cements and composites with 40 wt% HEMA in the liquid cement phase were produced. Furthermore, HEMA was modified with 10 wt% of PEG-PLLA cross-linker or a test series containing only 25% cross-linker was chosen for composites with a fully degradable polymeric phase. Hence, we developed suitable systems with increased elasticity and 5-6 times higher toughn ess values in comparison to pure inorganic cement matrix. Furthermore, conversion rate from alpha-tricalcium phosphate (alpha-TCP) to HA was still about 90% for all composite formulations, whereas crystal size decreased. Based on this material development and advancement for a dual setting system, we managed to overcome the drawback of brittleness for pure calcium phosphate cements.
Background
The spectrum of indications for the use of membranes and scaffolds in the field of oral and maxillofacial surgery includes, amongst others, guided bone regeneration (GBR). Currently available membrane systems face certain disadvantages such as difficult clinical handling, inconsistent degradation, undirected cell growth and a lack of stability that often complicate their application. Therefore, new membranes which can overcome these issues are of great interest in this field.
Methods
In this pilot study, we investigated polycaprolactone (PCL) scaffolds intended to enhance oral wound healing by means of melt electrospinning writing (MEW), which allowed for three-dimensional (3D) printing of micron scale fibers and very exact fiber placement. A singular set of box-shaped scaffolds of different sizes consisting of medical-grade PCL was examined and the scaffolds’ morphology was evaluated via scanning electron microscopy (SEM). Each prototype sample with box sizes of 225 μm, 300 μm, 375 μm, 450 μm and 500 μm was assessed for cytotoxicity and cell growth by seeding each scaffold with human osteoblast-like cell line MG63.
Results
All scaffolds demonstrated good cytocompatibility according to cell viability, protein concentration, and cell number. SEM analysis revealed an exact fiber placement of the MEW scaffolds and the growth of viable MG63 cells on them. For the examined box-shaped scaffolds with pore sizes between 225 μm and 500 μm, a preferred box size for initial osteoblast attachment could not be found.
Conclusions
These well-defined 3D scaffolds consisting of medical-grade materials optimized for cell attachment and cell growth hold the key to a promising new approach in GBR in oral and maxillofacial surgery.
In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP–2, BMP–7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP–2. In vitro, both scaffolds presented similar mechanical properties, rhBMP–2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP–2 mediated bone healing.
Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (µCT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage.
To facilitate true regeneration, a vascular graft should direct the evolution of a neovessel to obtain the function of a native vessel. For this, scaffolds have to permit the formation of an intraluminal endothelial cell monolayer, mimicking the tunica intima. In addition, when attempting to mimic a tunica media‐like outer layer, the stacking and orientation of vascular smooth muscle cells (vSMCs) should be recapitulated. An integral scaffold design that facilitates this has so far remained a challenge. A hybrid fabrication approach is introduced by combining solution electrospinning and melt electrowriting. This allows a tissue‐structure mimetic, hierarchically bilayered tubular scaffold, comprising an inner layer of randomly oriented dense fiber mesh and an outer layer of microfibers with controlled orientation. The scaffold supports the organization of a continuous luminal endothelial monolayer and oriented layers of vSM‐like cells in the media, thus facilitating control over specific and tissue‐mimetic cellular differentiation and support of the phenotypic morphology in the respective layers. Neither soluble factors nor a surface bioactivation of the scaffold is needed with this approach, demonstrating that heterotypic scaffold design can direct physiological tissue‐like cell organization and differentiation.