Pseudopod Extension Research Articles

Pseudopod Tracking and Statistics During Cell Movement in Buffer and Chemotaxis.

Amoeboid cells such as the protist Dictyostelium, human neutrophils, and the fungus B.d. chytrid move by extending pseudopods. The trajectories of cell movement depend on the size, rhythm, and direction of long series of pseudopods. These pseudopod properties are regulated by internal factors such as memory of previous directions and by external factors such as gradients of chemoattractants or electric currents. Here a simple method is described that defines the X, Y time coordinates of a pseudopod at the start and the end of the extension phase. The connection between the start and end of an extending pseudopod defines a vector, which is the input of different levels of analysis that defines cell movement. The primary information of the vector is its spatial length (pseudopod size), temporal length (extension time), extension rate (size divided by time), and direction. The second layer of information describes the sequence of two (or more) pseudopods: the direction of the second pseudopod relative to the direction of the first pseudopod, the start of the second pseudopod relative to the extension phase of the first pseudopod (the second starts while the first is still extending or after the first has stopped), and the alternating right/left extension of pseudopods. The third layer of information is provided by specific and detailed statistical analysis of these data and addresses question such as: is pseudopod extension in buffer in random direction or has the system internal directional memory, and how do shallow external electrical or chemical gradients bias the intrinsic pseudopod extension. The method is described for Dictyostelium, but has been used successfully for fast-moving neutrophils, slow-moving stem cells, and the fungus B.d. chytrid.

Amoeboid soft robot based on multi-material composite 3D printing technology

A core–shell structured, amoeba-like, magnetically controlled soft robot is proposed for 3D printing using magnetically controlled smart materials and silicone materials. The purpose of this robot is to overcome the limitations of existing soft robot kinematic mechanisms that rely on the elastic deformation of flexible materials. The printability of the magnetically controlled smart material was verified through rheological tests. The effect of different component ratios on the rheological properties of the material was investigated and a suitable material ratio for printing was selected by adjusting the amount of SiO2 in the silicone and conducting rheological tests. Mechanical simulation was used to select and optimize the design of the amoeboid soft robotic shell structures. Printing was then used to verify the stability of the shell structures. The self-supporting capability of the magnetically controlled smart materials was investigated under different magnetic field strengths. To solve the problem of the phase of the magnetron smart material and the silicone material, the composite printing of the two materials was accomplished by adjusting the printing process. Soft robots mimicking amoebas were then printed and fabricated. The magnetron array-based driving method is proposed, and the magnetic field of the array used to drive the amoeba-like soft robot is constructed. The magnetic field of the array surface was then simulated under several different driving modes. Based on the simulation results, a suitable driving mode was selected. Using the material mechanism of the magnetron-only material sol–gel transformation, the pseudopod extension gait of the amoeba-like soft robot was designed, and the localized deformation of the amoeba-like soft robot was realized.This paper leveraged the distinctive properties of magnetorheological smart materials to devise and fabricate an integrated 3D-printed soft robot. Such magnetorheological smart materials can function as both the supporting framework for hollow cavities and the propelling substance for the robot. During the course of this process, we investigated material properties pertinent to 3D printing and refined the design of the robot's structure and driving mechanism.

Cleavage furrow positioning in dividing Dictyostelium cells.

Accurate placement of the cleavage furrow is crucial for successful cell division. Recent advancements have revealed that diverse mechanisms have evolved across different branches of the phylogenetic tree. Here, we employed Dictyostelium cells to validate previous models. We observed that during metaphase and early anaphase, mitotic spindles exhibited random rotary movements which ceased when the spindle elongated by approximately 7 μm. At this point, astral microtubules reached the polar cell cortex and fixed the spindle axis, causing cells to elongate by extending polar pseudopods and divide along the spindle axis. Therefore, the position of the furrow is determined when the spindle orientation is fixed. The distal ends of astral microtubules stimulate the extension of pseudopods at the polar cortex. One signal for pseudopod extension may be phosphatidylinositol trisphosphate in the cell membrane, but there appears to be another unknown signal. At the onset of polar pseudopod extension, cortical flow began from both poles toward the equator. We suggest that polar stimulation by astral microtubules determines the furrow position, induces polar pseudopod extension and cortical flow, and accumulates the elements necessary for the construction of the contractile ring.

A rare case of progressive subretinal fibrosis in a young child – A case report

Progressive subretinal fibrosis and uveitis is a rare entity which usually affects young myopic females and causes significant visual loss. Though there is no proven therapy, it usually requires systemic immunosuppression. We report unique pseudopodial extension of subretinal fibrosis in a 9-year-old girl and document 3-year follow-up of the case.

Intelectin enhances the phagocytosis of macrophages via CDC42-WASF2-ARPC2 signaling axis in Megalobrama amblycephala

Intelectin has been identified in various vertebrates and plays an important role in the host immune system. In our previous studies, recombinant Megalobrama amblycephala intelectin (rMaINTL) protein with excellent bacterial binding and agglutination activities enhances the phagocytic and killing activities of macrophages in M. amblycephala; however, the underlying regulatory mechanisms remain unclear. The present study showed that treatment with Aeromonas hydrophila and LPS induced the expression of rMaINTL in macrophages, and its level and distribution in macrophages or kidney tissue markedly increased after incubation or injection with rMaINTL. The cellular structure of macrophages was significantly affected after incubation with rMaINTL, resulting in an increased surface area and pseudopodia extension, which might contribute to enhancing the phagocytic ability of macrophages. Then, digital gene expression profiling analysis of the kidneys from rMaINTL-treated juvenile M. amblycephala identified some phagocytosis-related signaling factors that were enriched in pathways involved in the regulation of the actin cytoskeleton. In addition, qRT-PCR and western blotting verified that rMaINTL upregulated the expression of CDC42, WASF2, and ARPC2 in vitro and in vivo; however, the expression of these proteins was inhibited by a CDC42 inhibitor in macrophages. Moreover, CDC42 mediated the promotion of rMaINTL on actin polymerization by increasing the F-actin/G-actin ratio, which led to the extension of pseudopodia and remodeling of the macrophage cytoskeleton. Furthermore, the enhancement of macrophage phagocytosis by rMaINTL was blocked by the CDC42 inhibitor. These results suggested that rMaINTL induced the expression of CDC42 as well as the downstream signaling molecules WASF2 and ARPC2, thereby facilitating actin polymerization to promote cytoskeletal remodeling and phagocytosis. Overall, MaINTL enhanced the phagocytosis activity of macrophages in M. amblycephala via activation of the CDC42-WASF2-ARPC2 signaling axis.

Value of pseudopod sign on high‐frequency ultrasound in predicting the pathological invasion of extramammary Paget's disease lesions

Vertical invasion of extramammary Paget's disease (EMPD) is associated with poor prognosis. The usual vertical invasion route is directly downward or along the skin appendages. High-frequency ultrasound (HFUS) can be used to measure the EMPD lesion thickness, and visualize the pseudopod extensions due to skin appendage involvement. It is a non-invasive method for evaluating the extent of vertical invasion in EMPD. To investigate the value of HFUS in predicting the extent of vertical invasion in EMPD. In this retrospective study, 85 patients with EMPD were divided into two groups based on the pathology: invasive EMPD (iEMPD) group (n = 13) and in situ EMPD group (n = 72). The clinical and HFUS features of both the groups were analysed. The different types of pseudopodia morphology on HFUS were as follows: no pseudopodia, irregular bottom, small sphere, short strip, long strip, vase shape and nodular convex. These were further stratified into low-risk and high-risk levels. The clinical features were comparable between the two groups (P > 0.05). There were significant differences between the two groups in the HFUS features (lesion thickness, lesion shape, bottom shape, layer involvement, pseudopodia morphology and colour Doppler blood flow signal; all P < 0.05). The distribution of the pseudopodia morphology types in the in situ EMPD and iEMPD groups was as follows: no pseudopodia, 30/72 and 0/13; irregular bottom, 5/72 and 0/13; small sphere, 5/72 and 0/13; short strip, 21/72 and 0/13; long strip, 8/72 and 3/13; vase shape, 3/72 and 3/13; and nodular convex, 0/72 and 7/13 (P < 0.05 for all). The sensitivity and specificity of high-risk pseudopodia in identifying iEMPD were 100% and 84.7%, respectively. High-frequency ultrasound provides morphological information regarding EMPD lesions. Risk stratification for pseudopodia can help to distinguish between iEMPD and in situ EMPD lesions.

Gradients of PI(4,5)P2 and PI(3,5)P2 Jointly Participate in Shaping the Back State of Dictyostelium Cells.

Polarity, which refers to the molecular or structural asymmetry in cells, is essential for diverse cellular functions. Dictyostelium has proven to be a valuable system for dissecting the molecular mechanisms of cell polarity. Previous studies in Dictyostelium have revealed a range of signaling and cytoskeletal proteins that function at the leading edge to promote pseudopod extension and migration. In contrast, how proteins are localized to the trailing edge is not well understood. By screening for asymmetrically localized proteins, we identified a novel trailing-edge protein we named Teep1. We show that a charged surface formed by two pleckstrin homology (PH) domains in Teep1 is necessary and sufficient for targeting it to the rear of cells. Combining biochemical and imaging analyses, we demonstrate that Teep1 interacts preferentially with PI(4,5)P2 and PI(3,5)P2 in vitro and simultaneous elimination of these lipid species in cells blocks the membrane association of Teep1. Furthermore, a leading-edge localized myotubularin phosphatase likely mediates the removal of PI(3,5)P2 from the front, as well as the formation of a back-to-front gradient of PI(3,5)P2. Together our data indicate that PI(4,5)P2 and PI(3,5)P2 on the plasma membrane jointly participate in shaping the back state of Dictyostelium cells.

Interpretable deep learning uncovers cellular properties in label-free live cell images that are predictive of highly metastatic melanoma.

Deep learning has emerged as the technique of choice for identifying hidden patterns in cell imaging data but is often criticized as "black box." Here, we employ a generative neural network in combination with supervised machine learning to classify patient-derived melanoma xenografts as "efficient" or "inefficient" metastatic, validate predictions regarding melanoma cell lines with unknown metastatic efficiency in mouse xenografts, and use the network to generate in silico cell images that amplify the critical predictive cell properties. These exaggerated images unveiled pseudopodial extensions and increased light scattering as hallmark properties of metastatic cells. We validated this interpretation using live cells spontaneously transitioning between states indicative of low and high metastatic efficiency. This study illustrates how the application of artificial intelligence can support the identification of cellular properties that are predictive of complex phenotypes and integrated cell functions but are too subtle to be identified in the raw imagery by a human expert. A record of this paper's transparent peer review process is included in the supplemental information. VIDEO ABSTRACT.

Unified control of amoeboid pseudopod extension in multiple organisms by branched F-actin in the front and parallel F-actin/myosin in the cortex.

The trajectory of moving eukaryotic cells depends on the kinetics and direction of extending pseudopods. The direction of pseudopods has been well studied to unravel mechanisms for chemotaxis, wound healing and inflammation. However, the kinetics of pseudopod extension-when and why do pseudopods start and stop- is equally important, but is largely unknown. Here the START and STOP of about 4000 pseudopods was determined in four different species, at four conditions and in nine mutants (fast amoeboids Dictyostelium and neutrophils, slow mesenchymal stem cells, and fungus B.d. chytrid with pseudopod and a flagellum). The START of a first pseudopod is a random event with a probability that is species-specific (23%/s for neutrophils). In all species and conditions, the START of a second pseudopod is strongly inhibited by the extending first pseudopod, which depends on parallel filamentous actin/myosin in the cell cortex. Pseudopods extend at a constant rate by polymerization of branched F-actin at the pseudopod tip, which requires the Scar complex. The STOP of pseudopod extension is induced by multiple inhibitory processes that evolve during pseudopod extension and mainly depend on the increasing size of the pseudopod. Surprisingly, no differences in pseudopod kinetics are detectable between polarized, unpolarized or chemotactic cells, and also not between different species except for small differences in numerical values. This suggests that the analysis has uncovered the fundament of cell movement with distinct roles for stimulatory branched F-actin in the protrusion and inhibitory parallel F-actin in the contractile cortex.

Depletion of gipc-1 and gipc-2 causes infertility in Caenorhabditis elegans by reducing sperm motility

The G-protein signaling pathway plays a key role in multiple cellular processes and is well conserved in eukaryotes. Although GIPC (G-protein α subunit interacting protein (GAIP)-interacting protein, C terminus) has been studied in several model organisms, little is known about its role in Caenorhabditis elegans. In the present study, we investigated the roles of gipc-1 and gipc-2 in C. elegans. We observed that they were exclusively expressed in sperm throughout the development and that gipc-1; gipc-2 double mutants were infertile. Further examination of sperm development in gipc-1; gipc-2 mutants revealed defective sperm activation and abnormal pseudopod extension that resulted in reduced sperm motility. Moreover, major sperm protein (MSP) was abnormally segregated between spermatids and residual bodies in gipc-1; gipc-2 mutants. Our findings indicate that gipc-1 and gipc-2 are required for the proper pseudopod extension of sperm during the terminal differentiation of spermatids. During this process, the segregation of MSP into spermatids is important for ensuring normal sperm motility during fertilization.

Amikacin Suppresses Human Breast Cancer Cell MDA-MB-231 Migration and Invasion.

(1) Background: Amikacin is an aminoglycoside antibiotic used for treating gram-negative bacterial infections in cancer patients. In this study, our aims are to investigate the migratory inhibition effects of amikacin in human MDA-MB-231 cells. (2) Methods: We used a wound-healing assay, trans-well analysis, Western blotting, immunostaining and siRNA knockdown approaches to investigate how amikacin influenced MDA-MB-231 cell migration and invasion. (3) Results: Wound healing showed that the MDA-MB-231 cell migration rates decreased to 44.4% in the presence of amikacin. Trans-well analysis showed that amikacin treatment led to invasion inhibition. Western blotting demonstrated that amikacin induced thioredoxin-interacting protein (TXNIP) up-regulation. TXNIP was knocked down using siRNA in MDA-MB-231 cell. Using immunostaining analysis, we found that inhibition of TXNIP expression led to MDA-MB-231 pseudopodia extension; however, amikacin treatment attenuated the cell extension formation. (4) Conclusions: We observed inhibition of migration and invasion in MDA-MB-231 cells treated with amikacin. This suggests inhibition might be mediated by up-regulation of TXNIP.

Promoted Bone Regeneration by 3D-Printed Porous Scaffolds with the Synergy of a Nanotopological Morphology and Amino Modification.

Three-dimensional (3D)-printed scaffolds have great advantages for bone repair, and the combination of physical and chemical modifications of the surface can improve deficient biological properties to promote bone regeneration. In this study, a nanotopological morphology and an amino group were introduced into scaffold surfaces in sequence by alkaline solution and amination, respectively. The surface properties and the ability for osteogenic induction were investigated. The nanotopological morphology of the surface slightly enhanced the hydrophilic property of the material, while amination obviously increased the hydrophilicity of the surface. The aminated surface improved cell adhesion and proliferation, while the nanotopological morphology was able to facilitate the spread of stem cells, pseudopod extension, and osteogenic differentiation by changing the cell skeleton. The study confirmed that a nanotopological morphology and an amino group can play synergistic roles in improving the osteogenic efficiency and hydrophilicity, which was also confirmed in vivo by showing that effective surface modification of polylactic acid scaffolds enhanced high-quality bone formation, thus demonstrating great potential for clinical applications. The results indicate that scaffolds with the synergy of a nanotopological morphology and amino modification improve the osteogenic induction ability of scaffolds.

EPIC3, a novel Ca2+ indicator located at the cell cortex and in microridges, detects high Ca2+ subdomains during Ca2+ influx and phagocytosis

The construction of a low affinity Ca2+-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca2+). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca2+-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca2+ influx on intra-wrinkle Ca2+ in the macrophage cell line, RAW 264.7. During experimentally–induced Ca2+influx, EPIC3 reported Ca2+ concentrations at the cell cortex in the region of 30−50 μM, with peak locations towards the tips of wrinkles reaching 80 μM. These concentrations were associated with cleavage of ezrin (a substrate for the Ca2+ activated protease calpain-1) and released the C-terminal fluors. The cortical Ca2+ levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50−80 μM) during phagocytosis. As phagocytosis was completed, hotspots of Ca2+ near the phagosome were also observed.

Observations on nascent matrix structures in embryonic cornea: Important in cell interactions, or merely vestiges of the lens surface?

Here we present some new observations on early stages in chick corneal development obtained by remining of datasets obtained via serial block face scanning electron microscopy. We focus on matrix cords, proteoglycan-rich structures of apparent ectodermal origin, emerging from the epithelial basal lamina, which extend proximally into the growing collagenous matrix destined to become the corneal stroma. Cords have no known function. In their earliest manifestation, we describe how they appear to run continuously from epithelium to the lens, in contact with both tissues and may therefore be simply vestigial structures, remaining from the earlier detachment of the lens from its parent ectoderm. However, neural crest cells migrating to form the corneal endothelial monolayer appear to form close associations with cords via elaborate pseudopodial extensions. Presumptive endothelium and keratocytes, in the subsequent wave of neural crest cell influx, may conceivably utilise cords, as well as utilising collagenous fibrils of the interstitial matrix, as substrate cues in cell guidance, attachment and migration. The possibility also exists that cords fulfil a functional role in corneal morphogenesis via mechanotransduction through cell matrix interactions.

Symmetry Breaking during Cell Movement in the Context of Excitability, Kinetic Fine-Tuning and Memory of Pseudopod Formation.

The path of moving eukaryotic cells depends on the kinetics and direction of extending pseudopods. Amoeboid cells constantly change their shape with pseudopods extending in different directions. Detailed analysis has revealed that time, place and direction of pseudopod extension are not random, but highly ordered with strong prevalence for only one extending pseudopod, with defined life-times, and with reoccurring events in time and space indicative of memory. Important components are Ras activation and the formation of branched F-actin in the extending pseudopod and inhibition of pseudopod formation in the contractile cortex of parallel F-actin/myosin. In biology, order very often comes with symmetry. In this essay, I discuss cell movement and the dynamics of pseudopod extension from the perspective of symmetry and symmetry changes of Ras activation and the formation of branched F-actin in the extending pseudopod. Combining symmetry of Ras activation with kinetics and memory of pseudopod extension results in a refined model of amoeboid movement that appears to be largely conserved in the fast moving Dictyostelium and neutrophils, the slow moving mesenchymal stem cells and the fungus B.d. chytrid.

Statin-induced GGPP depletion blocks macropinocytosis and starves cells with oncogenic defects

Cancer cells display novel characteristics which can be exploited for therapeutic advantage. Isolated studies have shown that 1) the mevalonate pathway and 2) increased macropinocytosis are important in tumorigenesis, but a connection between these two observations has not been envisioned. A library screen for compounds that selectively killed Dictyostelium pten- cells identified pitavastatin. Pitavastatin also killed human breast epithelial MCF10A cells lacking PTEN or expressing K-RasG12V, as well as mouse tumor organoids. The selective killing of cells with oncogenic defects was traced to GGPP (geranylgeranyl diphosphate) depletion. Disruption of GGPP synthase in Dictyostelium revealed that GGPP is needed for pseudopod extension and macropinocytosis. Fluid-phase uptake through macropinocytosis is lower in PTEN-deleted cells and, as reported previously, higher in cells expressing activated Ras. Nevertheless, uptake was more sensitive to pitavastatin in cells with either of these oncogenic mutations than in wild-type cells. Loading the residual macropinosomes after pitavastatin with high concentrations of protein mitigated the cell death, indicating that defective macropinocytosis leads to amino acid starvation. Our studies suggest that the dependence of cancer cells on the mevalonate pathway is due to the role of GGPP in macropinocytosis and the reliance of these cells on macropinocytosis for nutrient uptake. Thus, inhibition of the networks mediating these processes is likely to be effective in cancer intervention.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

Tunable Adhesion of Different Cell Types Modulated by Thermoresponsive Polymer Brush Thickness.

Tunable adhesion of different cell types on well-defined surfaces has attracted common interests in the field of biomaterial science and surface engineering. Herein, we demonstrate a new strategy for the regulation of cell adhesion by simply controlling the thickness of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) brushes via surface-initiated atom transfer radical polymerization (ATRP). The adhesion of different cell types (4T1, HEK293, H9C2, HUVEC, and L929) can be easily modulated by varying the thickness of PNIPAAm brushes from 5.9 ± 1.0 nm (PN1) to 69.0 ± 5.0 nm (PN6). The fluorescent staining of different cell types on a variety of surfaces reveals that the thickness of PNIPAAm brushes would regulate the assembly of F-actin and the expression of vinculin and fibronectin, which are essential in regulating the adherent status of cells. Moreover, the cellular morphologies revealed that the adherent cells are well-spread, and multiple pseudopod extensions and protrusions can be observed at the margin of cells. This work provides a facile strategy for regulating tunable adhesion of different cell types, which may find applications in tissue engineering and regenerative medicine.

Three-D Models for Megakaryopoiesis and Platelet Production

Access to human platelets is fundamental in the quest for understanding the mechanisms of platelet-related diseases and the clinical need for platelet transfusions for patients. Effective solutions for producing platelets ex vivo are essential to obtaining this goal. Circulating platelets are highly specialised cells produced by megakaryocytes through the extension of unique pseudopods called pro-platelets. Leading studies point to the bone marrow niche as the core of blood cell production, revealing interesting and complex environmental factors for consideration. Megakaryocytes take cues from the physic-chemical 3D environment in the bone marrow, which includes contact with extracellular matrix components, interaction with other cell types, particularly endothelial cells, and the shear stress generated by the blood circulation into the sinusoid lumen. The technical impossibility of obtaining an intact bone marrow organ prompted the investigation of potential models to extrapolate ex vivo structures and functions to gain new insight on its activity. Thus, future advancements in the study of megakaryopoiesis depend on the evolution of bioengineering techniques for developing physiologically relevant models that reproduce the conditions in the bone marrow niche environment. Silk fibroin, derived from Bombyx mori silkworm cocoons,1 is one of the most promising biomaterials for bone marrow tissue engineering due to its tunable architecture and mechanical properties. Silk is a naturally derived, biocompatible biomaterial, which can be prepared in a range of material formats and processed entirely in aqueous systems allowing the incorporation of labile compounds without loss of bioactivity (e.g., extracellular matrix components). Exploiting innovative silk biomaterials, we have developed a series of 3D scalable systems that reproduce structure and composition of the human bone marrow, supporting natural platelet release from megakaryocytes.2-4 Data demonstrate that 1) silk supports platelet production maintaining their function without premature activation; 2) silk surface topography and stiffness direct megakaryocyte behaviour by activating mechanosensors and calcium signalling; 3) bloodstream impact on megakaryocyte function can be mimicked by perfusing the silk models at different shear rate and flows; 4) silk functionalisation with fibronectin and/or endothelial cells enhances megakaryocyte function; 5) silk models can be used to reproduce and study the primary megakaryocyte related disorders such as Myeloproliferative Neoplasms and Inherited Thrombocytopenias; 6) the same silk models can be exploited to test drug efficacy and/or toxicology on a per patient basis. Our models provide valuable and unique technology to reproduce the complexity of human bone marrow and study megakaryocytes and platelet formation. The silk platform has potentially limitless applications for basic research, clinic, pharmaceutical industry and transfusion medicine. Omenetto FG and Kaplan DL. New opportunities for an ancient material. Science. 2010; 329: 528. Di Buduo CA, Wray LS, Tozzi L, et al. Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies. Blood. 2015; 125: 2254. Di Buduo CA, Soprano PM, Tozzi L, et al. Modular flow chamber for engineering bone marrow architecture and function. Biomaterials. 2017; 146: 60. Abbonante V, Di Buduo CA, Gruppi C, et al. A new path to platelet production through matrix sensing. Haematologica. 2017; 102: 1150. Disclosures No relevant conflicts of interest to declare.

Fibroin/peptide co-functionalized calcium titanate nanorods improve osteoinductivity of titanium via mimicking osteogenic niche

The surface-inspirable biomaterials have captured extensive attention in simulating microenvironments by mimicking nanoscale architectures and signal molecules in the extracellular matrix (ECM). To activate osteogenic differentiation through biomimetic nanostructures, osteoid-like calcium titanate nanorods (CT) were fabricated via anodization and hydrothermal treatment. SEM, EDS, and XPS were employed to confirm the morphology and chemical composition. The functional fibroin and peptide layers on CT (PFCT) were subsequently applied to provide binding sites and bioactive molecules for osteoblast growth and differentiation. The hydrophilicity obviously increased with the protein layers. PFCT presented enhanced cell viability with pronounced pseudopod extension and continuous proliferation. The osteogenic differentiation analysis revealed that the alkaline phosphatase expression improved in the early stage. The calcium nodes and the collagen secretion were also enhanced. Quantitative polymerase chain reaction assessment indicated that osteogenesis-related markers including ALP, Col1, and Runx2, were up-regulated. The in vitro results demonstrated that fibroin/peptide co-functionalized calcium titanate nanorods were efficient to stimulate osteoblasts and improve cell adhesion, proliferation, and differentiation. Therefore, the nanorods can create an osteogenic niche on titanium implants, which can be a promising modification strategy in bone tissue engineering application.