Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

The Elastic Basis for the Shape of Borrelia burgdorferi

The mechanisms that determine bacterial shape are in many ways poorly understood. A prime example is the Lyme disease spirochete, Borrelia burgdorferi (B. burgdorferi), which mechanically couples its motility organelles, helical flagella, to its rod-shaped cell body, producing a striking flat-wave morphology. A mathematical model is developed here that accounts for the elastic coupling of the flagella to the cell cylinder and shows that the flat-wave morphology is in fact a natural consequence of the geometrical and material properties of the components. Observations of purified periplasmic flagella show two flagellar conformations. The mathematical model suggests that the larger waveform flagellum is the more relevant for determining the shape of B. burgdorferi. Optical trapping experiments were used to measure directly the mechanical properties of these spirochetes. These results imply relative stiffnesses of the two components, which confirm the predictions of the model and show that the morphology of B. burgdorferi is completely determined by the elastic properties of the flagella and cell body. This approach is applicable to a variety of other structures in which the shape of the composite system is markedly different from that of the individual components, such as coiled-coil domains in proteins and the eukaryotic axoneme.     

Mechanical response and conformational amplification in α-helical coiled coils

α-Helical coiled coils (CCs) are ubiquitous tertiary structural domains that are often found in mechanoproteins. CCs have mechanical rigidity and are often involved in force transmission between protein domains. Although crystal structures of CCs are available, information about their conformational flexibility is limited. The role of hydrophobic interactions in determining the CC conformation is not clear. In this work we examined the mechanical responses of typical CCs and constructed a coarse-grained mechanical model to describe the conformation of the protein. The model treats α-helices as elastic rods. Hydrophobic bonds arranged in a repeated pattern determine the CC structure. The model is compared with molecular-dynamics simulations of CCs under force. We also estimate the effective bending and twisting persistence length of the CC. The model allows us to examine unconventional responses of the CC, including significant conformational amplification upon binding of a small molecule. We find that the CC does not behave as a simple elastic rod and shows complex nonlinear responses. These results are significant for understanding the role of CC structures in chemoreceptors, motor proteins, and mechanotransduction in general.     

Chromatin decondensation and DNA synthesis in human sperm activated in vitro by using Xenopus laevis egg extracts

An in vitro sperm activation system was used to study nuclear swelling-chromatin decondensation and DNA synthesis; processes that occur in vivo following fertilization. Lysolecithin-permeabilized human sperm were incubated in Xenopus laevis egg extract and examined by using phase-contrast light microscopy, electron microscopy, and autoradiography. During a 3-hour incubation, the activated sperm nuclear chromatin underwent a decondensation-recondensation cycle during which DNA was synthesized. This also occurred when egg extract was given a 3-hour preincubation before the addition of the sperm, suggesting that the factor(s) required for initiating the decondensation-recondensation cycle is associated with the sperm. Because both nuclear swelling and DNA synthesis were found to be reproducible and quantifiable, we studied the effect of various agents on the two processes, characterizing the critical component(s) in the egg extract that induces these events. EGTA was found to have no effect on the induced nuclear swelling or DNA synthesis that occurs in the activated sperm. Freezing and thawing the extract or treating the extract with aphidicolin also had no effect on subsequent nuclear swelling; however, the DNA synthesis activity was blocked. Sperm incubated in extract treated with alkaline phosphatase (AP) had both nuclear swelling and DNA synthesis blocked. However, if the sperm were pretreated with DTT, and then incubated with the AP-treated extract, only the DNA synthesis activity of the extract was blocked. When the extract was treated with serine protease inhibitors (PMSF, soybean trypsin inhibitor, or alpha-2-macroglobulin), nuclear swelling occurred; however, DNA synthesis was blocked. These data suggest that phosphoproteins are involved in one or more of the activation events and that a serine protease(s) is involved in the synthesis of DNA.