Differential expressions of the lysyl oxidase family and matrix metalloproteinases-1, 2, 3 in posterior cruciate ligament fibroblasts after being co-cultured with synovial cells.

PURPOSE: The adult human posterior cruciate ligament (PCL) has poor functional healing response. The synovial tissue, which surrounds the PCL ligament, might be the major regulator of the microenvironment in the joint cavity after PCL injury, thus affecting the healing process. Here we establish a novel co-culture system for PCL fibroblasts and synovial cells (SC) in vitro to explore the direct influence of paracrine on PCL cells by characterizing the different expressions of the lysyl oxidase family (LOXs) and matrix metalloproteinases (MMP-1, 2, 3), which respectively facilitate extracellular matrix (ECM) repair and degradation. METHODS: Total RNA was harvested, reverse transcribed and assessed by semi-quantitative PCR and real-time PCR for the expression of LOXs and MMP-1, 2, 3 messenger RNAs. MMP-2 activity was assayed from the collected culture media samples by using zymography. RESULTS: We found co-culture could promote gene expressions of the LOXs and MMP-1, 2, 3 in normal PCL fibroblasts. But in injured PCL, we found that matrix crosstalk induced an increase of the MMP-1, 2, 3 expressions and a down-regulation of the LOXs. CONCLUSION: Based on these results, the crosstalk between PCL and SC strongly modified homeostatic balance of ECM and appeared to have a significant impact on PCL wound healing; decreased expression of cross-linking enzymes (LOXs) and increased expression of ECM-degrading proteinases (MMP-1, 2, 3) might be of great contribution to poor healing ability of PCL ligament.

Quantification of magnetically induced changes in ECM local apparent stiffness.

The stiffness of the extracellular matrix (ECM) is known to influence cell behavior. The ability to manipulate the stiffness of ECM has important implications in understanding how cells interact mechanically with their microenvironment. This article describes an approach to manipulating the stiffness ECM, whereby magnetic beads are embedded in the ECM through bioconjugation between the streptavidin-coated beads and the collagen fibers and then manipulated by an external magnetic field. It also reports both analytical results (obtained by formal modeling and numerical simulation) and statistically meaningful experimental results (obtained by atomic force microscopy) that demonstrate the effectiveness of this approach. These results clearly suggest the possibility of creating desired stiffness gradients in ECM in vitro to influence cell behavior.

NDDepth: Normal-Distance Assisted Monocular Depth Estimation and Completion.

Over the past few years, monocular depth estimation and completion have been paid more and more attention from the computer vision community because of their widespread applications. In this paper, we introduce novel physics (geometry)-driven deep learning frameworks for these two tasks by assuming that 3D scenes are constituted with piece-wise planes. Instead of directly estimating the depth map or completing the sparse depth map, we propose to estimate the surface normal and plane-to-origin distance maps or complete the sparse surface normal and distance maps as intermediate outputs. To this end, we develop a normal-distance head that outputs pixel-level surface normal and distance. Afterthat, the surface normal and distance maps are regularized by a developed plane-aware consistency constraint, which are then transformed into depth maps. Furthermore, we integrate an additional depth head to strengthen the robustness of the proposed frameworks. Extensive experiments on the NYU-Depth-v2, KITTI and SUN RGB-D datasets demonstrate that our method exceeds in performance prior state-of-the-art monocular depth estimation and completion competitors.

Up-regulation expressions of lysyl oxidase family in Anterior Cruciate Ligament and Medial Collateral Ligament fibroblasts induced by Transforming Growth Factor-Beta 1.

PURPOSE: The lysyl oxidase (LOX) family plays a crucial role in the formation and stabilisation of extracellular matrix (ECM) by catalysing the cross-linking of collagen and elastin, implicating its important fundamental roles in injury healing. A high level of transforming growth factor-ß(1) (TGF-ß(1)) accompanies the inflammatory phase of an injury of the knee joint. Our purpose was to detect the expressions of the LOX family in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) response to TGF-ß(1). METHODS: This study used reversed transcript PCR, real time quantitative PCR and Western blot for analyses. RESULTS: The results showed significant increases in mRNA levels of LOX family members. At 5 ng/ml concentration of TGF-ß(1,) the gene profiles of LOXs showed most active, and LOX and LOXL-3 showed increasing peaks at 12 hours after TGF-ß(1) treatment (LOX: 7.2, 8.8-fold and LOXL-3: 3.8, 5.4-fold compared with normal controls in ACL and MCL, respectively); LOXL-1, LOXL-2 and LOXL-4 reached their highest amounts at six hours (LOXL-1: 1.9, 2.4-fold; LOXL-2: 14.8, 16.2-fold; LOXL-4: 2.5, 4.4-fold in ACL and MCL, respectively). Protein assays revealed that LOXs in ACL cells had relatively lower response to TGF-ß(1) compared with those in MCL cells. CONCLUSIONS: The differential expression and activities of LOXs might help to explain the intrinsic difference between ACL and MCL, and LOXs could imply a potential capability for ACL healing.

Comparison of real-time microvascular abnormalities in pediatric and adult sickle cell anemia patients.

The conjunctival microcirculation in 14 pediatric and eight adult sickle cell anemia (SCA) patients was studied using computer-assisted intravital microscopy. The bulbar conjunctiva in SCA patients in both age groups exhibited a blanched/avascular appearance characterized by decreased vascularity. SCA patients from both age groups had many of the same abnormal morphometric [vessel diameter, vessel distribution, morphometry (shape), tortuosity, arteriole:venule (A:V) ratio, and hemosiderin deposits] and dynamic [vessel sludging/sludged flow, boxcar blood (trickled) flow, and abnormal flow velocity] abnormalities. A severity index (SI) was computed to quantify the degree of vasculopathy for comparison between groups. The severity of vasculopathy differed significantly between the pediatric and adult patients (SI: 4.2 ± 1.8 vs. 6.6 ± 2.4; P = 0.028), indicative of a lesser degree of overall severity in the pediatric patients. Specific abnormalities that were less prominent in the pediatric patients included abnormal vessel morphometry and tortuosity. Sludged flow, abnormal vessel distribution, abnormal A:V ratio, and boxcar flow appeared in high prevalence in both age groups. The results indicate that SCA microvascular abnormalities develop in childhood and the severity of vasculopathy likely progresses with age. Intervention and effective treatment/management modalities should target pediatric patients to ameliorate, slow down, or prevent progressive microvascular deterioration.

Whole blood viscosity and microvascular abnormalities in Alzheimer's Disease.

We hypothesized that abnormalities in hemorheologic parameters, including vessel diameter, flow velocity, and whole blood viscosity (WBV), would be present in Alzheimer's Disease (AD) and would correlate with microvascular abnormalities (vasculopathy). Using the Rheolog, we obtained WBV profiles, measured at shear rates of 1-1,000 s-1, for 10 AD subjects and age matched non-AD controls. Vessel diameter, flow velocity, and microvascular abnormalities were quantified using computer-assisted intravital microscopy (CAIM) of the conjunctival microcirculation. A Severity Index (SI), scale 0-15, was computed to reflect degree/severity of vasculopathy. AD subjects compared to controls had significantly higher WBV (3.96+/-0.29 cP vs. 3.34+/-0.05 cP, sheared at 300 s-1; P<0.05) and SI (7.00+/-2.36 vs. 0.30+/-0.70; P<0.05). WBV was correlated (rhos=0.648; P<0.05) with SI in AD subjects. These results strongly suggest the simultaneous involvement of hemorheologic abnormalities and systemic vasculopathy in AD.

Effects of pegylated hamster red blood cells on microcirculation.

The objective of this study was to examine the effects of polyethylene glycol (PEG) treated red blood cells (RBCs) on the microcirculation in a hamster back skin window chamber model. Donor hamster RBCs were PEGylated through an incubation with an activated PEG solution, washed, resuspended, and infused through a 10% volume top loading procedure into the carotid artery in an awake Syrian Golden hamster. Eight hamster groups were treated with activated PEG different sizes and concentrations: 0.05 mM-5 kDa PEG, 0.5 mM-5 kDa PEG, 1.1 mM-5 kDa PEG, 2.2 mM-5 kDa PEG, 22 mM-5 kDa PEG, 0.05 mM-20 kDa PEG, 0.5 mM-20 kDa PEG, and 5 mM-20 kDa PEG. Non-treated RBCs were used as control. The microvascular bed under observation was videotaped 30 min before the infusion and followed for 30 min post infusion. The diameter of individual blood vessels and blood flow velocities in selected vessels was measured. Hematocrit and hemoglobin concentration were recorded before infusion and at the end of experiment. Tissue pO(2) was also monitored. Results showed the hamsters tolerated the PEGylated RBCs without apparent ill effects. No significant changes were recorded for the hematocrit, the hemoglobin concentration, the blood vessel diameters, blood flow velocities, and the interstitial partial oxygen pressure (pO(2)) before, during, and after the injections of PEG-RBCs (P > 0.05). Unlike most hemoglobin-based oxygen carrying compounds, which can cause vasoconstriction, the PEGylated RBCs did not produce any measurable vasoactivity. Together with the absence of rouleaux formation and the fact that PEG molecules can mask the surface antigens on RBCs, PEGylation appeared promising as a circulation enhancement treatment.

A Reconfigurable Pneumatic Bending Actuator with Replaceable Inflation Modules.

A fully reconfigurable, pneumatic bending actuator is fabricated by implementing the concept of modularity to soft robotics. The actuator features independent, removable, fabric inflation modules that are attached to a common flexible but non-inflating plastic spine. The fabric modules are individually fabricated by heat sealing a thermoplastic polyurethane-coated nylon fabric, whereas the spine is manufactured through fused deposition modeling 3D printing; the components can be assembled and dismantled without the aid of any external tools. The replacement of specific modules along the array facilitates the reconfiguration of the actuator's bending trajectory and torque output; likewise, the combination of inflation modules with dissimilar geometries translates to several different trajectories on a single spine and allows the actuator to bend into assorted, unique structures. A detailed description of the actuator's design is thoroughly presented. We explored how reconfiguration of the actuator's modular geometry affected both the steady state and the dynamic characteristics of the actuator. The torque output of the actuator is proportional to the magnitude of the pressure applied. The actuator was excited by sinusoidal and square pressure inputs, and a second-order linear fit was performed. There were no perceived changes in its performance even after 100,000 inflation and deflation cycles.

An Electromagnetic System for Inducing a Localized Force Gradient in an ECM and Its Influence on HMVEC Sprouting.

Mechanical properties of the extracellular matrix (ECM) have been observed to influence the behavior of cells. Investigations on such an influence commonly rely on using soluble cues to alter the global intrinsic ECM properties in order to study the subsequent response of cells. This article presents an electromagnetic system for inducing a localized force gradient in an ECM, and reports the experimentally observed effect of such a force gradient on in vitro angiogenic sprouting of human microvascular endothelial cells (HMVECs). This force gradient is realized through the induction of magnetic forces on the superparamagnetic microparticle-embedded ECM ( sECM). Both analytical and statistically meaningful experimental results demonstrate the effectiveness of this approach in influencing the behavior of a targeted HMVEC sprout without affecting that of other sprouts nearby. These results suggest the possibility of selectively controlling the in vitro behavior of cells by the induction of a localized force gradient in the ECM.

A Markovian approach to the control of genetic regulatory networks.

This paper presents an approach for controlling gene networks based on a Markov chain model, where the state of a gene network is represented as a probability distribution, while state transitions are considered to be probabilistic. An algorithm is proposed to determine a sequence of control actions that drives (without state feedback) the state of a given network to within a desired state set with a prescribed minimum or maximum probability. A heuristic is proposed and shown to improve the efficiency of the algorithm for a class of genetic networks.

Dynamic Surface Control for a Class of Nonlinear Feedback Linearizable Systems With Actuator Failures.

In this brief, we propose a dynamic surface control with actuator failure compensation for a class of feedback linearizable systems with locally Lipschitz nonlinearities. First, a dynamic surface state feedback control scheme is designed, which incorporates radial basis function networks in a novel approach, to compensate system uncertainties and dynamic changes induced by actuator failures. Then, an output feedback controller is obtained by means of high-gain observers. It is proved that our control schemes guarantee the uniform ultimate boundedness of the system, and that the output tracking error converges to an arbitrarily small residual set. Finally, a simulation is carried out to illustrate the performance of the designed control schemes.

A Magneto-Microfluidic System for Investigating the Influence of an Externally Induced Force Gradient in a Collagen Type I ECM on HMVEC Sprouting.

Advances in mechanobiology have suggested that physiological and pathological angiogenesis may be differentiated based on the ways in which the cells interact with the extracellular matrix (ECM) that exhibits partially different mechanical properties. This warrants investigating the regulation of ECM stiffness on cell behavior using angiogenesis assays. In this article, we report the application of the technique of active manipulation of ECM stiffness to study in vitro angiogenic sprouting of human microvascular endothelial cells (HMVECs) in a microfluidic device. Magnetic beads were embedded in the ECM through bioconjugation (between the streptavidin-coated beads and collagen fibers) in order to create a pretension in the ECM when under the influence of an external magnetic field. The advantage of using this magneto-microfluidic system is that the resulting change in the local deformability of the collagen fibers is only apparent to a cell at the pericellular level near the site of an embedded bead, while the global intrinsic material properties of the ECM remain unchanged. The results demonstrate that this system represents an effective tool for inducing noninvasively an external force on cells through the ECM, and suggest the possibility of creating desired stiffness gradients in the ECM for manipulating cell behavior in vitro.

A discrete-event approach to transcription control with dynamic event-controllability.

This paper presents a discrete-event approach to the synthesis of transcription control for a class of (computational) gene networks. Given a set of genes and protein-gene and/or protein-protein interaction patterns, this approach can be used to synthesize a transcription control induced by these interaction patterns, and as a consequence, determine the behavior of the gene network. This proposed approach is demonstrated in the synthesis of a transcription control that produces the observed (logical) behavior in the induction of a phage lambda lysogen.

Three-Dimensional Characterization of Mechanical Interactions between Endothelial Cells and Extracellular Matrix during Angiogenic Sprouting.

We studied the three-dimensional cell-extracellular matrix interactions of endothelial cells that form multicellular structures called sprouts. We analyzed the data collected in-situ from angiogenic sprouting experiments and identified the differentiated interaction behavior exhibited by the tip and stalk cells. Moreover, our analysis of the tip cell lamellipodia revealed the diversity in their interaction behavior under certain conditions (e.g., when the heading of a sprout is switched approximately between the long-axis direction of two different lamellipodia). This study marks the first time that new characteristics of such interactions have been identified with shape changes in the sprouts and the associated rearrangements of collagen fibers. Clear illustrations of such changes are depicted in three-dimensional views.

Controlled synthesis of target strings in a class of splicing systems.

This article presents an approach for synthesizing target strings in a class of computational models of DNA recombination. The computational models are formalized as splicing systems in the context of formal languages. Given a splicing system (of a restricted type) and a target string to be synthesized, we construct (i) a rule-embedded splicing automaton that recognizes languages containing strings embedded with symbols representing splicing rules, and (ii) an automaton that implicitly recognizes the target string. By manipulating these two automata, we extract all rule sequences that lead to the production of the target string (if that string belongs to the splicing language). An algorithm for synthesizing a certain type of target strings based on such rule sequences is presented.

A computational model of a class of gene networks with positive and negative controls.

This article proposes a computational framework for modelling the logical behavior of a class of gene networks. We characterize the basic behavior of genes in terms of a state-and-transition structure, and model the individual genes as language-generating automata. We consider positive and negative controls as the interaction mechanisms among the genes, and treat such controls as constraints (also expressed in automata) imposed on the behavior of the gene network. By computing the intersection of the languages generated by the gene models and the constraints, we obtain the complete set of pathways in a gene network. Implications and possible improvement of this work are discussed.

Characterization of uniaxial stiffness of extracellular matrix embedded with magnetic beads via bio-conjugation and under the influence of an external magnetic field.

In this paper, we study the deformation, and experimentally quantify the change in stiffness, of an extracellular matrix (ECM) embedded with magnetic beads that are bio-conjugated with the collagen fibers and under the influence of an external magnetic field. We develop an analytical model of the viscoelastic behavior of this modified ECM, and design and implement a stretch test to quantify (based on statistically meaningful experiment data) the resulting changes in its stiffness induced by the external magnetic field. The analytical results are in close agreement with that obtained from the experiments. We discuss the implication of these results that point to the possibility of creating desired stiffness gradients in an ECM in vitro to influence cell behavior.

TNF-α induced down-regulation of lysyl oxidase family in anterior cruciate ligament and medial collateral ligament fibroblasts.

BACKGROUND: The lysyl oxidase (LOX) family has the capacity to catalyze the cross-linking of collagen and elastin, implicating its important fundamental role in injury healing. Tumor necrosis factor alpha (TNF-α) is considered to be an important chemical mediator in the acute inflammatory phase of the ligament injury. The role of the lysyl oxidase family induced by TNF-α in the knee ligaments' wound healing process is poorly understood. Our purpose was to determine the different expressions of the LOXs in poorly self-healing anterior cruciate ligament (ACL) and well functionally self-healing medial collateral ligament (MCL) induced by TNF-α. METHODS: Semi-quantitative PCR, quantitative real-time PCR and western blot were performed for original research. RESULTS: The results showed that all LOX family members were expressed at higher levels in MCL than those in ACL fibroblasts; the significant differences existed in the down-regulations of the LOXs induced by TNF-α; and the TNF-α-mediated down-regulations of the LOXs were more prominent in ACL than those in MCL fibroblasts. 1-20 ng/ml TNF-α down-regulated mRNA levels in ACL and MCL fibroblasts by up to 76% and 58% in LOX; 90% and 45% in LOXL-1; 97.5% and 90% in LOXL-2; 89% and 68% in LOXL-3; 52% and 25% in LOXL-4, respectively. Protein assay also showed LOXs had lower expressions in ACL than those in MCL. CLINICAL RELEVANCE: Based on these results, the differential expressions of the LOXs might help to explain the intrinsic differences between the poorly self-healing ACL and well functionally self-healing MCL.

Slanted spiral microfluidics for the ultra-fast, label-free isolation of circulating tumor cells.

The enumeration and characterization of circulating tumor cells (CTCs), found in the peripheral blood of cancer patients, provide a potentially accessible source for cancer diagnosis and prognosis. This work reports on a novel spiral microfluidic device with a trapezoidal cross-section for ultra-fast, label-free enrichment of CTCs from clinically relevant blood volumes. The technique utilizes the inherent Dean vortex flows present in curvilinear microchannels under continuous flow, along with inertial lift forces which focus larger CTCs against the inner wall. Using a trapezoidal cross-section as opposed to a traditional rectangular cross-section, the position of the Dean vortex core can be altered to achieve separation. Smaller hematologic components are trapped in the Dean vortices skewed towards the outer channel walls and eventually removed at the outer outlet, while the larger CTCs equilibrate near the inner channel wall and are collected from the inner outlet. By using a single spiral microchannel with one inlet and two outlets, we have successfully isolated and recovered more than 80% of the tested cancer cell line cells (MCF-7, T24 and MDA-MB-231) spiked in 7.5 mL of blood within 8 min with extremely high purity (400-680 WBCs mL(-1); ~4 log depletion of WBCs). Putative CTCs were detected and isolated from 100% of the patient samples (n = 10) with advanced stage metastatic breast and lung cancer using standard biomarkers (CK, CD45 and DAPI) with the frequencies ranging from 3-125 CTCs mL(-1). We expect this simple and elegant approach can surmount the shortcomings of traditional affinity-based CTC isolation techniques as well as enable fundamental studies on CTCs to guide treatment and enhance patient care.

Differential expressions of lysyl oxidase family in ACL and MCL fibroblasts after mechanical injury.

Lysyl oxidase (LOX) family has the capacity to catalyse the cross-linking of collagen and elastin, implicating its important fundamental roles in tissue development and injury healing. However, the variations in expression of the LOX family in the normal and injured anterior cruciate ligament (ACL) are not fully known. To better understand the role of LOX family in the self-healing inability mechanism of injured ACL, this study is to measure the LOX family's differential expressions in ACL and medial collateral ligament (MCL) fibroblasts after mechanical injury induced by using an equi-biaxial stretching chamber. The cells received various degrees of mechanical stretch 0% (resting state), 6% (physiological state) and 12% (injurious state), respectively. The gene profile and protein expressions were analysed by semi-quantitative PCR, quantitative real-time PCR and Western blotting. At physiological state, gene expression showed LOX in ACL was 2.6-5.2 folds higher than that in MCL in all culture time periods, LOXL-4 1.2-3.6 folds, but LOXL-3 in MCL showed 1.1-4.8 folds higher than that in ACL. In injurious state, MCL gene expressions were 2.8-29.6 folds higher than ACL in LOX, LOXL-2, LOXL-3 and LOXL-4 at 2, 6 and 12h periods. These differential expression profiles of the LOX family in the two ligament tissues were further used to explain the intrinsic differences between ACL and MCL, and why injured ACL could not be amenable to repair itself, whereas MCL could.