Soft materials to treat central nervous system injuries: Evaluation of the suitability of non-mammalian fibrin gels

AbstractPolymeric scaffolds formed from synthetic or natural materials have many applications in tissue engineering and medicine, and multiple material properties need to be optimized for specific applications. Recent studies have emphasized the importance of the scaffolds' mechanical properties to support specific cellular responses in addition to considerations of biochemical interactions, material transport, immunogenicity, and other factors that determine biocompatibility. Fibrin gels formed from purified fibrinogen and thrombin, the final two reactants in the blood coagulation cascade, have long been shown to be effective in wound healing and supporting the growth of cells in vitro and in vivo. Fibrin, even without additional growth factors or other components has potential for use in neuronal wound healing in part because of its mechanical compliance that supports the growth of neurons without activation of glial proliferation. This review summarizes issues related to the use of fibrin gels in neuronal cell contexts, with an emphasis on issues of immunogenicity, and considers the potential advantages and disadvantages of fibrin prepared from non-mammalian sources

Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation

Most tissue cells grown in sparse cultures on linearly elastic substrates typically display a small, round phenotype on soft substrates and become increasingly spread as the modulus of the substrate increases until their spread area reaches a maximum value. As cell density increases, individual cells retain the same stiffness-dependent differences unless they are very close or in molecular contact. On nonlinear strain-stiffening fibrin gels, the same cell types become maximally spread even when the low strain elastic modulus would predict a round morphology, and cells are influenced by the presence of neighbors hundreds of microns away. Time lapse microscopy reveals that fibroblasts and human mesenchymal stem cells on fibrin deform the substrate by several microns up to five cell lengths away from their plasma membrane through a force limited mechanism. Atomic force microscopy and rheology confirm that these strains locally and globally stiffen the gel, depending on cell density, and this effect leads to long distance cell-cell communication and alignment. Thus cells are acutely responsive to the nonlinear elasticity of their substrates and can manipulate this rheological property to induce patterning

Differential cellular response to linear and strain stiffening hydrogel substrates

The mechanical properties of the substrate upon which cells are cultured have been shown to influence a variety of cell properties including cell adhesion, spreading, protein expression and differentiation. The work presented here examines how the nonlinear mechanical properties of biopolymer gels affect the cellular responses to substrate stiffness. Cell spread area decreases with decreasing substrate stiffness when cells are cultured on linearly elastic polyacrylamide gels but display no spread area sensitivity when cultured on fibrin gels of various moduli. Fibrin gels, and other semiflexible biopolymer networks, exhibit strain stiffening, whereby the elastic modulus of the gel increases with increasing applied strain. Mechanosensitive cells and strain stiffening gels engage in a mechanical feedback loop with cells increasing their applied force and the gel modulus increasing as a result until the cells reach their maximum spread area. Cell applied forces locally induce anisotropy in an initially isotropic matrix providing a mechanism for cell/cell communication over a distance of ∼5 cell lengths. This results in alignment of adjacent cells and formation of ring-like multicellular patterns. Finally, due in part to its mechanical properties, fibrin is an appealing scaffold for neural tissue repair. Initial animal studies confirm that salmon derived fibrin mitigates pain and inflammation after injury to the central nervous system

Fibrin gels and their clinical and bioengineering applications

Fibrin gels, prepared from fibrinogen and thrombin, the key proteins involved in blood clotting, were among the first biomaterials used to prevent bleeding and promote wound healing. The unique polymerization mechanism of fibrin, which allows control of gelation times and network architecture by variation in reaction conditions, allows formation of a wide array of soft substrates under physiological conditions. Fibrin gels have been extensively studied rheologically in part because their nonlinear elasticity, characterized by soft compliance at small strains and impressive stiffening to resist larger deformations, appears essential for their function as haemostatic plugs and as matrices for cell migration and wound healing. The filaments forming a fibrin network are among the softest in nature, allowing them to deform to large extents and stiffen but not break. The biochemical and mechanical properties of fibrin have recently been exploited in numerous studies that suggest its potential for applications in medicine and bioengineering

Plasma gelsolin modulates cellular response to sphingosine 1-phosphate

Hypogelsolinemia is observed in patients with different states of acute or chronic inflammation such as sepsis, rheumatoid arthritis, and multiple sclerosis. In animal models of sepsis, repletion of plasma gelsolin reduces septic mortality. However, the functions of extracellular gelsolin and the mechanisms leading to its protective nature are poorly understood. Potential mechanisms involve gelsolin's extracellular actin scavenging function or its ability to bind bioactive lipids or proinflammatory mediators, which would limit inflammatory responses and prevent tissue damage. Here we report that human plasma gelsolin binds to sphingosine 1-phosphate (S1P), a pleiotropic cellular agonist involved in various immune responses, and to its synthetic structural analog FTY720P (Gilenya). The fluorescence intensity of a rhodamine B-labeled phosphatidylinositol 4,5-bisphosphate binding peptide derived from gelsolin and the optical density of recombinant human plasma gelsolin (rhpGSN) were found to decrease after the addition of S1P or FTY720P. Gelsolin's ability to depolymerize F-actin also decreased progressively with increasing addition of S1P. Transient increases in phosphorylation of extracellular signal-regulated kinase in bovine aortic endothelial cells (BAECs) after S1P treatment were inhibited by rhpGSN. The ability of S1P to increase F-actin content and the elastic modulus of primary astrocytes and BAECs was also prevented by rhpGSN. Evaluation of S1P and gelsolin levels in cerebrospinal fluid reveals a low concentration of gelsolin and a high concentration of S1P in samples obtained from patients suffering from lymphatic meningitis. These findings suggest that gelsolin-mediated regulation of S1P bioactivity may be important to maintain immunomodulatory balance at inflammatory sites

Measurements of accuracy and precision of LiquidBiopsy capture of MCF7 and H1650 cells.

<p>Measurements of accuracy and precision of LiquidBiopsy capture of MCF7 and H1650 cells.</p

LiquidBiopsy automated platform.

<p>(A) Diagram of the LiquidBiopsy platform. (B) Closeup of the platform worksurface. A X-Y pipetting arm with 4 pipettor heads transfers sample, buffers and antibody stains into 4 flow cells in the manifold. Sheath buffers are controlled by pumps on the rear wall of the platform.</p

Validation of Automated CTC flow cell operation on LiquidBiopsy platform.

<p>(A) Plot of CTC recovery as a function of spike-in density for MCF7 and H1650 cells. (B) Plot of sample purity as a function of spike in density for MCF7 and H1650 cells. The orange dots indicate the predicted purity of MCF7 cells if the non target recovery is held constant at 55 cells/mL. (C) Raw data and linear fit to MCF7 recovery curve from 9 to 90 cells/mL. (D) Raw data and linear fit to H1650 recovery curve from 9 to 300 cells/mL.</p

CTC Flow cell operation and performance.

<p>(A) The LiquidBiopsy CTC flow cell. (B) A cartoon illustrating how target cells are pulled from the sheath flow while non targets move through the flow cell unhindered. (C) Efficiency of recovery of target cells using EpCAM based recovery: Incremental numbers of MCF7 (N = 34), HCC1419 (N = 27) or A549 (N = 65) cells were spiked into NHD blood and purified on the CTC flow cell. Recovered cells were enumerated in the flow cell. Graphs show number of targets spiked per mL of blood against the yield +/−1 SD from an average of between 7 and 27 experiments.</p