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•Single glycine substitutions in the triple helix impair structure and function.•Designer collagens are used as a model system to study collagen mutations.•Computational modeling reveals collagen properties at different hierarchy levels.•Designer collagens are promising biomaterials for biomedical applications.

Multicellular aggregates are an excellent model system to explore the role of tissue biomechanics in specifying multicellular reorganization during embryonic developments and malignant invasion. Tissue-like spheroids, when subjected to a compressive force, are known to exhibit liquid-like behaviors at long timescales (hours), largely because of cell rearrangements that serve to effectively dissipate the applied stress. At short timescales (seconds to minutes), before cell rearrangement, the mechanical behavior is strikingly different. The current work uses shape relaxation to investigate the structural characteristics of aggregates and discovers two coherent timescales: one on the order of seconds, the other tens of seconds. These timescales are universal, conserved across a variety of tested species, and persist despite great differences in other properties such as tissue surface tension and adhesion. A precise mathematical theory is used to correlate the timescales with mechanical properties and reveals that aggregates have a relatively strong envelope and an unusually “soft” interior (weak bulk elastic modulus). This characteristic is peculiar, considering that both layers consist of identical units (cells), but is consistent with the fact that this structure can engender both structural integrity and the flexibility required for remodeling. In addition, tissue surface tension, elastic modulus, and viscosity are proportional to each other. Considering that these tissue-level properties intrinsically derive from cellular-level properties, the proportionalities imply precise coregulation of the latter and in particular of the tension on the cell-medium and cell-cell interfaces.

Electroporation creates transient openings in the cell membrane, allowing for intracellular delivery of diagnostic and therapeutic substances. The degree of cell membrane permeability during electroporation plays a key role in regulating the size of the delivery payload as well as the overall cell viability. A microfluidic platform offers the ability to electroporate single cells with impedance detection of membrane permeabilization in a high-throughput, continuous-flow manner. We have developed a flow-based electroporation microdevice that automatically detects, electroporates, and monitors individual cells for changes in permeability and delivery. We are able to achieve the advantages of electrical monitoring of cell permeabilization, heretofore only achieved with trapped or static cells, while processing the cells in a continuous-flow environment. We demonstrate the analysis of membrane permeabilization on individual cells before and after electroporation in a continuous-flow environment, which dramatically increases throughput. We have confirmed cell membrane permeabilization by electrically measuring the changes in cell impedance from electroporation and by optically measuring the intracellular delivery of a fluorescent probe after systematically varying the electric field strength and duration and correlating the pulse parameters to cell viability. We find a dramatic change in cell impedance and propidium iodide (PI) uptake at a pulse strength threshold of 0.87 kV/cm applied for a duration of 1 ms or longer. The overall cell viability was found to vary in a dose dependent manner with lower viability observed with increasing electric field strength and pulse duration. Cell viability was greater than 83% for all cases except for the most aggressive pulse condition (1kV/cm for 5ms), where the viability dropped to 67.1%. These studies can assist in determining critical permeabilization and molecular delivery parameters while preserving viability.

Type-I collagen assembles in a stepwise, hierarchic fashion from the folding of the triple helix to the assembly of fibrils into fibers. The mature assembled fibers are crucial for tissue structure and mechanics, cell interactions, and other functions in vivo. Although triple helix folding can be followed with the use of optical methods such as circular dichroism (CD) spectroscopy, fibrillogenesis is typically measured by alternative methods such as turbidity, rheology, and microscopy. Together, these approaches allow for investigation of the mechanical properties and architectures of collagen-based scaffolds and excised tissues. Herein, we demonstrate that CD spectroscopy, a technique that is used primarily to evaluate the secondary structure of proteins, can also be employed to monitor collagen fibrillogenesis. Type-I collagen suspensions demonstrated a strong, negative ellipticity band between 204 and 210 nm under conditions consistent with fibrillogenesis. Deconvolution of CD spectra before, during, and after fibrillogenesis identified a unique fibril spectrum distinct from triple helix and random coil conformations. The ability to monitor multiple states of collagen simultaneously in one experiment using one modality provides a powerful platform for studying this complex assembly process and the effects of other factors, such as collagenases, on fibrillogenesis and degradation.

Matrix and cellular alignment are critical factors in the native function of many tissues, including muscle, nerve, and ligaments. Collagen is frequently a component of these aligned tissues, and collagen biomaterials are widely used in tissue engineering applications. However, the generation of aligned collagen scaffolds that maintain the native architecture of collagen fibrils has not been straightforward, with many methods requiring specialized equipment or technical procedures, extensive incubation times, or denaturing of the collagen. Herein, we present a simple, rapid method for fabrication of highly aligned collagen scaffolds. Collagen was assembled to form a fibrillar hydrogel in a cylindrical conduit with high aspect ratio and then frozen and lyophilized. The resulting collagen scaffolds demonstrated highly aligned topographical features along the scaffold surface. This presence of an initial fibrillar network and the high-aspect ratio vessel were both required to generate alignment. The diameter of fabricated scaffolds was found to vary significantly with both the collagen concentration of the hydrogel suspension and the diameter of conduits used for fabrication. Additionally, the size of individual aligned topographical features was significantly dependent on the conduit diameter and the freezing temperature. When cultured on aligned collagen scaffolds, both rat dermal fibroblasts and axons emerging from chick dorsal root ganglia explants demonstrated elongated, aligned morphology and growth on the aligned topographical features. Overall, this method presents a simple means for generating aligned collagen scaffolds that can be applied to a wide variety of tissue types, particularly those where such alignment is critical to native function.

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.

The biophysical interactions between cells and type I collagen are controlled by the level of cell adhesion, which is dictated primarily by the density of ligands on collagen and the density of integrin receptors on cells. The native adhesivity of collagen was modulated by covalently grafting glycine–arginine–glycine–aspartic acid–serine (GRGDS), which includes the bioactive RGD sequence, or glycine–arginine–aspartic acid–glycine–serine (GRDGS), which includes the scrambled RDG sequence, to collagen with the hetero-bifunctional coupling agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The peptide-grafted collagen self-assembled into a fibrillar gel with negligible changes in gel structure and rheology. Rat dermal fibroblasts (RDFs) and human smooth muscle cells demonstrated increased levels of adhesion on gels prepared from RGD-grafted collagen, and decreased levels of adhesion on RDG-grafted collagen. Both cell types demonstrated an increased ability to compact free-floating RGD-grafted collagen gels, and an impaired ability to compact RDG-grafted gels. RDF migration on and within collagen was increased with RDG-grafted collagen and decreased with RGD-grafted collagen, and dose–response experiments indicated a biphasic response of RDF migration to adhesion. Smooth muscle cells demonstrated similar, though not statistically significant, trends. The ability to both positively and negatively modulate cell adhesion to collagen increases the versatility of this natural biomaterial for regenerative therapies.

Although molecular and physical mechanisms of fibroblast matrix assembly have been widely investigated, the role of adhesive ligand presentation on matrix assembly has only been recently probed (Pereira et al.Tissue Eng., 2007). In the present study, various-sized albumin-derived nanocarriers (ANCs) were fabricated as nanoscale organization units for functionalization with the cell adhesion domain of fibronectin. The adhesion, morphology, and matrix assembly of human dermal fibroblasts were compared on substrate-deposited, ligand-ANCs of varying size. At early time points, fibroblast attachment, stress fiber formation, and spreading were higher on functionalized, larger-sized carriers than on smaller carriers. Matrix assembly was greatest at the highest ligand density on larger nanocarriers but was undetectable at the same ligand density on smaller carriers. Tracking of fluorophore-encapsulated ANCs showed that larger carriers were displaced less than smaller carriers and that atomic force microscopy of ligand-ANCs binding to adherent cells demonstrated that the larger ligand-ANCs required larger dissociation forces. Taken together, these data suggest that the greater inertia of larger adhesive nanocarriers may generate more cellular tension, which in turn, promotes up-regulation of matrix assembly. Thus, the size of the nanocarrier and the density of ligand on that nanocarrier combine to dictate the early kinetics of fibroblast matrix assembly. These insights may be useful for understanding cell–matrix interactions, as well as for development of bioactive materials with defined cell-adhesive activities such as wound repair and matrix remodeling events.

Current models used in our laboratory to assess the migration and traction of a population of cells within biopolymer gels are extended to investigate temporal changes in these parameters during compaction of mechanically constrained gels. The random cell migration coefficient, μ(t) is calculated using a windowing technique by regressing the mean-squared displacement of cells tracked at high magnification in three dimensions with a generalized least squares algorithm for a subset of experimental time intervals, and then shifting the window interval-by-interval until all time points are analyzed. The cell traction parameter, τ0(t), is determined by optimizing the solution of our anisotropic biphasic theory to tissue equivalent compaction. The windowing technique captured simulated sinusoidal and step changes in cell migration superposed on a persistent random walk in simulated cell movement. The optimization software captured simulated time dependence of compaction on cell spreading. Employment of these techniques on experimental data using rat dermal fibroblasts (RDFs) and human foreskin fibroblasts (HFFs) demonstrated that these cells exhibit different migration-traction relationships. Rat dermal fibroblast migration was negatively correlated to traction, suggesting migration was not the driving force for compaction with these cells, whereas human foreskin fibroblast migration was positively correlated to traction.

The dose–response effects of platelet-derived growth factor BB (PDGF-BB) on rat dermal fibroblast (RDF) behavior in mechanically stressed and unstressed type I collagen and fibrin were investigated using quantitative assays developed in our laboratory. In chemotaxis experiments, RDFs responded optimally (P< 0.05) to a gradient of 10 ng/ml PDGF-BB in both collagen and fibrin. In separate experiments, the migration of RDFs and the traction exerted by RDFs in the presence of PDGF-BB (0, 0.1, 1, 10, or 100 ng/ml) were assessed simultaneously in the presence or absence of stress. RDF migration increased significantly (P< 0.05) at doses of 10 and 100 ng/ml PDGF-BB in collagen and fibrin in the presence and absence of stress. In contrast, the effects of PDGF-BB on RDF traction depended on the gel type and stress state. PDGF-BB decreased fibroblast traction in stressed collagen, but increased traction in unstressed collagen (P< 0.05). No statistical conclusion could be inferred for stressed fibrin, but increasing PDGF-BB decreased traction in unstressed fibrin (P< 0.05). These results demonstrate the complex response of fibroblasts to environmental cues and suggest that mechanical resistance to compaction may be a crucial element in dictating fibroblast behavior.

A numerical and experimental hybrid approach is developed to study the constitutive behavior of the central nervous system white matter. A published transversely isotropic hyperelastic strain energy function is reviewed and used to determine stress–strain relationships for three idealized, simple loading scenarios. The proposed constitutive model is simplified to a three-parameter hyperelastic model by assuming the white matter's incompressibility. Due to a lack of experimental data in all three loading scenarios, a finite element model that accounts for microstructural axons and their kinematics is developed to simulate behaviors in simple shear loading scenarios to supplement existing uniaxial tensile test data. The parameters of the transversely isotropic hyperelastic material model are determined regressively using the hybrid data. The results highlight that a hybrid numerical virtual test coupled with experimental data, can determine the transversely isotropic hyperelastic model. It is noted that the model is not limited to small strains and can be applied to large deformations.

We used a new approach, termed dynamic cortical deformation (DCD), to study the neuronal, vascular, and glial responses that occur in focal cerebral contusions. DCD produces experimental contusion by rapidly deforming the cerebral cortex with a transient, nonablative vacuum pulse of short duration (25 milliseconds) to mimic the circumstances of traumatic injury. A neuropathological evaluation was performed on brain tissue from adult rats sacrificed 3 days following induction of either moderate (4 psi, n = 6) or high (8 psi, n = 6) severity DCD. In all animals, DCD produced focal hemorrhagic lesions at the vacuum site without overt damage to other regions. Examination of histological sections showed localized gross tissue and neuronal loss in the cortex at the injury site, with the volume of cell loss dependent upon the mechanical loading (p < 0.001). Axonal pathology shown with neurofilament immunostaining (SMI-31 and SMI-32) was observed in the subcortical white matter inferior to the injury site and in the ipsilateral internal capsule. No axonal injury was observed in the contralateral hemisphere or in any remote regions. Glial fibrillary acidic protein (GFAP) immunostaining revealed widespread reactive astrocytosis surrounding the necrotic region in the ipsilateral cortex. This analysis confirms that rapid mechanical deformation of the cortex induces focal contusions in the absence of primary damage to remote areas 3 days following injury. Although it is suggested that massive release of neurotoxic substances from a contusion may cause damage throughout the brain, these data emphasize the importance of combined injury mechanisms, e.g. mechanical distortion and excitatory amino acid mediated damage, that underlie the complex pathology patterns observed in traumatic brain injury.