Quantification of cell migration: metrics selection to model application.

Cell migration plays an essential role in physiological and pathological states, such as immune response, tissue generation and tumor development. This phenomenon can occur spontaneously or it can be triggered by an external stimuli, including biochemical, mechanical, or electrical cues that induce or direct cells to migrate. The migratory response to these cues is foundational to several fields including neuroscience, cancer and regenerative medicine. Various platforms are available to qualitatively and quantitatively measure cell migration, making the measurements of cell motility straight-forward. Migratory behavior must be analyzed by multiple metrics and then models to connect the measurements to physiological meaning. This review will focus on describing and quantifying cell movement for individual cell migration.

Effect of Intraoperative Electrical Stimulation on Recovery after Rat Sciatic Nerve Isograft Repair.

Peripheral nerve injuries, associated with significant morbidity, can benefit from electrical stimulation (ES), as demonstrated in animal studies through improved axonal growth. This study combined the clinical gold standard of isograft repair in a rat model of sciatic nerve injury to evaluate the effects of intraoperative ES on functional tests and histology. Forty rats underwent a surgically induced gap injury to the right sciatic nerve and subsequent repair with an isograft. Half of these rats were randomly selected to receive 10 min of intraoperative ES. Functional testing, including response time to a heat stimulus and motor functional tests, were conducted. Histology of the sciatic nerves and gastrocnemius muscles were analyzed after 6 and 12 weeks of recovery. Rats that underwent ES treatment showed incremental improvements in motor function between weeks 2 and 12, with a significantly higher push-off response than the no-ES controls after 6 weeks. Although no differences were detected between groups in the sensory testing, significant improvements over time were noted in the ES group. Histology parameters, sciatic nerve measures, and gastrocnemius muscle weights demonstrated nerve recovery over time for both the ES and no-ES control groups. Although ES promoted improvements in motor function comparable to that in previous studies, the benefits of intraoperative ES were not detectable in other metrics of this rat model of peripheral nerve injury. Future work is needed to optimize sensory testing in the rodent injury model and compare electrical activity of collagen scaffolds to native tissue to detect differences.

RGD-Modified Nanofibers Enhance Outcomes in Rats after Sciatic Nerve Injury.

Nerve injuries requiring surgery are a significant problem without good clinical alternatives to the autograft. Tissue engineering strategies are critically needed to provide an alternative. In this study, we utilized aligned nanofibers that were click-modified with the bioactive peptide RGD for rat sciatic nerve repair. Empty conduits or conduits filled with either non-functionalized aligned nanofibers or RGD-functionalized aligned nanofibers were used to repair a 13 mm gap in the rat sciatic nerve of animals for six weeks. The aligned nanofibers encouraged cell infiltration and nerve repair as shown by histological analysis. RGD-functionalized nanofibers reduced muscle atrophy. During the six weeks of recovery, the animals were subjected to motor and sensory tests. Sensory recovery was improved in the RGD-functionalized nanofiber group by week 4, while other groups needed six weeks to show improvement after injury. Thus, the use of functionalized nanofibers provides cues that aid in in vivo nerve repair and should be considered as a future repair strategy.

Novel microgel-based scaffolds to study the effect of degradability on human dermal fibroblasts.

For improved cell integration, tissue engineering scaffolds must be designed to degrade over time. Typically, the chemistry of scaffolds is modified to alter the degradation profile by using different hydrolytic or enzymatic sites within a material. It is more challenging, however, to fabricate self-assembling, injectable scaffolds that provide tunable degradation. Our laboratory has developed microgel-based scaffolds, where individual micron-sized hydrogels are crosslinked to make larger bulk scaffolds. The size of the individual microgels permits injection, and the microgels then self-assemble into a bulk structure and crosslink. We hypothesized that the microgel-based scaffolds can be used to tune degradability by mixing degradable and non-degradable microgels at various ratios within a self-assembling scaffold. Therefore, two types of microgels were fabricated, those composed of polyethylene glycol (PEG) and those composed of a PEG-lactic acid. Importantly, the microgels were similar in size and swelling and had a low polydispersity index due to their method of fabrication. Microgels were then mixed in four ratios to fabricate scaffolds and study how changes in scaffold composition altered the 3D proliferation and morphology of human dermal fibroblasts. Microgel-based scaffolds formed with 100% degradable microgels lost >60% of their mass over the 14 days of the study. Human dermal fibroblasts were mixed within the 3D scaffolds at the time of assembly and all scaffolds had cells with high viability and typical morphology. The scaffolds that had 25%-50% degradable microgels showed statistically increased proliferation of fibroblasts after 1 and 2 weeks over non-degradable scaffolds and those scaffolds with 75% or 100% degradable microgels. Overall, this work demonstrates the development and use of a tunable, self-assembled, microgel-based scaffold to investigate the effects of degradability on cellular response.

The interplay of peptide affinity and scaffold stiffness on neuronal differentiation of neural stem cells.

Cells are sensitive to physical cues in their environment, such as the stiffness of the substrate, peptide density, and peptide affinity. Understanding how neural stem cells (NSCs) sense and respond to these matrix cues has the potential to improve disease outcome, particularly if a regenerative response can be exploited. While the material properties are known to influence other stem cells, little is known about how NSC differentiation is altered by this interplay of mechanical, or bulk properties, with peptide concentration and affinity, or microscale properties. We are interested in the combined effect of bulk and microscale features in an in vitro hydrogel model and therefore we investigated NSC differentiation by focusing on integrin interactions via RGD peptide affinity and concentration. Our studies demonstrated that the peptide concentration affected adhesion as there were more cells on scaffolds with 1 mM RGD than 2.5 mM RGD. The hydrogel stiffness affected neurite length in differentiating NSCs, as 0.1-0.8 kPa substrates promoted greater neurite extension than 4.2-7.9 kPa substrates. The NSCs differentiated towards ß-ΙΙΙ tubulin positive cells on scaffolds with RGD after 7 days and those scaffolds containing 1 mM linear or cyclic RGD had longer neurite extensions than scaffolds containing 0.1 or 2.5 mM RGD. While peptide affinity had a lesser effect on the NSC response in our hydrogel system, blocking actin, myosin II, or integrin interactions resulted in changes to the cell morphology and focal adhesion assembly. Overall, these results demonstrated NSCs are more responsive to a change in tissue stiffness than peptide affinity in the range of gels tested, which may influence design of materials for neural tissue engineering.

Electrical Stimulation Increases Random Migration of Human Dermal Fibroblasts.

Exogenous electrical stimulation (ES) has been investigated as a therapy for chronic wounds, as the skin produces currents and electrical fields (EFs) during wound healing. ES therapies operate by applying small EFs to the skin to mimic the transepithelial potentials that occur during the granulation phase of wound healing. Here, we investigated the effect of short duration (10 min) ES on the migration of HDFs using various magnitudes of physiologically relevant EFs. We modeled cutaneous injury by culturing HDFs in custom chambers that allowed the application of ES and then performed timelapse microscopy on a standard wound model. Using MATLAB to process cell coordinate data, we determined that the cells were migrating randomly and fit mean squared displacement data to the persistent random walk equation using nonlinear least squares regression analysis. Results indicated that application of 25-100 mV/mm DC EFs to HDFs on either uncoated or FN-coated surfaces demonstrated no significant changes in viability or proliferation. Of significance is that the HDFs increased random migration behavior under some ES conditions even after 10 min, providing a mechanism to enhance wound healing.

Tuning the Mechanical Properties of Poly(Ethylene Glycol) Microgel-Based Scaffolds to Increase 3D Schwann Cell Proliferation.

2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel-based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol-ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈ 5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

Stimulation Frequency Alters the Dorsal Root Ganglion Neurite Growth and Directionality In Vitro.

OBJECTIVE: To improve peripheral nerve repair, new techniques to increase the speed of regeneration are required. Studies have shown that the electrical stimulation can enhance nerve regeneration; however, stimulation parameters that regulate the growth increases are unknown. The objective of this study was to examine dorsal root ganglion (DRG) neurite extension, directionality, and density after using methods to specifically control ac electrical field intensity and frequency exposure. METHODS: Chick DRG explants were exposed to 20-Hz, 200-Hz, 1-MHz, and 20-MHz sinusoidal electric field of 17.86 V/m, and tissue parameters were measured. RESULTS: Results show that neurite extension and directionality were influenced by frequency; however, the ratio of support cell emigration with respect to neurite extension from the DRG body was not. These results were further verified through finite-element modeling of intracellular calcium, which show that higher frequencies have minimal effect on intracellular calcium. CONCLUSION: In conclusion, these results demonstrate that 1) directional growth of neurites within EFs can be achieved, 2) high-frequency stimulation in megahertz does not enhance or impair the neurite growth, and 3) low-frequency stimulation affects the growth and directionality. SIGNIFICANCE: The significance of this study is the direct comparison of neurite extension after high stimulation frequencies (megahertz) with typical low-frequency fields (20 and 200 Hz), and modeling the results with finite-element modeling.

Mechanotransduction of Neural Cells Through Cell-Substrate Interactions.

Neurons and neural stem cells are sensitive to their mechanical and topographical environment, and cell-substrate binding contributes to this sensitivity to activate signaling pathways for basic cell functions. Many transmembrane proteins transmit signals into and out of the cell, including integrins, growth factor receptors, G-protein-coupled receptors, cadherins, cell adhesion molecules, and ion channels. Specifically, integrins are one of the main transmembrane proteins that transmit force across the cell membrane between a cell and its extracellular matrix, making them critical in the study of cell-material interactions. This review focuses on mechanotransduction, defined as the conversion of force a cell generates through cell-substrate bonds to a chemical signal, of neural cells. The chemical signals relay information via pathways through the cellular cytoplasm to the nucleus, where signaling events can affect gene expression. Pathways and the cellular response initiated by substrate binding are explored to better understand their effect on neural cells mechanotransduction. As the results of mechanotransduction affect cell adhesion, cell shape, and differentiation, knowledge regarding neural mechanotransduction is critical for most regenerative strategies in tissue engineering, where novel environments are developed to improve conduit design for central and peripheral nervous system repair in vivo.

Development of a High-Throughput Ultrasound Technique for the Analysis of Tissue Engineering Constructs.

Development of hydrogel-based tissue engineering constructs is growing at a rapid rate, yet translation to patient use has been sluggish. Years of costly preclinical tests are required to predict clinical performance and safety of these devices. The tests are invasive, destructive to the samples and, in many cases, are not representative of the ultimate in vivo scenario. Biomedical imaging has the potential to facilitate biomaterial development by enabling longitudinal noninvasive device characterization directly in situ. Among the various available imaging modalities, ultrasound stands out as an excellent candidate due to low cost, wide availability, and a favorable safety profile. The overall goal of this work was to demonstrate the utility of clinical ultrasound in longitudinal characterization of 3D hydrogel matrices supporting cell growth. Specifically, we developed a quantitative technique using clinical B-mode ultrasound to differentiate collagen content and fibroblast density within poly(ethylene glycol) (PEG) hydrogels and validated it in an in vitro phantom environment. By manipulating the hydrogel gelation, differences in ultrasound signal intensity were found between gels with collagen fibers and those with non-fiber forming collagen, indicating that the technique was sensitive to the configuration of the protein. At a collagen density of 2.5 mg/mL collagen, fiber forming collagen had a significantly increased signal intensity of 14.90 ± 2.58 × 10(-5) a.u. compared to non-fiber forming intensity at 2.74 ± 0.36 × 10(-5) a.u. Additionally, differences in intensity were found between living and fixed fibroblasts, with an increased signal intensity detected in living cells (5.00 ± 0.80 × 10(-5) a.u. in 1 day live cells compared to 2.26 ± 0.39 × 10(-5) a.u.in fixed cells at a concentration of 1 × 10(6) cells/mL in gels containing collagen). Overall, there was a linear correlation >0.90 for ultrasound intensity with increasing cell density. Results demonstrate the feasibility of using clinical ultrasound for characterization of PEG-based hydrogels in a tissue-mimicking phantom. The approach is clinically-relevant and could, with further validation, be utilized to nondestructively monitor in vivo performance of implanted tissue engineering scaffolds over time in preclinical and clinical settings.

Short-term electrical stimulation to promote nerve repair and functional recovery in a rat model.

PURPOSE: To evaluate the effect of duration of electrical stimulation on peripheral nerve regeneration and functional recovery. Based on previous work, we hypothesized that applying 10 minutes of electrical stimulation to a 10-mm rat sciatic nerve defect would significantly improve nerve regeneration and functional recovery compared with the non-electrical stimulation group. METHODS: A silicone tube filled with a collagen gel was used to bridge a 10-mm nerve defect in rats, and either 10 minutes or 60 minutes of electrical stimulation was applied to the nerve during surgery. Controls consisted of a silicone tube with collagen gel and no electrical stimulation or an isograft. We analyzed recovery over a 12-week period, measuring sciatic functional index and extensor postural thrust scores and concluding with histological examination of the nerve. RESULTS: Functional assessment scores at week 12 increased 24% in the 10-minute group as compared to the no stimulation control group. Electrical stimulation of either 10 or 60 minutes improved the number of nerve fibers over no stimulation. Additionally, the electrical stimulation group's histomorphometric analysis was not different from the isograft group. CONCLUSIONS: Several previous studies have demonstrated the effectiveness of 60-minute stimulations on peripheral nerve regeneration. This study demonstrated that an electrical stimulation of 10 minutes enhanced several functional and histomorphometric outcomes of nerve regeneration and was overall similar to a 60-minute stimulation over 12 weeks. CLINICAL RELEVANCE: Decreasing the electrical stimulation time from 60 minutes to 10 minutes provided a potential clinically feasible and safe method to enhance nerve regeneration and functional recovery.

Short duration electrical stimulation to enhance neurite outgrowth and maturation of adult neural stem progenitor cells.

New therapies are desperately needed for human central nervous system (CNS) regeneration to circumvent the lack of innate regenerative ability following traumatic injuries. Previously attempted therapies have been stymied by barriers to CNS regeneration largely because of protective mechanisms such as the blood brain barrier, inhibitory molecules, and glial scar formation. The application of electric stimulation (ES) has shown promise for enhancing peripheral nervous system regeneration, but is in its infancy in CNS regeneration. The objective of this study is to better understand how short duration ES can be harnessed to direct adult neural stem progenitor cell (NSPC) neurogenesis, neurite extension, and maturation. Herein, NSPCs were exposed to physiological levels of electrical stimulation of 0.53 or 1.83 V/m (applied power supply setting of 1.2 and 2.5 V) of direct current (DC) for 10 min/days for 2 days with a total differentiation time of 3 days. Culturing conditions consisted of either mitogenic growth factors or the neuronal differentiation factor interferon-γ (IFN-γ). Stimulated NSPCs showed lengths that were over five times longer than unstimulated controls (112.0 ± 88.8 µm at 0.53 V/m vs. 21.3 ± 8.5 µm for 0 V/m with IFN-γ) with the longest neurites reaching up to 600 µm. Additionally, ES resulted in mature neuronal morphologies and signs of differentiation through positive ßIII tubulin, neuronal nuclei (NeuN), and better organized filamentous-actin (f-actin) staining with growth cone formation. Additionally, the neurites and soma of stimulated NSPCs showed increases in intracellular Ca(2+) during stimulation, signifying the presence of functional neurons capable of electrical conductance and communication with other cells. Our study demonstrates that short stimulation times (10 min/ day) result in significant neurite extension of stem cells in a quick time frame (3 days). This ES modality is potentially advantageous for promoting axon re-growth at an injury site using delivered adult stem cells; however, significant work still remains to understand both the delivery approach of cells as well as ES application in vivo.

The dynamics of shrinking and expanding drug-loaded microspheres: A semi-empirical approach.

The dynamics of shrinking and expanding drug-loaded microspheres were studied using a diffusion equation in spherical coordinates. A movable boundary condition was incorporated as a convection term in the original model. The resulting convective-diffusive problem was solved using Laplace transform techniques with the Bromwich integral and the residue theorem. Analytical solutions were derived for the general case of shrinking or expanding microspheres and three particular kinetics expressions: linear growth, exponential swelling and exponential shrinking. Simulations show that microspheres with fast-swelling kinetics released their therapeutic cargo at a relatively slow rate in the first two cases. Ninety-nine percent of the medication was delivered at four times the effective time constant. In line with laboratory studies using bovine serum albumin, an increase in the shrinking rate led to a fast release of the medication from its carrier. The method was applied to analyze insulin transport through spherical Ca-alginate beads. A good agreement was noted between predicted and experimental data. The theoretical effective time constant was 114.0 min.

Comparison of neurite growth in three dimensional natural and synthetic hydrogels.

Extracellular matrix incorporated within a scaffold plays an important role in assisting cell behavior in neural tissue engineering. In this study, we investigated how the concentration of fibronectin (FN) affected neurite growth when incorporated within a synthetic polymer gel made of poly(ethylene glycol) (PEG) or a natural polymer gel of collagen I. Mechanical and chemical properties of the scaffold were varied by using a range of concentrations of gels and FN. Rheology was used to determine the mechanical stiffness of hydrogels and neurite length and viability were measured to evaluate cell response. In both types of gels, increasing the concentration of the base scaffold (PEG or collagen) increased the mechanical stiffness as denoted by G∗. Neurite lengths in PEG gels increased with increasing FN concentration and decreased with increasing G∗. In collagen gels, FN reduced neurite extension for the lowest concentrations of collagen (0.4-0.6 mg/mL) while FN increased neurite extension for mid and high collagen concentrations (1.0-2.0 mg/mL). The results from these two different scaffolds indicate that both stiffness and FN concentration impact the growth of the neurite and that the addition of small amounts of FN (100 µg/ml) permits PEG gels to perform on par with similar stiffness collagen gels.

Characteristics of precipitation-formed polyethylene glycol microgels are controlled by molecular weight of reactants.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e. low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

The impact of laminin on 3D neurite extension in collagen gels.

The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL(-1) supplemented with laminin at concentrations of 0, 1, 10, or 100 µg mL(-1). When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, α1, α3, α6 and ß1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.

Modular poly(ethylene glycol) scaffolds provide the ability to decouple the effects of stiffness and protein concentration on PC12 cells.

This research focused on developing a modular poly(ethylene glycol) (PEG) scaffold, assembled from PEG microgels and collagen I, to provide an environment to decouple the chemical and mechanical cues within a three-dimensional scaffold. We first characterized the microgel fabrication process, examining the size, polydispersity, swelling ratio, mesh size and storage modulus of the polymer particles. The resulting microgels had a low polydispersity index, PDI=1.08, and a diameter of ~1.6 µm. The mesh size of the microgels, calculated from the swelling ratio, was 47.53 Å. Modular hydrogels (modugels) were then formed by compacting N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimidyl group-activated microgels with PEG-4arm-amine and 0, 1, 10, or 100 µg ml(-1) collagen. The stiffness (G(∗)) of the modugels was not significantly altered with the addition of collagen, allowing for modification of the chemical environment independent from the mechanical properties of the scaffold. PC12 cell aggregation increased in modugels as collagen concentrations increased and cell viability in modugels was improved over bulk PEG hydrogels. Overall, these results indicate that further exploration of modular scaffolds formed from microgels could allow for a better understanding of the relationship between the chemical and mechanical properties and cellular behavior.

Student award winner in the undergraduate's degree category for the Society for Biomaterials 35th Annual Meeting, Orlando, Florida, April 13-16, 2011. Neurite growth in PEG gels: effect of mechanical stiffness and laminin concentration.

Within a 3D environment, the chemical and mechanical properties of a scaffold can significantly influence nerve behavior. How these properties influence with nerve cells is important for optimizing neurite extension within a scaffold. The purpose of this study was to investigate the effect of low concentration poly(ethylene glycol) (PEG) with added laminin on 3D growth of dissociated dorsal root ganglia. Because of its high affinity for neurite adhesion and ability to promote extension, laminin was conjugated to the PEG chain, as well as mixed in the gel, at various concentrations to provide chemical cues. Gel stiffness, as determined by G*, significantly decreased with decreasing PEG concentration and with increasing laminin conjugate. Extension within the gels increased as the concentration of laminin increased with no difference between how laminin was presented (mixed or conjugated) to the cells. For example, in 3% PEG, extension increased from 92.29 ± 5.27 µm to 146.35 ± 13.12 µm as laminin conjugate concentration increased from plain to 100 µg/ml. Results indicated that the chemical properties of the scaffold influenced neurite growth more than the mechanical properties as laminin concentration had a greater impact on growth than the stiffness of the gel over the range studied. Neurite length as a function of scaffold stiffness and adhesion properties was also characterized and demonstrated a positive linear relationship between rate of neurite extension and laminin concentration. This study further demonstrates the importance of characterizing interactions between cell behavior and the chemical and mechanical environment.

Poly(ethylene glycol) microparticles produced by precipitation polymerization in aqueous solution.

Methods were developed to perform precipitation photopolymerization of PEG-diacrylate. Previously, comonomers have been added to PEG when precipitation polymerization was desired. In the present method, the LCST of the PEG itself was lowered by the addition of the kosmotropic salt sodium sulfate to an aqueous solution. Typical of a precipitation polymerization, small microparticles or microspheres (1-5 µm) resulted with relatively low polydispersity. However, aggregate formation was often severe, presumably because of a lack of stabilization of the phase-separated colloids. Microparticles were also produced by copoymerization of PEG-diacrylate with acrylic acid or aminoethylmethacrylate. The comonomers affected the zeta potential of the formed microparticles but not the size. The carboxyl groups of acrylic-acid-containing PEG microparticles were activated, and scaffolds were formed by mixing with amine-containing PEG microparticles. Although the scaffolds were relatively weak, human hepatoma cells showed excellent viability when present during microparticle cross-linking.