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Measuring human mesenchymal stem cell remodeling in hydrogels with a step-change in elastic modulus
Abstract
Human mesenchymal stem cells (hMSCs) are instrumental in the wound healing process. They migrate to wounds from their native niche in response to chemical signals released during the inflammatory phase of healing. At the wound, hMSCs downregulate inflammation and regulate tissue regeneration. Delivering additional hMSCs to wounds using cell-laden implantable hydrogels has the potential to improve healing outcomes and restart healing in chronic wounds. For these materials to be effective, cells must migrate from the scaffold into the native tissue. This requires cells to traverse a step-change in material properties at the implant-tissue interface. Migration of cells in material with highly varying properties is not well characterized. We measure 3D encapsulated hMSC migration and remodeling in a well-characterized hydrogel with a step-change in stiffness. This cell-degradable hydrogel is composed of 4-arm poly(ethylene glycol)-norbornene cross-linked with an enzymatically-degradable peptide. The scaffold is made with two halves of different stiffnesses separated by an interface where stiffness changes rapidly. We characterize changes in structure and rheology of the pericellular region using multiple particle tracking microrheology (MPT). MPT measures Brownian motion of embedded particles and relates it to material rheology. We measure more remodeling in the soft region of the hydrogel than the stiff region on day 1 post-encapsulation and similar remodeling everywhere on day 6. In the interface region, we measure hMSC-mediated remodeling along the interface and migration towards the stiff side of the scaffold. These results can improve materials designed for cell delivery from implants to a wound to enhance healing.
... Degradation is essential for the formation of protrusions and we observe that Stiff-Deg materials promote longer and higher filopodia number compared to Soft-NoDeg materials. The higher initial stiffness of the degradable material, might also lead to a quicker degradation, as MMP production is increased in stiffer matrices [40]. The control hydrogels formed with a non-degradable version of the peptide (MMP-scramble), showed that cells do not form filopodia in nondegradable materials (Supplementary Fig. 7). ...
In nature, tissues are patterned, but most biomaterials used in human applications are not. Patterned biomaterials offer the opportunity to mimic spatially segregating biophysical and biochemical properties found in nature. Engineering such properties allows to study cell-matrix interactions in anisotropic matrices in great detail. Here, we developed alginate-based hydrogels with patterns in stiffness and degradation, composed of distinct areas of soft non-degradable (Soft-NoDeg) and stiff degradable (Stiff-Deg) material properties. The hydrogels exhibit emerging patterns in stiffness and degradability over time, taking advantage of dual crosslinking: Diels-Alder covalent crosslinking (norbornene-tetrazine, non degradable) and UV-mediated peptide crosslinking (matrix metalloprotease sensitive peptide, enzymatically degradable). The materials were mechanically characterized using rheology for single-phase and surface micro-indentation for patterned materials. 3D encapsulated mouse embryonic fibroblasts (MEFs) allowed to characterize the anisotropic cell-matrix interaction in terms of cell morphology by employing a novel image-based quantification tool. Live/dead staining showed no differences in cell viability but distinct patterns in proliferation, with higher cell number in Stiff-Deg materials at day 14. Patterns of projected cell area became visible already at day 1, with larger values in Soft-NoDeg materials. This was inverted at day 14, when larger projected cell areas were identified in Stiff-Deg. This shift was accompanied by a significant decrease in cell circularity in Stiff-Deg. The control of anisotropic cell morphology by the material patterns was also confirmed by a significant increase in filopodia number and length in Stiff-Deg materials. The novel image-based quantification tool was useful to spatially visualize and quantify the anisotropic cell response in 3D hydrogels with stiffness-degradation spatial patterns. Our results show that patterning of stiffness and degradability allows to control cell anisotropic response in 3D and can be quantified by image-based strategies. This allows a deeper understanding of cell-matrix interactions in a multicomponent material.
This paper describes the preparation of an environmentally friendly and biodegradable hydrogel type electromagnetic shielding material by using common salt to induce the self-assembly of sodium alginate.
The effect of nanoscale defects on nanoparticle dynamics in defective tetra-poly(ethylene glycol) (tetra-PEG) hydrogels is investigated using single particle tracking. In a swollen nearly homogeneous hydrogel, PEG-functionalized quantum dot (QD) probes with a similar hydrodynamic diameter (dh = 15.1 nm) to the mesh size (〈ξs〉 = 16.3 nm), are primarily immobile. As defects are introduced to the network by reaction-tuning, both the percentage of mobile QDs and the size of displacements increase as the number and size of the defects increase with hydrolysis time, although a large portion of the QDs remain immobile. To probe the effect of nanoparticle size on dynamics in defective networks, the transport of dh = 47.1 nm fluorescent polystyrene (PS) and dh = 9.6 nm PEG-functionalized QDs is investigated. The PS nanoparticles are immobile in all hydrogels, even in highly defective networks with an open structure. Conversely, the smaller QDs are more sensitive to perturbations in the network structure with an increased percentage of mobile particles and larger diffusion coefficients compared to the larger QDs and PS nanoparticles. The differences in nanoparticle mobility as a function of size suggests that particles of different sizes probe different length scales of the defects, indicating that metrics such as the confinement ratio alone cannot predict bulk dynamics in these systems. This study provides insight into designing hydrogels with controlled transport properties, with particular importance for degradable hydrogels for drug delivery applications.
Hydrogels serve as valuable tools for studying cell–extracellular matrix interactions in three-dimensional environments that recapitulate aspects of native extracellular matrix. However, the impact of early protein deposition on cell behaviour within hydrogels has largely been overlooked. Using a bio-orthogonal labelling technique, we visualized nascent proteins within a day of culture across a range of hydrogels. In two engineered hydrogels of interest in three-dimensional mechanobiology studies—proteolytically degradable covalently crosslinked hyaluronic acid and dynamic viscoelastic hyaluronic acid hydrogels—mesenchymal stromal cell spreading, YAP/TAZ nuclear translocation and osteogenic differentiation were observed with culture. However, inhibition of cellular adhesion to nascent proteins or reduction in nascent protein remodelling reduced mesenchymal stromal cell spreading and nuclear translocation of YAP/TAZ, resulting in a shift towards adipogenic differentiation. Our findings emphasize the role of nascent proteins in the cellular perception of engineered materials and have implications for in vitro cell signalling studies and application to tissue repair. The extracellular matrix surrounding cells plays a significant role in their behaviour. The spreading, mechanosensing and differentiation of mesenchymal stem cells are shown to be dependent on the early deposition and remodelling of local nascent proteins within degradable and viscoelastic hydrogels.
Significance
Mechanobiology is receiving an increasing amount of focus, but the mechanics of cell-substrate behavior are often neglected in cell biology. As such, novel materials and systems that are simple to build and share in a nonengineering laboratory are sorely needed. We have fabricated gradient hydrogels with continuous linear gradients above and below the durotactic threshold, making it possible to pinpoint optimal stiffness values for a wide range of biological phenomena without the confounding effects of durotaxis. This system has the potential for wide adoption in the cell biology community because of its ease of fabrication, simple material ingredients, and wide gradient possibilities in a single well.
Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel's initial elastic modulus, degradation, and cell-adhesion-ligand density. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture.
Studies focused on understanding the role of matrix biophysical signals on cells, especially those when cells are encapsulated in hydrogels that are locally remodelled, are often complicated by appropriate methods to measure differences between the bulk and local material properties. From this perspective, stress-relaxing materials that allow long-term culture of embedded cells provide an opportunity to elucidate aspects of this biophysical signalling. In particular, rheological characterization of the stress relaxation properties allows one to link a bulk material measurement to local aspects of cellular functions by quantifying the corresponding cellular forces that must be applied locally. Here, embryonic stem cell-derived motor neurons were encapsulated in a well-characterized covalently adaptable bis-aliphatic hydrazone crosslinked PEG hydrogel, and neurite outgrowth was observed over time. Using fundamental physical relationships describing classical mechanics and viscoelastic materials, we calculated the forces and energies involved in neurite extension, the results of which provide insight to the role of biophysical cues on this process.
Mesenchymal stem cells (MSC) are present throughout the body and are thought to play a role in tissue regeneration and control of inflammation. MSC can be easily expanded in vitro and their potential as a therapeutic option for degenerative and inflammatory disease is therefore intensively investigated. Whilst it was initially thought that MSC would replace dysfunctional cells and migrate to sites of injury to interact with inflammatory cells, experimental evidence indicates that the majority of administered MSC get trapped in capillary networks and have a short life span. In this review, we discuss current knowledge on the migratory properties of endogenous and exogenous MSC and confer on how culture-induced modifications of MSC may affect these properties. Finally, we will discuss how, despite their limited survival, administered MSC can bring about their therapeutic effects.
We present a new method for measuring the linear viscoelastic shear moduli of complex fluids. Using photodiode detection of laser light scattered from a thermally excited colloidal probe sphere, we track its trajectory and extract the moduli using a frequency-dependent Stokes-Einstein equation. Spectra obtained for polyethylene oxide in water are in excellent agreement with those found mechanically and using diffusing wave spectroscopy. Since only minute sample volumes are required, this method is well suited for biomaterials of high purity, as we demonstrate with a concentrated DNA solution.
Human mesenchymal stem cell (hMSC) migration and recruitment play a critical role during bone fracture healing. Within the complex 3D in vivo microenvironment, hMSC migration is regulated through a myriad of extracellular cues. Here, we use a thiol-ene photopolymerized hydrogel to recapitulate structural and bioactive inputs in a tunable manner to understand their role in regulating 3D hMSC migration. Specifically, peptide-functionalized poly(ethylene glycol) hydrogels were used to encapsulate hMSC while varying the crosslinking density, 0.18 ± 0.02 - 1.60 ± 0.04 mM, and the adhesive ligand density, 0.001 to 1.0 mM. Using live cell videomicroscopy migratory cell paths were tracked and fit to a persistent random walk model. It was shown that hMSC migrating through the lowest crosslinking density and highest adhesivity had more sustained polarization, higher migrating speeds (17.6 ± 0.9 μm/hr), and higher cell spreading (Elliptical Form Factor = 3.9 ± 0.2). However, manipulation of these material properties did not significantly affect migration persistence. Further, there was a monotonic increase in cell speed and spreading with increasing adhesivity showing a lack of the biphasic trend seen in two dimensional cell migration. Immunohistochemistry showed well-formed actin fibers and β1 integrin staining at the ends of stress fibers. This thiol-ene platform provides a highly tunable substrate to characterize 3D hMSC migration with application as an implantable cell carrier platform or for the recruitment of endogenous hMSC in vivo.
The primary function of the skin is to serve as a protective barrier against the environment. Loss of the integrity of large portions of the skin as a result of injury or illness may lead to major disability or even death. Every year in the United States more than 1.25 million people have burns1 and 6.5 million have chronic skin ulcers caused by pressure, venous stasis, or diabetes mellitus.2 The primary goals of the treatment of wounds are rapid wound closure and a functional and aesthetically satisfactory scar. Recent advances in cellular and molecular biology have greatly expanded our understanding . . .
Wound healing requires a coordinated interplay among cells, growth factors, and extracellular matrix proteins. Central to this process is the endogenous mesenchymal stem cell (MSC), which coordinates the repair response by recruiting other host cells and secreting growth factors and matrix proteins. MSCs are self-renewing multipotent stem cells that can differentiate into various lineages of mesenchymal origin such as bone, cartilage, tendon, and fat. In addition to multilineage differentiation capacity, MSCs regulate immune response and inflammation and possess powerful tissue protective and reparative mechanisms, making these cells attractive for treatment of different diseases. The beneficial effect of exogenous MSCs on wound healing was observed in a variety of animal models and in reported clinical cases. Specifically, they have been successfully used to treat chronic wounds and stimulate stalled healing processes. Recent studies revealed that human placental membranes are a rich source of MSCs for tissue regeneration and repair. This review provides a concise summary of current knowledge of biological properties of MSCs and describes the use of MSCs for wound healing. In particular, the scope of this review focuses on the role MSCs have in each phase of the wound-healing process. In addition, characterization of MSCs containing skin substitutes is described, demonstrating the presence of key growth factors and cytokines uniquely suited to aid in wound repair.
Methods to precisely control growth factor presentation in a local and sustained fashion are of increasing interest for a number of complex tissue engineering applications. The cytokine transforming growth factor beta (TGFβ) plays a key role in promoting the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Traditional chondrogenic approaches utilize soluble delivery, an approach with limited application for clinical translation. In this work, we introduce a reactive thiol onto TGFβ and covalently tether the growth factor into poly(ethylene glycol) (PEG) hydrogels using a photoinitiated thiol-acrylate polymerization mechanism. We demonstrate the bioactivity of thiolated TGFβ, before and after polymerization, using a SMAD2 reporter cell line. hMSCs were encapsulated in PEG hydrogels with and without tethered TGFβ, and subsequently assayed for glycosaminoglycan and collagen II production as indicators of chondrogenesis. Over a 21-day time course, tethered TGFβ promoted chondrogenesis at levels similar to a positive control using solubly dosed growth factor. These results provide evidence that tethered TGFβ materials can be successfully used to promote chondrogenic differentiation of MSCs.
In the knee joint, the purpose of the cartilage-bone interface is to maintain structural integrity of the osteochondral unit during walking, kneeling, pivoting, and jumping--during which tensile, compressive, and shear forces are transmitted from the viscoelastic articular cartilage layer to the much stiffer mineralized end of the long bone. Mature articular cartilage is integrated with subchondral bone through a approximately 20 to approximately 250 microm thick layer of calcified cartilage. Inside the calcified cartilage layer, perpendicular chondrocyte-derived collagen type II fibers become structurally cemented to collagen type I osteoid deposited by osteoblasts. The mature mineralization front is delineated by a thin approximately 5 microm undulating tidemark structure that forms at the base of articular cartilage. Growth plate cartilage is anchored to epiphyseal bone, sometimes via a thin layer of calcified cartilage and tidemark, while the hypertrophic edge does not form a tidemark and undergoes continual vascular invasion and endochondral ossification (EO) until skeletal maturity upon which the growth plates are fully resorbed and replaced by bone. In this review, the formation of the cartilage-bone interface during skeletal development and cartilage repair, and its structure and composition are presented. Animal models and human anatomical studies show that the tidemark is a dynamic structure that forms within a purely collagen type II-positive and collagen type I-negative hyaline cartilage matrix. Cartilage repair strategies that elicit fibrocartilage, a mixture of collagen type I and type II, are predicted to show little tidemark/calcified cartilage regeneration and to develop a less stable repair tissue-bone interface. The tidemark can be regenerated through a bone marrow-driven growth process of EO near the articular surface.
Step-growth, radically mediated thiol-norbornene photopolymerization is used to create versatile, stimuli-responsive poly(ethylene glycol)-co-peptide hydrogels (see image). The reaction is cytocompatible and allows for the encapsulation of human mesenchymal stem cells with a viability greater than 95%. Cellular spreading is dictated via three-dimensional biochemical photopatterning.
Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ∼8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ∼0.6±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior.
The mechanical properties of the extracellular matrix (ECM) can exert significant influence in determining cell fate. Human mesenchymal stem cells (MSCs) grown on substrates with varying stiffness have been shown to express various cell lineage markers, without the use of toxic DNA demethylation agents or complex cocktails of expensive growth factors. Here we investigated the myogenic and osteogenic potential of various polyacrylamide gel substrates that were coated with covalently bound tissue-specific ECM proteins (collagen I, collagen IV, laminin, or fibronectin). The gel-protein substrates were shown to support the growth and proliferation of MSCs in a stiffness-dependent manner. Higher stiffness substrates encouraged up to a 10-fold increase in cell number over lower stiffness gels. There appears to be definitive interplay between substrate stiffness and ECM protein with regard to the expression of both osteogenic and myogenic transcription factors by MSCs. Of the 16 gel-protein combinations investigated, osteogenic differentiation was found to occur significantly only on collagen I-coated gels with the highest modulus gel tested (80 kPa). Myogenic differentiation occurred on all gel-protein combinations that had stiffnesses >9 kPa but to varying extents as ascertained by MyoD1 expression. Peak MyoD1 expression was seen on gels with a modulus of 25 kPa coated in fibronectin, with similar levels of expression observed on 80-kPa collagen I-coated gels. The modulation of myogenic and osteogenic transcription factors by various ECM proteins demonstrates that substrate stiffness alone does not direct stem cell lineage specification. This has important implications in the development of tailored biomaterial systems that more closely mimic the microenvironment found in native tissues.
Due to mild reaction conditions and temporal and spatial control over material formation, photopolymerization has become a valuable technique for the encapsulation of living cells in three dimensional, hydrated, biomimetic materials. For such applications, 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (I2959) is the most commonly used photoinitiator (by virtue of its moderate water solubility), yet this initiator has an absorption spectrum that is poorly matched with wavelengths of light generally regarded as benign to living cells, limiting the rate at which it may initiate polymerization in their presence. In contrast, acylphosphine oxide photoinitiators, generally exhibit absorption spectra at wavelengths suitable for cell encapsulation, yet commercially available initiators of this class have low water solubility. Here, a water soluble lithium acylphosphinate salt is evaluated for its ability to polymerize diacrylated poly(ethylene glycol) (PEGDA) monomers rapidly into hydrogels, while maintaining high viability during direct encapsulation of cells. Through rheometric measurements, the time to reach gelation of a PEGDA solution with the phosphinate initiator is one tenth the time for that using I2959 at similar concentrations, when exposed to 365 nm light. Further, polymerization with the phosphinate initiator at 405 nm visible light exposure is achieved with low initiator concentrations and light intensities, precluded in polymerizations initiated with I2959 by its absorbance profile. When examined 24h after encapsulation, survival rates of human neonatal fibroblasts encapsulated in hydrogels polymerized with the phosphinate initiator exceed 95%, demonstrating the cytocompatibility of this initiating system.
Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (k(cat) and K(m)) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 microg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.
Biological materials have length scale dependent structure enabling complex cell-material interactions and driving cellular processes. Synthetic biomaterials are designed to mimic aspects of these biological materials for applications including enhancing cell delivery during wound healing. To mimic native microenvironments, we must understand how cells manipulate their surroundings over several length scales. Our work characterizes length scale dependent rheology in a well-established 3D cell culture platform for human mesenchymal stem cells (hMSCs). hMSCs re-engineer their microenvironment through matrix metalloproteinase (MMP) secretions and cytoskeletal tension. Remodeling occurs across length scales: MMPs degrade cross-links on nanometer scales resulting in micrometer-sized paths that hMSCs migrate through, eventually resulting in bulk scaffold degradation. We use multiple particle tracking microrheology (MPT) and bi-disperse MPT to characterize hMSC-mediated length scale dependent pericellular remodeling. MPT measures particle Brownian motion to calculate rheological properties. We use MPT to measure larger length scales with 4.5 µm particles. Bi-disperse MPT simultaneously measures two different length scales (0.5 and 2.0 µm). We measure that hMSCs preferentially remodel larger length scales measured as a higher mobility of larger particles. We inhibit cytoskeletal tension by inhibiting myosin-II and no longer measure this difference in particle mobility. This indicates that cytoskeletal tension is the source of cell-mediated length scale dependent rheological changes. Particle mobility correlates with cell speed across length scales, relating material rheology to cell behavior. These results quantify length scale dependent pericellular remodeling and provide insight into how these microenvironments can be designed into materials to direct cell behavior.
Traditional hydrogels are strong candidates for biomedical applications; however, they may suffer from drawbacks such as weak mechanics, static properties, and an inability to fully replicate aspects of the cellular microenvironment. These challenges can be addressed through the incorporation of second networks to form interpenetrating polymer network (IPN) hydrogels. The objective of this review is to establish clear trends on the enhanced functionality achieved by incorporating secondary networks into traditional, biopolymer-based hydrogels. These include mechanical reinforcement, 'smart' systems that respond to external stimuli, and the ability to tune cell-material interactions. Through attention to network structure and chemistry, IPN hydrogels may advance to meet challenging criteria for a wide range of biomedical fields.
During the wound healing process, human mesenchymal stem cells (hMSCs) are recruited to the injury where they regulate inflammation and initiate healing and tissue regeneration. To aid in healing, synthetic cell-laden hydrogel scaffolds are being designed to deliver additional hMSCs to wounds to enhance or restart the healing process. These scaffolds are being designed to mimic the native tissue environments, which includes physical cues, such as scaffold stiffness. In this work, we focus on how the initial scaffold stiffness hMSCs are encapsulated in changes cell-mediated remodeling and degradation and motility of the encapsulated cells. To do this, we encapsulate hMSCs in a well-defined synthetic hydrogel scaffold that recapitulates aspects of the native extracellular matrix (ECM). We then characterize cell-mediated degradation in the pericellular region as a function of the initial microenvironmental stiffness. Our hydrogel consists of a 4-arm poly(ethylene glycol) (PEG) end-functionalized with norbornene which is chemically cross-linked with a matrix metalloproteinase (MMP) degradable peptide sequence. This peptide sequence is cleaved by hMSC-secreted MMPs. The hydrogel elastic modulus is varied from 80 to 2400 Pa by changing the concentration of the peptide cross-linker. We use multiple particle tracking microrheology (MPT) to characterize the spatio-temporal cell-mediated degradation in the pericellular region. In MPT, fluorescently labeled particles are embedded in the material and their Brownian motion is measured. We measure an increase in cell-mediated degradation and remodeling as the post-encapsulation time increases. MPT also measures changes in the degradation profile in the pericellular region as hydrogel stiffness is increased. We hypothesize that the change in the degradation profile is due to a change in the amount and type of molecules secreted by hMSCs. We also measure a significant decrease in cell speed as hydrogel stiffness increases due to the increased physical barrier that needs to be degraded to enable motility. These measurements increase our understanding of the rheological changes in the pericellular region in different physical microenvironments which could lead to better design of implantable biomaterials for cell delivery to wounded areas.
Multiple particle tracking microrheology (MPT) is a passive microrheological technique that measures the Brownian motion of probe particles embedded in a sample to characterize material rheological properties. MPT is a powerful tool that quantifies material rheology in the low moduli range while requiring only small sample volumes and relatively simple data acquisition using video microscopy. MPT quantitatively characterizes spatiotemporal rheological properties and is particularly well suited for the investigation of evolving materials with complex microenvironments. MPT has expanded the study of a variety of materials including biofilms, colloidal gels, hydrogels, stimuli-responsive materials, and cell-laden biomaterials. The aim of this Tutorial is to summarize the fundamentals, illustrate the versatility, and highlight recent advances in MPT. In each application, we will highlight how MPT is uniquely positioned to gather rheological properties, which would be difficult, if not impossible, to attain with other rheological characterization techniques and highlight how MPT can be used to supplement other measurement techniques. This Tutorial should provide researchers with the fundamental basis and skills needed to use MPT and develop new MPT techniques to characterize materials for their unique applications.
Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodelling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free.
Using our advanced setup, we measured local, apparent elastic moduli G0,app in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G0,app by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G0,app was observed.
The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems.
In this Primer, Sunyer and Trepat introduce durotaxis, the mode of migration by which cells follow gradients of extracellular matrix stiffness.
Human mesenchymal stem cells (hMSCs) are motile cells that migrate from their native niche to wounded sites where they regulate inflammation during healing. New materials are being developed as hMSC delivery platforms to enhance wound healing. To act as an effective wound healing material, the hydrogel must degrade at the same rate as tissue regeneration while maintaining high cell viability. This work determines the kinetics and mechanism of cell-mediated degradation in hMSC-laden poly(ethylene glycol) (PEG) hydrogels. We use a well-established hydrogel scaffold that is composed of a backbone of four-arm star PEG functionalized with norbornene that is cross-linked with a matrix metalloproteinase (MMP) degradable peptide. This peptide sequence is cleaved by cell-secreted MMPs, which allow hMSCs to actively degrade the hydrogel during motility. Three mechanisms of degradation are characterized: hydrolytic, non-cellular enzymatic and cell-mediated degradation. We use bulk rheology to characterize hydrogel material properties and quantify degradation throughout the entire reaction. Hydrolysis and non-cellular enzymatic degradation are first characterized in hydrogels without hMSCs, and follow first-order and Michaelis-Menten kinetics, respectively. High cell viability is measured in hMSC-laden hydrogels, even after shearing on the rheometer. After confirming hMSC viability, bulk rheology characterizes cell-mediated degradation. When comparing cell-mediated degradation to non-cellular degradation mechanisms, cell-mediated degradation is dominated by enzymatic degradation. This indicates hydrogels with hMSCs are degraded primarily due to cell-secreted MMPs and very little network structure is lost due to hydrolysis. Modeling cell-mediated degradation provides an estimate of the initial concentration of MMPs secreted by hMSCs. By changing the concentration of hMSCs, we determine the initial MMP concentration increases with increasing hMSC concentration. This work characterizes the rate and mechanism of scaffold degradation, giving new insight into the design of these materials as implantable scaffolds.
We present a comprehensive overview of microrheology, emphasizing the underlying theory, practical aspects of its implementation, and current applications to rheological studies in academic and industrial laboratories. Key methods and techniques are examined, including important considerations to be made with respect to the materials most amenable to microrheological characterization and pitfalls to avoid in measurements and analysis. The fundamental principles of all microrheology experiments are presented, including the nature of colloidal probes and their movement in fluids, soft solids, and viscoelastic materials. Microrheology is divided into two general areas, depending on whether the probe is driven into motion by thermal forces (passive), or by an external force (active). We present the theory and practice of passive microrheology, including an in-depth examination of the Generalized Stokes-Einstein Relation (GSER). We carefully treat the assumptions that must be made for these techniques to work, and what happens when the underlying assumptions are violated. Experimental methods covered in detail include particle tracking microrheology, tracer particle microrheology using dynamic light scattering and diffusing wave spectroscopy, and laser tracking microrheology. Second, we discuss the theory and practice of active microrheology, focusing specifically on the potential and limitations of extending microrheology to measurements of non-linear rheological properties, like yielding and shear-thinning. Practical aspects of magnetic and optical tweezer measurements are preseted. Finally, we highlight important applications of microrheology, including measurements of gelation, degradation, high-throughput rheology, protein solution viscosities, and polymer dynamics.
Human mesenchymal stem cells (hMSCs) dynamically remodel their microenvironment during basic processes, such as migration and differentiation. Migration requires extracellular matrix invasion, necessitating dynamic cell-material interactions. Understanding these interactions is critical to advancing materials design that harness and manipulate these processes for applications including wound healing and tissue regeneration. In this work, we encapsulate hMSCs in a cell-degradable poly(ethylene glycol)-peptide hydrogel to determine how cell-secreted enzymes, specifically matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), create unique pericellular microenvironments. Using multiple particle tracking microrheology (MPT), we characterize spatio-temporal rheological properties in the pericellular region during cell-mediated remodeling. In MPT, the thermal motion of probes embedded in the network is measured. A newly designed sample chamber that limits probe drift during degradation and minimizes high value antibody volumes required for cell treatments enables MPT characterization. Previous MPT measurements around hMSCs show that directly around the cell the scaffold remains intact with the cross-link density decreasing as distance from the cell increases. This degradation profile suggests that hMSCs are simultaneously secreting TIMPs, which are inactivating MMPs through MMP−TIMP complexes. By neutralizing TIMPs using antibodies, we characterize the changes in matrix degradation. TIMP inhibited hMSCs create a reaction-diffusion type degradation profile where MMPs are actively degrading the matrix immediately after secretion. In this profile, the cross-link density increases with increasing distance from the cell. This change in material properties also increases the speed of migration. This simple treatment could increase delivery of hMSCs to injuries to aid wound healing and tissue regeneration.
Human mesenchymal stem cells (hMSCs) are encapsulated in synthetic matrix metalloproteinase (MMP) degradable poly(ethylene glycol)-peptide hydrogels to characterize cell-mediated degradation of the pericellular region using multiple particle tracking microrheology. The hydrogel scaffold is degraded by cell-secreted enzymes and cytoskeletal tension. We determine that cell-secreted enzymatic degradation can be the main contributor to changes in the pericellular region, with cytoskeletal tension playing a minimal role. Measured degradation profiles for untreated and myosin II inhibited hMSCs have with the highest cross-link density around the cell. We hypothesize that cells are also secreting tissue inhibitors of metalloproteinases (TIMPs) to inhibit MMPs, which allow cell spreading and attachment prior to motility. We develop a Michaelis-Menten competitive enzymatic inhibition model which accurately describes the degradation profile due to MMP-TIMP unbinding.
This study describes the use of a set of protein-based biomaterials that allow us to explore the mechanism of cell-mediated 3-D invasion associated with peripheral nerve regeneration. Hydrogels made from poly(ethylene glycol) (PEG) conjugated extracellular matrix proteins, including fibrinogen, gelatin and albumin were compared in their ability to support the neurite extension and glial cell migration from dorsal root ganglion (DRG) as compared to PEG only hydrogel controls. The synthetic polymer in the system provides a cross-linked network with controlled mechanical properties and degradation, whereas the protein components provide the unique extracellular matrix (ECM) for controlling neuronal cell morphogenesis. A range of hydrogel compositions were found to support DRG cell outgrowth, based on the mechanical properties, density and proteolytic degradation of the matrix. The 3-D invasion and morphogenesis of newly grown neurites and glial cells in the different materials were characterized and correlated to the properties of the scaffolds. The DRG cell outgrowth was highly correlated with the density of different hydrogel compositions.
Significance: Almost 7 million Americans have chronic cutaneous wounds and billions of dollars are spent on their treatment. The number of patients with nonhealing wounds keeps increasing worldwide due to an ever-aging population, increasing number of obese and diabetic patients, and cardiovascular disease. Recent Advances: Advanced treatments for difficult wounds are needed. Therapy with mesenchymal stem cells (MSCs) is attractive due to their differentiating potential, their immunomodulating properties, and their paracrine effects. Critical Issues: New technologies (including growth factors and skin substitutes) are now widely used for stimulating wound healing. However, in spite of these advances, the percentage of complete wound closure in most clinical situations is around 50–60%. Moreover, there is a high rate of wound recurrence. Future Directions: Recently, it has been demonstrated that MSCs speed up wound healing by decreasing inflammation, by promoting angiogenesis, and by decreasing scarring. However, there are some potential limitations to successful MSC therapy. These limitations include the need to improve cell delivery methods, cell viability, heterogeneity in MSC preparations, and suboptimal wound bed preparation. Further large, controlled clinical trials are needed to establish the safety of MSCs before widespread clinical application.
We describe a set of image processing algorithms for extracting quantitative data from digitized video microscope images of colloidal suspensions. In a typical application, these direct imaging techniques can locate submicrometer spheres to within 10 nn in the focal plane and 150 nn in depth. Combining information from a sequence of video images into single-particle trajectories makes possible measurements of quantities of fundamental and practical interest such as diffusion coefficients and pair-wise interaction potentials, The measurements we describe in detail combine the outstanding resolution of digital imaging with video-synchronized optical trapping to obtain highly accurate and reproducible results very rapidly. (C) 1996 Academic Press,Inc.
For over two decades, immunologists and biomaterials scientists have co-existed in parallel world with the rationale of understanding the molecular profile of immune responses to vaccination, implantation, and treating incurable diseases. Much of the field of biomaterial-based immunotherapy has relied on evaluating model antigens such as chicken egg ovalbumin in mouse models but their relevance to humans has been point of much discussion. Nevertheless, such model antigens have provided important insights into the mechanisms of immune regulation and served as a proof-of-concept for plethora of biomaterial-based vaccines. After years of extensive development of numerous biomaterials for immunomodulation, it is only recently that an experimental scaffold vaccine implanted beneath the skin has begun to use the human model to study the immune responses to cancer vaccination by co-delivering patient-derived tumor lysates and immunomodulatory proteins. If successful, this scaffold vaccine will change the way we approached untreatable cancers, but more importantly, will allow a faster and more rational translation of therapeutic regimes to other cancers, chronic infections, and autoimmune diseases. Most materials reviews have focused on immunomodulatory adjuvants and micro-nano-particles. Here we provide an insight into emerging hydrogel and scaffold based immunomodulatory approaches that continue to demonstrate efficacy against immune associated diseases.
The number of patients with nonhealing wounds has rapidly accelerated over the past 10 years in both the United States and worldwide. Some causative factors at the macro level include an aging population, epidemic numbers of obese and diabetic patients, and an increasing number of surgical procedures. At the micro level, chronic inflammation is a consistent finding.
A number of treatment modalities are currently used to accelerate wound healing, including energy-based modalities, scaffoldings, the use of mechano-transduction, cytokines/growth factors, and cell-based therapies. The use of stem cell therapy has been hypothesized as a potentially useful adjunct for nonhealing wounds. Specifically, mesenchymal stem cells (MSCs) have been shown to improve wound healing in several studies. Immune modulating properties of MSCs have made them attractive treatment options.
Current limitations of stem cell therapy include the potentially large number of cells required for an effect, complex preparation and delivery methods, and poor cell retention in targeted tissues. Comparisons of published in-vitro and clinical trials are difficult due to cell preparation techniques, passage number, and the impact of the micro-environment on cell behavior.
MSCs may be more useful if they are preactivated with inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma. This article will review the current literature with regard to the use of stem cells for wound healing. In addition the anti-inflammatory effects of MSCs will be discussed along with the potential benefits of stem cell preactivation.
Niches are local tissue microenvironments that maintain and regulate stem cells. Haematopoiesis provides a model for understanding mammalian stem cells and their niches, but the haematopoietic stem cell (HSC) niche remains incompletely defined and beset by competing models. Recent progress has been made in elucidating the location and cellular components of the HSC niche in the bone marrow. The niche is perivascular, created partly by mesenchymal stromal cells and endothelial cells and often, but not always, located near trabecular bone. Outstanding questions concern the cellular complexity of the niche, the role of the endosteum and functional heterogeneity among perivascular microenvironments.
The design of hydrogel matrices for cell encapsulation and tissue regeneration has become increasingly complex. Oftentimes, researchers seek to recapitulate specific biophysical and biochemical cues critical for the resident cell population and an in depth understanding of changes in the local microstructure and rheological properties of the synthetic matrix during enzymatic degradation would be extremely beneficial. Multiple particle tracking microrheology (MPT) enables simultaneous characterization of rheological properties and visualization of the microstructure in an evolving hydrogel scaffold. MPT measures the Brownian motion of fluorescently labeled probe particles embedded in the material, which is directly related to rheological properties using the Generalized Stokes-Einstein Relation (GSER). Here, we study a hydrogel scaffold consisting of a four-arm poly(ethylene glycol) (PEG) end functionalized with norbornene that is cross-linked with both a nondegradable PEG-dithiol and a matrix metalloproteinase (MMP) degradable peptide sequence (KCGPQG↓IWGQCK) using thiol-ene chemistry. The material degradation is measured as a function of time and extent of degradability, focusing on measuring the gel-sol transition. Using time-cure superposition, we determine the critical degradation time and critical extent of degradability for this specific gel formulation as tc = 1.85 h and pc = 0.589, respectively, and the critical relaxation exponent, n = 0.16. Finally, spatial information gained by MPT measurements quantifies the heterogeneity within the scaffold showing that these hydrogels degrade homogeneously when collagenase is introduced in solution at a concentration of 0.1-0.3 mg mL−1. Understanding the microstructural and rheological properties of a material near the gel-sol transition enables researchers to improve their insight as to how cells remodel their microenvironment when encapsulated in gels, and more precisely design and manipulate this parameter to improve three-dimensional culture systems.
Microrheology uses the motion of dispersed colloidal probe particles to measure the viscosity or viscoelastic moduli of soft materials. The distinct advantages of microrheology include small sample volume requirements, access to a large range of time scales for the dynamic response and short acquisition times. These advantages make microrheology important for studies of biomaterial hydrogelators. Recent advances have enabled the precise characterization of hydrogelator sol–gel transitions, measurements of rare and scarce materials and high-throughput screening of hydrogel rheology over a large composition space. In this review, we focus on multiple particle tracking microrheology, including the considerations that define its operating regimes and its recent applications. Those interested in biomaterial rheology will find these methods as accessible as bulk rheological measurements and straightforward to implement in their own work.
An inexpensive, biocompatible self-healing hydrogel as a new injectable cell therapy carrier has been facilely developed.
Recent observations suggest that cells on fibrous extracellular matrix materials sense mechanical signals over much larger distances than they do on linearly elastic synthetic materials. In this work, we systematically investigate the distance fibroblasts can sense a rigid boundary through fibrous gels by quantifying the spread areas of human lung fibroblasts and 3T3 fibroblasts cultured on sloped collagen and fibrin gels. The cell areas gradually decrease as gel thickness increases from 0 to 150 μm, with characteristic sensing distances of >65 μm below fibrin and collagen gels, and spreading affected on gels as thick as 150 μm. These results demonstrate that fibroblasts sense deeper into collagen and fibrin gels than they do into polyacrylamide gels, with the latter exhibiting characteristic sensing distances of <5 μm. We apply finite-element analysis to explore the role of strain stiffening, a characteristic mechanical property of collagen and fibrin that is not observed in polyacrylamide, in facilitating mechanosensing over long distances. Our analysis shows that the effective stiffness of both linear and nonlinear materials sharply increases once the thickness is reduced below 5 μm, with only a slight enhancement in sensitivity to depth for the nonlinear material at very low thickness and high applied traction. Multiscale simulations with a simplified geometry predict changes in fiber alignment deep into the gel and a large increase in effective stiffness with a decrease in substrate thickness that is not predicted by nonlinear elasticity. These results suggest that the observed cell-spreading response to gel thickness is not explained by the nonlinear strain-stiffening behavior of the material alone and is likely due to the fibrous nature of the proteins.
Mesenchymal stem cells (MSCs) have been investigated as a clinical therapy to promote tissue repair. However, the disappearance of grafted cells soon after engraftment suggests a possible role as initiators of repair rather than effectors. We evaluated the relative contribution of grafted human MSCs and host stem/progenitor cells in promoting wound healing by using a novel asymmetric wound model in normal and impaired healing diabetic (db/db) mice to discriminate between the effect of direct engraftment and the subsequent systemic response. Experimental animals received paired wounds, with one wound receiving human mesenchymal stem cells (hMSCs) and the other wound receiving vehicle to assess local and systemic effects, respectively. Control animals received vehicle in both wounds. Grafted hMSCs significantly improved healing in both normal and impaired healing animals; produced significant elevation of signals such as Wnt3a, vascular endothelial growth factor, and platelet-derived growth factor receptor-α; and increased the number of pre-existing host MSCs recruited to the wound bed. Improvement was also seen in both the grafted and nongrafted sides, suggesting a systemic response to hMSC engraftment. Healing was enhanced despite the rapid loss of hMSCs, suggesting that mobilizing the host response is the major outcome of grafting MSCs to tissue repair. We validate that hMSCs evoke a host response that is clinically relevant, and we suggest that therapeutic efforts should focus on maximizing the mobilization of host MSCs.
A new class of biodegradable polymeric biomaterials is described which exhibits degradation by specific enzymes that are localized near cell surfaces during cell migration. These materials are telechelic BAB block copolymers of the water-soluble biocompatible polymer, polyethylene glycol (A block) and short oligopeptide that are cleavage sequences for targeted enzymes (B block). These polymers are further capped at each end with reactive acrylate groups to allow polymerization to form crosslinked hydrogel networks. The combination of two types of bioactive signals, one for cell-mediated degradation, and one for cell adhesion, into one material should achieve a biomemitic polymeric hydrogel with properties of natural extracellular matrix.
Using a dynamic scaling theory for the viscoelasticity of cross-linking polymers near the gel point, we predict the superposition of viscoelastic functions at differing extents of reactions. For example, to superpose the stress relaxation modulus, a vertical shift is needed to account for the increase in the equilibrium modulus with cure, G∞ ∼ ∈8/3, and a horizontal shift for the divergence of the characteristic relaxation time, τz ∼ ∈-4, where ∈ is the critical extent of reaction. Experiments on model epoxies show excellent superposition both below and above the gel point and give G∞ ∼ ∈2.8±0.2 and τz ∼ ∈-3.9±0.2. The critical regime where this superposition is valid is surprisingly wide, encompassing over half of the reaction.
Local control of the stem cell microenvironment with biomaterial design is of critical importance for tissue engineering. Matrix mechanics is one aspect of biomaterial design that has received considerable attention recently due to the effect of mechanics on stem cell proliferation, morphology, and differentiation. In order to investigate the effect of locally controlled mechanics on human mesenchymal stem cells (hMSCs), a sequentially crosslinked hyaluronic acid hydrogel system was developed that permits spatial patterning of mechanics (distinct patterns and gradients). Methacrylated hyaluronic acid was synthesized to allow for crosslinking via both Michael-type addition using a dithiol and radical polymerization using light. By varying the initial methacrylate consumption through addition crosslinking, restricting UV light to specified regions, and varying UV exposure time, a wide range of mechanics (from 100 kPa) was possible in both uniform and patterned hydrogels. hMSCs exhibited increased spreading and proliferation on stiffer gels compared to cells cultured on softer gels. Furthermore, cells grown on gels with patterned mechanics exhibited spreading and proliferation behavior that correlated with the local mechanics. This method to spatially control matrix mechanics represents a novel hydrogel system to tune the stem cell microenvironment.
In microrheology, the local and bulk properties of a complex fluid are ex-tracted from the motion of probe particles embedded within it. In passive microrheology, particles are forced by thermal fluctuations and probe linear viscoelasticity, whereas active microrheology involves external probe forcing that can be extended out of equilibrium to the nonlinear regime. Here we review the development, present state, and future directions of this devel-oping field. We organize our review around the generalized Stokes-Einstein relation (GSER), which plays a central role in the interpretation of microrhe-ology. By discussing the Stokes and Einstein components of the GSER indi-vidually, we identify the key assumptions that underpin each, and the conse-quences (both positive and negative) that occur when they are violated. We conclude with a discussion of two techniques—multiple particle-tracking and nonlinear microrheology—that arose from systems where the GSER breaks down.
We obtain the linear viscoelastic shear moduli of complex fluids from the time-dependent mean square displacement, <Δr
2(t)>, of thermally-driven colloidal spheres suspended in the fluid using a generalized Stokes–Einstein (GSE) equation. Different
representations of the GSE equation can be used to obtain the viscoelastic spectrum, G˜(s), in the Laplace frequency domain, the complex shear modulus, G
*(ω), in the Fourier frequency domain, and the stress relaxation modulus, G
r
(t), in the time domain. Because trapezoid integration (s domain) or the Fast Fourier Transform (ω domain) of <Δr
2(t)> known only over a finite temporal interval can lead to errors which result in unphysical behavior of the moduli near the
frequency extremes, we estimate the transforms algebraically by describing <Δr
2(t)> as a local power law. If the logarithmic slope of <Δr
2(t)> can be accurately determined, these estimates generally perform well at the frequency extremes.
Thiol-ene photopolymerization offers a unique platform for the formation of peptide-functionalized poly(ethylene glycol) hydrogels and the encapsulation, culture and differentiation of cells. Specifically, this photoinitiated polymerization scheme occurs at neutral pH and can be controlled both spatially and temporally. Here, we have encapsulated human mesenchymal stem cells (hMSCs) in matrix metalloproteinase (MMP) degradable and cell-adhesive hydrogels using thiol-ene photopolymerization. We find that hMSCs survive equally well in this system, regardless of MMP-degradability. When hMSCs are encapsulated in these cell-degradable hydrogels, they survive and are able to proliferate. In classic hMSC differentiation medias, hMSCs locally remodel their microenvironment and take on characteristic morphologies; hMSCs cultured in growth or osteogenic differentiation media are less round, as measured by elliptical form factor, and are smaller than hMSCs cultured in chondrogenic or adipogenic differentiation media. In addition, hMSCs encapsulated in completely cell-degradable hydrogels and cultured in osteogenic, chondrogenic, or adipogenic differentiation media generally express increased levels of specific differentiation markers as compared to cells in hydrogels that are not cell-degradable. These studies demonstrate the ability to culture and differentiate hMSCs in MMP-degradable hydrogels polymerized via a thiol-ene reaction scheme and that increased cell-mediated hydrogel degradability facilitates directed differentiation of hMSCs.
Poly(ethylene glycol) (PEG) hydrogels hold great promise as in vivo cell carriers for tissue engineering. To ensure appropriate performance of these materials when implanted, the host response must be well understood. The objectives for this study were to characterize the temporal evolution of the foreign body reaction (FBR) to acellular PEG-based hydrogels prepared from PEG diacrylate precursors when implanted subcutaneously in immunocompentent c57bl/6 mice by (immuno)histochemical analysis and gene expression. Compared with a normal FBR elicited by silicone (SIL), PEG hydrogels without or with a cell adhesion ligand RGD elicited a strong early inflammatory response evidenced by a thick band of macrophages as early as day 2, persisting through two weeks, and by increased interleukin-1β expression. PEG-only hydrogels showed a slower, but more sustained progression of inflammation over PEG-RGD. Temporal changes in gene expression were observed in response to PEG-based materials and in general exhibited, elevated expression of inflammatory and wound healing genes in the tissues surrounding the implants, while the expression patterns were more stable in response to SIL. While a stabilized FBR was achieved with SIL and to a lesser degree with PEG-RGD, the PEG-only hydrogels had not yet stabilized after 4 weeks. In summary, PEG-only hydrogels elicit a strong early inflammatory reaction, which persists throughout the course of the implantation even as a collagenous capsule begins to form. However, the incorporation of RGD tethers partially attenuates this response within 2 weeks leading to an improved FBR to PEG-based hydrogels.
Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.
Interface tissue engineering (ITE) is a rapidly developing field that aims to fabricate biological tissue alternates with the goal of repairing or regenerating the functions of diseased or damaged zones at the interface of different tissue types (also called "interface tissues"). Notable examples of the interface tissues in the human body include ligament-to-bone, tendon-to-bone and cartilage-to-bone. Engineering interface tissues is a complex process, which requires a combination of specialized biomaterials with spatially organized material composition, cell types and signaling molecules. Therefore, the use of conventional biomaterials (monophasic or composites) for ITE has certain limitations to help stimulate the tissue integration or recreating the structural organization at the junction of different tissue types. The advancement of micro- and nanotechnologies enable us to develop systems with gradients in biomaterials properties that encourage the differentiation of multiple cell phenotypes and subsequent tissue development. In this review we discuss recent developments in the fabrication of gradient biomaterials for controlling cellular behavior such as migration, differentiation and heterotypic interactions. Moreover, we give an overview of potential uses of gradient biomaterials in engineering interface tissues such as soft tissues (e.g. cartilage) to hard tissues (e.g. bone), with illustrated experimental examples. We also address fundamentals of interface tissue organization, various gradient biomaterials used in ITE, micro- and nanotechnologies employed for the fabrication of those gradients, and certain challenges that must be met in order for ITE to reach its full potential.
We present a novel experimental method to measure linear viscoelastic moduli of complex fluids using dynamic light scattering. A generalized Langevin equation is used to relate the mean square displacement of a probe particle to the storage and loss moduli of the bulk complex fluid. We confirm the experimental validity of this technique by comparing the light scattering results with mechanical measurements for several complex fluids. This method probes the moduli over a greatly extended frequency range and provides significant new insight into the elastic susceptibility of complex fluids.