Chitosan-polygalacturonic acid complex dressing improves diabetic wound healing and hair growth in diabetic mice.

Chronic skin wounds decrease the quality of life of millions of diabetic patients worldwide. Chitosan has previously been shown to possess hemostatic properties, decrease inflammation, promote fibroblast proliferation, and hair growth. We developed a relatively low-cost polyelectrolyte complex (PEC) film dressing made of chitosan and polygalacturonic acid and tested it for its ability to accelerate diabetic wound healing. Genetically diabetic male mice were shaved on the dorsum, and one day later a 1 cm diameter full-thickness excisional wound was created. The PEC film was applied immediately after wounding and left in place for 14 days. Controls consisted of wounds treated with a fibrin gel. Wounds covered with the PEC film had closed completely by post-wounding day 42, while untreated wounds were only half-way closed. Histological analysis of wounds confirmed that PEC-treated wounds had fully re-epithelialized, while control wounds lacked a continuous epidermis at the wound center. We also observed that the area of skin under the PEC film experienced much more rapid hair growth. Histologically, there were significantly more hair follicles around the scar area (p < 0.05) in the PEC-treated group as compared to the control group. Thus, chitosan-polygalacturonic acid PEC films can accelerate both wound healing and hair growth in diabetic mice, and should be further investigated as a potential future treatment for diabetic chronic wounds.

Preparation of DNA-crosslinked polyacrylamide hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

The effects of substrate elastic modulus on neural precursor cell behavior.

The spinal cord has a limited capacity to self-repair. After injury, endogenous stem cells are activated and migrate, proliferate, and differentiate into glial cells. The absence of neuronal differentiation has been partly attributed to the interaction between the injured microenvironment and neural stem cells. In order to improve post-injury neuronal differentiation and/or maturation potential, cell-cell and cell-biochemical interactions have been investigated. However, little is known about the role of stem cell-matrix interactions on stem cell-mediated repair. Here, we specifically examined the effects of matrix elasticity on stem cell-mediated repair in the spinal cord, since spinal cord injury results in drastic changes in parenchyma elasticity and viscosity. Spinal cord-derived neural precursor cells (NPCs) were grown on bis-acrylamide substrates with various rigidities. NPC growth, proliferation, and differentiation were examined and optimal in the range of normal spinal cord elasticity. In conclusion, limitations in NPC growth, proliferation, and neuronal differentiation were encountered when substrate elasticity was not within normal spinal cord tissue elasticity ranges. These studies elucidate the effect injury mediated mechanical changes may have on tissue repair by stem cells. Furthermore, this information can be applied to the development of future neuroregenerative biomaterials for spinal cord repair.

Mechanical Properties of DNA-Crosslinked Polyacrylamide Hydrogels with Increasing Crosslinker Density.

DNA-cross-linked polyacrylamide hydrogels (DNA gels) are dynamic mechanical substrates. The addition of DNA oligomers can either increase or decrease the crosslinker density to modulate mechanical properties. These DNA-responsive gels show promise as substrates for cell culture and tissue-engineering applications, since the gels allow time-dependent mechanical modulation. Previously, we reported that fibroblasts plated on DNA gels responded to modulation in elasticity via an increase or decrease in crosslinker density. To better characterize fibroblast mechanical signals, changes in stress and elastic modulus of DNA gels were measured over time as crosslinker density altered. In a previous study, we observed that as crosslinker density decreased, stress was generated, and elasticity changed over time; however, we had not evaluated stress and elastic modulus measurements of DNA gels as crosslinker density increased. Here, we completed this set of fibroblast studies by reporting stress and elastic modulus measurements over time as the crosslinker density increased. We found that the stress generated and the elastic modulus alterations were correlated. Hence, it seemed impossible to separate the effect of stress from the effect of modulus changes for fibroblasts plated on DNA gels. Yet, previous results and controls revealed that stress contributed to fibroblast behavior.

Fibroblast morphology on dynamic softening of hydrogels.

Despite cellular environments having dynamic characteristics, many laboratories utilized static polyacrylamide hydrogels to study the ECM-cell relationship. To attain a more in vivo like environment, we have developed a dynamic, DNA-crosslinked hydrogel (DNA gel). Through the controlled delivery of DNA, we can temporally decrease or increase gel stiffness while expanding or contracting the gel, respectively. These dual mechanical changes make DNA gels a cell-ECM model for studying dynamic mechano-regulated processes, such as wound healing. Here, we characterized DNA gels on a mechanical and cellular level. In contrast to our previous publication, in which we examined the increasing stiffness effects on fibroblast morphology, we examined the effects of decreased matrix stiffness on fibroblast morphology. In addition, we quantified the bulk and/or local stress and strain properties of dynamic gels. Gels generated about 0.5 Pa stress and about 6-11% strain upon softening to generate larger and more circular fibroblasts. These results complemented our previous study, where dynamic gels contracted upon stiffening to generate smaller and longer fibroblasts. In conclusion, we developed a biomaterial that increases and decreases in stiffness while contracting and expanding, respectively. We found that the dynamic deformation directionality of the matrix determined the fibroblast morphology and possibly influences function.

Neural lineage differentiation of embryonic stem cells within alginate microbeads.

Cell replacement therapies, using renewable stem cell sources, hold tremendous potential to treat a wide range of degenerative diseases. Although many studies have established techniques to successfully differentiate stem cells into different mature cell lineages using growth factors or extracellular matrix protein supplementation in both two and three-dimensional configurations, they are often limited by lack of control and low yields of differentiated cells. Previously, we developed a scalable murine embryonic stem cell differentiation environment which maintained cell viability and supported ES cell differentiation to hepatocyte lineage cells. Differentiated hepatocyte function was contingent upon aggregate formation within the alginate microbeads. The present studies were designed to determine the feasibility of adapting the alginate encapsulation technique to neural lineage differentiation. The results of our studies indicate that by incorporating the soluble inducer, retinoic acid (RA), into the permeable microcapsule system, cell aggregation was decreased and neural lineage differentiation enhanced. In addition, we demonstrated that even in the absence of RA, differentiation could be directed away from the hepatocyte and toward the neural lineage by physical cell-cell aggregation blocking. In conjunction with the mechanical and physical characterization of the alginate crosslinking network, we determined that 2.2% alginate microencapsulation can be optimally adapted to ES neural differentiation. This study offers insights into targeting cellular differentiation toward both endodermal and ectodermal cell lineages, and could potentially be adaptable to differentiation of other stem cell types given the correct inducible factors and material properties.

Probing mechanical adaptation of neurite outgrowth on a hydrogel material using atomic force microscopy.

In this study, we describe the design and initial results of probing mechanical adaptation of neurite growth of lightly fixed neurons on a hydrogel substrate by using atomic force microscopy (AFM). It has been shown previously that cells are responsive to the physical conditions of their micro-environment, and that certain cells can adjust their own stiffness as part of the adaptation to the substrate. AFM, a powerful tool to probe micro- and nano-scale structures, has been utilized in assessing topography, morphology, and structural change of neuronal cells. We used AFM with a robust force analysis approach in this study to probe the mechanical properties of both neurites and the substrate at close proximity. We first confirmed the robustness and consistency of the approach specific to soft materials by comparing measurements made on the same reference material using different methods. Subsequently, it was found that the primary spinal cord neurons that were lightly fixed exhibited different stiffnesses between the cell body and neurites. Furthermore, in comparison to the rigidity of the substrate, the stiffness of the neurites was lower, whereas that of the neuronal cell body was higher.

Regulation of dendrite arborization by substrate stiffness is mediated by glutamate receptors.

Brain injury or disease can initiate changes in local or global stiffness of brain tissue. While stiffness of the extracellular environment is known to affect the morphology and function of many cell types, little is known about how the dendrites of neurons respond to changes in brain stiffness. To assess how extracellular stiffness affects dendrite morphology, we took biomaterial and biomedical engineering approaches. We cultured mixed and pure hippocampal neurons on hydrogels composed of polyacrylamide (PA) of varying stiffnesses to mimic the effects of extracellular matrix stiffness on dendrite morphology. The majority of investigations of cortical and spinal cord neurons on soft hydrogels examined branching at early time points (days in vitro (DIV) 2-7), an important distinction from our study, where we include later time points that encompass the peak of branching (DIV 10-12). At DIV 12, dendrite branching was altered by stiffness for both pure and mixed neuronal cultures. Furthermore, we treated hippocampal cultures with glutamate receptor antagonists and with astrocyte-conditioned media. Blocking AMPA and NMDA receptors affected the changes in dendrite branching seen at varying rigidities. Moreover, extracellular factors secreted by astrocytes also change dendrite branching seen at varying rigidities. Thus, astrocytes and ionotropic glutamate receptors contribute to mechanosensing.

Effect of dynamic stiffness of the substrates on neurite outgrowth by using a DNA-crosslinked hydrogel.

Central nervous system tissues, like other tissue types, undergo constant remodeling, which potentially leads to changes in their mechanical stiffness. Moreover, mechanical compliance of central nervous system tissues can also be modified under external load such as that experienced in traumatic brain or spinal cord injury, and during pathological processes. Thus, the neuronal responses to the dynamic stiffness of the microenvironment are of significance. In this study, we induced decrease in stiffness by using a DNA-crosslinked hydrogel, and subjected rat spinal cord neurons to such dynamic stiffness. The neurons respond to the dynamic cues as evidenced by the primary neurite structure, and the response from each neurite property (e.g., axonal length and primary dendrite number) is consistent with the behavior on static gels of same substrate rigidity, with one exception of mean primary dendrite length. The results on cell population distribution confirm the neuronal responses to the dynamic stiffness. Quantification on the focal adhesion kinase expression in the neuronal cell body on dynamic gels suggests that neurons also modify adhesion in coping with the dynamic stiffnesses. The results reported here extend the neuronal mechanosensing capability to dynamic stiffness of extracellular matrix, and give rise to a novel way of engineering neurite outgrowth in time dimension.

The relationship between fibroblast growth and the dynamic stiffnesses of a DNA crosslinked hydrogel.

The microenvironment of cells is dynamic and undergoes remodeling with time. This is evident in development, aging, pathological processes, and at tissue-biomaterial interfaces. But in contrast, the majority of the biomimetic materials have static properties. Here, we show that a previously developed DNA crosslinked hydrogel circumvents the need of environmental factors and undergoes controlled stiffness change via DNA delivery, a feasible approach to initiate property changes in vivo, different from previous attempts. Two types of fibroblasts, L929 and GFP, were subject to the alterations in substrate rigidity presented in the hydrogels. Our results show that exogenous DNA does not cause appreciable cell shape change. Cells do respond to mechanical alterations as demonstrated in the cell projection area and polarity (e.g., Soft vs. Soft-->Medium), and the responses vary depending on magnitude (e.g., Soft-->Medium vs. Soft-->Stiff) and range of stiffness changes (e.g., Soft-->Medium vs. Medium-->Stiff). The two types of fibroblasts share specific responses in common (e.g., Soft-->Medium), while differ in others (e.g., Medium-->Stiff). For each cell type, the projection area and polarity respond differently. This approach provides insight into pathology (e.g., cancer) and tissue functioning, and assists in designing biomaterials with controlled dynamic stiffness by choosing the range and magnitude of stiffness change.

A nonintrusive method of measuring the local mechanical properties of soft hydrogels using magnetic microneedles.

Soft hydrogels serving as substrates for cell attachment are used to culture many types of cells. The mechanical properties of these gels influence cell morphology, growth, and differentiation. For studies of cell growth on inhomogeneous gels, techniques by which the mechanical properties of the substrate can be measured within the proximity of a given cell are of interest. We describe an apparatus that allows the determination of local gel elasticity by measuring the response of embedded micron-sized magnetic needles to applied magnetic fields. This microscope-based four-magnet apparatus can apply both force and torque on the microneedles. The force and the torque are manipulated by changing the values of the magnetic field at the four poles of the magnet using a feedback circuit driven by LABVIEW. Using Hall probes, we have mapped out the magnetic field and field gradients produced by each pole when all the other poles are held at zero magnetic field. We have verified that superposition of these field maps allows one to obtain field maps for the case when the poles are held at arbitrary field values. This allows one to apply known fields and field gradients to a given microneedle. An imaging system is employed to measure the displacement and rotation of the needles. Polyacrylamide hydrogels of known elasticity were used to determine the relationship between the field gradient at the location of the needles and the force acting on the needles. This relationship allows the force on the microneedle to be determined from a known field gradient. This together with a measurement of the displacement of the needle in a given gel allows one to determine the stiffness (Fdelta) of the gel and the elastic modulus, provided Poison's ratio is known. Using this method, the stiffness and the modulus of elasticity of type-I collagen gels were found to be 2.64+/-0.05 nNmicrom and 284.6+/-5.9 Pa, respectively. This apparatus is presently being employed to track the mechanical stiffness of the DNA-cross-linked hydrogels, developed by our group, whose mechanical properties can be varied on demand by adding or removing cross-linker strands. Thus a system that can be utilized to track the local properties of soft media as a function of time with minimum mechanical disturbance in the presence of cells is presented.

Comparison of kinematics, kinetics, and EMG throughout wheelchair propulsion in able-bodied and persons with paraplegia: an integrative approach.

A systematic integrated data collection and analysis of kinematic, kinetic, and electromyography (EMG) data allow for the comparison of differences in wheelchair propulsion between able-bodied individuals and persons with paraplegia. Kinematic data from a motion analysis system, kinetic data from force-sensing push rims, and electromyography data from four upper-limb muscles were collected for ten push strokes. Results are as follows: Individuals with paraplegia use a greater percentage of their posterior deltoids, biceps, and triceps in relation to maximal voluntary contraction. These persons also reached peak anterior deltoid firing nearly 10 deg earlier on the push rim, while reaching peak posterior deltoid nearly 10 deg later on the push rim. Able-bodied individuals had no triceps activity in the initial stages of propulsion while their paraplegic groups had activity throughout. Able-bodied participants also had, on average, peak resultant, tangential, and radial forces occurring later on the push rim (in degrees). There are two main conclusions that can be drawn from this integrative investigation: (1) A greater "muscle energy," as measured by the area under the curve of the percentage of EMG throughout propulsion, results in a greater resultant joint force in the shoulder and elbow, thus potentially resulting in shoulder pathology. (2) Similarly, a greater muscle energy may result in fatigue and play a factor in the development of shoulder pain and pathology over time; fatigue may compromise an effective propulsive stroke placing undue stresses on the joint capsule. Muscle activity differences may be responsible for the observed kinematic and kinetic differences between the two groups. The high incidence of shoulder pain in manual wheelchair users as compared to the general population may be the result of such differences, although the results from this biomedical investigation should be examined with caution. Future research into joint forces may shed light on this. Further investigation needs to focus on whether the pattern of kinematics, kinetics, and muscle activity during wheelchair propulsion is compensatory or evolutionary by tracking individuals longitudinally.

Validation of a musculoskeletal model of wheelchair propulsion and its application to minimizing shoulder joint forces.

The majority of manual wheelchair users (MWUs) will inevitably develop some degree of shoulder pain over time. Previous research has suggested a link between the shoulder joint forces associated with the repetition of wheelchair (WC) propulsion and pain. The objective of this work is to present and validate a rigid-body musculoskeletal model of the upper limb for calculation of shoulder joint forces throughout WC propulsion. It is anticipated that when prescribing a WC, the use of a patient-specific computational model will aide in determining an axle placement in which shoulder joint forces are at a minimum, thus potentially delaying or reducing the shoulder pain that so many MWUs experience. During the validation experiment, 3 subjects (2 individuals with paraplegia and one able-bodied individual) propelled a WC at a self-selected speed, during which, kinematics, kinetics, and electromyography (EMG) activity were measured for the contact phase of 10 consecutive push strokes. The measured forces at the push rim and the 3-D propulsion kinematics drove the model, and the computationally calculated muscle activities were compared with the experimental muscle activities, resulting in an average mean absolute error (MAE) of 0.165. Further investigation of the shoulder joint forces throughout propulsion demonstrate the effect of axle placement on the magnitude of these forces. The present work serves to validate the patient-specific upper limb model for use as a prescriptive tool for fitting a subject to their WC. Minimizing joint forces from injury onset may prolong a MWU's pain-free way of life.

Neurite outgrowth on a DNA crosslinked hydrogel with tunable stiffnesses.

Mechanical cues arising from extracellular matrices greatly affect cellular properties, and hence, are of significance in designing biomaterials. In this study, a DNA crosslinked hydrogel was employed to examine cellular responses of spinal cord neurons to substrate compliances. Using DNA as crosslinkers in polymeric hydrogel formation has given rise to a new class of hydrogels with a number of attractive properties (e.g., reversible gelation and controlled crosslinking). Here, it was demonstrated that by varying length of crosslinker, monomer concentration, and level of crosslinking, DNA gel stiffnesses span from approximately 100 Pa to 30 kPa. Assessment of neurite outgrowth on functionalized DNA gels showed that although primary dendrite length is not significantly affected, spinal cord neurons extend more primary dendrites and shorter axons on stiffer gels. Additionally, a greater proportion of neurons have more primary dendrites and shorter axons on stiffer gels. There is a pronounced reduction in focal adhesion kinase (FAK) when neurons are exposed to stiffer substrates, suggesting its involvement in neuronal mechanosensing and neuritogenesis in response to stiffness. These results demonstrate the importance of mechanical aspects of the cell-ECM interactions, and provide guidance for the design of mechanical properties of bio-scaffolds for neural tissue engineering applications.

Functional modulation of ES-derived hepatocyte lineage cells via substrate compliance alteration.

Pluripotent embryonic stem cells represent a promising renewable cell source to generate a variety of differentiated cell types including hepatocyte lineage cells, and may ultimately be incorporated into extracorporeal bioartificial liver devices and cell replacement therapies. Recently, we and others have utilized sodium butyrate to directly differentiate hepatocyte-like cells from murine embryonic stem cells cultured in a monolayer configuration. However, to incorporate stem cell technology into clinical and pharmaceutical applications, and hopefully increase the therapeutic potential of these differentiated cells for liver disease treatment, a major challenge remains in sustaining differentiated functions for an extended period of time in their secondary culture environment. In the present work, we have investigated the use of polyacrylamide hydrogels with defined mechanical compliances as a cell culture platform for improving and/or stabilizing functions of these hepatocyte-like cells. Several functional assays, e.g., urea secretion, intracellular albumin content, and albumin secretion, were performed to characterize hepatic functions of cells on polyacrylamide gels with stiffnesses of 5, 46.6, and 230 kPa. In conjunction with the mechanical and cell morphological characterization, we showed that hepatic functions of sodium butyrate differentiated cells were sustained and further enhanced on compliant substrates. This study promises to offer insights into regulating stem cell differentiation via mechanical stimuli, and assist us with designing a variety of dynamic culture systems for applications in tissue and cellular engineering.

Biomechanical alterations in intact osteoporotic spine due to synthetic augmentation: finite element investigation.

A three-dimensional nonlinear finite element model (FEM) was developed for a parametric study that examined the effect of synthetic augmentation on nonfractured vertebrae. The objective was to isolate those parameters primarily responsible for the effectiveness of the procedure; bone cement volume and bone density were expected to be highly important. Injection of bone cement was simulated in the FEM of a vertebral body that included a cellular model for the trabecular core. The addition of 10% and 20% cement by volume resulted in an increase in failure load, and the larger volume resulted in an increase in stiffness for the vertebral body. Placement of cement within the vertebral body was not as critical a parameter as cement amount. Simulated models of very poor bone quality saw the best therapeutic benefits.

Measurement and analyses of the effects of adjacent end plate curvatures on vertebral stresses.

BACKGROUND: Vertebral end plates of the lumbosacral spine have various degrees of concavity and convexity. It is believed that the shape of the end plates alters the distribution of loads transferred along the spine, between the vertebrae. Animal models have been regularly used in the design and development of vertebral disc implants and cages; to date, very little information is known about the animal vertebral end plate curvature. PURPOSE: The purpose was to measure and analyze the end plate curvature in the cadaver human male-female, chimpanzee, and canine lumbar vertebral bones. STUDY DESIGN/SETTING: Nondestructive and nontouching scanning method was designed to obtain curvature in anterior-posterior and medial-lateral directions in the cadaver bones. Statistical analysis was performed on the data collected, and this data was then used to create a biomechanical model to evaluate the load transmission. METHOD: Measurements in anterior-posterior and medial-lateral directions were performed on human, canine, and chimpanzee cadaver lumbar bones to obtain accurate data for the end plate curvatures. Six sets of measurements (on human male-female L4 lower to S1 upper end plates) were performed. A parametric vertebral motion segment model (with and without posterior elements) that includes the experimental curvature information was developed. The characteristic kidney-shaped cross-sectional model was created using a parametric equation. This model was used to perform finite element analyses investigating the effects of the location of maximum curvature on the stress distributions. RESULTS: The measurements for different species showed that the canine and chimpanzees, the quadrupeds, have entirely different curvature of their upper end plates compared with those in humans, the bipeds. Also, the curvatures of the human S1 upper end plates are significantly different from the rest of the vertebrae. This is a very useful piece of information in the comparison of these species. The stress distribution varied as the location of the maximum curvature shifted from the center to a more posterior position. The stresses in the vertebral core were found to decrease, with the shell taking more loads. CONCLUSIONS: This provides essential information for rehabilitation and surgical techniques, including designs for various interbody devices such as fusion cages, bone grafts, and disc prosthesis.

Use of rigid spherical inclusions in Young's moduli determination: application to DNA-crosslinked gels.

Current techniques for measuring the bulk shear or elastic (E) modulus of small samples of soft materials are usually limited by materials handling issues. This paper describes a nondestructive testing method based on embedded spherical inclusions. The technique simplifies materials preparation and handling requirements and is capable of continuously monitoring changes in stiffness. Exact closed form derivations of E as functions of the inclusion force-displacement relationship are presented. Analytical and numerical analyses showed that size effects are significant for medium dimensions up to several times those of the inclusion. Application of the method to DNA-crosslinked gels showed good agreement with direct compression tests.

A review of spinal fusion for degenerative disc disease: need for alternative treatment approach of disc arthroplasty?

This article reviews the history of spinal fusion. The most common indication for fusion may be painful disc degeneration although the exact determination of this condition remains controversial. In the analysis of surgical fusion techniques, literature review has documented a higher rate of success in attaining successful fusion than in obtaining excellent clinical outcomes. Significant morbidity can be seen in the performance of spinal fusion. A limited number of patients presently undergoing spinal fusion will be candidates for disc arthroplasty, though indications for spinal arthroplasty are still evolving.

Mechanical properties of a reversible, DNA-crosslinked polyacrylamide hydrogel.

Mechanical properties of a polyacrylamide gel with reversible DNA crosslinks are presented. In this system, three DNA strands replace traditional chemical crosslinkers. In contrast to thermoset chemically crosslinked polyacrylamide, the new hydrogel is thermoreversible; crosslink dissociation without the addition of heat is also feasible by introducing a specific removal DNA strand. This hydrogel is characterized by a critical crosslink concentration at which gelation occurs. Below the critical point, a characteristic temperature exists at which a transition in viscosity is observed. Both temperature-dependent viscosity and elastic modulus of the material are functions of crosslink density.