Samad Ahadian

Gelatin methacrylate as a promising hydrogel for 3D microscale organization and proliferation of dielectrophoretically patterned cells

Establishing the 3D microscale organization of cells has numerous practical applications, such as in determining cell fate (e.g., proliferation, migration, differentiation, and apoptosis) and in making functional tissue constructs. One approach to spatially pattern cells is by dielectrophoresis (DEP). DEP has characteristics that are important for cell manipulation, such as high accuracy, speed, scalability, and the ability to handle both adherent and non-adherent cells. However, widespread application of this method is largely restricted because there is a limited number of suitable hydrogels for cell encapsulation. To date, polyethylene glycol-diacrylate (PEG-DA) and agarose have been used extensively for dielectric patterning of cells. In this study, we propose gelatin methacrylate (GelMA) as a promising hydrogel for use in cell dielectropatterning because of its biocompatibility and low viscosity. Compared to PEG hydrogels, GelMA hydrogels showed superior performance when making cell patterns for myoblast (C2C12) and endothelial (HUVEC) cells as well as in maintaining cell viability and growth. We also developed a simple and robust protocol for co-culture of these cells. Combined application of the GelMA hydrogels and the DEP technique is suitable for creating highly complex microscale tissues with important applications in fundamental cell biology and regenerative medicine in a rapid, accurate, and scalable manner.

Dielectrophoretically aligned carbon nanotubes to control electrical and mechanical properties of hydrogels to fabricate contractile muscle myofibers.

Dielectrophoresis is used to align carbon nanotubes (CNTs) within gelatin methacrylate (GelMA) hydrogels in a facile and rapid manner. Aligned GelMA-CNT hydrogels show higher electrical properties compared with pristine and randomly distributed CNTs in GelMA hydrogels. The muscle cells cultured on these materials demonstrate higher maturation compared with cells cultured on pristine and randomly distributed CNTs in GelMA hydrogels.

Hybrid hydrogels containing vertically aligned carbon nanotubes with anisotropic electrical conductivity for muscle myofiber fabrication

Biological scaffolds with tunable electrical and mechanical properties are of great interest in many different fields, such as regenerative medicine, biorobotics, and biosensing. In this study, dielectrophoresis (DEP) was used to vertically align carbon nanotubes (CNTs) within methacrylated gelatin (GelMA) hydrogels in a robust, simple, and rapid manner. GelMA-aligned CNT hydrogels showed anisotropic electrical conductivity and superior mechanical properties compared with pristine GelMA hydrogels and GelMA hydrogels containing randomly distributed CNTs. Skeletal muscle cells grown on vertically aligned CNTs in GelMA hydrogels yielded a higher number of functional myofibers than cells that were cultured on hydrogels with randomly distributed CNTs and horizontally aligned CNTs, as confirmed by the expression of myogenic genes and proteins. In addition, the myogenic gene and protein expression increased more profoundly after applying electrical stimulation along the direction of the aligned CNTs due to the anisotropic conductivity of the hybrid GelMA-vertically aligned CNT hydrogels. We believe that platform could attract great attention in other biomedical applications, such as biosensing, bioelectronics, and creating functional biomedical devices.

Interdigitated array of Pt electrodes for electrical stimulation and engineering of aligned muscle tissue

Engineered skeletal muscle tissues could be useful for applications in tissue engineering, drug screening, and bio-robotics. It is well-known that skeletal muscle cells are able to differentiate under electrical stimulation (ES), with an increase in myosin production, along with the formation of myofibers and contractile proteins. In this study, we describe the use of an interdigitated array of electrodes as a novel platform to electrically stimulate engineered muscle tissues. The resulting muscle myofibers were analyzed and quantified in terms of their myotube characteristics and gene expression. The engineered muscle tissues stimulated through the interdigitated array of electrodes demonstrated superior performance and maturation compared to the corresponding tissues stimulated through a conventional setup (i.e., through Pt wires in close proximity to the muscle tissue). In particular, the ES of muscle tissue (voltage 6 V, frequency 1 Hz and duration 10 ms for 1 day) through the interdigitated array of electrodes resulted in a higher degree of C2C12 myotube alignment (∼80%) as compared to ES using Pt wires (∼65%). In addition, higher amounts of C2C12 myotube coverage area, myotube length, muscle transcription factors and protein biomarkers were found for myotubes stimulated through the interdigitated array of electrodes compared to those stimulated using the Pt wires. Due to the wide array of potential applications of ES for two- and three-dimensional (2D and 3D) engineered tissues, the suggested platform could be employed for a variety of cell and tissue structures to more efficiently investigate their response to electrical fields.

Bioconjugated Hydrogels for Tissue Engineering and Regenerative Medicine

Hydrogels are hydrophilic polymer networks with high water content, which have played an important role as scaffolds for cells, as carriers for various biomolecules (e.g., drugs, genes, and soluble factors), and as injectable biomaterials in tissue engineering (TE) and regenerative medicine. Bioconjugation is an approach for improving the performance of hydrogels using cell-responsive components, such as proteins and peptides, which have high affinity to regulate cellular behaviors and tissue morphogenesis. However, the current knowledge on the role of those bioconjugated moieties in controlling cellular functions and tissue morphogenesis and bioconjugation methods are limited in the context of TE and organogenesis. Moreover, micro- and nanofabrication techniques have been used to manipulate bioconjugated hydrogels for regulating cell behaviors and function. This Review therefore describes synthesis, characteristics, and manipulation of various bioconjugated hydrogels and their potential in TE applications with special emphasis on preclinical/clinical translation.

Electrical stimulation as a biomimicry tool for regulating muscle cell behavior.

There is a growing need to understand muscle cell behaviors and to engineer muscle tissues to replace defective tissues in the body. Despite a long history of the clinical use of electric fields for muscle tissues in vivo, electrical stimulation (ES) has recently gained significant attention as a powerful tool for regulating muscle cell behaviors in vitro. ES aims to mimic the electrical environment of electroactive muscle cells (e.g., cardiac or skeletal muscle cells) by helping to regulate cell-cell and cell-extracellular matrix (ECM) interactions. As a result, it can be used to enhance the alignment and differentiation of skeletal or cardiac muscle cells and to aid in engineering of functional muscle tissues. Additionally, ES can be used to control and monitor force generation and electrophysiological activity of muscle tissues for bio-actuation and drug-screening applications in a simple, high-throughput, and reproducible manner. In this review paper, we briefly describe the importance of ES in regulating muscle cell behaviors in vitro, as well as the major challenges and prospective potential associated with ES in the context of muscle tissue engineering.

Hybrid hydrogel-aligned carbon nanotube scaffolds to enhance cardiac differentiation of embryoid bodies

UNLABELLED Carbon nanotubes (CNTs) were aligned in gelatin methacryloyl (GelMA) hydrogels using dielectrophoresis approach. Mouse embryoid bodies (EBs) were cultured in the microwells fabricated on the aligned CNT-hydrogel scaffolds. The GelMA-dielectrophoretically aligned CNT hydrogels enhanced the cardiac differentiation of the EBs compared with the pure GelMA and GelMA-random CNT hydrogels. This result was confirmed by Troponin-T immunostaining, the expression of cardiac genes (i.e., Tnnt2, Nkx2-5, and Actc1), and beating analysis of the EBs. The effect on EB properties was significantly enhanced by applying an electrical pulse stimulation (frequency, 1Hz; voltage, 3V; duration, 10ms) to the EBs for two continuous days. Taken together, the fabricated hybrid hydrogel-aligned CNT scaffolds with tunable mechanical and electrical characteristics offer an efficient and controllable platform for electrically induced differentiation and stimulation of stem cells for potential tissue regeneration and cell therapy applications. STATEMENT OF SIGNIFICANCE Dielectrophoresis approach was used to rapidly align carbon nanotubes (CNTs) in gelatin methacryloyl (GelMA) hydrogels resulting in hybrid GelMA-CNT hydrogels with tunable and anisotropic electrical and mechanical properties. The GelMA-aligned CNT hydrogels may be used to apply accurate and controllable electrical pulses to cell and tissue constructs and thereby regulating their behavior and function. In this work, it was demonstrated that the GelMA hydrogels containing the aligned CNTs had superior performance in cardiac differentiation of stem cells upon applying electrical stimulation in contrast with control gels. Due to broad use of electrical stimulation in tissue engineering and stem cell differentiation, it is envisioned that the GelMA-aligned CNT hydrogels would find wide applications in tissue regeneration and stem cell therapy.

Flexible shape-memory scaffold for minimally invasive delivery of functional tissues

Despite great progress in engineering functional tissues for organ repair, including the heart, an invasive surgical approach is still required for their implantation. Here, we designed an elastic and microfabricated scaffold using a biodegradable polymer (poly(octamethylene maleate (anhydride) citrate)) for functional tissue delivery via injection. The scaffolds shape memory was due to the microfabricated lattice design. Scaffolds and cardiac patches (1 cm × 1 cm) were delivered through an orifice as small as 1 mm, recovering their initial shape following injection without affecting cardiomyocyte viability and function. In a subcutaneous syngeneic rat model, injection of cardiac patches was equivalent to open surgery when comparing vascularization, macrophage recruitment and cell survival. The patches significantly improved cardiac function following myocardial infarction in a rat, compared with the untreated controls. Successful minimally invasive delivery of human cell-derived patches to the epicardium, aorta and liver in a large-animal (porcine) model was achieved.

Electrically regulated differentiation of skeletal muscle cells on ultrathin graphene-based films

The electrical conductivity of graphene provides a unique opportunity to modify the behavior of electrically sensitive cells. Here, we demonstrate that C2C12 myoblasts that were cultured on ultrathin thermally reduced graphene (TR-Graphene) films had more favorable cell adhesion and spreading compared to those on graphene oxide (GO) and glass slide substrates, comparable with conventional Petri dish. More importantly, we demonstrate that electrical stimulation significantly enhanced myoblast cell differentiation on a TR-Graphene substrate compared to GO and glass slide surfaces as confirmed by the expression of myogenic genes and proteins. These results highlight the potential applications of graphene-based materials for cell-based studies, bioelectronics, and biorobotics.

A contactless electrical stimulator: application to fabricate functional skeletal muscle tissue

Engineered skeletal muscle tissues are ideal candidates for applications in drug screening systems, bio-actuators, and as implantable constructs in tissue engineering. Electrical field stimulation considerably improves the differentiation of muscle cells to muscle myofibers. Currently used electrical stimulators often use direct contact of electrodes with tissue constructs or their culture medium, which may cause hydrolysis of the culture medium, joule heating of the medium, contamination of the culture medium due to products of electrodes corrosion, and surface fouling of electrodes. Here, we used an interdigitated array of electrodes combined with an isolator coverslip as a contactless platform to electrically stimulate engineered muscle tissue, which eliminates the aforementioned problems. The effective stimulation of muscle myofibers using this device was demonstrated in terms of contractile activity and higher maturation as compared to muscle tissues without applying the electrical field. Due to the wide array of potential applications of electrical stimulation to two- and three-dimensional (2D and 3D) cell and tissue constructs, this device could be of great interest for a variety of biological applications as a tool to create noninvasive, safe, and highly reproducible electric fields.