Cell Shape and Matrix Stiffness Impact Schwann Cell Plasticity via YAP/TAZ and Rho GTPases.

Schwann cells (SCs) are a highly plastic cell type capable of undergoing phenotypic changes following injury or disease. SCs are able to upregulate genes associated with nerve regeneration and ultimately achieve functional recovery. During the regeneration process, the extracellular matrix (ECM) and cell morphology play a cooperative, critical role in regulating SCs, and therefore highly impact nerve regeneration outcomes. However, the roles of the ECM and mechanotransduction relating to SC phenotype are largely unknown. Here, we describe the role that matrix stiffness and cell morphology play in SC phenotype specification via known mechanotransducers YAP/TAZ and RhoA. Using engineered microenvironments to precisely control ECM stiffness, cell shape, and cell spreading, we show that ECM stiffness and SC spreading downregulated SC regenerative associated proteins by the activation of RhoA and YAP/TAZ. Additionally, cell elongation promoted a distinct SC regenerative capacity by the upregulation of Rac1/MKK7/JNK, both necessary for the ECM and morphology changes found during nerve regeneration. These results confirm the role of ECM signaling in peripheral nerve regeneration as well as provide insight to the design of future biomaterials and cellular therapies for peripheral nerve regeneration.

The impact of physical, biochemical, and electrical signaling on Schwann cell plasticity.

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.

Mechanical stimulation of a bioactive, functionalized PVDF-TrFE scaffold provides electrical signaling for nerve repair applications.

Traumatic nerve injuries have limited success in achieving full functional recovery, with current clinical solutions often including implementation of nerve grafts or the use of nerve conduits to guide damaged axons across injury gaps. In search of alternative, and complimentary solutions, piezoelectric biomaterials demonstrate immense potential for tissue engineering applications. Piezoelectric poly(vinylidene fluoride-triflouroethylene) (PVFD-TrFE) scaffolds can be harnessed to non-invasively stimulate and direct function of key peripheral nervous system (PNS) cells in regeneration strategies. In this study, electrospun PVDF-TrFE was characterized, fabricated into a 3D scaffold, and finally rendered bioactive with the incorporation of a cell-secreted, decellularized extracellular matrix (dECM). PVDF-TrFE scaffolds were characterized extensively for piezoelectric capacity, mechanical properties, and cell-material interactions with fibroblasts and Schwann cells. Through functionalization of PVDF-TrFE scaffolds with a native, cell-assembled dECM, the ability to promote cell adhesion and enhanced viability was also demonstrated. Additionally, incorporation of bioactive functionalization improved the assembly of key regenerative ECM proteins and regenerative growth factors. PVDF-TrFE scaffolds were then fabricated into a conduit design that retained key physical, chemical, and piezoelectric properties necessary for PNS repair. This work shows great promise for multi-cue, electrospun biomaterials for regeneration of the PNS in traumatic injury.

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

Extracellular matrix cues modulate Schwann cell morphology, proliferation, and protein expression.

Peripheral nerve injuries require a complex set of signals from cells, macrophages, and the extracellular matrix (ECM) to induce regeneration across injury sites and achieve functional recovery. Schwann cells (SCs), the major glial cell in the peripheral nervous system (PNS), are critical to nerve regeneration due to their inherent capacity for altering phenotype postinjury to facilitate wound healing. The ECM plays a vital role in wound healing as well as regulating cell phenotype during tissue repair. To examine the underlying mechanisms between the ECM and SCs, this work sought to determine how specific ECM cues regulate the phenotype of SCs. To address this, SCs were cultured on polydimethylsiloxane substrates of a variable Young's modulus coated with ECM proteins. Cells were analyzed for spreading area, proliferation, cell and nuclear shape, and c-Jun expression. It was found that substrates with a stiffness of 8.67 kPa coated with laminin promoted the highest expression of c-Jun, a marker signifying a "regenerative" SC. Microcontact printed, cell adhesive areas were then utilized to precisely control the geometry and spreading of SCs and by controlling spreading area and cellular elongation; expression of c-Jun was either promoted or downregulated. These results begin to address the significant interplay between ECM cues and phenotype of SCs, while offering a potential means to enhance PNS regeneration through cellular therapies.

Micropatterning Decellularized ECM as a Bioactive Surface to Guide Cell Alignment, Proliferation, and Migration.

Bioactive surfaces and materials have displayed great potential in a variety of tissue engineering applications but often struggle to completely emulate complex bodily systems. The extracellular matrix (ECM) is a crucial, bioactive component in all tissues and has recently been identified as a potential solution to be utilized in combination with biomaterials. In tissue engineering, the ECM can be utilized in a variety of applications by employing the biochemical and biomechanical cues that are crucial to regenerative processes. However, viable solutions for maintaining the dimensionality, spatial orientation, and protein composition of a naturally cell-secreted ECM remain challenging in tissue engineering. Therefore, this work used soft lithography to create micropatterned polydimethylsiloxane (PDMS) substrates of a three-dimensional nature to control cell adhesion and alignment. Cells aligned on the micropatterned PDMS, secreted and assembled an ECM, and were decellularized to produce an aligned matrix biomaterial. The cells seeded onto the decellularized, patterned ECM showed a high degree of alignment and migration along the patterns compared to controls. This work begins to lay the groundwork for elucidating the immense potential of a natural, cell-secreted ECM for directing cell function and offers further guidance for the incorporation of natural, bioactive components for emerging tissue engineering technologies.

Development of a Piezoelectric PVDF-TrFE Fibrous Scaffold to Guide Cell Adhesion, Proliferation, and Alignment.

Severe peripheral nervous system injuries currently hold limited therapeutic solutions. Existing clinical techniques such as autografts, allografts, and newer nerve guidance conduits have shown variable outcomes in functional recovery, adverse immune responses, and in some cases low or minimal availability. This can be attributed in part to the lack of chemical, physical, and electrical cues directing both nerve guidance and regeneration. To address this pressing clinical issue, electrospun nanofibers and microfibers composed of piezoelectric polyvinylidene flouride-triflouroethylene (PVDF-TrFE) have been introduced as an alternative template for tissue engineered biomaterials, specifically as it pertains to their relevance in soft tissue and nerve repair. Here, biocompatible scaffolds of PVDF-TrFE are fabricated and their ability to generate an electrical response to mechanical deformations and produce a suitable regenerative microenvironment is examined. It is determined that 20% (w/v) PVDF-TrFE in (6:4) dimethyl formamide (DMF):acetone solvent maintains a desirable piezoelectric coefficient and the proper physical and electrical characteristics for tissue regeneration. Further, it is concluded that scaffolds of varying thickness promoted the adhesion and alignment of Schwann cells and fibroblasts. This work offers a prelude to further advancements in nanofibrous technology and a promising outlook for alternative, autologous remedies to peripheral nerve damage.