Phosphodiesterase (PDE) 4 inhibition boosts Schwann cell myelination in a 3D regeneration model.

Phosphodiesterase 4 (PDE4) inhibitors have been extensively researched for their anti-inflammatory and neuroregenerative properties. Despite the known neuroplastic and myelin regenerative properties of nonselective PDE4 inhibitors on the central nervous system, the direct impact on peripheral remyelination and subsequent neuroregeneration has not yet been investigated. Therefore, to examine the possible therapeutic effect of PDE4 inhibition on peripheral glia, we assessed the differentiation of primary rat Schwann cells exposed in vitro to the PDE4 inhibitor roflumilast. To further investigate the differentiation promoting effects of roflumilast, we developed a 3D model of rat Schwann cell myelination that closely resembles the in vivo situation. Using these in vitro models, we demonstrated that pan-PDE4 inhibition using roflumilast significantly promoted differentiation of Schwann cells towards a myelinating phenotype, as indicated by the upregulation of myelin proteins, including MBP and MAG. Additionally, we created a unique regenerative model comprised of a 3D co-culture of rat Schwann cells and human iPSC-derived neurons. Schwann cells treated with roflumilast enhanced axonal outgrowth of iPSC-derived nociceptive neurons, which was accompanied by an accelerated myelination speed, thereby showing not only phenotypic but also functional changes of roflumilast-treated Schwann cells. Taken together, the PDE4 inhibitor roflumilast possesses a therapeutic benefit to stimulate Schwann cell differentiation and, subsequently myelination, as demonstrated in the biologically relevant in vitro platform used in this study. These results can aid in the development of novel PDE4 inhibition-based therapies in the advancement of peripheral regenerative medicine.

A Humanized In Vitro Model of Innervated Skin for Transdermal Analgesic Testing.

Sensory innervation of the skin is essential for its function, homeostasis, and wound healing mechanisms. Thus, to adequately model the cellular microenvironment and function of native skin, in vitro human skin equivalents (hSE) containing a sensory neuron population began to be researched. In this work, a fully human 3D platform of hSE innervated by induced pluripotent stem cell-derived nociceptor neurospheres (hNNs), mimicking the native mode of innervation, is established. Both the hSE and nociceptor population exhibit morphological and phenotypical characteristics resembling their native counterparts, such as epidermal and dermal layer formation and nociceptor marker exhibition, respectively. In the co-culture platform, neurites develop from the hNNs and navigate in 3D to innervate the hSE from a distance. To probe both skin and nociceptor functionality, a clinically available capsaicin patch (Qutenza) is applied directly over the hSE section and neuron reaction is analyzed. Application of the patch causes an exposure time-dependent neurite regression and degeneration. In platforms absent of hSE, axonal degeneration is further increased, highlighting the role of the skin construct as a barrier. In sum, an in vitro tool of functional innervated skin with high interest for preclinical research is established.

Development of an In Vitro Biomimetic Peripheral Neurovascular Platform.

Nerves and blood vessels are present in most organs and are indispensable for their function and homeostasis. Within these organs, neurovascular (NV) tissue forms congruent patterns and establishes vital interactions. Several human pathologies, including diabetes type II, produce NV disruptions with serious consequences that are complicated to study using animal models. Complex in vitro organ platforms, with neural and vascular supply, allow the investigation of such interactions, whether in a normal or pathological context, in an affordable, simple, and direct manner. To date, a few in vitro models contain NV tissue, and most strategies report models with nonbiomimetic representations of the native environment. To this end, we have established here an NV platform that contains mature vasculature and neural tissue, composed of human microvascular endothelial cells (HMVECs), induced pluripotent stem cell (iPSCs)-derived sensory neurons, and primary rat Schwann cells (SCs) within a fibrin-embedded polymeric scaffold. First, we show that SCs can induce the formation of and stabilize vascular networks to the same degree as the traditional and more thoroughly studied human dermal fibroblasts (HDFs). We also show that through SC prepatterning, we are able to control vessel orientation. Using our NV platform, we demonstrate the concomitant formation of three-dimensional neural and vascular tissue, and the influence of different medium formulations and cell types on the NV tissue outcome. Finally, we propose a protocol to form mature NV tissue, via the integration of independent neural and vascular constituents. The platform described here provides a versatile and advanced model for in vitro research of the NV axis.

Modular mixing of benzene-1,3,5-tricarboxamide supramolecular hydrogelators allows tunable biomimetic hydrogels for control of cell aggregation in 3D.

Few synthetic hydrogels can mimic both the viscoelasticity and supramolecular fibrous structure found in the naturally occurring extracellular matrix (ECM). Furthermore, the ability to control the viscoelasticity of fibrous supramolecular hydrogel networks to influence cell culture remains a challenge. Here, we show that modular mixing of supramolecular architectures with slow and fast exchange dynamics can provide a suitable environment for multiple cell types and influence cellular aggregation. We employed modular mixing of two synthetic benzene-1,3,5-tricarboxamide (BTA) architectures: a small molecule water-soluble BTA with slow exchange dynamics and a telechelic polymeric BTA-PEG-BTA with fast exchange dynamics. Copolymerisation of these two supramolecular architectures was observed, and all tested formulations formed stable hydrogels in water and cell culture media. We found that rational tuning of mechanical and viscoelastic properties is possible by mixing BTA with BTA-PEG-BTA. These hydrogels showed high viability for both chondrocyte (ATDC5) and human dermal fibroblast (HDF) encapsulation (>80%) and supported neuronal outgrowth (PC12 and dorsal root ganglion, DRG). Furthermore, ATDC5s and human mesenchymal stem cells (hMSCs) were able to form spheroids within these viscoelastic hydrogels, with control over cell aggregation modulated by the dynamic properties of the material. Overall, this study shows that modular mixing of supramolecular architectures enables tunable fibrous hydrogels, creating a biomimetic environment for cell encapsulation. These materials are suitable for the formation and culture of spheroids in 3D, critical for upscaling tissue engineering approaches towards cell densities relevant for physiological tissues.

3D culture platform of human iPSCs-derived nociceptors for peripheral nerve modeling and tissue innervation.

Functional humanizedin vitronerve models are coveted as an alternative to animal models due to their ease of access, lower cost, clinical relevance and no need for recurrent animal sacrifice. To this end, we developed a sensory nerve model using induced pluripotent stem cells-derived nociceptors that are electrically active and exhibit a functional response to noxious stimuli. The differentiated neurons were co-cultured with primary Schwann cells on an aligned microfibrous scaffold to produce biomimetic peripheral nerve tissue. Compared to glass coverslips, our scaffold enhances tissue development and stabilization. Using this model, we demonstrate that myelin damage can be induced from hyperglycemia exposure (glucose at 45 mM) and mitigated by epalrestat (1µM) supplementation. Through fibrin embedding of the platform, we were able to create 3D anisotropic myelinated tissue, reaching over 6.5 mm in length. Finally, as a proof-of-concept, we incorporated pancreatic pseudoislets and endometrial organoids into our nerve platform, to demonstrate the potential in generating nociceptor innervation models. In summary, we propose here an improved tool for neurobiology research with potential applications in pathology modeling, drug screening and target tissue innervation.

Peripheral neurovascular link: an overview of interactions and in vitro models.

Nerves and blood vessels (BVs) establish extensive arborized networks to innervate tissues and deliver oxygen/metabolic support. Developmental cues direct the formation of these intricate and often overlapping patterns, which reflect close interactions within the peripheral neurovascular system. Besides the mutual dependence to survive and function, nerves and BVs share several receptors and ligands, as well as principles of differentiation, growth and pathfinding. Neurovascular (NV) interactions are maintained in adult life and are essential for certain regenerative mechanisms, such as wound healing. In pathological situations (e.g., type 2 diabetes mellitus), the NV system can be severely perturbed and become dysfunctional. Unwanted neural growth and vascularization are also associated with the progression of some pathologies, such as cancer and endometriosis. In this review, we describe the fundamental NV interactions in development, highlighting the similarities between both networks and wiring mechanisms. We also describe the NV contribution to regenerative processes and potential pathological dysfunctions. Finally, we provide an overview of current in vitro models used to replicate and investigate the NV ecosystem, addressing present limitations and future perspectives.

A three-dimensional biomimetic peripheral nerve model for drug testing and disease modelling.

In vitro peripheral nerve models provide valuable tools to study neurobiology questions and assess drug performance, in a regenerative or pathology context. To this end, we have developed a representative model of the peripheral nerve that displays three-dimensional (3D) neural anisotropy and myelination, which we showcase here as a simple and low-cost platform for drug screening. The model is composed of three main parts, including rat primary Schwann cells (SCs) seeded onto an electrospun scaffold to create bands of Büngner (BoB), primed PC12 cells as neuronal cell population, and a fibrin hydrogel to provide three-dimensionality. We also validated the use of primed PC12 as a neuron population by comparing it to rat dorsal root ganglions (DRGs) neurons. In both models we could obtain well aligned neurites and mature myelin segments. In short term cultures (7 days), we found that the addition of exogenous SCs enhanced neurite length and neurite growth area, compared to scaffolds with a laminin coating only. Addition of fibrin also lead to increased outgrowth of DRG and primed PC12 neurites, compared to 2D cultures. Moreover, neurite outgrowth in fibrin cultures was simultaneously multiplanar and anisotropic, suggesting that the SC-seeded scaffold can direct not only the growth of adjacent neurites, but also those growing above it. These results highlight the feasibility of the combination of a SC pre-seeded scaffold with a fibrin hydrogel, to direct and improve neurite growth in 3D. To demonstrate the model potential, we tested our platform at an immature (7 days in vitro) and mature state (28 days in vitro) of development. At the immature stage we could inhibit neurite growth through protein blocking (via antibody binding) and show suramin (200 µM) neurotoxicity on cells. At the mature stage, when myelin is compact, we exposed cells to hyperglycemic conditions (45 mM glucose) to mimic diabetic conditions and showed that myelin deforms consequently. Moreover, we demonstrated that by supplementing cultures with epalrestat (1 µM), myelin deformation can be partly prevented. In sum, we developed a biomimetic nerve platform using an affordable and accessible cell line as neuronal population, which displays similar results to primary neurons, but does not require recurrent animal sacrifice. This platform holds great promise as it can be used to conveniently and inexpensively perform drug screenings on peripheral nerve-like tissue, in a normal or pathological state.

Multivalency Enables Dynamic Supramolecular Host-Guest Hydrogel Formation.

Supramolecular and dynamic biomaterials hold promise to recapitulate the time-dependent properties and stimuli-responsiveness of the native extracellular matrix (ECM). Host-guest chemistry is one of the most widely studied supramolecular bonds, yet the binding characteristics of host-guest complexes (ß-CD/adamantane) in relevant biomaterials have mostly focused on singular host-guest interactions or nondiscrete multivalent pendent polymers. The stepwise synergistic effect of multivalent host-guest interactions for the formation of dynamic biomaterials remains relatively unreported. In this work, we study how a series of multivalent adamantane (guest) cross-linkers affect the overall binding affinity and ability to form supramolecular networks with alginate-CD (Alg-CD). These binding constants of the multivalent cross-linkers were determined via NMR titrations and showed increases in binding constants occurring with multivalent constructs. The higher multivalent cross-linkers enabled hydrogel formation; furthermore, an increase in binding and gelation was observed with the inclusion of a phenyl spacer to the cross-linker. A preliminary screen shows that only cross-linking Alg-CD with an 8-arm-multivalent guest results in robust gel formation. These cytocompatible hydrogels highlight the importance of multivalent design for dynamically cross-linked hydrogels. These materials hold promise for development toward cell- and small molecule-delivery platforms and allow discrete and fine-tuning of network properties.

Direct Writing Electrospinning of Scaffolds with Multidimensional Fiber Architecture for Hierarchical Tissue Engineering.

Nanofibrous structures have long been used as scaffolds for tissue engineering (TE) applications, due to their favorable characteristics, such as high porosity, flexibility, high cell attachment and enhanced proliferation, and overall resemblance to native extracellular matrix (ECM). Such scaffolds can be easily produced at a low cost via electrospinning (ESP), but generally cannot be fabricated with a regular and/or complex geometry, characterized by macropores and uniform thickness. We present here a novel technique for direct writing (DW) with solution ESP to produce complex three-dimensional (3D) multiscale and ultrathin (∼1 µm) fibrous scaffolds with desirable patterns and geometries. This technique was simply achieved via manipulating technological conditions, such as spinning solution, ambient conditions, and processing parameters. Three different regimes in fiber morphologies were observed, including bundle with dispersed fibers, bundle with a core of aligned fibers, and single fibers. The transition between these regimes depended on tip to collector distance (Wd) and applied voltage (V), which could be simplified as the ratio V/Wd. Using this technique, a scaffold mimicking the zonal organization of articular cartilage was further fabricated as a proof of concept, demonstrating the ability to better mimic native tissue organization. The DW scaffolds directed tissue organization and fibril matrix orientation in a zone-dependent way. Comparative expression of chondrogenic markers revealed a substantial upregulation of Sox9 and aggrecan (ACAN) on these structures compared to conventional electrospun meshes. Our novel method provides a simple way to produce customized 3D ultrathin fibrous scaffolds, with great potential for TE applications, in particular those for which anisotropy is of importance.

Patterning Vasculature: The Role of Biofabrication to Achieve an Integrated Multicellular Ecosystem.

In regenerative medicine (RM), creating engineered tissues with functionally relevant vasculature is a critical goal. Recent technological advances in biofabrication and bioprinting have been reported which present significant steps toward achieving this aim. While many approaches to address this challenge derive from microfabrication techniques, progress in the material science field and 3D printing technologies fields have introduced exciting new possibilities for the creation of increasingly complex and functional vascularized tissues. Here, we provide a brief overview of the process of vascularization and its importance within the fields of RM and tissue engineering (TE). We give a brief synopsis of various strategies that have been reported to induce cell patterning for a designed vascular network within a TE construct, including material-based strategies, structural molding approaches, and direct cell-patterning techniques. As well as highlighting advances in the field, we discuss possible areas for further development; in particular, we advocate a combination of strategies to successfully overcome current limitations in developing functional artificial tissues. Overall, the technological innovations in new bioprinting approaches and complementary progress in materials development are recognized as having critical roles as TE matures toward broadly applicable, clinically relevant applications.