Bioprinting of Stem Cell Spheroids Followed by Post-Printing Chondrogenic Differentiation for Cartilage Tissue Engineering.

Cartilage tissue presents low self-repair capability and lesions often undergo irreversible progression. Structures obtained by tissue engineering, such as those based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for research and therapeutic purposes. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. Herein, this work reports a reproducible bioprinting process followed by a successful post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs are bioprinted, ionically crosslinked, and post chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV are observed. After 56 days in culture, the bioprinted constructs are still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results show a promising procedure to obtain 3D models for cartilage research and ultimately, an in vitro proof-of-concept of their potential use as stable chondral tissue implants.

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.

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.