Human 3D nucleus pulposus microtissue model to evaluate the potential of pre-conditioned nasal chondrocytes for the repair of degenerated intervertebral disc.

Introduction: An in vitro model that appropriately recapitulates the degenerative disc disease (DDD) microenvironment is needed to explore clinically relevant cell-based therapeutic strategies for early-stage degenerative disc disease. We developed an advanced 3D nucleus pulposus (NP) microtissues (µT) model generated with cells isolated from human degenerating NP tissue (Pfirrmann grade: 2-3), which were exposed to hypoxia, low glucose, acidity and low-grade inflammation. This model was then used to test the performance of nasal chondrocytes (NC) suspension or spheroids (NCS) after pre-conditioning with drugs known to exert anti-inflammatory or anabolic activities. Methods: NPµTs were formed by i) spheroids generated with NP cells (NPS) alone or in combination with ii) NCS or iii) NC suspension and cultured in healthy or degenerative disc disease condition. Anti-inflammatory and anabolic drugs (amiloride, celecoxib, metformin, IL-1Ra, GDF-5) were used for pre-conditioning of NC/NCS. The effects of pre-conditioning were tested in 2D, 3D, and degenerative NPµT model. Histological, biochemical, and gene expression analysis were performed to assess matrix content (glycosaminoglycans, type I and II collagen), production and release of inflammatory/catabolic factors (IL-6, IL-8, MMP-3, MMP-13) and cell viability (cleaved caspase 3). Results: The degenerative NPµT contained less glycosaminoglycans, collagens, and released higher levels of IL-8 compared to the healthy NPµT. In the degenerative NPµT, NCS performed superior compared to NC cell suspension but still showed lower viability. Among the different compounds tested, only IL-1Ra pre-conditioning inhibited the expression of inflammatory/catabolic mediators and promoted glycosaminoglycan accumulation in NC/NCS in DDD microenvironment. In degenerative NPµT model, preconditioning of NCS with IL-1Ra also provided superior anti-inflammatory/catabolic activity compared to non-preconditioned NCS. Conclusion: The degenerative NPµT model is suitable to study the responses of therapeutic cells to microenvironment mimicking early-stage degenerative disc disease. In particular, we showed that NC in spheroidal organization as compared to NC cell suspension exhibited superior regenerative performance and that IL-1Ra pre-conditioning of NCS could further improve their ability to counteract inflammation/catabolism and support new matrix production within harsh degenerative disc disease microenvironment. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of IVD repair.

Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc.

Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.

Engineered Magnetic Nanocomposites to Modulate Cellular Function.

Magnetic nanoparticles (MNPs) have various applications in biomedicine, including imaging, drug delivery and release, genetic modification, cell guidance, and patterning. By combining MNPs with polymers, magnetic nanocomposites (MNCs) with diverse morphologies (core-shell particles, matrix-dispersed particles, microspheres, etc.) can be generated. These MNCs retain the ability of MNPs to be controlled remotely using external magnetic fields. While the effects of these biomaterials on the cell biology are still poorly understood, such information can help the biophysical modulation of various cellular functions, including proliferation, adhesion, and differentiation. After recalling the basic properties of MNPs and polymers, and describing their coassembly into nanocomposites, this review focuses on how polymeric MNCs can be used in several ways to affect cell behavior. A special emphasis is given to 3D cell culture models and transplantable grafts, which are used for regenerative medicine, underlining the impact of MNCs in regulating stem cell differentiation and engineering living tissues. Recent advances in the use of MNCs for tissue regeneration are critically discussed, particularly with regard to their prospective involvement in human therapy and in the construction of advanced functional materials such as magnetically operated biomedical robots.

Nose to Spine: spheroids generated by human nasal chondrocytes for scaffold-free nucleus pulposus augmentation.

Cell-based strategies for nucleus pulposus (NP) regeneration that adequately support the engraftment and functionality of therapeutic cells are still lacking. This study explores a scaffold-free approach for NP repair, which is based on spheroids derived from human nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. We generated NC spheroids (NCS) in two types of medium (growth or chondrogenic) and analyzed their applicability for NP repair with regard to injectability, biomechanical and biochemical attributes, and integration potential in conditions simulating degenerative disc disease (DDD). NCS engineered in both media were compatible with a typical spinal needle in terms of size (lower than 600µm), shape (roundness greater than 0.8), and injectability (no changes in morphology and catabolic gene expression after passing through the needle). While growth medium ensured stable elastic modulus (E) at 5 kPa, chondrogenic medium time-dependently increased E of NCS, in correlation with gene/protein expression of collagen. Notably, DDD-mimicking conditions did not impair NCS viability nor NCS fusion with NP spheroids simulating degenerated NP in vitro. To assess the feasibility of this approach, NCS were injected into an ex vivo-cultured bovine intervertebral disc (IVD) without damage using a spinal needle. In conclusion, our data indicated that NC cultured as spheroids can be compatible with strategies for minimally invasive NP repair in terms of injectability, tuneability, biomechanical features, and resilience. Future studies will address the capacity of NCS to integrate within degenerated NP under long-term loading conditions. STATEMENT OF SIGNIFICANCE: Current regenerative strategies still do not sufficiently support the engraftment of therapeutic cells in the nucleus pulposus (NP). We present an injectable approach based on spheroids derived from nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. NC spheroids (NCS) generated with their own matrix and demonstrated injectability, tuneability of biomechanical/biochemical attributes, and integration potential in conditions simulating degenerative disc disease. To our knowledge, this is the first study that explored an injectable spheroid-based scaffold-free approach, which showed potential to support the adhesion and viability of therapeutic cells in degenerated NP. The provided information can be of substantial interest to a wide audience, including biomaterial scientists, biomedical engineers, biologists and medical researchers.