Engineering human nasal chondrocyte spheroids, towards regeneration of the intervertebral disc

Chronic low back pain (LBP) is a leading cause of disability worldwide and is strongly associated with degeneration of the intervertebral disc (IVD) [1, 2]. Current therapies for the treatment of discogenic LBP (termed degenerative disc disease, DDD) include conservative or surgical methods which have their limitations and drawbacks. These may include discectomy, fusion surgery and disc arthroplasty [3]. Nevertheless, in recent years, scientific knowledge on human physiology and cell biology as well as interactions has greatly increased creating a tremendous impact in the advancement of tissue engineering and prominently regenerative techniques for IVD repair [4]. Cell therapy has shown promising results for IVD regeneration.
Regenerative IVD therapies target minimal-invasive application of autologous cells amongst others that should ideally differentiate to IVD-like cells and/or secrete trophic and anti-inflammatory factors with the aim to repair the IVD [3, 5-7]. A typical approach taken is to combine cell suspension with biocompatible materials which are supposed to provide mechanical stability and protection of the cells after implantation. Nevertheless, none of these strategies have been widely accepted for clinical implementation. Several publications have indicated that therapeutic cells suffer within the harsh microenvironment of the DDD [5, 8-12]. Human nasal chondrocytes (NC) are a promising cell source for the treatment of DDD. NC can be harvested from nasoseptal cartilage biopsy obtained under local anaesthesia and with minimal donor site morbidity [13-15]. They were shown to possess high proliferation and post-expansion differentiation capacity and can adapt to the heterotopic transplantation sites [16]. Moreover, they showed superior viability over articular chondrocytes (AC) and mesenchymal stromal/stem cells (MSC) in simulated DDD microenvironment thus represent a robust cell population with the potential of survival in the IVD post injection [17].
Within other tissue engineering fields, it has been demonstrated that spheroid-based strategies might help to overcome hurdles associated with DDD due to their superior regenerative performance and/or resistance compared to single cells [18, 19]. Spheroids can self-assemble by forming intercellular contacts on non-adhesive substrates [20-23]. Their 3D organization mimics closer the physiological conditions and allows the cells to differentiate toward target tissue. The 3D structure allows the cells to accumulate ECM that might protect them from a surrounding harsh micromilieu [1, 24]. Furthermore, by priming the spheroids using biomimetic environment [25-27], genetically modifying the cells [28, 29], and/or combining the spheroids with an instructive biomaterial [30] is hypothesized to further increase their performance within a harsh microenvironment. Therefore, taking a spheroid-based therapeutic approach for IVD repair could be a promising strategy.
Currently applied in vitro models to study cell treatment strategies for IVD repair use 2D and 3D culture systems among others [31]. The use of an in vitro 3D model mimicking the degenerated IVD environment, would allow to study the regenerative performance of the therapeutic cells. The harsh IVD microenvironment is characterized by avascular, hypoxia, low glucose level, acidic, inflamed, high osmolality and non-physiological biomechanics [32]. However, it has been shown that less than 15% of the in vitro studies include either one of these parameters thus not mimic the harsh IVD microenvironment properly [31].
In my PhD thesis I give an introduction into the field of my research (Chapter 1: Introduction) and highlight the areas of future research in spheroid-based regeneration of IVD in the form of a review (Chapter 2: Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc). I 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.
I report my findings on the suitability of nasal chondrocyte derived spheroids (NCS) for use as grafts in cell-based and scaffold-free regeneration of the NP. In particular, I investigate whether human NCS (1) can be generated in a feasible and reproducible way (2) possess the biomechanical and biochemical properties relevant for the target NP tissue, and (3) can be injected into the IVD via a spinal needle and engraft within a DDD-mimicking microenvironment (Chapter 3: Nose to Spine: spheroids generated by human nasal chondrocytes for scaffold-free nucleus pulposus augmentation).
Furthermore, I introduce a novel 3D in vitro degenerative NP micro-tissue (NPµT) model that allows researchers to study the reaction of NCS to the harsh DDD microenvironment. I investigate how pre-conditioning of the NCS with FDA approved drugs could enhance their performance within DDD microenvironment (Chapter 4: Human 3D nucleus pulposus micro-tissue model to evaluate the potential of pre-conditioned nasal chondrocytes for the repair of degenerated intervertebral disc).