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Background: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions.Aims: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models.

Minimally invasive delivery of "living cell factories" consisting of cells and therapeutic agents has gained wide attention for next generation biomaterial device systems for multiple applications including musculoskeletal tissue regeneration, diabetes and cancer. Cellularbased microcapsules and microcarrier systems offer several attractive features for this particular purpose. One such technology capable of generating these types of systems is electrohydrodynamic (EHD) spraying. Depending on various parameters including applied voltage, biomaterial properties (viscosity, conductivity) and needle geometry, complex structures and arrangements can be fabricated for therapeutic strategies. In this paper, we outline the advances in the use of EHD technology specifically in the manipulation of bioactive and dynamic material systems to control size, composition and configuration in the development of minimally invasive micro-scaled biopolymeric systems. The exciting therapeutic applications of this technology, future perspectives and associated challenges are also presented.

Discogenic back pain is a common condition without approved intervertebral disc (IVD) repair therapies. Cell delivery using injectable biomaterial carriers offers promise to restore disc height and biomechanical function, while providing a functional niche for delivered cells to repair degenerated tissues. This systematic review advances the injectable IVD cell delivery biomaterials field by characterising its current state and identifying themes of promising strategies. Preferred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) guidelines were used to screen the literature and 183 manuscripts met the inclusion criteria. Cellular and biomaterial inputs, and biological and biomechanical outcomes were extracted from each study. Most identified studies targeted nucleus pulposus (NP) repair. No consensus exists on cell type or biomaterial carrier, yet most common strategies used mesenchymal stem cell (MSC) delivery with interpenetrating network/co-polymeric (IPN/CoP) biomaterials composed of natural biomaterials. All studies reported biological outcomes with about half the studies reporting biomechanical outcomes. Since the IVD is a load-bearing tissue, studies reporting compressive and shear moduli were analysed and two major themes were found. First, a competitive balance, or ‘seesaw’ effect, between biomechanical and biological performance was observed. Formulations with higher moduli had inferior cellular performance, and vice versa. Second, several low-modulus biomaterials had favourable biological performance and matured throughout culture duration with enhanced extracellular matrix synthesis and biomechanical moduli. Findings identify an opportunity to develop next-generation biomaterials that provide high initial biomechanical competence to stabilise and repair damaged IVDs with a capacity to promote cell function for long-term healing.

Collagen plays a key structural role in both the annulus fibrosus (AF) and nucleus pulposus (NP) of intervertebral disks (IVDs). Changes in collagen content with degeneration suggest a shift from collagen type II to type I within the NP, and the activation of pro-inflammatory factors is indicative of fibrosis throughout. While IVD degeneration is considered a fibrotic process, an increase in collagen content with degeneration, reflective of fibrosis, has not been demonstrated. Additionally, changes in collagen content and structure in human IVDs with degeneration have not been characterized with high spatial resolution. The collagen content of 23 human lumbar L2/3 or L3/4 IVDs was quantified using second harmonic generation imaging (SHG) and multiple image processing algorithms, and these parameters were correlated with the Rutges histological degeneration grade. In the NP, SHG intensity increased with degeneration grade, suggesting fibrotic collagen deposition. In the AF, the entropy of SHG intensity was reduced with degeneration indicating increased collagen uniformity and suggesting less-organized lamellar structure. Collagen orientation entropy decreased throughout most IVD regions with increasing degeneration grade, further supporting a loss in collagen structural complexity. Overall, SHG imaging enabled visualization and quantification of IVD collagen content and organization with degeneration. There was an observed shift from an initially complex structure to more uniform structure with loss of microstructural elements and increased NP collagen polarity, suggesting fibrotic remodeling.

Priming towards a discogenic phenotype and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs) may offer an attractive therapeutic approach for disc repair. It potentially obviates the need for in vivo administration of exogenous growth factors, otherwise required to promote matrix synthesis, in addition to providing 'off-the-shelf' availability. Cryopreserved and primed BMSC microcapsules were evaluated in an in vitro surrogate co-culture model system with nucleus pulposus (NP) cells under intervertebral disc (IVD)-like culture conditions and in an ex vivo bovine organ culture disc model.BMSCs were microencapsulated in alginate microcapsules and primed for 14 d with transforming growth factor beta-3 (TGF-β3) under low oxygen conditions prior to cryopreservation. For the in vitro phase, BMSC microcapsules (unprimed or primed) were cultured for 28 d in a surrogate co-culture model system mimicking that of the IVD. For the ex vivo phase, microcapsules (unprimed or primed) were injected into the NP of bovine discs that underwent nucleotomy.In vitro results revealed that although NP cells produced significantly more matrix components in co-culture with BMSC microcapsules regardless of the differentiation state, unprimed microcapsules were inadequate at synthesising matrix as compared to primed microcapsules. However, this difference was diminished when evaluated in the ex vivo organ culture model, with both unprimed and primed BMSC microcapsules accumulating large amounts of sulphated glycosaminoglycan (sGAG) and collagen and filling the defect cavity. Both models demonstrated that cryopreservation of BMSC microcapsules may offer a feasible strategy for predesigned delivery through cryobanking for on-demand regeneration of the IVD.

Cell delivery and leakage during injection remains a challenge for cell-based intervertebral disc regeneration strategies. Cellular microencapsulation may offer a promising approach to overcome these limitations by providing a protective niche during intradiscal injection. Electrohydrodynamic spraying (EHDS) is a versatile one-step approach for microencapsulation of cells using a high voltage electric field. The primary objective of this work was to characterise key processing parameters such as applied voltage (0, 5, 10 or 15 kV), emitter needle gauge (21, 26 or 30 G), alginate concentration (1%, 2% or 3%) and flow rate (50, 100, 250 or 500 μl min) to regulate the size and morphology of alginate microcapsules as well as subsequent cell viability when altering these parameters. The effect of initial cell seeding density (5, 10 and 20 × 10 cells ml) on subsequent matrix accumulation of microencapsulated articular chondrocytes was also evaluated. Results showed that increasing alginate concentration and thus viscosity increased overall microcapsule size but also affected the geometry towards ellipsoidal-shaped gels. Altering the electric field strength and needle diameter regulated microcapsule size towards a smaller diameter with increasing voltage and smaller needle diameter. Needle size did not appear to affect cell viability when operating with lower alginate concentrations (1% and 2%), although higher concentrations (3%) and thus higher viscosity hydrogels resulted in diminished viability with decreasing needle diameter. Increasing cell density resulted in decreased cell viability and a concomitant decrease in DNA content, perhaps due to competing nutrient demands as a result of more closely packed cells. However, higher cell densities resulted in increased levels of extracellular matrix accumulated. Overall, this work highlights the potential of EHDS as a controllable and versatile approach to fabricate microcapsules for injectable delivery which can be used in a variety of applications such as drug development or cell therapies.

A challenge in using stromal cells for intervertebral disc (IVD) regeneration is their limited differentiation capacity in vivo without exogenous growth factor (GF) supplementation. Priming of stromal cells prior to transplantation may offer a feasible strategy to overcome this limitation. Furthermore, the ability to cryopreserve cells could help alleviate logistical issues associated with storage and transport. With these critical translational challenges in mind, we aimed to develop a strategy involving priming and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs). In phase one, we utilised the electrohydrodynamic atomisation process to fabricate BMSC-encapsulated microcapsules that were primed with TGF-β3 for 14 d after which they were cultured for a further 21 d under basal or GF supplemented media conditions. Results showed that priming induced differentiation of BMSC microcapsules such that they synthesised significant amounts of sGAG (61.9 ± 2.0 μg and 55.3 ± 6.1 μg for low and high cell densities) and collagen (24.4 ± 1.9 μg and 55.3 ± 4.6 μg for low and high cell densities) in continued culture without GF supplementation compared to Unprimed microcapsules. Phase two of this work assessed the extracellular matrix forming capacity of Primed BMSC microcapsules over 21 d after cryopreservation. Notably, primed and cryopreserved BMSCs successfully retained the ability to synthesise both sGAG (24.8 ± 2.7 μg and 75.1 ± 11.6 μg for low and high cell densities) and collagen (26.4 ± 7.8 μg and 93.1 ± 10.2 μg for low and high cell densities) post-cryopreservation. These findings demonstrate the significant potential of priming and cryopreservation approaches for IVD repair and could possibly open new horizons for pre-designed, 'off-the-shelf' injectable therapeutics.