Priming as a strategy to overcome detrimental pH effects on cells for intervertebral disc regeneration.

Disc disease is characterised by degeneration of the nucleus pulposus (NP), the central gelatinous tissue of the intervertebral disc (IVD). As degeneration progresses, the microenvironment of the IVD becomes more hostile (i.e. decrease in oxygen, glucose and pH), providing a significant challenge for regeneration using cell-based therapies. Tissue engineering strategies such as priming cells or micro tissues with growth factors prior to implantation may overcome some of these issues by providing a pre-formed protective niche composed of extracellular matrix. The present study investigated the effect of priming on bone-marrow-derived stem cells (BMSCs) and articular chondrocytes (ACs) using transforming growth factor ß3 (TGF-ß3), cultured at different pH levels (pH 7.1, 6.8 and 6.5) representative of the in vivo disc microenvironment. Low pH was found to have a detrimental effect on both cell viability and matrix accumulation, which could be mitigated by priming cells using TGF-ß3. Investigating the activation of the transmembrane acid-sensing ion channels (ASIC-1 and -3) showed an increased expression of ASIC-1 in BMSCs and ASIC-3 in ACs at lower pH levels post-priming. Metabolic activity in terms of lactic acid production was also found to be affected significantly by priming, whereas oxygen and glucose consumptions did not change considerably. Overall, the study demonstrated that cells could be equipped to sustain the harsh environment of the IVD and promote accumulation of NP-like matrix through priming. Such an approach may open new avenues to engineer tissues capable of sustaining challenging microenvironments such as those found in the IVD.

Balancing biological and biomechanical performance in intervertebral disc repair: a systematic review of injectable cell delivery biomaterials.

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.

In vitro co-culture and ex vivo organ culture assessment of primed and cryopreserved stromal cell microcapsules for intervertebral disc regeneration.

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-ß3) 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,withboth 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.