CD146/MCAM distinguishes stem cell subpopulations with distinct migration and regenerative potential in degenerative intervertebral discs.

OBJECTIVE: This study aimed to characterize the mesenchymal stem cell (MSC) subpopulation migrating towards a degenerated intervertebral disc (IVD) and to assess its regenerative potential. DESIGN: Based on initial screening for migration towards C-C motif chemokine ligand 5 (CCL5), the migration potential of CD146+ and CD146- mesenchymal stem cells (MSCs) was evaluated in vitro and in a degenerated organ culture model (degeneration by high-frequency loading in a bioreactor). Discogenic differentiation potential of CD146+ and CD146- MSCs was investigated by in vitro pellet culture assay with supplementation of growth and differentiation factor-6 (GDF6). Furthermore, trypsin degenerated IVDs were treated by either homing or injection of CD146+ or CD146- MSCs and glycosaminoglycan synthesis was evaluated by Sulphur 35 incorporation after 35 days of culture. RESULTS: Surface expression of CD146 led to a higher number of migrated MSCs both in vitro and in organ culture. CD146+ and CD146- pellets responded with a similar up-regulation of anabolic markers. A higher production of sulfated glycosaminoglycans (sGAG)/DNA was observed for CD146+ pellets, while in organ cultures, sGAG synthesis rate was higher for IVDs treated with CD146- MSCs by either homing or injection. CONCLUSIONS: The CD146+ MSC subpopulation held greater migration potential towards degenerative IVDs, while the CD146- cells induced a stronger regenerative response in the resident IVD cells. These findings were independent of the application route (injection vs migration). From a translational point of view, our data suggests that CD146+ MSCs may be suitable for re-population, while CD146- MSCs may represent the primary choice for stimulation of endogenous IVD cells.

Mechanical loading of intervertebral disc modulates microglia proliferation, activation, and chemotaxis.

OBJECTIVE: The aim of the study is to assess the effects of the neuroinflammatory microenvironment of a mechanically-induced degenerating intervertebral disc (IVD) on neuroinflammatory like cells such as microglia, in order to comprehend the role of microglial cells in degenerative disc disease. METHODS: Bovine caudal IVDs were kept in culture in an ex vivo bioreactor under high frequency loading and limited nutrition or in free swelling conditions as control samples. Conditioned media (CM) were collected, analysed for cytokine and neurotrophin content and applied to microglial cells for neuroinflammatory activation assessment. RESULTS: Degenerative conditioned medium (D-CM) induced a higher production of interleukin (IL)-8, nerve growth factor (NGF), interferon (IFN)-γ, IL-17 from IVD cells than unloaded control conditioned medium (U-CM). Upon 48 h of co-incubation with microglia, D-CM stimulated microglia proliferation, activation, with increased expression of ionized calcium binding adaptor molecule 1 (IBA1) and CD68, and chemotaxis. Moreover, an increment of nitrite production was observed. Interestingly, D-CM caused an upregulation of IL-1ß, IL-6, tumour necrosis factor α (TNFα), inducible NO synthase (iNOS), IBA1, and vascular endothelial growth factor (VEGF) genes in microglia. Similar results were obtained when microglia were treated with the combination of the measured cytokines. CONCLUSIONS: Our findings show that in IVD degenerative microenvironment, IL-8, NGF, IFN-γ, IL-17 drive activation of microglia in the spinal cord and increase upregulation of neuroinflammatory markers. This, in turn, enhances the inflammatory milieu within IVD tissues and in the peridiscal space, aggravating the cascade of degenerative events. This study provides evidence for an important role of microglia in maintaining IVD neuroinflammatory microenvironment and probably inducing low back pain.

Cobalt-containing bioactive glasses reduce human mesenchymal stem cell chondrogenic differentiation despite HIF-1α stabilisation.

Bioactive glasses (BGs) are excellent delivery systems for the sustained release of therapeutic ions and have been extensively studied in the context of bone tissue engineering. More recently, due to their osteogenic properties and expanding application to soft tissue repair, BGs have been proposed as promising materials for use at the osteochondral interface. Since hypoxia plays a critical role during cartilage formation, we sought to investigate the influence of BGs releasing the hypoxia-mimicking agent cobalt (CoBGs) on human mesenchymal stem cell (hMSC) chondrogenesis, as a novel approach that may guide future osteochondral scaffold design. The CoBG dissolution products significantly increased the level of hypoxia-inducible factor-1 alpha in hMSCs in a cobalt dose-dependent manner. Continued exposure to the cobalt-containing BG extracts significantly reduced hMSC proliferation and metabolic activity, as well as chondrogenic differentiation. Overall, this study demonstrates that prolonged exposure to cobalt warrants careful consideration for cartilage repair applications.

CCL5/RANTES is a key chemoattractant released by degenerative intervertebral discs in organ culture.

Release of chemotactic factors in response to tissue damage has been described for different musculoskeletal tissues, including the intervertebral disc (IVD). This study investigated the chemoattractants that are released by induced degenerative IVDs and may be involved in recruiting mesenchymal stem cells (MSCs). Bovine caudal discs were cultured within a bioreactor and loaded under conditions that mimicked physiological or degenerative settings. Between days 4-6, medium was replaced by PBS, which was subsequently used for proteomic, ELISA and immunoprecipitation analyses of secreted chemokines and cytokines. A Boyden chamber assay was used to observe human MSC migration towards native and chemokine depleted media. Gene expression levels of chemokine receptors in human MSCs were analysed, and CCL5 was localised in bovine and human IVD by immunohistochemistry. Proteomic analysis revealed the presence of CCL5 and CXCL6 within conditioned media. Higher concentrations of CCL5 were found in the degenerative media, and a relationship was found between interleukin-1ß and CCL5 concentration. Chemokine immunoprecipitation showed that MSCs had a significantly reduced chemotactic migration towards CCL5-immunoprecipitated and CCL5/CXCL6 co-immunoprecipitated media, whilst CXCL6 depletion did not change MSC chemotaxis. MSCs showed a significant increase in mRNA expression of the CCL5 receptors, CCR1 and CCR4, upon culture in degenerative media. Furthermore, CCL5 was identified in bovine and human disc tissue by immunohistochemistry. Hence, CCL5 may be a key chemoattractant that is produced and released by the intervertebral disc cells. Therefore, these factors could be used to enhance stem/progenitor cell mobilisation in regenerative therapies for early stages of disc degeneration.

Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production.

PURPOSE: Platelet-rich plasma (PRP) contains growth factors and creates a 3D structure upon clotting; PRP or platelet lysate (PL) might be considered for annulus fibrosus (AF) repair. METHODS: Bovine AF cells were cultured with 25% PRP, 50% PRP, 25% PL, 50% PL, or 10% FBS. After 2 and 4 days, DNA, glycosaminoglycan (GAG), and mRNA levels were analyzed. Histology was performed after injection of PRP into an AF defect in a whole disc ex vivo. RESULTS: By day 4, significant increases in DNA content were observed in all treatment groups. All groups also showed elevated GAG synthesis, with highest amounts at 50% PL. Collagen I and II expression was similar between groups; aggrecan, decorin, and versican expression was highest at 25% PL. Injection of PRP into the AF defect resulted in an increased matrix synthesis. CONCLUSIONS: Platelet-rich preparations increased the matrix production and cell number and may therefore be considered to promote AF repair.

Evaluation of a new press-fit in situ setting composite porous scaffold for cancellous bone repair: towards a "surgeon-friendly" bone filler?

In this study, a composite porous material obtained by coating a poly(ester urethane) foam with a calcium phosphate cement is proposed as novel cancellous bone filler with easy handling, in situ hardening and press-fitting properties. The coating can be applied to the foam in the surgical theater, allowing refinement of scaffold shape to the needs of the ongoing surgery. An innovative experiment was developed in order to determine the setting curve of the composite scaffold as well as the time of manipulation available to the surgeon without risk of material damage. This composite material is soft and can be press-fit in a cavity without damaging the scaffold in the first 5 min after coating application. The composite scaffold hardens quickly (22 min) and, once the cement has set, its compressive strength and fracture energy are increased by over an order of magnitude as compared to the initial poly(ester urethane) foam. This set of interesting properties makes calcium phosphate cement-coated elastomeric scaffolds a new promising strategy for cancellous bone filling.