Staphylococcus aureus is the main causative pathogen of subcutaneous, bone, and implant-related infections, forming structures known as staphylococcal abscess communities (SACs) within tissues that also contain immunosuppressive myeloid-derived suppressor cells (MDSCs). Although both SACs and MDSCs are present in chronic S. aureus infections, it remains unknown whether SACs directly trigger MDSC expansion. To investigate this, a previously developed 3D in vitro SAC model was co-cultured with murine and human bone marrow cells. Subsequently, it was shown that SAC-exposed human CD11blow/- myeloid cells or SAC-exposed murine CD11b+ Gr-1+ cells were immunosuppressive mainly by reducing absolute CD4+ and CD8α+ T cell numbers, as shown in T cell proliferation assays and with flow cytometry. Monocytic MDSCs from mice with an S. aureus bone infection also strongly reduced CD4+ and CD8α+ T cell numbers. Using protein biomarker analysis and an immunoassay, we detected in SAC-bone marrow co-cultures high levels of GM-CSF, IL-6, VEGF, IL-1β, TNFα, IL-10, and TGF-β. Furthermore, SAC-exposed neutrophils expressed Arg-1 and SAC-exposed monocytes expressed Arg-1 and iNOS, as shown via immunofluorescent stains. Overall, this study showed that SACs cause MDSC expansion from bone marrow cells and identified possible mediators to target as an additional strategy for treating chronic S. aureus infections.

Low back pain is a clinically highly relevant musculoskeletal burden and is associated with inflammatory as well as degenerative processes of the intervertebral disc. However, the pathophysiology and cellular pathways contributing to this devastating condition are still poorly understood. Based on previous evidence, we hypothesize that tissue renin-angiotensin system (tRAS) components, including the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2), are present in human nucleus pulposus (NP) cells and associated with inflammatory and degenerative processes. Experiments were performed with NP cells from four human donors. The existence of angiotensin II, angiotensin II type 1 receptor (AGTR1), AGTR2, MAS-receptor (MasR), and ACE2 in human NP cells was validated with immunofluorescent staining and gene expression analysis. Hereafter, the cell viability was assessed after adding agonists and antagonists of the target receptors as well as angiotensin II in different concentrations for up to 48 h of exposure. A TNF-α-induced inflammatory in vitro model was employed to assess the impact of angiotensin II addition and the stimulation or inhibition of the tRAS receptors on inflammation, tissue remodeling, expression of tRAS markers, and the release of nitric oxide (NO) into the medium. Furthermore, protein levels of IL-6, IL-8, IL-10, and intracellular as well as secreted angiotensin II were assessed after exposing the cells to the substances, and inducible nitric oxide synthase (iNOS) levels were evaluated by utilizing Western blot. The existence of tRAS receptors and angiotensin II were validated in human NP cells. The addition of angiotensin II only showed a mild impact on gene expression markers. However, there was a significant increase in NO secreted by the cells. The gene expression ratios of pro-inflammatory/anti-inflammatory cytokines IL-6/IL-10, IL-8/IL-10, and TNF-α/IL-10 were positively correlated with the AGTR1/AGTR2 and AGTR1/MAS1 ratios, respectively. The stimulation of the AGTR2 MAS-receptor and the inhibition of the AGTR1 receptor revealed beneficial effects on the gene expression of inflammatory and tissue remodeling markers. This finding was also present at the protein level. The current data showed that tRAS components are expressed in human NP cells and are associated with inflammatory and degenerative processes. Further characterization of the associated pathways is warranted. The findings indicate that tRAS modulation might be a novel therapeutic approach to intervertebral disc disease.

Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix.

Inflammation plays an important role in the pathogenesis of intervertebral disc (IVD) degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has shown markedly higher expression in degenerated human disc tissue compared with healthy controls. Anti-inflammatory treatment targeting TNF-α has shown to alleviate discogenic pain in patients with low back pain. Therefore, in vitro and ex vivo inflammatory models utilizing TNF-α provide relevant experimental conditions for drug development in disc degeneration research. The current method article addressed several specific questions related to the model establishment. (a) The effects of bovine and human recombinant TNF-α on bovine nucleus pulposus (NP) cells were compared. (b) The required dose for an inflammatory IVD organ culture model with intradiscal TNF-α injection was studied. (c) The effect of TNF-α blocking at different stages of inflammation was evaluated. Outcomes revealed that bovine and human recombinant TNF-α induced equivalent inflammatory effects in bovine NP cells. A bovine whole IVD inflammatory model was established by intradiscal injection of 100 ng TNF-α/ cm3 disc volume, as indicated by increased nitric oxide, glycosaminoglycan, interleukin 6 (IL-6), and interleukin 8 (IL-8) release in culture media, and upregulation of MMP3, ADAMTS4, IL-8, IL-6, and cyclooxygenase (COX)-2 expression in NP tissue. However, results in human NP cells showed that the time point of anti-inflammatory treatment was crucial to achieve significant effects. Furthermore, anticatabolic therapy in conjunction with TNF-α inhibition would be required to slow down the pathologic cascade of disc degeneration.

Lower back pain is a leading cause of disability worldwide. The recovery of nucleus pulposus (NP) progenitor cells (NPPCs) from the intervertebral disc (IVD) holds high promise for future cell therapy. NPPCs are positive for the angiopoietin-1 receptor (Tie2) and possess stemness capacity. However, the limited Tie2+ NPC yield has been a challenge for their use in cell-based therapy for regenerative medicine. In this study, we attempted to expand NPPCs from the whole NP cell population by spheroid-formation assay. Flow cytometry was used to quantify the percentage of NPPCs with Tie2-antibody in human primary NP cells (NPCs). Cell proliferation was assessed using the population doublings level (PDL) measurement. Synthesis and presence of extracellular matrix (ECM) from NPC spheroids were confirmed by quantitative Polymerase Chain Reaction (qPCR), immunostaining, and microscopy. Compared with monolayer, the spheroid-formation assay enriched the percentage of Tie2+ in NPCs' population from ~10% to ~36%. Moreover, the spheroid-formation assay also inhibited the proliferation of the Tie2- NPCs with nearly no PDL. After one additional passage (P) using the spheroid-formation assay, NPC spheroids presented a Tie2+ percentage even further by ~10% in the NPC population. Our study concludes that the use of a spheroid culture system could be successfully applied to the culture and expansion of tissue-specific progenitors.

The blood-brain barrier is necessary to provide an optimal environment for cerebral function. It consists of endothelial cells that interact through interendothelial tight junctions and form a barrier with low permeability. Therefore, the infiltration of lymphocytes into the central nervous system is limited. Pathological conditions, such as chronic-inflammatory diseases and viral infections, induce a breakdown in the blood-brain barrier, which facilitates the accumulation of immune cells in the brain.

Bone marrow mesenchymal stromal cells (BMSCs) are promising for therapeutic use in cartilage repair, because of their capacity to differentiate into chondrocytes. Often, in vitro differentiation protocols employ the use of high amount of glucose, which does not reflect cartilage physiology. For this reason, we investigated how different concentrations of glucose can affect the chondrogenic differentiation of BMSCs in cell culture pellets. Additionally, we investigated how fructose could influence the chondrogenic differentiation in vitro.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG-HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells.

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the β1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether β1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1β (IL-1β)-induced inflammatory in vitro BBB model. We demonstrate that IL-1β increases cell-matrix adhesion and induces a redistribution of active β1 integrins to the basal surface. In particular, binding of α5β1 integrin to its ligand fibronectin is enhanced and α5β1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5β1 integrin reduces the IL-1β-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1β-induced inflammation not only destabilizes tight junctions but also increases α5β1 integrin-dependent cell-matrix adhesion to fibronectin.

Low back pain (LBP) is a global health concern. Increasing evidence implicates intervertebral disk (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, a novel progenitor cell population was described in the nucleus pulposus (NP) that is positive for Tie2 marker. These cells have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies.

Introduction: Low back pain (LBP) is a significant cause of disability in many countries, affecting more than half a billion people worldwide. In the past, progenitor cells have been found within the nucleus pulposus (NP) of the human intervertebral disc (IVD). However, in the context of cell therapy, little is known about the effect of cryopreservation and expansion on here called "heterogenic" human NP cells (hNPCs), and whether commercially available cryopreservation media are more efficient than "commonly used" media in terms of cell viability. Materials: In this study, hNPCs from four trauma patients (age 40.5 ± 14.3 years) and two patients with degenerated IVDs (age 24 and 46 years), undergoing spinal surgery, were collected. To isolate hNPCs, the tissue was digested with a mild two-step protocol. After subsequent expansion, hNPCs at passages 2-5 were separated and either cryo-preserved for 1 week at -150°C or differentiated into osteogenic, adipogenic, or chondrogenic lineages for 21 days. Cryopreservation was performed with five different media to compare their effect on the cell's viability and differentiation potential. Cell viability was determined with flow cytometry using propidium iodide and the trilineage differentiation potential was assessed by quantitative polymerase chain reaction and histological analysis. Results: After 1 week of cryopreservation, the hNPC's cell viability was comparable for all conditions, that is, independent of the cryopreservation medium used (82.3 ± 0.8% of cell viability). Furthermore, hNPCs from trauma patients showed some evidence for adipogenic and chondrogenic differentiation and at lower levels, this and evidence of osteogenic differentiation could be confirmed with hNPCs from degenerated discs. Moreover, cryopreservation did not affect the cell's differentiation potential in the majority of the cases tested. Conclusion: "Commonly used" cryopreservation media seem to perform just as well as commercially available media in terms of cell viability and the overall maintenance of the hNPCs trilineage differentiation potential.

Mesenchymal stromal cells (MSCs) have been introduced as promising cell source for regenerative medicine. Besides their multilineage differentiation capacity, MSCs release a wide spectrum of bioactive factors. This secretome holds immunomodulatory and regenerative capacities. In intervertebral disc (IVD) cells, application of MSC secretome has been shown to decrease the apoptosis rate, induce proliferation, and promote production of extracellular matrix (ECM). For clinical translation of secretome-based treatment, characterization of the secretome composition is needed to better understand the induced biological processes and identify potentially effective secretomes.