Senescent response in inner annulus fibrosus cells in response to TNFα, H2O2, and TNFα-induced nucleus pulposus senescent secretome.

Senescence, particularly in the nucleus pulposus (NP) cells, has been implicated in the pathogenesis of disc degeneration, however, the mechanism(s) of annulus fibrosus (AF) cell senescence is still not well understood. Both TNFα and H2O2, have been implicated as contributors to the senescence pathways, and their levels are increased in degenerated discs when compared to healthy discs. Thus, the objective of this study is to identify factor(s) that induces inner AF (iAF) cell senescence. Under TNFα exposure, at a concentration previously shown to induce senescence in NP cells, bovine iAF cells did not undergo senescence, indicated by their ability to continue to proliferate as demonstrated by Ki67 staining and growth curves and lack of expression of the senescent markers, p16 and p21. The lack of senescent response occurred even though iAF express higher levels of TNFR1 than NP cells. Interestingly, iAF cells showed no increase in intracellular ROS or secreted H2O2 in response to TNFα which contrasted to NP cells that did. Following TNFα treatment, only iAF cells had increased expression of the superoxide scavengers SOD1 and SOD2 whereas NP cells had increased NOX4 gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2 (50 µM) induced senescence, however unlike TNFα, H2O2 did not induce degenerative-like changes as there was no difference in COL2, ACAN, MMP13, or IL6 gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells may have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling by senescent NP cells, iAF and TNFα-treated NP cells were co-cultured. In contact co-culture the NP cells induced iAF senescence. Thus, senescent NP cells may secrete soluble factors that induce degenerative and senescent changes within the iAF. This may contribute to a positive feedback loop of disc degeneration. It is possible these factors may include H2O2 and cytokines (such as TNFα). Further studies will investigate if human disc cells respond similarly.

Immunomagnetic Isolation and Enrichment of Microvascular Endothelial Cells from Human Adipose Tissue.

Human adipose tissue-resident microvascular endothelial cells are not only garnering attention for their emergent role in the pathogenesis of obesity-related metabolic disorders, but are also of considerable interest for vascular tissue engineering due, in part, to the abundant, accessible, and uniquely dispensable nature of the tissue. Here, we delineate a protocol for the acquisition of microvascular endothelial cells from human fat. A cheaper, smaller, and simpler alternative to fluorescence-assisted cell sorting for the immunoselection of cells, our protocol adapts magnet-assisted cell sorting for the isolation of endothelial cells from enzymatically digested adipose tissue and the subsequent enrichment of their primary cultures. Strategies are employed to mitigate the non-specific uptake of immunomagnetic microparticles, enabling the reproducible acquisition of human adipose tissue-resident microvascular endothelial cells with purities ≥98%. They exhibit morphological, molecular, and functional hallmarks of endothelium, yet retain a unique proteomic signature when compared with endothelial cells derived from different vascular beds. Their cultures can be expanded for >10 population doublings and can be maintained at confluence for at least 28 days without being overgrown by residual stromal cells from the cell sorting procedure. The isolation of human adipose tissue-resident microvascular endothelial cells can be completed within 6 hours and their enrichment within 2 hours, following approximately 7 days in culture. Graphical abstract.

Induced senescence of healthy nucleus pulposus cells is mediated by paracrine signaling from TNF-α-activated cells.

Intervertebral disc degeneration is an irreversible process associated with accumulation of senescent nucleus pulposus (NP) cells. This study investigates the hypothesis that Tumor necrosis factor-α (TNF-α)-treated senescent NP cells propagate senescence of neighboring healthy cells via a paracrine effect that involves p-Stat3 signaling and the cytokine interleukin-6 (IL-6). NP cells isolated from bovine caudal intervertebral disc (IVD) were treated with TNF-α to induce senescence which was confirmed by demonstrating upregulation of senescence-associated ß-galactosidase and p16. This was correlated with downregulation of NP-associated markers, Aggrecan, Col2A1, and Sox9. Direct contact and non-contact co-culture of healthy and senescent cells showed that TNF-α-treated cells increased the senescence in healthy cells via a paracrine effect. The senescent cells have a secretory phenotype as indicated by increased gene and protein levels of IL-6. Phosphorylated Signal Transducer and Activator of Transcription 3 (pStat3) levels were also high in treated cells and appeared to upregulate IL-6 as inhibition of Stat3 phosphorylation by StatticV downregulated IL-6 mRNA expression in cells and protein levels in the culture media. All trans retinoic acid, an IL-6 inhibitor, also decreased the secretion of IL-6 and reduced the paracrine effect of senescent cells on healthy cells. Decreased pStat3 levels and inhibition of IL-6 secretion did not fully restore NP gene expression of Col2A1 but importantly, appeared to cause senescent cells to undergo apoptosis and cell death. This study demonstrated the paracrine effect of senescent NP cells which involves Stat3 and IL-6 and may explain why senescent NP cells accumulate in IVD with age. The role of pSTAT3 and IL-6 in mediating NP senescence requires further study as it may be a novel strategy for modulating the senescent-inducing effects of TNF-α.

Transforming Growth Factor ß Enhances Tissue Formation by Passaged Nucleus Pulposus Cells In Vitro.

The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor ß (TGFß) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFß1 or TGFß3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFß1 nor TGFß3 were able to restore the notochordal phenotype. Although TGFß1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:438-449, 2020.

Biomaterials for polynucleotide delivery to anchorage-independent cells.

Anchorage-independent cells possess morphological features and cell membrane compositions that are distinct from adherent cells. They display minimal surface area, have a low rate of endocytosis and generally possess few proteoglycans which make it a challenge to deliver nucleic acids into them. Wide ranges of methods and materials have been developed to tackle the delivery obstacles for the polynucleotide-based therapeutics in modifying non-adherent cells. This article summarizes the techniques and biomaterials that have been utilized for transfection of anchorage-independent cells. First, physical techniques are briefly described along with particular applications for which they are well-suited. The structure-activity relationship of various biomaterial carriers of polynucleotides are then discussed with strategies employed to enhance their capability to transfect anchorage-independent cells. In conclusion, the authors' perspectives on different methods for polynucleotide delivery to primary human cells are compared, along with a discussion of their progression towards clinical trials.

Short-term cytotoxicity assessment of components of the epiphany resin-percha obturating system by indirect and direct contact millipore filter assays.

The Epiphany Resin-Percha Obturating System was assessed for cytotoxicity, compared with gutta-percha and AH-Plus sealer. Specimen disks (Resilon, gutta-percha), filled glass rings (sealers), or imbibed cellulose disks (primer, thinning resin) were placed over Millipore filters in direct or indirect contact with HeLa cell monolayer, incubated for 2 hours, and stained with tetrazolium blue. Cytotoxicity was rated by the surrounding unstained zone: none (0 mm), mild (12 mm). Data were analyzed with one-way ANOVA and post hoc pairwise t tests. Unstained zones indicating moderate cytotoxicity were significantly larger (p < 0.05) for Epiphany primer than for thinning resin and for freshly mixed AH-Plus than for Epiphany sealer. Set sealers (24 and 48 hours), gutta-percha, and Resilon elicited noncytotoxic responses. In conclusion, cytotoxicity of set Epiphany sealer and Resilon was comparable with that of set AH-Plus and gutta-percha. Cytotoxicity of freshly mixed Epiphany sealer, primer, and thinning resin did not exceed that of freshly mixed AH-Plus.

Role of protein kinase C in the monocyte-derived macrophage-mediated biodegradation of polycarbonate-based polyurethanes.

Polycarbonate-polyurethanes (PCNUs) elicit a foreign body reaction during the initial tissue contact, partly mediated by the respiratory burst in monocytes, during which protein kinase C (PKC) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase. Using an in vitro cell system, monocytes were differentiated into monocyte-derived macrophages (MDMs) and then reseeded onto three PCNUs (HDI431, HDI321, or MDI321): hexane (HDI) or 4,4-methylene bis-phenyl (MDI) diisocyanates synthesized with poly(1,6-hexyl 1,2-ethyl carbonate) diol (PCN) and 14C-labeled butanediol (BD) in the ratios 4:3:1 or 3:2:1 (diisocyanate/PCN/BD). MDM-mediated degradation was assessed by radiolabel release in the presence of a PKC activator (phorbol myristate acetate), inhibitor (H7), and a catalase/peroxidase inhibitor (NaN3). Activating PKC decreased biodegradation and esterase activity in MDMs on HDI431 and HDI321 but not MDI321, whereas H7 and NaN3 inhibited the MDM degradation of MDI321 only. Pretreatment of the PCNUs with H2O2 inhibited esterase-mediated radiolabel release from HDI431 and HDI321 but stimulated radiolabel release from MDI321. The difference in the effect of H2O2 on the HDI versus MDI PCNUs contributes to explaining the effect of PKC activation on material degradation. Understanding the mechanism by which this pathway is linked to PCNU chemistry may assist in designing materials with tailored biodegradation rates.