Hyaluronic Acid Based Hydrogels Attenuate Inflammatory Receptors and Neurotrophins in Interleukin-1ß Induced Inflammation Model of Nucleus Pulposus Cells.

Inflammation plays an important role in symptomatic intervertebral disc degeneration and is associated with the production of neurotrophins in sensitizing innervation into the disc. The use of high molecular weight (HMw) hyaluronic acid (HA) hydrogels offers a potential therapeutic biomaterial for nucleus pulposus (NP) regeneration as it exerts an anti-inflammatory effect and provides a microenvironment that is more suitable for NP. Therefore, it was hypothesized that cross-linked HMw HA hydrogels modulate the inflammatory receptor of IL-1R1, MyD88 and neurotrophin expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in an in vitro inflammation model of NP. HA cross-linking was optimized using various concentrations of 4-arm PEG-amine by determination of free carboxyl groups of HA and unreacted free amine groups of PEG-amine. The optimally cross-linked HA hydrogels were characterized for hydrolytic stability, enzymatic degradation and cytotoxicity on NP cells. The therapeutic effect of HA hydrogels was further investigated in IL-1ß induced inflammation on NP cell cultures and the mechanism of HA by examining the expression of cell surface receptor of CD44. Hydrogel was optimally cross-linked at 75 mM PEG, stable in phosphate buffered saline, and showed greater than 40% resistance to enzymatic degradation. No cytotoxic effect of NP cells was observed in the presence of hydrogels for 1, 3, and 7 days. IL-1R1 and MyD88 were significantly suppressed. Additionally, NGF and BDNF mRNA were down-regulated after treatment with cross-linked HA hydrogel. Possible protective mechanism of HA is shown by high expression of CD44 receptor of NP cells after HA treatment in which suggest the binding of HA to CD44 receptor and prevent NP cells from further undergoing inflammation. These results indicate that optimally stabilized cross-linked HMw HA hydrogel has a therapeutic effect in response to inflammation-associated pain and becomes an ideal matrices hydrogel for NP regeneration.

Notch-1 mediates endothelial cell activation and invasion in psoriasis.

Notch receptor-ligand interactions are critical for cell proliferation, differentiation and survival; however, the role of Notch signalling in psoriasis remains to be elucidated. Serum amyloid A (A-SAA) is an acute-phase protein with cytokine-like properties, regulates cell survival pathways and is implicated in many inflammatory conditions. To examine the role of Notch-1 signalling in the pathogenesis of psoriasis, Notch-1, DLL-4, Jagged-1, Hrt-1/Hrt-2, A-SAA, Factor VIII and vascular endothelial growth factor (VEGF) mRNA and/or protein expression in psoriasis skin biopsies, serum and dHMVEC were assessed by immunohistology, dual-immunofluorescence, real-time PCR, ELISA and Western blotting. A-SAA-induced angiogenesis and invasion in the presence of Notch-1 siRNA was assessed by matrigel tube formation assays and Transwell invasion assay. Increased Notch-1, its ligand DLL-4 and Hrt-1 expression were demonstrated in lesional skin compared with non-lesional skin, with greatest expression observed in the dermal vasculature (P < 0.05). Dual-immunofluorescent staining demonstrated co-localization of Notch-1 to endothelial cell marker Factor VIII. A significant increase in A-SAA levels was demonstrated in psoriasis serum compared with healthy control serum (P < 0.05), and A-SAA expression was higher in lesional skin compared with non-lesional. In dHMVEC, A-SAA significantly induced Jagged-1, Hrt-1 and VEGF mRNA expression (P < 0.05) and activated Notch-1 IC indicative of transcriptional regulation. In contrast, A-SAA significantly inhibited DLL-4 mRNA expression (P < 0.05). Finally A-SAA-induced angiogenesis and invasion were inhibited by Notch-1 siRNA (P < 0.05). Notch receptor-ligand interactions mediate vascular dysfunction in psoriasis and may represent a potential therapeutic target.

Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2.

OBJECTIVE: Notch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether Notch signalling mediates vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2)-induced vascular function. METHODS: Notch-1 intracellular domain (Notch-1 IC), Notch-4 IC, Delta-like-ligand 4, Hes-related transcriptional repressors-1 and 2 (Hrt-1, Hrt-2) mRNA and/or protein expression was measured by Real-time PCR and/or western blot. VEGF/Ang2 induced EC function was assessed using transwell invasion chambers, matrigel tube formation assays and wound repair scratch assays±Notch-1 siRNA or an γ-secretase inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanly))-S-phenylglycine-t-Butyl Ester (DAPT) in RA synovial explants or human microvascular EC. Interleukin (IL)-6 and IL-8 were measured by ELISA and MMP2 and 9 by gelatine zymography. RESULTS: Notch-1 IC and Notch-4 IC protein expressions were demonstrated in RA and psoriatic arthritis synovial biopsies, with minimal expression observed in Osteoarthritis (OA). VEGF and Ang2 induced Notch-1 IC/ Notch-4 IC protein expression in synovial explant cultures and human microvascular EC levels were further potentiated by VEGF/Ang2 stimulation in combination. Notch-1, Delta-like-ligand 4, and Hrt-2 mRNA expression were significantly induced by VEGF and Ang2 alone and in combination. Furthermore VEGF/Ang2-induced EC invasion, angiogenesis and migration were inhibited by Notch-1 siRNA or DAPT. Conditioned media from VEGF/Ang2 stimulated RA synovial explants induced EC tube formation, an effect that was inhibited by DAPT. Finally, DAPT significantly decreased VEGF/Ang2 induced IL-6, IL-8, MMP2 and 9 expressions in RA synovial explants. CONCLUSIONS: Notch-1 mediates VEGF/Ang2-induced angiogenesis and EC invasion in inflammatory arthritis.

Histamine contributes to increased RANKL to osteoprotegerin ratio through altered nuclear receptor 4A activity in human chondrocytes.

OBJECTIVE: To elucidate histamine receptor-mediated signaling pathways, transcriptional events, and target gene expression in human cartilage. METHODS: Histamine modulation of cartilage destruction was assessed by Safranin O staining and proteoglycan release. H(1) , H(2) , H(3) , and H(4) histamine receptor-dependent regulation of transcription factors (nuclear receptor 4A1 [NR4A1], NR4A2, and NR4A3), RANKL, and osteoprotegerin (OPG) messenger RNA (mRNA) levels were measured in primary and SW-1353 chondrocyte cells using quantitative polymerase chain reaction and selective histamine receptor antagonists. Soluble RANKL and OPG protein levels were determined using enzyme-linked immunosorbent assays. NR4A protein levels and transactivity were evaluated by Western blot analysis, immunocytochemistry, and luciferase reporter assays. Stable depletion of NR4A1-3 was achieved by lentiviral transduction of NR4A short hairpin RNA. RESULTS: Primary human chondrocyte cells expressed differential steady-state levels of H(1) -H(4) histamine receptor mRNA. In combination with tumor necrosis factor α, histamine significantly promoted cartilage proteoglycan depletion and release. Histamine modulated the expression of NR4A1-3 orphan receptors in primary and immortalized human chondrocyte cells in a time- and concentration-dependent manner. Histamine selectively signaled through H(1) and H(2) histamine receptors in chondrocytes to modulate RANKL and NR4A2 expression. The temporal effects of histamine on NR4A2 gene transcription were reduced in cells pretreated with inhibitors directed against protein kinase A, MAPK, and NF-κB signaling pathways. Histamine modulated the expression of RANKL with modest effects on OPG levels, leading to increased RANKL:OPG mRNA and protein ratios. Stable knockdown of NR4A1-3 expression resulted in reduced endogenous OPG levels and the loss of histamine-dependent regulation of RANKL expression. CONCLUSION: Our findings indicate that histamine, via H(1) and H(2) histamine receptors, contributes to joint disease by enhancing the ratio of RANKL to OPG expression through altered NR4A activity in human chondrocyte cells.

SARS-CoV-2 epidemiology, antibody dynamics, and neutralisation capacity in Irish healthcare workers in the era of booster COVID-19 vaccinations.

Background: The PRECISE Study, a multi-phase cross-sectional seroprevalence study of anti-SARS-CoV-2 antibodies in Irish healthcare workers (HCW) investigated: (1) risk factors for SARS-CoV-2 seropositivity, (2) the durability of antibody responses in a highly vaccinated HCW cohort, and (3) the neutralisation capacity of detected antibodies, prior to booster COVID-19 vaccination. Materials and methods: Serology samples were collected across two hospital sites in November 2021 and analysed using the Roche Elecsys Anti-SARS-CoV-2/Elecsys-S Anti-SARS-CoV-2 assays to detect anti-nucleocapsid (N) and anti-spike (S) antibodies respectively. Paired serology results from prior study phases were used to analyse changes in individual HCW serostatus over time. Risk-factors for SARS-CoV-2 infection were assessed for demographic and work-related factors. Antibody neutralisation capacity was assessed in a subset of samples via an in vitro ACE2 binding enzyme-linked immunosorbent assay. Results: 2,344 HCW samples were analysed. Median age was 43 years (IQR 33-50) with 80.5% (n = 1,886) female participants. Irish (78.9%, n = 1,850) and Asian (12.3%, n = 288) were the most commonly reported ethnicities. Nursing/midwifery (39.3%, n = 922) was the most common job role. 97.7% of participants were fully vaccinated, with Pfizer (81.1%, n = 1,902) and AstraZeneca (16.1%, n = 377) the most common vaccines received. Seroprevalence for anti-SARS-CoV-2 antibodies indicating prior infection was 23.4%, of these 33.6% represented previously undiagnosed infections. All vaccinated participants demonstrated positive anti-S antibodies and in those with paired serology, no individual demonstrated loss of previously positive anti-S status below assay threshold for positivity. Interval loss of anti-N antibody positivity was demonstrated in 8.8% of previously positive participants with paired results. Risk factors for SARS-CoV-2 seropositivity suggestive of previous infection included age 18-29 years (aRR 1.50, 95% CI 1.19-1.90, p < 0.001), India as country of birth (aRR 1.35, 95% CI 1.01-1.73, p = 0.036), lower education level (aRR 1.35, 95% CI 1.11-1.66, p = 0.004) and HCA job role (aRR 2.12, 95% CI 1.51-2.95, p < 0.001). Antibody neutralisation varied significantly by anti-SARS-CoV-2 antibody status, with highest levels noted in those anti-N positive, in particular those with vaccination plus previous SARS-CoV-2 infection. Conclusion: All vaccinated HCWs maintained anti-S positivity prior to COVID-19 booster vaccination, however anti-N positivity was more dynamic over time. Antibody neutralisation capacity was highest in participants with COVID-19 vaccination plus prior SARS-CoV-2 infection.

The synergistic effect of physicochemical in vitro microenvironment modulators in human bone marrow stem cell cultures.

Modern bioengineering utilises biomimetic cell culture approaches to control cell fate during in vitro expansion. In this spirit, herein we assessed the influence of bidirectional surface topography, substrate rigidity, collagen type I coating and macromolecular crowding (MMC) in human bone marrow stem cell cultures. In the absence of MMC, surface topography was a strong modulator of cell morphology. MMC significantly increased extracellular matrix deposition, albeit in a globular manner, independently of the surface topography, substrate rigidity and collagen type I coating. Collagen type I coating significantly increased cell metabolic activity and none of the assessed parameters affected cell viability. At day 14, in the absence of MMC, none of the assessed genes was affected by surface topography, substrate rigidity and collagen type I coating, whilst in the presence of MMC, in general, collagen type I α1 chain, tenascin C, osteonectin, bone sialoprotein, aggrecan, cartilage oligomeric protein and runt-related transcription factor were downregulated. Interestingly, in the presence of the MMC, the 1000 kPa grooved substrate without collagen type I coating upregulated aggrecan, cartilage oligomeric protein, scleraxis homolog A, tenomodulin and thrombospondin 4, indicative of tenogenic differentiation. This study further supports the notion for multifactorial bioengineering to control cell fate in culture.

Physicochemical cues are not potent regulators of human dermal fibroblast trans-differentiation.

Due to their inherent plasticity, dermal fibroblasts hold great promise in regenerative medicine. Although biological signals have been well-established as potent regulators of dermal fibroblast function, it is still unclear whether physiochemical cues can induce dermal fibroblast trans-differentiation. Herein, we evaluated the combined effect of surface topography, substrate rigidity, collagen type I coating and macromolecular crowding in human dermal fibroblast cultures. Our data indicate that tissue culture plastic and collagen type I coating increased cell proliferation and metabolic activity. None of the assessed in vitro microenvironment modulators affected cell viability. Anisotropic surface topography induced bidirectional cell morphology, especially on more rigid (1,000 kPa and 130 kPa) substrates. Macromolecular crowding increased various collagen types, but not fibronectin, deposition. Macromolecular crowding induced globular extracellular matrix deposition, independently of the properties of the substrate. At day 14 (longest time point assessed), macromolecular crowding downregulated tenascin C (in 9 out of the 14 groups), aggrecan (in 13 out of the 14 groups), osteonectin (in 13 out of the 14 groups), and collagen type I (in all groups). Overall, our data suggest that physicochemical cues (such surface topography, substrate rigidity, collagen coating and macromolecular crowding) are not as potent as biological signals in inducing dermal fibroblast trans-differentiation.

A high molecular weight hyaluronic acid biphasic dispersion as potential therapeutics for interstitial cystitis.

Interstitial cystitis (IC) is a progressive bladder disease characterized by increased urothelial permeability, inflammation of the bladder with abdominal pain. While there is no consensus on the etiology of the disease, it was believed that restoring the barrier between urinary solutes and (GAG) urothelium would interrupt the progression of this disease. Currently, several treatment options include intravesical delivery of hyaluronic acid (HA) and/or chondroitin sulfate solutions, through a catheter to restore the urothelial barrier, but have shown limited success in preclinical, clinical trials. Herein we report for the first time successful engineering and characterization of biphasic system developed by combining cross-linked hyaluronic acid and naïve HA solution to decrease inflammation and permeability in an in vitro model of interstitial cystitis. The cross-linking of HA was performed by 4-arm-polyethyeleneamine chemistry. The HA formulations were tested for their viscoelastic properties and the effects on cell metabolism, inflammatory markers, and permeability. Our study demonstrates the therapeutic effects of different ratios of the biphasic system and reports their ability to increase the barrier effect by decreasing the permeability and alteration of cell metabolism with respect to relative controls. Restoring the barrier by using biphasic system of HA therapy may be a promising approach to IC.

Effect of Glycosaminoglycan Replacement on Markers of Interstitial Cystitis In Vitro.

Aims: To examine the effect of three commercial intravesical formulations of glycosaminoglycan on in vitro inflammatory models of IC/BPS to better understand there effect on specific markers of disease. Methods: Human urothelial cells (HTB-4) were cultured under four conditions in the presence or absence of commercial GAG formulations. Cells were cultured under a basal condition or pre-treated with protamine sulfate (100 ng/ml) (damages the endogenous glycosaminoglycan layer), hydrogen peroxide (1%) (a metabolic stressor) or TNFα (10 ng/ml) (creating an inflammatory environment). Each of these four culture conditions was then treated with one of three GAG formulations, CystistatⓇ, iAluRilⓇ and HyacystⓇ. Assays were then performed to examine the effect of the exogenous GAGs on cell viability, cell migration, sGAG production, cytokine and gene expression. Results: All GAG formulations were well tolerated by the HTB-4 cells and supported cell growth and migration. iAluRilⓇ was most effective at stimulating endogenous sGAG production under all conditions, increasing sGAGs by up to 15-fold. All GAG formulations significantly reduced the production of the pro-inflammatory cytokine IL-8 under basal conditions, while no GAG treatment suppressed cytokine production under any other condition. Only CystistatⓇ had a significant effect on HA receptor expression, significantly increasing ICAM-1 expression at 3 h that returned to basal levels at 24 h. No GAG treatment significantly changed the expression of GAG synthesis enzymes (CSGALNACT1, CSGALNACT2) or markers of tissue remodeling (MMP2, TIMP1) and pain (COX-1/PTGS-1, NGF). Conclusions: The data presented in this study reveal that commercial intravesical formulation support cell viability and migration. In addition, the commercial GAG formulations have a mild anti-inflammatory effect in the in vitro model of interstitial cystitis/bladder pain syndrome.

Development and characterization of an immunomodulatory and injectable system composed of collagen modified with trifunctional oligourethanes and silica.

Immunomodulatory biomaterials have emerged as a promising approach to engineer wound healing. To achieve this task, the bioactivity of the biomaterials and an easy application are two key desirable characteristics. This work reports an injectable gel system containing immune cells primed for wound healing. By combining the self-assembly of type I collagen, cross-linked with trifunctional oligourethanes, and silica particle entrapment, the structured collagen network acts as a delivery vehicle for macrophages. This structured collagen network primes the macrophages for an anti-inflammatory response. Rheological measurements suggest that the mixture of liquid precursors can be safely stored at low temperatures and low pH (4 °C, pH 3) for at least one month. After pH neutralization and injection, gels with a storage modulus of 50-80 Pa are obtained in five minutes. Several immunocytochemistry and ELISA tests strongly suggest that mouse and human macrophages are stimulated by the material to up-regulate the production of anti-inflammatory cytokines, while down-regulating the production of pro-inflammatory cytokines. The injection of gel in an ex vivo inflammation model of intervertebral discs demonstrated that it is possible to transit from a pro-inflammatory to an anti-inflammatory microenvironment. Altogether, the results suggest that this gel can polarize the macrophage response and promote a surrounding anti-inflammatory microenvironment ready for injection for wound healing applications.

Bioengineered three-dimensional diseased intervertebral disc model revealed inflammatory crosstalk.

Without an appropriate disease model, the understanding of the pathophysiology of intervertebral disc degeneration and inflammation is limited. The lack of understanding limits the potential discovery of therapeutic targets as viable treatment options. Here, we report a versatile method to develop a three-dimensional intervertebral disc (IVD) model to study the response of nucleus pulposus (NP) and annulus fibrosus (AF) cells to inflammatory (IL-1ß-induced) stimulation. The cell shape regulated IVD model was engineered by modulating the crosslinking of a self-assembled collagen hydrogel. The developed model has provided us with an understanding of the molecular changes that occur at genetic level which modulate the production of extracellular matrix components and key inflammatory pathways in the inflamed IVD. We have identified the role of the suppressor of cytokine proteins (SOCS) family in combating detrimental effects of pro-inflammatory cytokines in degenerated human NP tissue as predicted by the developed diseased model. The model could also provide an understanding of the expression of glycans implicated in the diseased IVD.

Biomimetic Lipid-Based Nanosystems for Enhanced Dermal Delivery of Drugs and Bioactive Agents.

Clinical utility of conventional oral therapies is limited by their inability to deliver therapeutic molecules at the local or targeted site, causing a variety of side effects. Transdermal delivery has made a significant contribution in the management of skin diseases with enhanced therapeutic activities over the past two decades. In the modern era, various biomimetic and biocompatible polymer-lipid hybrid systems have been used to augment the transdermal delivery of therapeutics such as dermal patches, topical gels, iontophoresis, electroporation, sonophoresis, thermal ablation, microneedles, cavitational ultrasound, and nano or microlipid vesicular systems. Nevertheless, the stratum corneum still represents the main barrier to the delivery of vesicles into the skin. Lipid based formulations applied to the skin are at the center of attention and are anticipated to be increasingly functional as the skin offers many advantages for the direction of such systems. Accordingly, this review provides an overview of the development of conventional to advanced biomimetic lipid vesicles for skin delivery of a variety of therapeutics, with special emphasis on recent developments in this field including the development of transferosomes, niosomes, aquasomes, cubosomes, and other new generation lipoidal carriers.

Hyaluronic acid decreases IL-6 and IL-8 secretion and permeability in an inflammatory model of interstitial cystitis.

Hyaluronic acid (HA) has received a lot of attention recently as a biomaterial with applications in wound healing, drug delivery, vascular repair and cell and/or gene delivery. Interstitial cystitis (IC) is characterised by an increase in the permeability of the bladder wall urothelium due to loss of the glycosaminoglycan (GAG) layer. The degradation of the urothelium leads to chronic pain and urinary dysfunction. The aetiology of the degradation of the GAG layer in this instance is currently unknown. At a clinical level, GAG replacement therapy using a HA solution is currently utilised as a treatment for IC. However, there is a significant lack of data on the mechanism of action of HA in IC. The current study investigates the mechanistic effect of clinically relevant HA treatment on an in vitro model of IC using urothelial cells, examining cytokine secretion, GAG secretion and trans-epithelial permeability. This study demonstrates that HA can significantly decrease induced cytokine secretion (4-5 fold increase), increase sulphated GAG production (2-fold increase) and without altering tight junction expression, decrease trans-epithelial permeability, suggesting that the HA pathway is a clinical target and potential treatment vector.

Regulation of Inflammation and Angiogenesis in Giant Cell Arteritis by Acute-Phase Serum Amyloid A.

OBJECTIVE: Giant cell arteritis (GCA) is pathologically characterized by dysfunctional angiogenesis and inflammatory cell infiltration. Acute-phase serum amyloid A (A-SAA) is an acute-phase reactant, but is also produced at sites of inflammation and may contribute to vascular inflammation in atherosclerosis. This study was undertaken to examine the effect of A-SAA on proinflammatory pathways and angiogenesis in GCA, using a novel ex vivo temporal artery tissue explant model. METHODS: Serum A-SAA levels were measured by enzyme-linked immunosorbent assay (ELISA). Temporal artery explants and peripheral blood mononuclear cell (PBMC) cultures were established from patients with GCA. Temporal artery explant morphology, viability, and spontaneous release of proinflammatory mediators following 24-hour culture were assessed by hematoxylin and eosin, calcein viability staining, and ELISA. Temporal artery explants and PBMC cultures were stimulated with A-SAA (10 µg/ml), and interleukin-6 (IL-6), IL-8, vascular endothelial growth factor, Ang2, and matrix metalloproteinase 2 (MMP-2)/MMP-9 were quantified by ELISA and gelatin zymography. The effect of conditioned medium from temporal artery explants on angiogenesis was assessed using endothelial cell Matrigel tube-formation assays. Temporal artery explants were also embedded in Matrigel, and myofibroblast outgrowth was assessed. RESULTS: Serum A-SAA levels were significantly higher in GCA patients versus healthy controls (P < 0.0001). Intact tissue morphology, cell viability, and spontaneous cytokine secretion were demonstrated in temporal artery explants. A-SAA treatment induced a significant increase in the levels of IL-6 and IL-8 from temporal artery explants (P < 0.05) and IL-8 from PBMCs (P < 0.05) compared to basal conditions. Conditioned medium from A-SAA-treated explants significantly induced angiogenic tube formation (P < 0.05 versus basal controls). Finally, A-SAA induced myofibroblast outgrowth and MMP-9 activation. CONCLUSION: Our findings demonstrate a functional role for A-SAA in regulating temporal artery inflammation, angiogenesis, and invasion, all key processes in the pathogenesis of GCA.