Nitric oxide-generating hydrogels inhibit neointima formation

This study evaluated the effects of localized delivery of nitric oxide (NO) from hydrogels covalently modified with S-nitrosocysteine (Cys-NO) on neoinitma formation, a key component of restenosis, in a rat balloon-injury model. Soluble Cys-NO was used in preliminary studies to identify dosage ranges that were able to simultaneously inhibit smooth muscle cell proliferation, enhance endothelial cell proliferation, and reduce platelet adhesion. Photo-cross-linked PEG-based hydrogels were formed with covalently immobilized Cys-NO. These materials release NO for approximately 24 h and can be applied to tissues and photo-cross-linked in situ to form local drug-delivery systems. Localized delivery of NO from hydrogels containing Cys-NO inhibited neointima formation in a rat balloon-injury model by approximately 75% at 14 days.     

Localized delivery of nitric oxide from hydrogels inhibits neointima formation in a rat carotid balloon injury model

Using novel nitric oxide (NO)-generating polymeric hydrogels that can be rapidly photopolymerized in situ, we can deliver NO locally at the site of vascular injury. Depending on material design, these poly(ethylene glycol) (PEG)-based hydrogels can generate NO for up to 50 d. This study demonstrates the ability of nitric oxide-generating hydrogels (PEG-Cys-NO) to influence key components of the restenosis cascade both in vitro and in vivo. PEG-Cys-NO hydrogels inhibited smooth muscle cell proliferation, increased endothelial cell proliferation, and inhibited platelet adhesion in vitro. Moreover, in vivo, PEG-Cys-NO hydrogels inhibited intimal thickening in a rat carotid balloon injury model. The perivascular application of NO-generating polymers post-injury reduced neointima formation at 14 d by approximately 80% compared to controls (intimal area/medial area (I/M): PEG-Cys-NO = 0.20 ± 0.17, control = 0.84 ± 0.19, p < 0.00002; intimal thickness: PEG-Cys-NO = 12 ± 10 μm, control = 60 ± 18 μm, p < 0.00002). Treatment with the PEG-Cys-NO hydrogels caused a significant decrease in the per cent of proliferating cell nuclear antigen positive medial cells (29 ± 5%) at 4 d as compared to treatment with the control hydrogels (51 ± 1%, p < 0.02). Additionally, vessel re-endothelialization at 14 d was slightly enhanced in the presence of the NO-generating hydrogels. These data indicate that localized delivery of NO from these hydrogels can significantly inhibit neointima formation in a rat carotid balloon injury model and suggest that these materials may be useful in preventing restenosis.     

大鼠主动脉内膜球囊剥脱后再生内皮细胞表型改变的研究

目的 探索大鼠主动脉内膜球囊剥脱后,内皮修复过程中再生内皮细胞结构和功能的改变,方法 大鼠主动脉内膜球囊剥脱２周和６周对内膜进行形态学观察,同时测定血管反应性,亚硝酸盐量和ＮＯＳ的活性,结果：剥脱２周和６周后,再生内皮细胞不仅结构与正常内皮细胞不同,而且血管再生内皮依赖性舒张反应９减弱,ＮＯ的释放减少,血管组织ｉＮＯＳ和ｃＮＯＳ活性均比正常降低,剥脱６周后的ｉＮＯＳ比剥脱２周时有所增加,但ｃＮＯＳ     

Nitric oxide-generating polymers reduce platelet adhesion and smooth muscle cell proliferation

We have developed polymeric biomaterials capable of providing localized and sustained production of nitric oxide (NO) for the prevention of thrombosis and restenosis. In the current study, we have characterized the kinetics of NO production by these materials and investigated their efficacy in reducing platelet adhesion and smooth muscle cell proliferation in vitro. Three nitric oxide donors with different half-lives were covalently incorporated into photopolymerized polyethylene glycol hydrogels. Under physiological conditions, NO was produced by these hydrogels over periods ranging from hours to months, depending upon the polymer formulation. NO production was inhibited at acidic pH, which may be useful for storage of the materials. The NO-releasing materials successfully inhibited smooth muscle cell growth in culture. Platelet adhesion to collagen-coated surfaces was also inhibited following exposure of whole blood to NO-producing hydrogels. The effects of NO production by these hydrogels on platelet adhesion and the proliferation of smooth muscle cells suggest that these materials could reduce thrombosis and restenosis following procedures such as balloon angioplasty.     

The modulation of cardiac progenitor cell function by hydrogel-dependent Notch1 activation

Myocardial infarction is the leading cause of death worldwide and phase I clinical trials utilizing cardiac progenitor cells (CPCs) have shown promising outcomes. Notch1 signaling plays a critical role in cardiac development and in the survival, cardiogenic lineage commitment, and differentiation of cardiac stem/progenitor cells. In this study, we functionalized self-assembling peptide (SAP) hydrogels with a peptide mimic of the Notch1 ligand Jagged1 (RJ) to evaluate the therapeutic benefit of CPC delivery in the hydrogels in a rat model of myocardial infarction. The behavior of CPCs cultured in the 3D hydrogels in vitro including gene expression, proliferation, and growth factor production was evaluated. Interestingly, we observed Notch1 activation to be dependent on hydrogel polymer density/stiffness with synergistic increase in presence of RJ. Our results show that RJ mediated Notch1 activation depending on hydrogel concentration differentially regulated cardiogenic gene expression, proliferation, and growth factor production in CPCs in vitro. In rats subjected to experimental myocardial infarction, improvement in acute retention and cardiac function was observed following cell therapy in RJ hydrogels compared to unmodified or scrambled peptide containing hydrogels. This study demonstrates the potential therapeutic benefit of functionalizing SAP hydrogels with RJ for CPC based cardiac repair.     

Regulation of cell proliferation by multi-layered phospholipid polymer hydrogel coatings through controlled release of paclitaxel

We fabricated multi-layered hydrogels on titanium alloy (Ti) surfaces by applying alternating layers of a water-soluble phospholipid polymer (PMBV) and polyvinyl alcohol (PVA). This was accomplished by a layer-by-layer (LbL) process that is based on the formation of reversible covalent bonds between the boronic acid subunits in the PMBV and the hydroxyl groups in the PVA. When placed in an aqueous medium, PMBV acquires a polymeric aggregate structure with hydrophobic domains that can effectively solubilize hydrophobic molecules such as the anticancer drug paclitaxel (PTX) used in this study. The PTX-containing PMBV layer acted as a reservoir in the multi-layered hydrogels. To obtain diverse release profiles, the PTX was loaded in either the top layer (top-type) or the bottom layer (bottom-type) of the hydrogels; additional layers of PMBV and PVA, without PTX, functioned as a diffusion-barrier. In cell culture experiments, top-type hydrogels demonstrated excessive suppression of human epidermal carcinoma A431 cell proliferation over 5 days due to the initial high concentration of released PTX. However, bottom-type hydrogels were able to maintain a constant cell number profile. The release of PTX from multi-layered hydrogels was governed by both diffusion through the diffusion-barrier and dissociation of the hydrogel through an exchange reaction of phenylboronic acid subunits with the low-molecular weight d-glucose in the cell culture medium. In the cell culture experiments, the cell cycle was arrested in S and G2/M phases, as expected following PTX-mediated growth inhibition; control hydrogels did not demonstrate any appreciable cell cycle arrest. We concluded that cell proliferation could be controlled by the concentration of PTX released from the multi-layered hydrogels prepared through the LbL process. This system when used to solubilize bioactive agents at an appropriate layer within the hydrogel has potential for localized and surface-mediated delivery of bioactive molecules from biomedical devices.     

Functionalized self-assembling peptide nanofiber hydrogels mimic stem cell niche to control human adipose stem cell behavior in vitro

A class of designer functionalized self-assembling peptide nanofiber scaffolds developed from self-assembling peptide RADA16-I (AcN-RADARADARADARADA-CONH₂) has become increasingly attractive not only for studying spatial behaviors of cells, but also for developing approaches for a wide range of medical applications including regenerative medicine, rapid hemostasis and cell therapy. In this study, we report three functionalized self-assembling peptide hydrogels that serve as a three-dimensional (3-D) artificial microenvironment to control human adipose stem cell (hASC) behavior in vitro. Short peptide motifs SKPPGTSS (bone marrow homing motif), FHRRIKA (heparin-binding motif) and PRGDSGYRGDS (two-unit RGD cell adhesion motif) were used to extend the C-terminus of RADA16-I to obtain functionalized peptides. Atomic force microscopy confirmed the formation of self-assembling nanofibers in the mixture of RADA16-I peptide and functionalized peptides. The behaviors of hASCs cultured in 3-D peptide hydrogels, including migration, proliferation and growth factor-secretion ability, were studied. Our results showed that the functionalized peptide hydrogels were suitable 3-D scaffolds for hASC growth with higher cell proliferation, migration and the secretion of angiogenic growth factors compared with tissue culture plates and pure RADA16-I scaffolds. The present study suggests that these functionalized designer peptide hydrogels not only have promising applications for diverse tissue engineering and regenerative medicine applications as stem cell delivery vehicles, but also could be a biomimetic 3-D system to study nanobiomaterial–stem cell interactions and to direct stem cell behaviors.     

A vanadium/aspirin complex controlled release using a poly(β - propiolactone) film. Effects on osteosarcoma cells

A delivery system for vanadium was developed using poly(β-propiolactone) (PβPL)films. The release kinetics of a complex of vanadium (IV) with aspirin (VOAspi) was evaluated with films prepared from polymers of different molecular weights, as well as with variable drug load. A sustained release of vanadium over 7 days was achieved. The drug release kinetics depends on contributions from two factors: (a) diffusion of the drug; and (b) erosion of the PβPL film. The experimental data at an early stage of release were fitted with a diffusion model, which allowed determination of the diffusion coefficient of the drug. VOAspi does not show strong interaction with the polymer, as demonstrated by the low apparent partition coefficient (approximately 10-2). UMR106 osteosarcoma cells were used as a model to evaluate the anticarcinogenic effects of the VOAspi released from the PβPL film. VOAspi-PβPL film inhibited cell proliferation in a dose-response manner and induced formation of approximately half of the thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, compared to that with free VOAspi in solution. The unloaded PβPL film did not generate cytotoxicity, as evaluated by cell growth and TBARS. Thus, the polymer-embedded VOAspi retained the antiproliferative effects showing lower cytotoxicity than the free drug. Results with VOAspi-PβPL films suggest that this delivery system may have promising biomedical and therapeutic applications.     

Thermal and photochemical nitric oxide release from S-nitrosothiols incorporated in Pluronic F127 gel: potential uses for local and controlled nitric oxide release

The local delivery of nitric oxide (nitrogen monoxide, NO) by thermal or photochemical means to target cells or organs has a great potential in several biomedical applications, especially if the NO donors are incorporated into non-toxic viscous matrices. In this work, we have shown that the NO donors S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) can be incorporated into F127 hydrogels, from where NO can be released thermally or photochemically (with lambda(irr)>480nm). High sensitivity differential scanning calorimetry (HSDSC) and a new spectrophotometric method, were used to characterize the micellization and the reversal thermal gelation processes of the F127 hydrogels containing NO donors, and to modulate the gelation temperatures to the range 29-32 degrees C. Spectral monitoring of the S-NO bond cleavage showed that the initial rates of thermal and photochemical NO release (ranging from 2 to 45 micromoll(-1)min(-1)) are decreased in the hydrogel matrices, relative to those obtained in aqueous solutions. This stabilization effect was assigned to a cage recombination mechanism and offers an additional advantage for the storage and handling of S-nitrosothiols. These results indicate that F127 hydrogels might be used for the thermal and photochemical delivery of NO from S-nitrosothiols to target areas in biomedical applications.     

Degradability of hydrogels containing azoaromatic crosslinks

A novel type of biodegradable, pH-sensitive hydrogels was synthesized by crosslinking of N,N-dimethylacrylamide copolymer precursors. These hydrogels contained azoaromatic crosslinks which may be degraded by the azoreductase activities in the colon. Hydrogel degradation experiments were performed both in vitro and in vivo, using rat cecum contents and implantation into the rat cecum, respectively. In order to evaluate the influence of the detailed network structure on the degradation properties, the degradability of hydrogels synthesized by two different methods, i.e., crosslinking of polymeric precursors and crosslinking copolymerization, were compared. Two different patterns of the degradation of hydrogels were observed, i. e., a surface erosion process and a bulk-degradation-like process. The azo bond cleavage rate of hydrogels prepared by crosslinking of polymeric precursors was faster than that of hydrogels prepared by crosslinking copolymerization. The differences in the gel degradation pattern and the cleavage rate of azo bonds were attributed to the differences in the structure of the hydrogel network, i. e., the molecular weight of primary chains and the formation of chain entanglements. Under the experimental conditions used, the degradation rate in vivo was 3 to 5 times faster than that in vitro. It appears that these hydrogels have a potential for colon-specific drug delivery.     

Generation of monodisperse alginate microbeads and <Emphasis Type="Italic">in situ</Emphasis> encapsulation of cell in microfluidic device

A microfluidic method for the in situ production of monodispersed alginate hydrogels using chaotic mixing is described. Aqueous droplets comprising of alginate and calcium as a cross-linking agent were formed as an immiscible continuous phase, and then the alginate and calcium in the droplet came into contact and were rapidly mixed. Gelation of the hydrogel was achieved in situ by the chaotic mixing of the droplets in the microfluidic device. Important operating parameters included: the capillary number (Ca) and the flow rate of the continuous phase, which mainly influenced the formation of three distinctive flow regimes, such as fluctuation, stable droplets, and laminar flow. Under the stable formation of droplets regime, monodispersed alginate microbeads having a narrow size distribution (below 3% of CV) were produced in the microfluidic device and the size of the microbeads, ranging from 60 to 95 μm, could be easily modulated by varying the flow rate, viscosity, and interfacial tension. In addition, this approach can be applied to the encapsulation of yeast cells in alginate hydrogels with a high monodispersity. This simple microfluidic technique for the production of monodispersed hydrogels and encapsulation of biomolecules shows strong potential for use in biosensors, cell sensors, drug delivery systems, and cell transplantation applications.     

Porous Agarose-Based Semi-IPN Hydrogels: Characterization and Cell Affinity Studies

Abstract

Hydrogels are frequently considered for medical applications due to the ease of preparation in different forms and high water content that makes them comparable to natural tissues. However, these general properties are not sufficient to make any hydrogel suitable for cell attachment and growth which are necessary for their use in tissue regeneration. Besides, the high water content makes the hydrogels mechanically weak. The formation of semi-interpenetrating networks (semi-IPNs) can be used in attempts to enhance physical, mechanical and thermal properties. In this study, semi-IPNs of agarose were prepared with chitosan and alginate, two polyelectrolytes that are positively and negatively charged under physiological conditions, respectively. Zeta potential was used to confirm the formation of charged hydrogels. All hydrogels had ultimate compression strengths in the range of 91–210 Pa where the value for pure agarose was about 103 Pa. Chitosan increased the compressive strength about two folds whereas the alginate had opposite effects. The amount of strongly bound water present in the hydrogels were estimated from TGA and DSC analysis and the highest value was found for alginate-agarose hydrogels as about 15%. The attachment and the migration of L929 fibroblasts were monitored in vitro using the MTS assay and confocal microscopy. The highest cell proliferation and penetration were observed for positively charged chitosan-agarose semi-IPN hydrogels.     

Dynamical release nanospheres containing cell growth factor from biopolymer hydrogel via reversible covalent conjugation

For practical adipose regeneration, the challenge is to dynamically deliver the key adipogenic insulin-like growth factors in hydrogels to induce adipogenesis. In order to achieve dynamic release, smart hydrogels to sense the change in the blood glucose concentration is required when glucose concentration increases. In this study, a heparin-based hydrogel has been developed for use in dynamic delivery of heparin nanospheres containing insulin-like growth factor. The gel scaffold was facilely prepared in physiological conditions by the formation of boronate-maltose ester cross-links between boronate and maltose groups of heparin derivatives. Due to its intrinsic glucose-sensitivity, the exposure of gel scaffold to glucose induces maltose functionalized nanospheres dissociation off hydrogel network and thereby could dynamically move into the microenvironment. The potential of the hydrogel as a cell scaffold was demonstrated by encapsulation of human adipose-derived stem cells (ASCs) within the gel matrix in vitro. Cell culture showed that this dynamic hydrogel could support survival and proliferation of ASCs. This biocompatible coupling chemistry has the advantage that it introduces no potentially cytotoxic groups into injectable gel scaffolds formed and can create a more biomimetic microenvironment for drug and cell delivery, rendering them more suitable for potential in vivo biomedical applications. All these results indicate that this biocompatible gel scaffold can render the formulation of a therapeutically effective platform for diabetes treatment and adipose regeneration.     

Assessment of the cytotoxicity of photocrosslinked dextran and hyaluronan-based hydrogels to vascular smooth muscle cells

The cytotoxicity of polysaccharide-based hydrogels and solutions was studied in vitro after 48h of indirect exposure of the materials with vascular smooth muscle cells. Dextran and/or hyaluronan were derivatized using glycidyl methacrylate, and hydrogels were formed in the presence of photoinitiators and ultraviolet radiation in multiwell inserts to avoid direct contact with cell monolayers. Observation of cell morphology indicated that dextran hydrogels, a blend of non-derivatized hyaluronan into dextran hydrogel. and a hyaluronan solution were highly cytocompatible. However, hydrogels made of derivatized hyaluronan were cytotoxic when compared to unexposed sham controls that contained multiwell inserts but no hydrogels. Results from quantitative assays for proliferation and viability corroborated the qualitative observations, and scrape wound assays revealed a significant increase in smooth muscles cell migration/proliferation after indirect exposure to several of the polysaccharide-based materials. Results from this study demonstrate that hydrogels made of dextran and hyaluronan solution show good cytocompatibility in vitro. making these degradable matrices interesting candidates for drug delivery purposes.     

Role of Thermo-responsiveness and Poly(ethylene glycol) Diacrylate Cross-link Density on Protein Release from Poly(N-isopropylacrylamide) Hydrogels

Thermo-responsive hydrogels have shown promise as injectable materials for local drug delivery. However, the phase-induced changes in polymer properties of N-isopropylacrylamide (NIPAAm) can pose additional challenges for achieving controlled protein release. In this work, thermo-responsive hydrogels derived from NIPAAm and cross-linked with poly(ethylene glycol) diacrylate (PEG-DA) were synthesized via free radical polymerization. The volume phase transition temperature (VPTT) of the hydrogels ranged from 32.9°C to 35.9°C. Below the VPTT, swelling ratios of the hydrogels decreased with cross-linker concentration, and showed a sharp drop (at least 4-fold) upon phase change. Protein encapsulation efficiency was high (84–90%) and decreased with cross-linker concentration. Release of bovine serum albumin, a model protein, at body temperature was significantly higher than at room temperature (67% at 37°C compared to 44% at 23°C after 48 h). The release kinetics of proteins from the hydrogels were initially expected to be a function of cross-link density. However, at the hydrogel compositions explored in this work, protein release did not change significantly with cross-linker mol fraction. The thermo-responsive hydrogels offer a promising platform for the localized delivery of proteins.     

The use of chitosan based hydrogel for enhancing the therapeutic benefits of adipose-derived MSCs for acute kidney injury

Transplantation of mesenchymal stem cells (MSCs) has been reported a great therapeutic potential for acute kidney injury (AKI). However, the therapeutic benefits are limited due to the low retention and survival of transplanted cells within target sites. In this study, thermosensitive chitosan chloride (CSCl) hydrogel was explored as injectable scaffold for adipose-derived MSCs (ADMSCs) delivery into ischemia/reperfusion (I/R) induced acute kidney injury (AKI). Thermosensitive CSCl hydrogels with/without ADMSCs were injected into the I/R site of rat AKI models. Dihydroethidium staining was used to detect the number of ROS in vivo. In order to track ADMSCs in vivo, ADMSCs were transfected with firefly luciferase and monomeric red fluorescent protein reporter genes (fluc-mrfp). The retention and survival of ADMSC were assessed using bioluminescence imaging, differentiation behaviors of ADMSCs were investigated using immunofluorescent and immunohistochemical staining. Proliferation and apoptosis of host renal cell in vivo were characterized by PCNA and TUNEL staining. Results suggested that CSCl hydrogels could improve the retention and survival of grafted ADMSCs, moreover, CSCl hydrogels could enhance the proliferation activity and reduce apoptosis of host renal cells. At 4 weeks, significant improvement of the renal function, microvessel density and tubular cell proliferation were observed in CSCl hydrogels with ADMSCs groups. Therefore, the application of thermosensitive CSCl hydrogel as scaffold for ADMSCs delivery into renal region could resolve the main obstacle of cell transplantation for acute kidney injury (AKI). Therefore, CSCl hydrogel is a potential cell carrier for treatment of AKI.     

Nanostructured PEG-based hydrogels with tunable physical properties for gene delivery to human mesenchymal stem cells

Effective delivery of DNA to direct cell behavior in a well defined three dimensional scaffold offers a superior approach in tissue engineering. In this study, we synthesized biodegradable nanostructured hydrogels with tunable physical properties for cell and gene delivery. The hydrogels were formed via Michael addition chemistry by reacting a four-arm acrylate-terminated PEG with a four-arm thiol-functionalized PEG. Nanosized micelles self-assembled from the amphiphilic PEG-b-polycarbonate diblock copolymer, having reactive end-groups, were chemically incorporated into the hydrogel networks at various contents. The use of Michael addition chemistry allows for in situ hydrogel formation under the physiological conditions. Mechanical property analysis of the hydrogels revealed a correlation between the content of micelles and the storage modulus of the hydrogels. Internal morphology of hydrogels was observed using a field emission scanning electron microscope, which showed that the number and/or size of the pores in the hydrogel increased with increasing micelle content due to reduced crosslinking degree. There exists an optimal micelle content for cell proliferation and gene transfection. MTT assays demonstrated the highest cell viability in the hydrogel with 20% micelles. The gene expression level in hMSCs in the hydrogel with 20% micelles was also significantly higher than that in the hydrogel without micelles. The enhanced cell viability and gene expression in the hydrogel with the optimized micelle content are likely attributed to the physical properties that provide a better environment for cell-matrix interactions. Therefore, incorporating micelles into the hydrogel is a good strategy to control cellular behavior in 3-D through changes in physical properties of the microenvironment.     

Towards a quantitative understanding of oxygen tension and cell density evolution in fibrin hydrogels

The in vitro culture of hydrogel-based constructs above a critical size is accompanied by problems of unequal cell distribution when diffusion is the primary mode of oxygen transfer. In this study, an experimentally-informed mathematical model was developed to relate cell proliferation and death inside fibrin hydrogels to the local oxygen tension in a quantitative manner. The predictive capacity of the resulting model was tested by comparing its outcomes to the density, distribution and viability of human periosteum derived cells (hPDCs) that were cultured inside fibrin hydrogels in vitro. The model was able to reproduce important experimental findings, such as the formation of a multilayered cell sheet at the hydrogel periphery and the occurrence of a cell density gradient throughout the hydrogel. In addition, the model demonstrated that cell culture in fibrin hydrogels can lead to complete anoxia in the centre of the hydrogel for realistic values of oxygen diffusion and consumption. A sensitivity analysis also identified these two parameters, together with the proliferation parameters of the encapsulated cells, as the governing parameters for the occurrence of anoxia. In conclusion, this study indicates that mathematical models can help to better understand oxygen transport limitations and its influence on cell behaviour during the in vitro culture of cell-seeded hydrogels.     

The role of nitric oxide in cellular response to hyperbaric conditions

Nitric oxide (NO) acts as a regulator in cell proliferation and expression of growth factors and forms peroxynitrite (ONOO⁻) in oxidative conditions. The aim of the study was to investigate the role of NO in cellular response to hyperbaric oxygen (HBO). NO and nitrotyrosine (NT), biochemical marker for ONOO⁻, cell proliferation and growth factors, were ex-vivo studied in cell cultures under HBO and normobaric (NOR) conditions. A549 (epithelial), L929 (fibroblast) and SVEC (endothelial) were exposed to 100% O₂, at P = 280 kPa for t = 60 min, once daily for five sessions. Cell proliferation was determined as the incorporation of bromodeoxyuridine (BrdU) into cells and NO as nitrates/nitrites (NO₃ ⁻/ NO₂ ⁻) Gries reaction product in cell culture supernatant (CCSP). NT, vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGFb1) were measured with enzyme-inked immunosorbent assay (ELISA) in CCSP. The time course of total NO was opposite to that of cell proliferation in HBO conditions, peaking after the second HBO session, while cell proliferation showed a reverse trend, minimizing at the same time, suggesting a reverse and transient anti-proliferative effect. Released growth factors were significantly increased in late HBO sessions. NT peaked after second treatment, indicating the formation of ONOO⁻. In control cultures (NOR), proliferation rate was downward and no significant differences were found for the other parameters. In conclusion, the data suggested a key role for NO in the beneficial HBO action, depending on its concentration, which fluctuated with the time of HBO exposure and the activation of oxidant–antioxidant (REDOX) mechanisms, regardless of cell type.