Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence.

The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-ß signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-ß signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-ß signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

Blue light irradiation and its beneficial effect on Dupuytren's fibroblasts.

Dupuytren's contracture is a fibroproliferative disorder affecting the palmar fascia of the hand. Most affected are the ring fingers, and little fingers of middle-aged men. Symptomatic for this disease is the increased proliferation and differentiation of fibroblasts to myofibroblasts, which is accompanied by an elevated α-SMA expression. The present study evaluated the therapeutic benefit of blue light (λ = 453 nm, 38 mW/cm2, continuous radiance, spot size 10-12 cm2) as well as the molecular mechanism mediating this effect. It could be determined that blue light significantly diminished the induced α-SMA protein expression in both normal palmar fibroblasts and Duypuytren's fibroblasts. The beneficial effect mediated by this irradiance, radiant exposure and wavelength was associated with an elevated reactive oxygen species generation. Furthermore, the data underlines the potential usefulness of blue light irradiation as a promising therapy option for Dupuytren's disease, especially for relapse prevention, and may represent a useful strategy to treat further fibrotic diseases, such as keloids, hypertrophic scarring, and scleroderma.

Effect of hyperbaric oxygen therapy (HBO) on implant-associated osteitis in a femur fracture model in mice.

Hyperbaric oxygen therapy (HBO) is applied very successfully in treatment of various diseases such as chronic wounds. It has been already suggested as adjunctive treatment option for osteitis by immune- and fracture modulating effects. This study evaluates the importance of HBO in an early implant-associated localized osteitis caused by Staphylococcus aureus (SA) compared to the standard therapy. In a standardized murine model the left femur of 120 BALB/c mice were osteotomized and fixed by a titanium locking plate. Osteitis has been induced with a defined amount of SA into the fracture gap. Debridément and lavages were progressed on day 7, 14, 28 and 56 to determine the local bacterial growth and the immune reaction. Hyperbaric oxygen (2 ATA, 90%) was applied for 90 minutes on day 7 to 21 for those mice allocated to HBO therapy. To evaluate the effect of HBO therapy the following groups were analyzed: Two sham-groups (12 mice / group) with and without HBO therapy, two osteotomy groups (24 mice / group) with plate osteosynthesis of the femur with and without HBO therapy, and two osteotomy SA infection groups (24 mice / group) with and without HBO therapy. Fracture healing was also quantified on day 7, 14, 28 and 56 by a.p. x-ray and bone healing markers from blood samples. Progression of infection was assessed by estimation of colony-forming units (CFU) and immune response was analyzed by determination of polymorphonuclear neutrophils (PMN), Interleukin (IL) - 6, and the circulating free DNA (cfDNA) in lavage samples. Osteitis induced significantly higher IL-6, cfDNA- and PMN-levels in the lavage samples (on day 7 and 14, each p < 0.05). HBO-therapy did not have a significant influence on the CFU and immune response compared to the standard therapy (each p > 0.05). At the same time HBO-therapy was associated with a delayed bone healing assessed by x-ray radiography and a higher rate of non-union until day 28. In conclusion, osteitis led to significantly higher bacterial count and infection parameters. HBO-therapy neither had a beneficial influence on local infection nor on immune response or fracture healing compared to the standard therapy in an osteitis mouse model.

Synergistic effects of HBO and PRP improve bone regeneration with autologous bone grafting.

Bone defects remain a challenge for patients and orthopaedic surgeons. Autologous transfer of cancellous bone grafts remains the standard of care. However, in recent years various osteoinductive substitute materials, such as platelet rich plasma (PRP) and hyperbaric oxygen therapy (HBO) have been shown to improve bone healing. This study evaluates the effects of a combined application of PRP and HBO with autologous bone grafting in an animal model. In 48 New Zealand White rabbits bone defects at the radius were filled with autologous bone harvested at the iliac crest. This was combined with application of autologous PRP and/or HBO treatment for the duration of this study. After 3 and 6 weeks histomorphometric, immunohistochemical and radiologic evaluations were performed. All animals tolerated the treatment well. Improved bone regeneration was shown in all groups at 6 weeks compared to 3 weeks. Additional application of PRP and HBO resulted in an increase in new bone formation and increased neovascularization at 3 and 6 weeks. There was no statistical significant difference between PRP and HBO application in these regards. A combinatory use of PRP and HBO resulted in an increased bone regeneration and neovascularization compared to all other groups. This study provides evidence for an improvement of bone regeneration with the combinatory application of PRP and HBO to autologous cancellous bone grafts in a model of weight bearing bone defects in rabbits. Also synergistic effects of these two measures on angiogenesis were evident.

Hyperbaric Oxygen Reduces Production of Reactive Oxygen Species in Neutrophils from Polytraumatized Patients Yielding in the Inhibition of p38 MAP Kinase and Downstream Pathways.

Trauma represents the leading cause of death among young people in western countries. Among the beneficial role of neutrophils in host defence, excessive priming and activation of neutrophils after major trauma lead to an overwhelming inflammatory response and secondary host tissue injury due to the release of toxic metabolites and enzymes. Hyperbaric oxygen (HBO) therapy has been proposed to possess antiinflammatory effects and might represent an appropriate therapeutic option to lower inflammation in a broad range of patients. Here, we studied the effects of HBO on the activity of neutrophils isolated from severely injured patients (days 1-2 after trauma), in fact on the production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs). We found exposure to HBO therapy to significantly diminish phorbol-12-myristate-13-acetate (PMA)-induced ROS production in neutrophils isolated from patients and healthy volunteers. At the same time, marked decrease in NETs release was found in control cells and a less pronounced reduction in patient neutrophils. Impaired ability to produce ROS following exposure to HBO was demonstrated to be linked to a strong downregulation of the activity of p38 MAPK. Only slight suppression of ERK activity could be found. In addition, HBO did not influence neutrophil chemotaxis or apoptosis, respectively. Collectively, this study shows for the first time that HBO therapy suppresses ROS production in inflammatory human neutrophils, and thus might impair ROS-dependent pathways, e.g. kinases activation and NETs release. Thus, HBO might represent a feasible therapy for patients suffering from systemic inflammation, including those with multiple trauma.

Assessment of hemostaseologic alterations induced by hyperbaric oxygen therapy using point-of-care analyzers.

BACKGROUND: Exposure to hyperbaric conditions influences the coagulation system. Thromboembolic events and disseminated intravascular coagulation were observed. OBJECTIVES: To detect the effects of a hyperbaric environment on the human coagulation system using the point-of-care coagulation analyzers Multiplate and ROTEM. PATIENTS/METHODS: 20 patients were included. Each received 90 minutes of oxygen intermittently at 2.4 atmospheres absolute, as per the TS 240-90 wound-healing protocol. Blood samples were taken before and after hyperbaric exposure and ROTEM, Multiplate and standard laboratory assays were subsequently performed. RESULTS: ROTEM showed a significant increase of the maximum clot firmness (EXTEM MCF; p < 0.05) and the thromboelastometric platelet component of the clot firmness (MCF(EXTEM) - MCF(FIBTEM); p < 0.01). Multiplate showed a platelet activation mediated by thrombin (AU TRAP-test; p < 0.05) and by arachidonic acid (AUC ASPI-test; p < 0.01). Standard laboratory assays revealed a lower activated partial thromboplastin time (p < 0.05) and a higher leukocyte count (p < 0.05). No further changes were detected. A t-test was performed after testing if data followed normal distribution. CONCLUSIONS: ROTEM and Multiplate were able to detect an activation of platelets after HBO2 therapy via thrombin and arachidonic acid pathways. Previously reported fibrinolysis could not be confirmed.

Mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic nitric oxide generation from photolabile nitric oxide derivates in human skin in vitro and in vivo.

Human skin contains photolabile nitric oxide (NO) derivates such as nitrite and S-nitrosothiols, which upon UVA radiation decompose under high-output NO formation and exert NO-specific biological responses such as increased local blood flow or reduced blood pressure. To avoid the injurious effects of UVA radiation, we here investigated the mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic NO generation from photolabile nitric oxide derivates in human skin in vitro and in vivo. As quantified by chemiluminescence detection (CLD), at physiological pH blue light at 420 or 453 nm induced a significant NO formation from S-nitrosoalbumin and also from aqueous nitrite solutions by a to-date not entirely identified Cu(1+)-dependent mechanism. As detected by electron paramagnetic resonance spectrometry in vitro with human skin specimens, blue light irradiation significantly increased the intradermal levels of free NO. As detected by CLD in vivo in healthy volunteers, irradiation of human skin with blue light induced a significant emanation of NO from the irradiated skin area as well as a significant translocation of NO from the skin surface into the underlying tissue. In parallel, blue light irradiation caused a rapid and significant rise in local cutaneous blood flow as detected noninvasively by using micro-light-guide spectrophotometry. Irradiation of human skin with moderate doses of blue light caused a significant increase in enzyme-independent cutaneous NO formation as well as NO-dependent local biological responses, i.e., increased blood flow. The effects were attributed to blue-light-induced release of NO from cutaneous photolabile NO derivates. Thus, in contrast to UVA, blue-light-induced NO generation might be therapeutically used in the treatment of systemic and local hemodynamic disorders that are based on impaired physiological NO production or bioavailability.

Selection of proangiogenic ascorbate derivatives and their exploitation in a novel drug-releasing system for wound healing.

The pathophysiology leading to delayed wound healing is complex and efficient therapeutic approaches for accelerated wound healing currently do not exist. We developed a novel drug-eluting platform for the potential use in wound dressings. Here, we report on the potential of eluting ascorbic acid-2-phosphate (ASC-2P), a highly stable variant of ascorbic acid, to induce angiogenesis and to promote collagen synthesis by fibroblasts. The drug-eluting platform device (DEPD) consists of biocompatible polymeric layers comprising polyethylene terephtalate, polyvinyl alcohol (PVA), and polyurethane with PVA as the solvent for ASC-2P. The angiogenic potential of ASC-2P was evaluated in the endothelial cell tube formation assay (TFA) and in the chorion allantoic membrane (CAM) model. Collagen synthesis by ASC-2P-stimulated fibroblasts was determined by Sirius Red staining. ASC-2P significantly induced angiogenesis in five independent TFA and CAM assays and induced collagen synthesis in two different fibroblast cell lines. The eluting kinetics of ASC-2P was determined by the ultraviolet NanoDrop method and the functional 2,2'-Azinobis-(3-ethylbenzthiazolin-6-sulfonic acid) method. Eluting profiles showed a continuous release in the range of biologically effective concentrations >10 days. This is the first report showing the proangiogenic- and collagen-promoting features of ASC-2P. DEPD loaded with ASC-2P ought to be further evaluated as wound dressings or as supplementary pads for topical treatment of delayed wound healing in preclinical studies.