Systematic Review of the Preclinical Technology Readiness of Orthopedic Gene Therapy and Outlook for Clinical Translation.

Bone defects and improper healing of fractures are an increasing public health burden, and there is an unmet clinical need in their successful repair. Gene therapy has been proposed as a possible approach to improve or augment bone healing with the potential to provide true functional regeneration. While large numbers of studies have been performed in vitro or in vivo in small animal models that support the use of gene therapy for bone repair, these systems do not recapitulate several key features of a critical or complex fracture environment. Larger animal models are therefore a key step on the path to clinical translation of the technology. Herein, the current state of orthopedic gene therapy research in preclinical large animal models was investigated based on performed large animal studies. A summary and an outlook regarding current clinical studies in this sector are provided. It was found that the results found in the current research literature were generally positive but highly methodologically inconsistent, rendering a comparison difficult. Additionally, factors vital for translation have not been thoroughly addressed in these model systems, and the risk of bias was high in all reviewed publications. These limitations directly impact clinical translation of gene therapeutic approaches due to lack of comparability, inability to demonstrate non-inferiority or equivalence compared with current clinical standards, and lack of safety data. This review therefore aims to provide a current overview of ongoing preclinical and clinical work, potential bottlenecks in preclinical studies and for translation, and recommendations to overcome these to enable future deployment of this promising technology to the clinical setting.

A biomimetic self-assembling peptide promotes bone regeneration in vivo: A rat cranial defect study.

Rationally designed, pH sensitive self-assembling ß-peptides (SAPs) which are capable of reversibly switching between fluid and gel phases in response to environmental triggers are potentially useful injectable scaffolds for skeletal tissue engineering applications. SAP P11-4 (CH3COQQRFEWEFEQQNH2) has been shown to nucleate hydroxyapatite mineral de novo and has been used in dental enamel regeneration. We hypothesised that addition of mesenchymal stromal cells (MSCs) would enhance the in vivo effects of P11-4 in promoting skeletal tissue repair. Cranial defects were created in athymic rats and filled with either Bio-Oss® (anorganic bone chips) or P11-4 ±â€¯human dental pulp stromal cells (HDPSCs). Unfilled defects served as controls. After 4 weeks, only those defects filled with P11-4 alone showed significantly increased bone regeneration (almost complete healing), compared to unfilled control defects, as judged using quantitative micro-CT, histology and immunohistochemistry. In silico modelling indicated that fibril formation may be essential for any mineral nucleation activity. Taken together, these data suggest that self-assembling peptides are a suitable scaffold for regeneration of bone tissue in a one step, cell-free therapeutic approach.

Delivery of the improved BMP-2-Advanced plasmid DNA within a gene-activated scaffold accelerates mesenchymal stem cell osteogenesis and critical size defect repair.

Gene-activated scaffolds have been shown to induce controlled, sustained release of functional transgene both in vitro and in vivo. Bone morphogenetic proteins (BMPs) are potent mediators of osteogenesis however we found that the delivery of plasmid BMP-2 (pBMP-2) alone was not sufficient to enhance bone formation. Therefore, the aim of this study was to assess if the use of a series of modified BMP-2 plasmids could enhance the functionality of a pBMP-2 gene-activated scaffold and ultimately improve bone regeneration when implanted into a critical sized bone defect in vivo. A multi-cistronic plasmid encoding both BMP-2 and BMP-7 (BMP-2/7) was employed as was a BMP-2-Advanced plasmid containing a highly truncated intron sequence. With both plasmids, the highly efficient cytomegalovirus (CMV) promoter sequence was used. However, as there have been reports that the elongated factor 1-α promoter is more efficient, particularly in stem cells, a BMP-2-Advanced plasmid containing the EF1α promoter was also tested. Chitosan nanoparticles (CS) were used to deliver each plasmid to MSCs and induced transient up-regulation of BMP-2 protein expression, in turn significantly enhancing MSC-mediated osteogenesis when compared to untreated controls (p < 0.001). When incorporated into a bone mimicking collagen-hydroxyapatite scaffold, the BMP-2-Advanced plasmid, under the control of the CMV promotor, induced MSCs to produce approximately 2500 µg of calcium per scaffold, significantly higher (p < 0.001) than all other groups. Just 4 weeks post-implantation in vivo, this cell-free gene-activated scaffold induced significantly more bone tissue formation compared to a pBMP-2 gene-activated scaffold (p < 0.001) as indicated by microCT and histomorphometry. Immunohistochemistry revealed that the BMP-2-Advanced plasmid accelerated differentiation of osteoprogenitor cells to mature osteoblasts, thus causing rapid healing of the bone defects. This study confirms that optimising the plasmid construct can enhance the functionality of gene-activated scaffolds and translate to accelerated bone formation in a critical sized defect.

Oestrogen receptor ß (ERß) regulates osteogenic differentiation of human dental pulp cells.

Estradiol (E2) has many important actions in the tissues of the oral cavity. Disruption of E2 metabolism or alterations in systemic E2 concentrations have been associated with compromised periodontal health. In many instances such changes occur secondarily to the well characterised effects of E2 on bone physiology -especially maintenance of bone mineral density (BMD). Despite these important epidemiological findings, little is known about the mechanism of action of E2 in oral tissues or the expression and function of oestrogen receptor (ER) isoforms in these tissues. We have isolated human dental pulp cells (hDPCs), which are able to differentiate towards an osteogenic lineage under appropriate culture conditions. We show that hDPCs express ERα, ERß1, ERß2 and the cell membrane associated G protein-coupled ER (GPR30). Following osteogenic differentiation of hDPCs, ERß1 and ERß2 were up regulated approximately 50-fold while ERα and GPR30 were down regulated, but to a much lesser degree (approximately 2-fold). ERß was characterised as a 59kDa protein following Western blot analysis with validated antibodies and ERß was detected in both nuclear and cytoplasmic cell compartments following immunofluorescence (IF) and immunohistochemical (IHC) analysis of cultured cells. Furthermore isoform specific antibodies detected both ERß1 and ERß2 in DPC cultures and in situ analysis of ERß expression in decalcified tooth/pulp sections identified the odontoblast layer of pulp cells juxtaposed to the tooth enamel as strongly reactive for both ERß isoforms. Finally the use of isoform specific agonists identified ERß as the main receptor responsible for the pro-osteogenic effect of oestrogenic hormones in this tissue. Our data suggest that oestrogens stimulated osteogenic differentiation in hDPCs and that this action is mediated principally through the ERß isoform. These findings may have important consequences for the investigation and treatment of oral and periodontal pathologies which are associated with imbalances in oestrogen concentrations and action.

Induced Hypothermia Preserves the Functional Enterocyte Mass in A Porcine Multiple Trauma Model: Retracted.

INTRODUCTION: Multiple organ dysfunction syndrome (MODS) and the resulting multiple organ failure (MOF) following severe trauma are associated with increased morbidity and mortality. Due to intestinal mucosal lesions and gut barrier disorders, the intestine contributes decisively to how post-traumatic MOF develops. As mild therapeutic hypothermia has been found to have protective effects on post-traumatic organ injuries, we analysed its effects on the intestine. METHODS: In a porcine model, Forty pigs were assigned to four groups: sham or trauma groups each with two sub-groups receiving either hypothermia or normothermia. The trauma was a combined trauma of blunt chest trauma, liver laceration and haemorrhagic shock. Functional enterocyte mass and enterocyte necrosis were evaluated by measuring plasma citrulline and iFABP. Mucosal lesions were assessed using a semi-quantitative histological scoring system. RESULTS: In normothermic trauma animals, citrulline decreased significantly compared to both sham groups and to the hypothermic trauma group. However, citrulline levels did not differ significantly between the hypothermic trauma group and the hypothermic sham group. Although histological analysis demonstrated subepithelial lifting and mucosal oedema in the ileal mucosa of all trauma animals, the semi-quantitative score of the group treated with hypothermia was comparable to that of the hypothermic sham group. However, the score was significantly elevated in normothermic trauma animals in comparison to sham and hypothermic trauma animals. CONCLUSION: Induced hypothermia preserves the functional enterocyte mass after severe trauma. Therefore induced hypothermia might represent a therapeutic strategy to avoid posttraumatic organ dysfunction, although further studies regarding the safety and long-term effects are required. LEVEL OF EVIDENCE: Level III; therapeutic study.

A Luciferase-Based Quick Potency Assay to Predict Chondrogenic Differentiation.

Chondrogenic differentiation of adipose-derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore, quick potency assays are required to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation capacity are time-consuming, and thus, a novel enhancer and tissue-specific promoter combination was used for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were cotransfected with the Metridia luciferase-based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three-dimensional culture conditions and, in a second approach, also under two-dimensional (2D) culture conditions. With our newly developed quick potency bioassay, we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies in a very short time and at low costs.

Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration.

It has been suggested that gene transfer into donor cells is an efficient and practical means of locally supplying requisite growth factors for applications in tissue regeneration. Here we describe, for the first time, an ultrasound-mediated system that can non-invasively facilitate gene transfer into cells entrapped within fibrin-based matrices. Since ultrasound-mediated gene transfer is enhanced using microbubbles, we compared the efficacy of neutral and cationic forms of these reagents on the ultrasound-stimulated gene transfer process in gel matrices. In doing so we demonstrated the beneficial effects associated with the use of cationic microbubble preparations that interact directly with cells and nucleic acid within matrices. In some cases, gene expression was increased two-fold in gel matrices when cationic microbubbles were compared with neutral microbubbles. In addition, incorporating collagen into fibrin gels yielded a 25-fold increase in gene expression after application of ultrasound to microbubble-containing matrices. We suggest that this novel system may facilitate non-invasive temporal and spatial control of gene transfer in gel-based matrices for the purposes of tissue regeneration.

Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation.

Tissue engineering (TE) strategies aim at imitating the natural process of regeneration by using bioresorbable scaffolds that support cellular attachment, migration, proliferation, and differentiation. Based on the idea of combining a fully degradable polymer [poly(ɛ-caprolactone)] with a thermoresponsive polymer (polyethylene glycol methacrylate), a scaffold was developed, which liquefies below 20°C and solidifies at 37°C. In this study, this scaffold was evaluated for its ability to support C2C12 cells and human adipose-derived stem cells (ASCs) to generate an expandable three-dimensional (3D) construct for soft or bone TE. As a first step, biomaterial seeding was optimized and cellular attachment, survival, distribution, and persistence within the 3D material were characterized. C2C12 cells were differentiated toward the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic, or osteogenic differentiation media. Differentiation was examined using quantitative real-time PCR for the expression of osteogenic, myogenic, and adipogenic markers and by enzyme activity and immunoassays. Both cell types attached and were found evenly distributed within the material. C2C12 cells and ASCs demonstrated the potential to differentiate in all tested lineages under 2D conditions. Under 3D osteogenic conditions for C2C12 cells, only osteocalcin expression (fold induction: 16.3±0.2) and alkaline phosphatase (ALP) activity (p<0.001) were increased compared with the control C2C12 cells. Three-dimensional osteogenic differentiation of ASC was limited and donor dependent. Only one donor showed an increase in the osteogenic markers osteocalcin (p=0.027) and osteopontin (p=0.038). In contrast, differentiation toward the myogenic or adipogenic lineage showed expression of specific markers in 3D, at least at the level of the 2D culture. In 3D culture, strong induction of myogenin (p<0.001) as well as myoD (p<0.001) was found in C2C12 cells. The adipogenic differentiation of one donor showed greater expression of peroxisome proliferative-activated receptor gamma (PPARγ) (p=0.004), fatty acid binding protein 4 (FABP4) (p=0.008), and adiponectin (p=0.045) in 3D compared with 2D culture. Leptin levels in the supernatant of the ASC cultures were elevated in the 3D cultures in both donors at day 14 and 21. In conclusion, the thermoresponsive scaffold was found suitable for 3D in vitro differentiation toward soft tissue.

Evaluation of fibrin-based gene-activated matrices for BMP2/7 plasmid codelivery in a rat nonunion model.

PURPOSE: Treatment of large-segmental bone defects still is a challenge in clinical routine. Application of gene-activated matrices (GAMs) based on fibrin, bone morphogenic protein (BMP) 2/7 plasmids and nonviral transfection reagents (cationic polymers) could be an innovative treatment strategy to overcome this problem. The aim of this study was to determine the therapeutic efficacy of fibrin GAMs with or without additional transfection reagents for BMP2 and 7 plasmid codelivery in a femur nonunion rat model. METHODS: In this experimental study, a critical-sized femoral defect was created in 27 rats. At four weeks after the surgery, animals were separated into four groups and underwent a second operation. Fibrin clots containing BMP2/7 plasmids with and without cationic polymer were implanted into the femoral defect. Fibrin clots containing recombinant human (rh) BMP2 served as positive and clots without supplement as negative controls. RESULTS: At eight weeks, animals that received GAMs containing the cationic polymer and BMP2/7 plasmids showed decreased bone volume compared with animals treated with GAMs and BMP2/7 only. Application of BMP2/7 plasmids in fibrin GAMs without cationic polymer led to variable results. Animals that received rhBMP2 protein showed increased bone volume, and osseous unions were achieved in two of six animals. CONCLUSIONS: Cationic polymers decrease therapeutic efficiency of fibrin GAM-based BMP2/7 plasmid codelivery in bone regeneration. Nonviral gene transfer of BMP2/7 plasmids needs alternative promoters (e.g. by sonoporation, electroporation) to produce beneficial clinical effects.

Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.

Cytokine signaling by grafted neuroectodermal stem cells rescues motoneurons destined to die.

Following an injury to their axons close to the cell body, adult motoneurons generally die. This type of injury, typically caused by avulsion of the spinal ventral root, initiates the activation of astrocytes and microglial cells and the extracellular space becomes loaded with excessive amounts of excitotoxic glutamate. We have provided evidence that, following ventral root avulsion and reimplantation, murine embryonic neuroectodermal stem cells (NE-GFP-4C) grafted into the rat spinal cord rescue the vast majority of the motoneurons that would otherwise die, and enable them to reinnervate peripheral targets. Stem cell grafts produced the modulatory cytokines IL-1-alpha, IL-6, IL-10, TNF-alpha and MIP-1-alpha, but not neurotrophic factors. The neurons and astrocytes in the ventral horn of grafted animals also produced IL-6 and MIP-1-alpha, indicating a strong interaction between the graft and the host tissue. The infusion of function-blocking antibodies against all cytokines into the grafted cords completely abolished their motoneuron-rescuing effect, while neutralization of only IL-10 suggested its strong effectivity as concerns motoneuron survival and a milder effect on reinnervation. It is suggested that, apart from the anti-inflammatory function of IL-10, the pro-inflammatory cytokines produced exert a strong modulatory function in the CNS, promoting the prevention of neuronal cell death.

Spatiotemporally limited BDNF and GDNF overexpression rescues motoneurons destined to die and induces elongative axon growth.

Axonal injury close to cell bodies of motoneurons induces the death of the vast majority of affected cells. Neurotrophic factors, such as brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF), delivered close to the damaged motor pool in a non-regulated manner induce good survival of injured motoneurons and sprouting of their axons but fail to induce functional reinnervation. To avoid these drawbacks of high levels of neurotrophic expression, we devised an ex vivo gene therapy system to induce transient expression of BDNF/GDNF in transfected rat adipose tissue-derived stem cells (rASCs) which were grafted around the reimplanted ventral root, embedded in collagen gel. Strong BDNF/GDNF expression was induced in vitro in the first days after transfection with a significant decline in expression 10-14 days following transfection. Numerous axons of injured motoneurons were able to enter the reimplanted root following reimplantation and BDNF or GDNF treatment (192±17 SEM vs 187±12 SEM, respectively) and produce morphological and functional reinnervation. Treatment with a combined cell population (BDNF+GDNF-transfected rASCs) induced slightly improved reinnervation (247±24 SEM). In contrast, only few motoneurons regenerated their axons in control animals (63±4 SEM) which received untransfected cells. The axons of surviving motoneurons showed elongative growth typical of regenerative axons, without aberrant growth or coil formation of sprouting axons. These findings provide evidence that damaged motoneurons require limited and spatially directed amounts of BDNF and GDNF to support their survival and regeneration. Moreover, neurotrophic support appears to be needed only for a critical period of time not longer than for two weeks after injury.

Sonoporation increases therapeutic efficacy of inducible and constitutive BMP2/7 in vivo gene delivery.

An ideal novel treatment for bone defects should provide regeneration without autologous or allogenous grafting, exogenous cells, growth factors, or biomaterials while ensuring spatial and temporal control as well as safety. Therefore, a novel osteoinductive nonviral in vivo gene therapy approach using sonoporation was investigated in ectopic and orthotopic models. Constitutive or regulated, doxycycline-inducible, bone morphogenetic protein 2 and 7 coexpression plasmids were repeatedly applied for 5 days. Ectopic and orthotopic gene transfer efficacy was monitored by coapplication of a luciferase plasmid and bioluminescence imaging. Orthotopic plasmid DNA distribution was investigated using a novel plasmid-labeling method. Luciferase imaging demonstrated an increased trend (61% vs. 100%) of gene transfer efficacy, and micro-computed tomography evaluation showed significantly enhanced frequency of ectopic bone formation for sonoporation compared with passive gene delivery (46% vs. 100%) dependent on applied ultrasound power. Bone formation by the inducible system (83%) was stringently controlled by doxycycline in vivo, and no ectopic bone formation was observed without induction or with passive gene transfer without sonoporation. Orthotopic evaluation in a rat femur segmental defect model demonstrated an increased trend of gene transfer efficacy using sonoporation. Investigation of DNA distribution demonstrated extensive binding of plasmid DNA to bone tissue. Sonoporated animals displayed a potentially increased union rate (33%) without extensive callus formation or heterotopic ossification. We conclude that sonoporation of BMP2/7 coexpression plasmids is a feasible, minimally invasive method for osteoinduction and that improvement of bone regeneration by sonoporative gene delivery is superior to passive gene delivery.

A New Visual Trap for Rhagoletis cerasi (L.) (Diptera: Tephritidae).

The European cherry fruit fly, Rhagoletis cerasi (L.) (Diptera: Tephritidae), is the most important pest of sweet cherries in Europe. The aim of our experiments was to develop a new, cost-efficient, lead chromate-free and more eco-friendly trap for monitoring and mass trapping of R. cerasi. Five different-colored yellow panels and three different trap shapes were compared to a standard Rebell(®) amarillo trap in three experimental orchards in 2012. Trap color F, with a strong increase in reflectance at 500-550 nm and a secondary peak in the UV-region at 300-400 nm, captured significantly more flies than the standard Rebell(®) amarillo trap. Yellow traps with increased reflectance in the blue region (400-500 nm) were least attractive. Trap shape was of minor importance, as long as the object was three-dimensional and visible from all directions. Based on economic and practical considerations, a cylinder-shaped trap "UFA-Samen Kirschenfliegenfalle" was developed for commercial use and is currently under on-farm evaluation.

Compartment-specific expression of plasminogen activator inhibitor-1 correlates with severity/outcome of murine polymicrobial sepsis.

INTRODUCTION: Plasminogen activator inhibitor type 1 (PAI-1) co-induces septic coagulopathy. We aimed to characterize spatiotemporal PAI-1 gene/protein changes occurring in acute sepsis and tested whether PAI-1 fluctuations correlate with sepsis severity and early outcome. MATERIALS AND METHODS: Female mice underwent cecal ligation and puncture (CLP) in three experiments. I: mild (23 G needle) CLP to compare circulating PAI-1 to its organ gene expression within 0-24h. II: mild or severe (17 G) CLP to asses differences in PAI-1 organ-specific expression and in coagulation/fibrinolysis. III: moderate (18 G) CLP to characterize circulating PAI-1 in survivors (SUR), and to retrospectively compare it to dying (DIE) mice. RESULTS: In mild sepsis, the trajectory of circulating PAI-1 (1089 ng/ml peak at 24h) was identical to PAI-1 gene expression in the left cranial vena cava (LCVC; 39-fold peak at 24h). PAI-1 expression rise was immediate (60-fold at 6h) and sustained in the liver, but marginal in the kidney, lungs and heart. Body temperature decrease correlated with the PAI-1 expression increase in the liver (rho = -0.79), and blood (protein, rho = -0.53). Regardless of severity, PAI-1 gene expression remained unaltered except the LCVC where it was >3-fold higher in 17G (vs. 23 G). Severe sepsis extended activated partial thromboplastin/pro-thrombin time and increased circulating PAI-1, while antithrombin and fibrinogen decreased at 6 and/or 24h (vs. 23 G). Within 24h of death, circulating PAI-1 in DIE was >3-fold higher versus SUR. CONCLUSIONS: Polymicrobial sepsis caused a gradual circulating PAI-1 release and highly variable gene expression response pattern in organs. Only circulating PAI-1 and PAI-1 expression in the LCVC correlated with response severity and/or outcome.

Anti-alpha-Gal antibody titres remain unaffected by the consumption of fermented milk containing Lactobacillus casei in healthy adults.

Alpha-Gal is a glycoconjugate present on cell membranes of non-primate mammals and bacteria, but not in humans, who display anti-Gal antibodies (ABs) in high titres. Probiotics contain bacterial strains which colonize the intestinal tract. In the present study, we investigated whether intake of fermented milk containing Lactobacillus casei (FML) affects anti-Gal AB titres. Serum was drawn from healthy probands (n = 19) for 6 weeks. After the second week, the probands consumed 125 ml of FML per day. Anti-Gal ABs of all isotypes and cytokines were measured. Bacterial cultures were bred from FML and bacteria were stained for alpha-Gal. Concentration of bacteria in FML was manifold higher than in conventional yoghurt (2 × 10(5)/g yoghurt vs. 1.1 × 10(7)/g FML). Both stained highly positive for Alpha-Gal. Alpha-Gal-specific ABs and cytokines remained unaffected by FML intake. Our results indicated that the consumption of FML does not elicit a humoral immune response in healthy adults.

Light therapy by blue LED improves wound healing in an excision model in rats.

BACKGROUND: Low level light therapy (LLLT) is an attractive alternative to enhance wound healing. So far most studies are performed with red or infrared irradiation. However, we recently showed that blue light (470 nm) can significantly influence biological systems, improving perfusion by release of nitric oxide from nitrosyl complexes with haemoglobin in a skin flap model in rats. Here, we compared the effects of blue and red low level light by light-emitting diodes (LEDs) on in vivo wound healing in an excision wound model in rats. METHODS: Circular excision wounds were surgically created on the dorsum of each rat. Excisions on either the left or right side were illuminated post-OP and on five consecutive days for 10 min by LED at 470 nm or 630 nm with an intensity of 50 mW/cm(2),while protecting the contralateral side from exposure. In the control group, neither side was illuminated. On day 7 post-OP, we analysed planimetric and histological parameters, as well as expression of keratin-1, keratin-10 and keratin-17 on mRNA level. RESULTS: Illumination substantially influenced wound healing. Blue light significantly decreased wound size on day 7, which correlated with enhanced epithelialisation. Light also affected mRNA expression. Both wavelengths decreased keratin-1 mRNA on day 7 post-OP, while keratin-10 mRNA level was elevated in both light treated group compared to control. Keratin-17 mRNA was also elevated in the red light group, but was unchanged in the blue light group. CONCLUSION: In contrast to previous studies, we showed that also blue light significantly influences wound healing. Furthermore, our data suggest that light therapy can play an important role in normotrophic wound healing by affecting keratin expression. Illumination would provide an easily applicable, safe and cost-effective treatment of surface wounds.

Enhanced reporter gene assay for the detection of osteogenic differentiation.

Detection of osteogenic differentiation is crucial for bone tissue engineering. Despite established standard end point assays, there is increasing demand for methods allowing noninvasive kinetic differentiation monitoring. Reporter gene assays employing tissue-specific promoters and suitable reporter genes fulfill these requirements. Many promoters, however, exhibit only weak cis-activating potential, thus limiting their application to generate sensitive reporter gene assays. Therefore, the aim of this study was to design a reporter gene assay employing elements of the murine osteocalcin promoter coupled to a viral enhancer for signal amplification. Additionally, the system's practicability was enhanced by introducing a secreted luciferase as a quantifiable reporter gene. The constructs were tested in C2C12 cells stimulated with recombinant human bone morphogenetic protein 2 for osteogenic differentiation in two-dimensional and three-dimensional culture. Osteogenic differentiation was confirmed by standard assays for osteogenesis. The reporter gene signal was detected through a secreted luciferase or fluorescence microscopy for enhanced yellow fluorescent protein. The constructs exhibited strong activation upon treatment with recombinant human bone morphogenetic protein 2. Weak background expression was observable in negative controls, attributed to the pan-active viral enhancer. In conclusion, a novel enhancer/tissue-specific promoter combination allows specific signal-amplified, kinetic monitoring of osteogenic differentiation in a nonsample-destructive manner.

Mycotoxigenic Fusarium and deoxynivalenol production repress chitinase gene expression in the biocontrol agent Trichoderma atroviride P1.

Mycotoxin contamination associated with head blight of wheat and other grains caused by Fusarium culmorum and F. graminearum is a chronic threat to crop, human, and animal health throughout the world. One of the most important toxins in terms of human exposure is deoxynivalenol (DON) (formerly called vomitoxin), an inhibitor of protein synthesis with a broad spectrum of toxigenicity against animals. Certain Fusarium toxins have additional antimicrobial activity, and the phytotoxin fusaric acid has recently been shown to modulate fungus-bacterium interactions that affect plant health (Duffy and Défago, Phytopathology 87:1250-1257, 1997). The potential impact of DON on Fusarium competition with other microorganisms has not been described previously. Any competitive advantage conferred by DON would complicate efforts to control Fusarium during its saprophytic growth on crop residues that are left after harvest and constitute the primary inoculum reservoir for outbreaks in subsequent plantings. We examined the effect of the DON mycotoxin on ecological interactions between pathogenic Fusarium and Trichoderma atroviride strain P1, a competitor fungus with biocontrol activity against a wide range of plant diseases. Expression of the Trichoderma chitinase genes, ech42 and nag1, which contribute to biocontrol activity, was monitored in vitro and on crop residues of two maize cultivars by using goxA reporter gene fusions. We found that DON-producing F. culmorum and F. graminearum strains repressed expression of nag1-gox. DON-negative wild-type Fusarium strains and a DON-negative mutant with an insertional disruption in the tricothecene biosynthetic gene, tri5, had no effect on antagonist gene expression. The role of DON as the principal repressor above other pathogen factors was confirmed. Exposure of Trichoderma to synthetic DON or to a non-DON-producing Fusarium mutant resulted in the same level of nag1-gox repression as the level observed with DON-producing FUSARIUM: DON repression was specific for nag1-gox and had no effect, either positive or negative, on expression of another key chitinase gene, ech42. This is the first demonstration that a target pathogen down-regulates genes in a fungal biocontrol agent, and our results provide evidence that mycotoxins have a novel ecological function as factors in Fusarium competitiveness.