Effect of sonic hedgehog/β-TCP composites on bone healing within the critical-sized rat femoral defect

The creation of entirely synthetically derived bone substitute materials which are as effective as autologous bone grafts is desirable. Osteogenesis involves the concerted action of several proteins within a signaling cascade. Hedgehog proteins act upstream of this cascade, inducing the expression of various bone morphogenetic proteins (BMPs) and promoting physiological bone healing. Therefore, the hypothesis that hedgehog signaling in bone defects improves bone healing more than BMP signaling alone was tested. Recombinant N-terminal sonic hedgehog protein (N-SHh), BMP-2 or a combination of the two was added to β-tricalcium phosphate (β-TCP) and 5-mm femoral midshaft defects in nude rats were filled with these composites. The defects were stabilized with mini-plates. After eight weeks, the animals were sacrificed and the femora were explanted. The radiological evaluation was followed by a three-point bending test and histological examination. BMP-2/β-TCP composites showed a trend of increased stiffness compared with the controls (β-TCP without protein). N-SHh/β-TCP composites had lower stiffness compared with the control group and the N-SHh/BMP-2/β-TCP composites also had lower average stiffness compared with the controls (all not significant). Histomorphometry, however, revealed abundant cartilage and bone core formation in the N-SHh-composite groups. The sum of the new cartilage and bone was highest in the combination group N-SHh/BMP-2 (not significant). The addition of N-SHh to bone substitute materials appears to delay bone healing at the applied concentration and observation time but also showed a trend for higher amounts of ossifying cartilage

In-vivo- und In-vitro-Untersuchungen zum Einfluß einer Enzym- bzw. Vitamin-A-Therapie auf das periphere Immunsystem bei Patienten mit Plattenepithelkarzinom im Kopf-, Halsbereich

Die doppelblinde Pilotstudie ''Wirksamkeit und Verträglichkeit einer Enzym­ oder Vitamin­ A­Therapie bei Patienten mit Plattenepithelkarzinom im Kopf­/Halsbereich" wurde in Zusammenarbeit mit der HNO­Klinik in dem Zeitraum von Oktober 1994 bis Juni 1996 mit 25 Patienten mit Plattenepithelkarzinomen und 15 gesunden Kontrollpersonen durchgeführt. Den Patienten wurde neben der konventionellen Therapie entweder Vitamin­A (Retinolpalmitat, 150,000 IE/d), die Enzympräparation (proteolytische Gesamtaktivität 20,880 F.I.P.­E/d) oder Placebo zugeteilt. In regelmäßigen Abständen wurde das Allgemeinbefinden/ Nebenwirkungen protokolliert, Blutbild und Leberwerte bestimmt, die peripheren Lymphozyten phänotypisiert und die NK­ LAK, bzw. LAK­Zell­Aktivität gemessen. Zur Auswertung gelangten 16 Patienten. Statistisch signifikant war eine Erhöhung der CD2 und CD3­Werte der Enzym­Gruppe (41 Messungen) gegenüber den Patienten der Gruppen Vitamin­A (18 Messungen), Placebo (12 Messungen) und den Ausgangswerten der Patienten (25 Messungen). Auch im Verlauf konnte die Erhöhung der CD3­Werte bei der Enzymgruppe gegenüber den anderen Patientengruppen bestätigt werden. Der Anteil der T­Zellen im peripheren Blut lag im Bereich der gesunden Kontrollen. Um die Wirkungen der Prüfmedikationen auf Leukozyten (PBL, PMNL, Monozyten) in vitro zu untersuchen, wurde ein Panel durchflußzytometrischer Tests entwickelt, bzw. etabliert, die die Messung der Funktionen NK­Zellaktivität, LAK­Aktivität, ADCC, Phagozytose und Sauerstoffradikalbildung ermöglichten bzw. vereinfachten. Die untersuchte Enzympräparation enthielt Papain, Trypsin und Chymotrypsin im Verhältnis 2,5:1:1. In vitro­Versuche ergaben eine dosisabhängige Stimulierung der NK­, NK­LAK, CTL­ bzw. LAK­Aktivität und der ADCC durch NK­Zellen durch die Enzympräparation. Es war möglich, die Lymphozyten mehrfach zu stimulieren, eine Enzymdauergabe, bzw. hohe Enzymdosen führten zu einem Aktivitätsrückgang. Die Enzymmischung verbesserte die Phagozytose und den oxidativen Burst bei PMNL und erhöhte die CD18­ und CD54­ bzw. erniedrigte die CD16­Expression auf PMNL. Mit all­trans­Retinsäure bzw. Retinol konnten die Lymphozytenaktivitäten in vitro mit den gewählten Versuchsbedingungen kaum beeinflußt werden, Änderungen konnten meist 48h bis 72h nach Zugabe beobachtet werden, die jedoch nicht signifikant waren. Beobachtungen zu Vitamin­A­Wirkungen auf PMNL waren nicht möglich, da diese nur maximal 24h kultiviert werden konnten

High calcium bioglass enhances differentiation and survival of endothelial progenitor cells, inducing early vascularization in critical size bone defects

Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca2+ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects

Human endothelial-like differentiated precursor cells maintain their endothelial characteristics when cocultured with mesenchymal stem cell and seeded onto human cancellous bone

Introduction. Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. Materials and Methods. We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding. Results. The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC’s collagen-1α gene expression increased significantly. Conclusion. EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter’s collagen-1α gene expression

Delayed wound repair in sepsis is associated with reduced local pro-inflammatory cytokine expression

Sepsis is one of the main causes for morbidity and mortality in hospitalized patients. Moreover, sepsis associated complications involving impaired wound healing are common. Septic patients often require surgical interventions that in-turn may lead to further complications caused by impaired wound healing. We established a mouse model to the study delayed wound healing during sepsis distant to the septic focus point. For this reason cecal ligation and puncture (CLP) was combined with the creation of a superficial wound on the mouse ear. Control animals received the same procedure without CPL. Epithelialization was measured every second day by direct microscopic visualization up to complete closure of the wound. As interplay of TNF-α, TGF-β, matrix metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) is important in wound healing in general, TNF-α, TGF-β, MMP7, and TIMP1 were assessed immunohistochemical in samples of wounded ears harvested on days 2, 6, 10 and 16 after wounding. After induction of sepsis, animals showed a significant delay in wound epithelialization from day 2 to 12 compared to control animals. Complete wound healing was attained after mean 12.2± standard deviation (SD) 3.0 days in septic animals compared to 8.7± SD 1.7 days in the control group. Septic animals showed a significant reduction in local pro-inflammatory cytokine level of TNF-α on day 2 and day 6 as well as a reduced expression of TGF-β on day 2 in wounds. A significant lower expression of MMP7 as well as TIMP1 was also observed on day 2 after wounding. The induction of sepsis impairs wound healing distant to the septic focus point. We could demonstrate that expression of important cytokines for wound repair is deregulated after induction of sepsis. Thus restoring normal cytokine response locally in wounds could be a good strategy to enhance wound repair in sepsis

Increasing global competition and labor productivity: lessons from the U.S. automotive industry

Increasing global competition is changing the environment facing most companies today. As trade barriers fall and transaction costs decline, new global competitors are entering previously more isolated domestic markets. In response to this intensified competitive pressure, local companies are pushed to enhance performance by innovating and adopting process and product improvements. This domestic sector dynamic leads to higher productivity, which, in turn, can create sustainable competitive advantages for companies, as well as being the most important driver of job creation and per-capita income growth for the economy. This link has been established in McKinsey Global Institute’s extensive country productivity research. ; Our new study goes further than previous research by focusing on how increasing global competition leads to productivity growth, using the U.S. automotive manufacturing sector as a case example. More specifically, we have focused on the production of new vehicles in the U.S., including parts assembly. We have chosen this example because of the globally competitive nature of the automotive market and the size of the U.S. in this market over our period of analysis. As we shall see, some of the non-US original equipment manufacturers (OEMs) had clear productivity advantages which enabled them to create significant competitive pressure in the U.S. market.

Beschleunigte Knochenheilung durch frühe Vaskularisierung mittels Implantation von EPC und MSC auf beta-TCP im Knochendefektmodell der Ratte

Fragestellung: In einem ausgedehnten Knochendefekt kann das Einwachsen von knochenbildenden Zellen limitiert sein, da ohne Gefässe die Ernährung der regenerativen Zellen im Knochenkonstrukt insuffizient ist. Endotheliale Progenitorzellen (EPC) sind wichtig bei der Neovaskularisierung. Die frühe Vaskularisierung von grossen Knochendefekten kann für das Überleben und die Funktion von mesenchymalen Stammzellen (MSC) und knochenbildenden Zellen entscheidend sein. Kann die Implantation von EPC und MSC auf osteokonduktiven beta-Tricalciumphosphat (beta-TCP) in einem "critical-size" Knochendefekt des Femur von athymischen Ratten die frühe Vaskularisierung und die Knochenheilung in vivo verbessern? Methodik: Humane EPC wurden aus Buffy-Coat und humane MSC aus Knochenmarkaspirat durch Dichtezentrifugation isoliert. 2.5 x 105 kultivierte und differenzierte EPC und MSC wurden in vitro auf beta-TCP geladen. In 145 athymischen, männlichen Ratten wurde das Femur osteotomiert, ein 5 mm Knochendefekt erzeugt und mit Fixateur externe stabilisiert. Die Knochendefekte wurden mit beta-TCP (Gruppe 1), beta-TCP und MSC (Gruppe 2), beta-TCP und EPC (Gruppe 3), beta-TCP und EPC und MSC (Gruppe 4) oder autologem Knochen (Gruppe 5) gefüllt. Nach 1 Woche (n=40), 4 Wochen (n=40), 8 Wochen (n=40) und 12 Wochen (n=25) wurden die Ratten getötet. Bei Pinlockerung wurde die Ratte ausgeschlossen. Die (immun)histologische Analyse (Färbung mit HE, VEGF-R2, vWF) der Vaskularisierung und Knochenneubildung erfolgte mit Image-Analysis-System. Nach 8 und 12 Wochen erfolgte ein µCT und ein 4-Punkte-Biegungstest. Für die statistische Analyse wurde der Kruskal-Wallis-Test verwendet. Ergebnisse und Schlussfolgerungen: Nach 1 Woche zeigte sich bei der Implantation von EPC/MSC und EPC allein signifikant mehr primitive vaskuläre Plexus (p=0.01;p=0.048) als in Vergleichsgruppen. Im Vergleich zur TCP Gruppe war in allen anderen Versuchsgruppen signifikant mehr Knochenneubildung zu sehen (p<0.01). Ausserdem war in der EPC/MSC-Gruppe signifikant mehr Knochenbildung zu erkennen als in der MSC-Gruppe (p=0.03). Nach 12 Wochen zeigten alle Gruppen eine knöcherne Durchbauung des Defektes, jedoch zeigten bereits 8 Wochen nach Implantation von MSC/EPC 83% der Defekte eine stabile, knöcherne Durchbauung. Bei der Implantation von MSC kam es in 18% der Knochendefekte zum knöchernen Durchbau. Alle anderen experimentellen Gruppen zeigten nach 8 Wochen keine knöcherne Durchbauung. Diese Resultate konnten im µCT, biomechanischen Test und in der Histologie quantifiziert werden. EPC scheinen die frühe Vaskularisierung innerhalb eines Knochenkonstrukt in vivo zu stimulieren und das Einwachsverhalten von MSC zu verbessern, was zu einer beschleunigten Knochenheilung im Knochendefektmodell der Ratte führt

Kagami‐Ogata syndrome: an important differential diagnosis to Beckwith‐Wiedemann syndrome

We report the case of a fetus with sonographic characteristics of Beckwith-Wiedemann syndrome (BWS). A 30-year-old gravida 2 para 1 was referred to our fetal medicine unit with an omphalocele. Fetal macrosomia, organomegaly, and polyhydramnios but no macroglossia were detected and BWS was suspected. Genetic testing for BWS did not confirm the suspected diagnosis as the karyotype was normal. Symptomatic polyhydramnios led to repeated amnioreductions. At 35 + 5 weeks of gestation, a female neonate of 3660 g was delivered with APGAR scores of 6/7/8, after 1/5/10 min, respectively. The abnormal shape of the thorax, facial dysmorphism, need for ventilation, and generalized muscular hypotonia led to the suspicion of Kagami-Ogata syndrome (KOS), which was confirmed by genetic testing. KOS in our patient was caused by a large deletion in the MEG3-region on chromosome 14q32 affecting the maternal allele. In this report, we highlight the notion that when sonographic signs suggestive of BWS such as macrosomia, polyhydramnios, and omphalocele are present and genetic testing does not confirm the suspected diagnosis, KOS should be tested for

Circulating Leukotriene B4 Identifies Respiratory Complications after Trauma

Background. Leukotriene B4 (LTB4), a proinflammatory lipid mediator correlates well with the acute phase of Acute Respiratory Distress Syndrome (ARDS). Therefore, LTB4-levels were investigated to determine whether they might be a useful clinical marker in predicting pulmonary complications (PC) in multiply traumatized patients. Methods: Plasma levels of LTB4 were determined in 100 patients on admission (ED) and for five consecutive days (daily). Twenty healthy volunteers served as control. LTB4-levels were measured by ELISA. Thirty patients developed PC (pneumonia, respiratory failure, acute lung injury (ALI), ARDS, pulmonary embolism) and 70 had no PC (ØPC). Results. LTB4-levels in the PC-group [127.8 pg/mL, IQR: 104–200pg/ml] were significantly higher compared to the ØPC-group on admission [95.6 pg/mL, IQR: 55–143 pg/mL] or control-group [58.4 pg/mL, IQR: 36–108 pg/mL]. LTB4 continuously declined to basal levels from day 1 to 5 without differences between the groups. The cutoff to predict PC was calculated at 109.6 pg/mL (72% specificity, 67% sensitivity). LTB4 was not influenced by overall or chest injury severity, age, gender or massive transfusion. Patients with PC received mechanical ventilation for a significantly longer period of time, and had prolonged intensive care unit and overall hospital stay. Conclusion. High LTB4-levels indicate risk for PC development in multiply traumatized patients

Effects of green tea catechins on the pro-inflammatory response after haemorrhage/resuscitation in rats

Plant polyphenols, i.e. green tea extract (GTE), possess high antioxidative and anti-inflammatory capacity, thus being protective in various models of acute inflammation. However, their anti-inflammatory effect and a feasible mechanism in haemorrhage/resuscitation (H/R)-induced liver injury remain unknown. We investigated the effects of GTE and the role of NF-κB in the pathogenesis of liver injury induced by H/R, and their effects on intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil infiltration. Female Lewis rats were fed a standard chow diet (control, ctrl) or a diet containing 0·1% polyphenolic GTE for five consecutive days before H/R. Rats were haemorrhaged to a mean arterial pressure of 30 (sem 2)mmHg for 60min and resuscitated. Control groups (sham_ctrl and sham_GTE) underwent surgical procedures without H/R. At 2h after resuscitation, tissues were harvested. Serum alanine aminotransferase (ALT) and IL-6 were measured. Hepatic necrosis, ICAM-1 expression and polymorphonuclear leucocyte (PMNL) infiltration were assessed. Hepatic expression of IκBα (phospho) was measured. H/R induced strong liver damage with increased necrosis and serum ALT levels. Compared with both sham groups, inflammatory markers (serum IL-6 and hepatic PMNL infiltration) were elevated after H/R (P<0·05). Also, H/R increased IκBα phosphorylation. GTE administration markedly (P<0·05) decreased serum ALT and IL-6 levels, hepatic necrosis as well as PMNL infiltration and the expression of ICAM-1 and phosphorylated IκBα compared with H/R. In conclusion, we observed that NF-κB activation plays an important role in the pathogenesis of liver injury after H/R through the up-regulation of hepatic ICAM-1 expression and subsequent PMNL infiltration. GTE pre-treatment prevents liver damage in this model of acute inflammation through a NF-κB-dependent mechanis