Proof of concept: predicting the onset of meat spoilage by an integrated oxygen sensor spot in MAP packages.

During storage of modified atmosphere packaged (MAP) meat, the initial microbiota grows to high cell numbers, resulting in perceptible spoilage after exceeding a specific threshold level. This study analyses, whether elevated oxygen consumption in the headspace of MA-packages would enable a prediction method for meat spoilage. We monitored the growth of single spoiling species inoculated on high-oxygen MAP beef and poultry, performed sensorial analysis and determined oxygen concentrations of the headspace via a non-invasive sensor spot technology. We detected microbial headspace oxygen consumption occurring prior to perceptible meat spoilage for certain species inoculated on beef steaks. However, headspace oxygen consumption and cell counts at the onset of spoilage were highly species-dependent, which resulted in a strong (Brochothrix thermosphacta) and moderate (Leuconostoc gelidum subspecies) decrease of the headspace oxygen content. No linear decrease of the headspace oxygen could be observed for Carnobacterium divergens and Carnobacterium maltaromaticum inoculated on poultry meat. We demonstrate the applicability of an incorporated oxygen sensor spot technology in MAP meat packages for detection of spoilage in individual packages prior to its perceptible onset. This enables individual package evaluation and sorting within retail, and consequently reduces meat disposal as waste.

Acid stress response of Staphylococcus xylosus elicits changes in the proteome and cellular membrane.

AIMS: Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami. METHODS AND RESULTS: A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness. CONCLUSION: This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci.

Motivation of trauma patients to stop smoking after admission to the emergency department.

Every smoker should be offered smoking cessation treatment when they present for clinical care. The Readiness to Change-Smokers (RTC-S) questionnaire and the Heidelberg Smoking History (HSH) are brief questionnaires that divide patients into three stages. The purpose of this study was to prospectively compare the performance of each questionnaire at identifying patients who will successfully quit smoking within one year of Emergency Department (ED) discharge. Out of 1292 injured ED patients nearly half (n = 599, 46.4%) were identified as current smokers. Both questionnaires were given to all 599 subjects, and used to divide patients into three stages. At 12-months postdischarge 306 patients (51.1%) were contacted to determine smoking status. Patients were similarly classified by both tests in only 36% of cases. Concordance between tests was poor (kappa = 0.33). The RTC-S classified fewer patients as ready to quit (A = 13% vs. 22.2%). At 12 month follow-up, 55 patients (17.9%) had stopped smoking. The HSH was more successful to predict quitters. Multivariate logistic regression with respect to smoking cessation resulted in significant impact of HSH (p = 0.024).

Homologous growth hormone accelerates bone healing--a biomechanical and histological study.

The purpose of this study was to prove whether homologous growth hormone has a beneficial effect in the early phase of bone healing. Therefore the left tibias of 24 Yucatan micropigs were osteotomized and stabilized by plate fixation. The treatment group (12 animals) received 100 microg of recombinant porcine growth hormone (rpGH)/kg body w/day sc, whereas the control pigs (12 animals) received 1 ml sodium chloride as placebo. After a healing period of 4 weeks the animals were sacrificed and destructive torsional testing was performed. For histological evaluation 6 microm serial slices of the tibiae were stained with von Kossa. The total area of callus formation (CA) and the mineralized bone area (BA) were quantified by image analysis. The fraction of mineralized bone tissue within the callus area, the bone density (BD), was calculated as follows: BD = (BA/CA) x 100. Torsional failure load was 91% higher and torsional stiffness 61% higher in the treatment group than in the control group (P < 0.05). The histomorphometric measurements revealed an advance for the CA (GH: 127.6 +/- 38.9 mm(2); placebo: 75.9 +/- 50.7 mm(2); P < 0.005) as well as for the BA (GH: 89.3 +/- 25.8 mm(2); placebo: 55.9 +/- 38.5 mm(2); P < 0.001) for the GH-treated animals in comparison to the control animals. The BD was similar in both groups (GH: 70.6 +/- 8.4%; placebo: 74.0 +/- 6.24%; P = 0.28). These data indicate that administration of homologous GH stimulates callus formation and ossification in the early phase of bone healing, which consequently results in an increased mechanical strength and stiffness.

Systemic application of growth hormone enhances the early healing phase of osteochondral defects--a preliminary study in micropigs.

Healing of osteochondral defects following trauma remains a significant clinical problem, often leading to osteoarthritis. Growth hormone (GH) has been shown to accelerate formation of bone and cartilage tissue in the growth plates and in cell cultures. To investigate the influence of systemically administered recombinant porcine growth hormone (r-pGH) on the healing of osteochondral defects we performed a histomorphometrical analysis of full-thickness cartilage defects in the femoral condyle of micropigs. Forty-eight mature female Yucatan micropigs were divided into two groups, one receiving a daily injection of r-pGH (100 microg/kg), the other receiving sodium chloride as placebo. A circular 6-mm-diameter full-thickness defect of the cartilage was created, extending 1.5 mm into the subchondral bone. The animals were sacrificed after 4 (n = 24) and 6 (n = 24) weeks. The von-Kossa stain was used to visualise the calcified structures; cartilage and the fibrous tissue were marked with a combined Safranin-O/light-green stain. The defect filling and the percentage of bone, cartilage, and fibrous tissue into the defect were evaluated using an image analysis system. Furthermore, histological grading was performed using the modified Wakitani score. After 4 weeks no differences were observed between both groups. The defect filling after 6 weeks with newly formed bone was significantly higher in the r-pGH-treated group. The formation of cartilage and fibrous tissue showed a trend towards better healing in the GH-treated group; however, there was no significant difference. In the r-pGH-treated group, the percentage of total defect filling was significantly higher. The evaluation of the vascularity showed a significantly lower number of vessels in the GH-treated group after 6 weeks. Histomorphological grading revealed a significantly lower total Wakitani score in the GH-treated group, which represents a better healing result compared to the controls. The results of the present study suggest that circulating r-pGH or one of its mediators may accelerate osteochondral defect healing by stimulating the formation of osseous and chondral tissue. The analysis of the vascularity leads to the assumption of an advanced maturation of the osteochondral defects under the influence of GH.

[Palmar plating with the locking compression plate for dorsally displaced fractures of the distal radius--first clinical experiences]. / Die winkelstabile palmare Plattenosteosynthese bei der dorsal dislozierten distalen Radiusfraktur--Anwendung und erste klinische Erfahrungen.

Injuries and irritation of extensor tendons are common problems in the treatment of fractures of the distal radius when plating is used via a dorsal approach. By the development of locking compression plates the possibility of palmar plating for dorsally displaced fractures of the distal radius is available. In this study our first clinical experiences using the 3.5 mm radius locking compression plate (LCP) are reported. Between February 2002 and September 2002 24 patients with dorsally displaced fractures of the distal radius were treated using a palmar approach with the LCP and included in a prospective study. The mean age of the patients was 52 years (28-87 years). Six weeks and six months after surgery a clinical assessment was done, the range of motion of the injured wrist was measured, and an X-ray control of the injured joint was performed. The preliminary results demonstrate the option of early functional treatment using the locking compression plate. Most of the patients had a good to excellent range of motion of the injured wrist which resulted in an early return to former activity. There were few intra- and postoperative complications. No irritation of the median nerve, no infection was observed. In one case a screw which was placed intraarticularly was removed prematurely. The palmar locking compression plate has been proven as a safe and effective implant for the treatment of dorsally displaced fractures of the distal radius.

Recombinant species-specific growth hormone increases hard callus formation in distraction osteogenesis.

The effect of growth hormone (GH) on secondary fracture healing and callus formation has been demonstrated in several previously investigated animal models. The aim of this study was to investigate and quantify the effects of GH on bone regenerates in a distraction osteogenesis model. In 20 mature female Yucatan micropigs, the tibia and fibula were osteotomized, stabilized with an external fixator, and distracted at 2 mm/day for 10 days after a 4 day latency period. The regenerates were allowed to consolidate for 10 days. Micropigs in the study group (ten animals) received a daily injection of 100 microg per kilogram body weight of recombinant porcine growth hormone (r-pGH). Micropigs in the control group (ten animals) received sodium chloride as placebo. After killing on day 25, a quantitative histomorphometrical analysis of the formed callus and the adjacent cortical bone was performed and the results of polychrome in vivo labeling were assessed. The regenerates of the r-pGH-treated animals showed a significantly larger callus area but no change in callus structure. We found islands of cartilage tissue in the regenerates of both groups; the calli from the control group exhibited a higher fraction of cartilage compared with the r-pGH group, but this was not significant. Quantification of the fluorescent in vivo labeling revealed that the distraction gap in GH-treated group showed significant ossification even during distraction. These results demonstrate that growth hormone can accelerate the maturation of the regenerate in distraction osteogenesis without changing the callus microstructure. This may prove to be a useful clinical tool for shortening the healing time in limb lengthening and bone segment transport.

The effect of growth hormone on insulin-like growth factor I and bone metabolism in distraction osteogenesis.

Limb lengthening in the left tibia of 30 mature female Yucatan micropigs was performed using distraction osteogenesis. A treatment group of 15 animals received recombinant porcine growth hormone (r-pGH) (100 microg/kg/day) while the others served as controls. Serial serum measurements of total insulin-like growth factor I (IGF-I), free IGF-I, IGF binding proteins -1, -2, -3 and -4 (IGFBP-1 to -4) were performed. Bone-specific alkaline phosphatase (bone-ALP) and the serum carboxyl-terminal telopeptide of type I collagen (ICTP) were measured as bone turnover markers. The GH-treated animals showed a significant increase in total IGF-I, free IGF-I and IGFBP-3 after surgery (P<0.001). Similarly, the treated animals showed a significantly higher level of bone-ALP (P<0.001) throughout the experiment compared to the controls. There was a significant correlation between bone-ALP and total IGF-I (r=0.76) in the GH-treated group and an even higher correlation for free IGF-I (r=0.90). There was no difference in the ICTP serum levels between the two groups. These data indicate that the application of species-specific growth hormone results in a stimulation of bone formation in distraction osteogenesis which may be mediated by IGF-I. The stronger correlation between free IGF-I and bone-ALP indicates that the anabolic effect of IGF-I may be regulated through the IGFBPs by binding and inactivating IGF-I.

Homologous growth hormone accelerates healing of segmental bone defects.

The effect of homologous recombinant porcine growth hormone (r-pGH) on secondary fracture healing was investigated in a diaphyseal defect of the tibia in Yucatan micropigs. A 1 cm defect of the tibia was created surgically and stabilized with an AO 3.5 mm DCP plate. The treatment group (12 animals) received 100 microg of r-pGH per kilogram of body weight subcutaneously once per day, whereas the control pigs (12 animals) received 1 mL of sodium chloride as placebo. For evaluation of the GH-axis, serum levels of insulin-like growth factor-I (IGF-I) were sampled every fourth day. The animals were killed 6 weeks after surgery. Quantitative computed tomography (qCT) was performed to determine bone mineral density (BMD) and bone mineral content (BMC) of the defect zone. The torsional stiffness and the torsional failure load were measured by destructive torsional testing of the defect and contralateral tibiae. qCT measurements revealed a significant increase in the BMC of the defect zone in the treatment group compared with controls (GH BMC = 2833 +/- 679 mg, placebo BMC = 2215 +/- 636 mg; p < 0.05), whereas the BMD values were similar in both groups (GH BMD = 668 +/- 60 mg/mm(2), placebo BMD = 629 +/- 52 mg/mm(2), p = 0.12). Torsional failure load was 70% higher and torsional stiffness 83% higher in the treatment group than in the control group (p < 0.05). The mean serum level of IGF-I in the treatment group increased to 382% of the preoperative basal level and decreased to 69% in the control group, and this difference was highly significant (p < 0.001). Our data indicate that daily administration of recombinant GH leads to an increase of serum IGF-I levels and stimulates secondary fracture healing, resulting in increased mechanical strength and stiffness of the callus.

Large-scale isolation of sinusoidal endothelial cells from pig and human liver.

OBJECTIVE: Hepatic in vitro studies, like those on hypoxia/reperfusion injury in liver transplants, demand large numbers of cultivated sinusoidal endothelial cells (SECs). In this article, we present and evaluate a new method for the isolation of SECs from porcine and human livers. METHODS: SECs were isolated employing a four-step collagenase perfusion. The sinusoidal character of the cells was validated by transmission and scanning electron microscopy, exclusion of Weibel-Palade bodies and factor VIII-related antigen, expression of scavenger receptor, and incorporation of latex beads. RESULTS: In 23 pigs, an average of 9 x 10(4) SECs were harvested from each liver. Cells were cultivated under standard conditions, as well as in multilayer cocultures of isolated SECs and hepatocytes in a "sandwich" configuration. Standard cultures showed an average of 90% SECs in primary cultures and 100% SECs after the first passage. The possibility of isolation of SECs from human livers was demonstrated in eight cases. CONCLUSION: With the four-step collagenase perfusion it is possible to easily isolate large numbers of viable and pure SECs from one organ. A further advantage is the possibility of isolating hepatocytes from the same organ.

[Recombinant growth hormone accelerates regenerate consolidation in distraction osteosynthesis]. / Rekombinantes Wachstumshormon beschleunigt die Regeneratkonsolidierung bei der Distraktionsosteogenese.

Distraction osteogenesis is a good model for evaluation of fracture healing with intramembranous bone formation. The purpose of the present study was to investigate whether homologous GH has a stimulating effect on bone healing in distraction osteogenesis. The left tibiae of 30 micropigs were osteomized and distracted with an external fixator 2 mm daily over a period of 10 days. Animals were killed after an additional healing time of 10 days. The treatment group received 100 micrograms r-pGH per kg bodyweight per day. A newly developed device allowed non-destructive torsional biomechanical evaluation of the regenerate strength as in vivo measurements. After killing, destructive torsional strength testing of the sites of distraction was performed. To determine the endocrine response to the administration of r-pGH, serum levels of IGF-I were determined. The non-destructive in vivo testing showed that torsional stiffness of the regenerate was significantly higher in the treatment group than in the control group. Final regenerate torsional failure load was 131% higher and ultimate torsional stiffness was 231% higher in the treatment group than in the control group. The mean serum level of IGF-I increased to 440% of the preoperative base level in the treatment group and remained unchanged in the control group. Our data indicate that systemic administration of recombinant homologous growth hormone significantly accelerates ossification of bone regenerate in distraction osteogenesis.

The influence of screw geometry on hamstring tendon interference fit fixation.

We used a standardized model of calf tibial bone to investigate the influence of screw diameter and length on interference fit fixation of a three-stranded semitendinosus tendon graft for anterior cruciate ligament reconstruction. Biodegradable poly-(L-lactide) interference screws with a diameter of 7, 8, and 9 mm and a length of 23 and 28 mm were used. We examined results in three groups of 10 specimens each: group 1, screw diameter equaled graft diameter and screw length was 23 mm; group 2, screw diameter equaled graft diameter plus 1 mm and screw length was 23 mm; group 3, screw diameter equaled graft diameter and screw length was 28 mm. The mean pull-out forces in groups 1, 2, and 3 were 367.2+/-78 N, 479.1+/-111.1 N, and 537.4+/-139.1 N, respectively. The force data from groups 2 and 3 were significantly higher than those from group 1. These results indicate that screw geometry has a significant influence on hamstring tendon interference fit fixation. Increasing screw length improves fixation strength more than oversizing the screw diameter. This is important, especially for increasing tibial fixation strength because the tibial graft fixation site has been considered to be the weak link of such a reconstruction.

Quantitative assessment of in vivo bone regeneration consolidation in distraction osteogenesis.

We present a new method for quantitatively assessing the consolidation of bone regeneration by performing distraction osteogenesis in micropigs. We measured in vivo stiffness using a newly developed, revolving, bone-healing meter. After the micropigs were killed, we obtained maximum torsional moment data for the regenerated bones by destructive mechanical testing, and we then correlated these data with the data for stiffness. We found a highly significant regression between in vivo stiffness and maximum torsional moment (r2 = 0.80), suggesting that monitoring stiffness may be useful for the prediction of bone regeneration in distraction osteogenesis. Therefore, our method may be a reliable tool for future quantitative monitoring of healing progress in patients with healing bones or in animal studies addressing treatments to increase bone formation in long-bone defects.

A new device to quantify regenerate torsional stiffness in distraction osteogenesis.

We present a newly developed torsional stiffness measurement device with the potential to quantitatively assess the in vivo torsional stiffness of bone regenerate during distraction osteogenesis. We describe the form and function of this device and its application in a model of regenerate consolidation. The device was able to produce data to assess stiffness of the regenerate with an accuracy between +/- 3 and +/- 9% for material stiffness ranging between 0.1 and 2.4 Nm/o and with a precision of +/- 3.6%. This method provides advantages over similar methods of bone fracture healing assessment with guaranteed maintenance of bone axis, minimized risk of bone misalignment during the bone healing process and a close relation to the functional loading pattern in torsion of bones such as tibia and femora.

Recombinant growth hormone accelerates bone regenerate consolidation in distraction osteogenesis.

The purpose of the present study was to prove whether homologous GH has a stimulating effect on bone healing. Therefore, left tibiae of 30 micropigs were osteomized and distracted over an external fixator at the rate of 2 mm/day on each of 10 consecutive days. Animals were killed after a healing period of another 10 days. The treatment group received 100 microg of recombinant porcine growth hormone (rpGH) per kilogram of body weight per day. Serial torsional nondestructive biomechanical tests were performed in vivo using a newly developed measurement device. After killing, destructive torsional strength testing of the sites of distraction was performed. To determine the endocrine response to the administration of rpGH, serum levels of insulin-like growth factor-I (IGF-I) were determined. Nondestructive in vivo testing showed that torsional stiffness of the regenerate was significantly higher in the treatment group than in the control group. Final regenerate torsional failure load was 131% higher and ultimate torsional stiffness was 231% higher in the treatment group than in the control group. The mean serum level of IGF-I increased to 440% of preoperative basal level in the treatment group and remained unchanged in the control group. Our data indicate that systemic administration of recombinant homologous growth hormone greatly accelerates ossification of bone regenerate in distraction osteogenesis.

Digital radiography. A predictor of regenerate bone stiffness in distraction osteogenesis.

The newly formed regenerate bone during limb lengthening usually is assessed by observing standard plain radiographs. However, no objective data correlate the radiographic appearance with the quantitative strength of the newly formed bone. A noninvasive method was developed to assess the regenerate bone using digital radiography. The investigations were performed in a micropig animal model. The right tibia was distracted for 10 days at 2 mm per day. Digital radiography was performed after the distraction during a 10-day consolidation period. For quantitative analysis, an aluminum step wedge was placed on all images, and a region of interest was outlined in the regenerate bone. A calibration curve was calculated by measuring the corresponding gray values of the aluminum phantom. The calibration equation was used for determining the mean density in the region of interest. This method was sensitive in assessing day to day healing progress. The measurements taken the day before sacrifice were compared with torsional mechanical data using a material testing machine. The high correlation between the densimetric evaluations and the biomechanical data suggests this method is a useful tool for in vivo assessment of the regenerate bone during the consolidation period in distraction osteogenesis.

[Recombinant growth hormone increasus callus maturation time in distraction osteogenesis--a histomorphometric study]. / Rekombinantes Wachstumshormon beschleunigt die Kallusreifung bei der Distraktionsosteogenese--Eine histomorphometrische Untersuchung.

Purpose of the present study was, to proof whether homologous recombinant GH has an stimulating effect on bone healing in distraction osteogenesis. Therefore the left tibiae of 20 micropigs were osteotomized and distracted over an external fixator 2 mm daily on each of ten consecutive days. Animals were sacrificed after a healing time of another ten days. The treatment group received 100 micrograms r-pGH per kg bodyweight per day. After sacrifice, the regenerates were embedded undecalcified and cut into 6 microns thick sagittal oriented slices. Callus area, bone area, cartilagee area and bone perimeter in the regenerates were measured using an image analysis system. The results revealed, that GH promotes bone formation and maturation of the regenerate without disturbing the callus structure.

Origin of neurons supplying the vas deferens of the rat.

Retrograde axonal tracing methods using Fluoro-Gold were used to examine the neuronal input to the vas deferens in the adult Wistar rat. The greatest number of labelled efferent neurons were found in the ipsilateral pelvic accessory ganglion (PAG) (68%) and the major pelvic ganglion (MPG) (15%). Fewer than 3% of labelled neurons were localized to the inferior mesenteric and sympathetic chain ganglia. Labelled neurons were also located in the ipsilateral L1, L2, and L6, S1 dorsal root ganglia (DRG), corresponding to afferents that travel in the hypogastric and pelvic nerves, respectively. Contributions from contralateral neurons in the PAG, MPG and L1 DRG were also documented. The role of afferents supplying the vas deferens is not known but they may relay nociceptive or mechanoreceptive input. Efferent input from peripheral ganglia probably contributes to contractility of the vas deferens based on previous investigations.

Sonographic detection of ureteral calculi in patients with normal excretory urography.

We report 3 cases of nonobstructing distal ureteral calculi diagnosed by sonography in which excretory urography was normal. In each case sonography was performed because of high clinical suspicion for a ureteral stone. In 2 patients a stone was impacted at an edematous ureteral orifice. All 3 patients subsequently passed the stone spontaneously. A high resolution ultrasound examination should be performed on patients with a history suggestive of ureteral calculus despite a normal excretory urogram.

Neural regulation of the vas deferens in the rat: an electrophysiological analysis.

Electrophysiological analysis of the neural control of the vas deferens was performed in urethan-anesthetized rats. Intraluminal distension (0.2 ml/min) or electrical stimulation of hypogastric (threshold 1-5 V, 20 Hz) and pelvic nerves (2-5 V, 20 Hz) produced contractions of the vas deferens. Distension-evoked contractile activity was not abolished by nicotinic ganglionic blockade or ipsilateral hypogastric and pelvic nerve transection. Contractions following hypogastric nerve stimulation were abolished by prazosin, while pelvic nerve-evoked responses were partially blocked by atropine. Hypogastric nerve, pelvic nerve, and sympathetic chain stimulation evoked volleys with latencies of 10-30 ms in vasal nerves. Crude estimates for conduction velocities for these responses (less than 0.5 m/s) corresponded to activation of unmyelinated C-fibers. Stimulation of the dorsal nerve of the penis (DNP) (10-16 V, 10-40 Hz) or administration of 5-methoxy-N,N-dimethyltryptamine, both of which produce seminal emission, elicited reflex discharges in nerves to the vas deferens. Hypogastric nerve but not pelvic nerve transection abolished both spontaneous and evoked (105- to 380-ms latency) reflex activity. These experiments provide insight into the organization of afferents in the DNP and efferents conveyed by autonomic pathways that regulate male reproduction.