Biomedical subjects

Ulrich Schneider

Publications and source records attributed to Ulrich Schneider.

18 recordsLinked to original sources

PubMed2007

BMP-7 loaded microspheres as a new delivery system for the cultivation of human chondrocytes in a collagen type-I gel.

In recent years, interest in chondrocyte cultures for transplantation has gained increasing attention. We investigated the use of PGLA microspheres as a new delivery system for BMP-7 and the effects on human chondrocytes cultivated in a 3D collagen gel culture. In an in vitro study, human chondrocytes obtained from osteoarthritic knee joints were released, transferred into a collagen type-I gel, and cultivated up to 14 days. In the treatment group PGLA microspheres loaded with human recombinant BMP-7 protein were added to the matrix. After the cultivation period, histological and immunohistochemical investigations were performed. In addition, the aggrecan core protein and type-II collagen mRNA concentrations were measured by real-time PCR. Histological staining for proteoglycan and collagen type-II protein and quantification via digital image processing revealed a significantly higher content in the samples cultivated with BMP-7 loaded microspheres in comparison to the control samples. Moreover, the collagen gel scaffold was partially remodeled by the chondrocytes and replaced by newly synthesized extracellular matrix. Cellular proliferation as well as apoptosis were low. In conclusion, we consider the PGLA microsphere system to be a functional device for the delivery of growth factors during the cultivation of articular chondrocytes leading to an increased content of type-II collagen and proteoglycan in the extracellular matrix.

Aged↗

PubMed2005

Ingrowth of osteochondral grafts under the influence of growth factors: 6-month results of an animal study.

INTRODUCTION: In the age of growth factors and gene therapy, the induction of cartilage healing remains an unsolved problem. Even in autologous grafting, one of the preferred methods of treatment for focal osteochondral lesions, chondral integration remains difficult. This study aims to define a possible positive influence of growth factor augmentation on the ingrowth of these transplants. MATERIALS AND METHODS: In an ovine model, questions regarding the healing of osteochondral transplants under the influence of two different growth factors were to be addressed. Two osteochondral autologous transplantations (OAT), one in the weight-bearing surface of each femoral condyle, were performed on the ovine knee using the standard operative protocol. One of the grafts was bathed in augmented PBS containing 50 microg bFGF or bone morphogenetic protein (BMP)-2 directly prior to implantation, while the other condyle served as the control. Two groups, consisting of eight sheep each, were evaluated for each growth factor after 6 months. RESULTS: During the evaluation of all the specimens, neither osteophytes nor synovial changes were observed. The mechanical consistency of the cartilaginous tissue began to reach a level equivalent to the surrounding tissue at 6 months, independent of the use of growth factor. Macroscopically, the superficial border of the transplanted osteochondral plug could easily be outlined in all groups, while the cartilage interface of the bFGF specimens was determined to be less demarcated than the BMP augmented plugs or the controls. Radiographically, a solid osteointegration of the graft could be documented at 6 months in the native and augmented groups. In contrast, integration of the chondral surface of the OAT was not seen macro- or microscopically in any specimen, even though cartilage surfaces remained viable. A firm physical interdigitation of the reconstructed joint surface could not be demonstrated in either of the two augmented groups or the control population. The augmentation with bFGF and BMP-2 stimulated the osseous ingrowth and seems to expedite the remodelling process, but was not able to improve chondral healing. CONCLUSION: The lack of integration of the cartilaginous portion of the transplanted plugs into the reconstructed joint surface, even following the augmentation with bFGF and BMP-2, does not bode well for the long-term survival of the joint itself.

Animals↗

PubMed2005

Nuclear oncoprotein prothymosin alpha is a partner of Keap1: implications for expression of oxidative stress-protecting genes.

Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin alpha. The in vivo and in vitro data indicated that the prothymosin alpha-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin alpha was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin alpha by using prothymosin alpha overproduction and mRNA interference approaches. Our data attribute to prothymosin alpha the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin alpha and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.

Active Transport, Cell Nucleus↗

PubMed2004

In-gel digestion of proteins using a solid-phase extraction microplate.

We present a new procedure for in-gel digestion of proteins introducing a combination of two different 96-well microplates. The two plates have incorporated small capillaries with a length of 2.4 mm in each well, one of which has 75-microm-inner diameter capillaries, whereas the second plate has reversed-phase-type capillaries fixed to it. The initial steps of the in-gel digestion process, comprising destaining, reduction/alkylation, dehydration, and digestion, was carried out in the plate containing 75-microm capillaries. Capillaries containing C18 reversed-phase modified monolithic silica rods of a 200-microm diameter were used for the second plate in which extraction and cleanup of peptides were carried out. Peptides were eluted directly from the solid-phase extraction plate onto the MALDI sample support. The separation of the process into two plates led to increased process stability, without compromising sensitivity, i.e. peptide recovery, making it suitable for true high-throughput protein identification. The handling of proteinases could easily be optimized, and no restrictions were made on chosen pH range through the absence of the solid phase in the initial steps of the protocol. Efficient binding of peptides to the solid phase and subsequent direct elution onto the MALDI sample support led to sensitivities in the attomole range. Performance of the process was demonstrated with tryptic digests of proteins stained with colloidal coomassie blue, silver, and the fluorescent stain SYPRO Ruby.

Animals↗

PubMed2004

Osteonecrosis 15 years after femoral neck fracture and long-term low-dose inhaled corticosteroid therapy.

Posttraumatic avascular necrosis of the femoral head typically occurs immediately or within a few years after a femoral neck injury, and non-traumatic avascular necrosis is often related to systemic glucocorticoid therapy. We report an unusual case in which avascular necrosis of the femoral head occurred 15 years after a transcervical femoral fracture in a woman with a 20-year history of daily inhaled glucocorticoid therapy for chronic bronchitis. She had not taken glucocorticoids by any other route and had no other risk factors for osteonecrosis. To our knowledge, this is the first report of osteonecrosis associated with inhaled glucocorticoid therapy in a patient with a local cause of diminished vascular reserve. Inhaled glucocorticoid therapy should be added to the list of risk factors for osteonecrosis.

Administration, Inhalation↗

PubMed2003

Serum levels of interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF) and elastase in aseptic prosthetic loosening.

In synovial-like membranes from failed total hip prostheses, an increased level of cytokines and cellular mediators has been identified. We compared two matched groups of patients with total hip arthroplasty (THA)--one with surgically proven component loosening and one without. We measured serum levels of interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF) and elastase. Soluble interleukin 2 receptor (sIL-2r) was also measured to exclude any hypersensitivity reaction. We found no significant difference in serum values between groups. Neither were there any differences with respect to implant material, mode of fixation, and periprosthetic osteolysis. In contrast to previous reports, our results suggest that serum levels of cytokines and cellular mediators may not be affected in aseptic loosening.

Aged↗

PubMed2003

Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis.

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.

Electrophoresis↗

PubMed2003

Molecular markers in the evaluation of autologous chondrocyte implantation.

PURPOSE: With the help of molecular markers, it has become possible to quantify cartilage repair and degradation in joints. In this study, we attempt to determine whether or not molecular markers in synovial fluid can be helpful in defining the repair process following autologous chondrocyte implantation (ACI). TYPE OF STUDY: As part of a prospective clinical pilot study, 17 patients were evaluated before, as well as 6 weeks, 3, 6, and 12 months after the ACI. A synovial analysis was performed and molecular markers for bone and cartilage metabolism were determined. METHODS: A number of parameters, including pyridinium crosslink (PY), deoxypyridinolin (DPD), n-telopeptide (NTX) from type I collagen, MMP-1, MMP-3; TIMP-1, PICP, proteoglycan, and YKL-40 were analyzed. The levels were referenced to the total protein concentration of the synovial fluid. The synovial analyses were compared with clinical parameters (Larson score) and magnetic resonance imaging (MRI) examinations. RESULTS: The analysis of the data revealed differing trends for the various synovial markers over time. The most remarkable marker was found to be DPD, which increased continuously between surgery and week 12, only to disappear after the repair process had ceased 1 year after surgery. All molecular markers for cartilage degradation increased initially after surgery and dropped off below the original levels 3 to 6 months later. CONCLUSIONS: The evaluation revealed that the determination of marker levels can provide valuable information regarding the metabolism of bone and cartilage in a joint. They seem to provide a method for monitoring the repair process associated with the various treatment forms for chondral lesions.

Adipokines↗

PubMed2002

Correlation of a novel matrix protein with the degree of cartilage degradation.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP), also referred to as melanoma-inhibiting activity (MIA), is detected physiologically only in cartilage tissue and pathologically in malignant melanoma. Recent studies indicated that this protein might be useful as a marker for altered joint metabolism or damage. The purpose of this study was to evaluate whether there is any correlation between the degree of cartilage degradation and levels of this protein. In a prospective cross-sectional study, synovial fluid samples were obtained from 67 consecutive patients undergoing surgery of the knee joint. The degree of cartilage degradation was determined intraoperatively using the Outerbridge and Noyes classifications. MIA/CD-RAP was measured by a commercially available enzyme-linked immunosorbent assay (ELISA). The marker levels were compared against the Outerbridge and Noyes classifications and the correlation coefficients were calculated. At advanced stages of cartilage degradation, there were decreased synovial fluid levels of MIA/CD-RAP. The correlation coefficients were -0.449 and -0.418 for the Outerbridge and Noyes classifications, respectively, with large 95% confidence intervals. While the physiological function of MIA/CD-RAP is still unclear, the results indicate that MIA/CD-RAP levels depend on the degree of cartilage degradation. Despite the high interindividual variations, MIA/CD-RAP might be useful as a marker to monitor degenerative joint lesions.

Adolescent↗

PubMed2002

Correlation of bone and cartilage markers in the synovial fluid with the degree of osteoarthritis.

In recent years, many biochemical markers were tested with the aim of developing a tool for the early detection and monitoring of osteoarthritis (OA). Apart from chondral markers, we also evaluated osseous markers from the synovial fluid to obtain more comprehensive information, and we compared the levels in relation to the severity of OA. In this prospective, cross-sectional study, synovial fluid samples were obtained from 73 patients with OA of the knee joint prior to operation and after the joint was flushed with 50 ml of normal saline. All patients underwent surgery and were classified in accordance with the Outerbridge and the Noyes classification. The measured biochemical markers included pyridinoline (PD), deoxypyridinoline (DPD), N-telopeptide (NTx), carboxyterminal propeptide of collagen-1 (PICP), matrix metalloproteinases (MMP-1 and MMP-3), and tissue inhibitor of MMPs (TIMP-1). The marker levels were normalized against the total protein content and were compared with the Outerbridge and the Noyes classification. For the majority of markers, the correlation coefficient was below r=0.3, with large 95% confidence intervals. The highest correlation coefficients were obtained from DPD and TIMP-1. In relation to the Outerbridge classification, DPD revealed high values only in stage IV. The overall results indicate that the majority of the tested markers are unspecific with regard to the different stages of OA. The two markers with the highest correlation coefficients showed no major sensitivity to detect OA at an initial stage. However, they might give additional information to clinical and radiological findings with regard to the severity of the disease.

Amino Acids↗

PubMed2002

Instability and variability of urinary telopeptides and free crosslinks.

BACKGROUND: The use of the urinary bone degradation markers, NTx and free Dpd, in the diagnosis and monitoring of pathological bone resorption has been studied intensely. Urinary NTx and Dpd are normalized by creatinine to account for differences in diuresis. METHODS: We investigated the effects of storage (-20 degrees C for 119 days) on concentrations of NTx, Dpd and creatinine in the urine of 40 subjects. Precipitates (visible upon thawing) were resuspended, but specimens were not recentrifuged (in contrast to previous experiments). RESULTS: After storage at -20 degrees C, the NTx and creatinine concentrations in urine specimens were significantly decreased by about 18% and 22% (p < 0.01 and p < 0.0001), respectively. Thus, the quotient of NTx/creatinine was slightly increased after storage (p < 0.05). Free Dpd and free Dpd/creatinine were both significantly increased after storage (p < 0.0001)--by about 20% and 60%, respectively. CONCLUSIONS: We suggest that storage at -20 degrees C frees Dpd and denatures the epitope used in NTx quantification, and that these changes are usually masked by the removal of free Dpd via recentrifugation and by the decrease in creatinine in NTx/creatinine. Additionally, we suggest that the quotient Dpd/NTx should be evaluated as an alternative to normalizing via creatinine excretion.

Aged↗

PubMed2002

Urinary creatinine instability falsely increases the deoxypyridinoline/creatinine quotient.

BACKGROUND: The concentration of urinary deoxypyridinoline crosslinks (Dpd) (as in line 17)-normalized with respect to the urinary creatinine concentration is used as a biochemical marker of pathological bone resorption. METHODS: The effect of various storage conditions on the stability of Dpd/creatinine in urine specimens was examined in a sample of 14 individuals without aseptic endoprosthetic loosening (control) and 14 patients with aseptic endoprosthetic loosening (AEL). RESULTS: In the control group, values of Dpd/creatinine measured after 1 day of storage at -20 degrees C were significantly greater (p < 0.005) than values measured in fresh urine specimens. In the AEL group, increases in urinary Dpd/creatinine values were less pronounced (NS to p < 0.05). Increases in Dpd/creatinine were primarily the mathematical consequence of significant decreases in urinary creatinine concentration in the control group (p < 0.0001) and the AEL group (p < 0.05, after at least 7 days of storage). The decrease in urinary creatinine concentration appears to reach a plateau--between 75% and 80% of the original value--after approximately 10 days of storage. CONCLUSION: To prevent falsely increased Dpd/creatinine quotients, we suggest that urinary creatinine should be measured exclusively in fresh urine specimens or consistently in specimens stored for at least 14 days at -20 or -80 degrees C.

Adult↗

PubMed2002

Mechanically induced electrical potentials of articular cartilage.

While there is increasing evidence that chondrocytes are affected by mechanically induced stimuli, endogenous force-related electrical potentials within articular cartilage have been so far observed only in-vitro. Using a porcine ex-vivo model (German Land Race), 8 knee joints were explanted and exposed to mechanical force (up to 800 N) using a special device. Electrodes were inserted into the cartilage matrix. With an amplifier and an A/D transducer the changes of electrical voltage between the electrodes as well as those of the force were recorded online and simultaneously on a computer. Additionally, we located one pair of electrodes on the surface of the cartilage tissue to detect electrical fields outside the cartilage tissue. In relation to the applied force we observed that electrical potentials derived from inside and outside the articular cartilage showed a correspondence. When an alternating force with an amplitude of 360 N and a frequency of about 0.2 Hz was periodically applied, we measured peak amplitudes ranging from 2.1 to 5.5 mV within the cartilage tissue with electrical negativity within the weight bearing area of the cartilage tissue. The measured voltages depended on the applied force, the location of the electrodes, and on anatomical variations. We found an almost linear relation between the magnitude of the applied force and the recorded voltage. With the help of the electrodes located outside and within the cartilage tissue, we were able to show that force dependent fields are generated inside the cartilage. There are several theories explaining the origin of these electrical phenomena, many of them focusing on the negative charges of the proteoglycans in relation to the flow of interstitial fluid and ions under compression. However, the consequences of these phenomena are yet not clear.

Animals↗

PubMed2002

Effects upon bone metabolism following total hip and total knee arthroplasty.

OBJECTIVE: In a prospective study we evaluated the course of markers for bone formation and bone resorption in patients undergoing elective total hip and total knee arthroplasty due to osteoarthritis. The aim was to evaluate which changes in bone metabolism take place in the immediate postoperative course following arthroplasty. METHODS: Serum and urine samples were obtained preoperatively and in the postoperative course from patients undergoing total arthroplasty over a 90- or 180-day period. The study included a total of 63 patients with cemented hip prostheses (n = 20), uncemented hip prostheses (n = 23) and cemented knee prostheses (n = 20). Different biochemical markers of bone metabolism were assessed including the serum markers bone-specific alkaline phosphatase, and carboxyterminal propeptide of type I procollagen, and the urine markers n-telopeptide (NTx) and deoxypyridinoline (DPD). RESULTS: For all four markers, there were changes over time which were statistically significant. Markers indicating bone formation were slightly elevated after a 3-month period. In contrast, the two markers indicating bone resorption were back to normal after a 6-month period. There were differences between the groups with cemented and uncemented total hip arthroplasty in the postoperative course with higher values of the markers indicating bone resorption in the cemented group. However, these differences were not statistically significant. CONCLUSIONS: Our results indicate that the two markers of bone resorption, NTx and DPD, are elevated during the first 6 months after hip and knee arthroplasty. Therefore, during this period they cannot be used as a marker for aseptic loosening. However, if these two markers are still elevated after this period, they may reflect an impaired bone metabolism. Further studies are required to assess the potential value of these markers for the diagnosis of aseptic loosening.

Adult↗

PubMed2002

Large-scale plant proteomics.

Large-scale and high throughput approaches increasingly play an essential role in the study of biological systems, which are per se highly complex. Therefore, they need to be examined by these extensive methods to receive information about the large genomic and proteomic networks. In plant biology, this purpose has a strong support through the accessability of the complete genome sequence of the model plant Arabidopsis thaliana. This brief review intends to focus on the basics and the state-of-the-art of these high-throughput technologies and their application to plant proteomics. It describes protein microarrays, the use of antibodies, 2-DE and MS methods and the yeast two hybrid system, which are emerging as the major technologies for plant proteomics.

Arabidopsis↗

PubMed2002

New potential markers for cartilage degradation of the knee joint.

OBJECTIVE: To determine the correlation of biochemical markers with the degree of cartilage degradation. METHODS: In a cross-sectional study, synovial fluid samples were obtained from 65 patients with cartilage lesions of the knee joint. The measured biochemical markers included MMP-1, MMP-3, and MMP-13, the tissue inhibitor of MMPs (TIMP-1), COMP, YKL-40, and tenascin. The marker levels were compared with the Outerbridge and the Noyes classification. RESULTS: For the majority of markers, the correlation coefficient was below r = 0.3. The highest correlation coefficients were obtained from tenascin (r = 0.66 and 0.67) and MMP-13 (r = 0.44 and 0.41). CONCLUSION: The overall results indicate that the majority of the tested markers is unspecific with regard to the different stages of the two classifications. However, tenascin and MMP-13 could be of clinical importance to indicate advanced stages. Yet the values of these markers in longitudinal studies are not known.

Adipokines↗

PubMedDate not supplied

Reciprocal translocation between Y chromosome long arm euchromatin and the short arm of chromosome 1.

A case with an apparently balanced reciprocal translocation between the long arm of the Y chromosome and the short arm of chromosome 1 t(Y;1)(q11.2;p34.3) is described. The translocation was found in a phenotypically normal male ascertained by infertility and presenting for intra-cytoplasmatic sperm injection treatment. Histological examination of testicular biopsies revealed spermatogenic failure. Chromosome painting with probes for chromosome 1 and for the euchromatic part of the Y chromsome confirmed the translocation of euchromatic Y chromosomal material onto the short arm of chromosome 1 and of a substantial part of the short arm of chromosome 1 onto the Y chromosome. Among the Y/autosome translocations, the rearrangements involving long arm euchromatin of the Y chromosome are relatively rare and mostly associated with infertility. Microdeletion screening at the azoospermia locus revealed no deletions, suggesting another mechanism causing infertility in this translocation carrier.

Adult↗

PubMedDate not supplied

In vitro comparison of six different matrix systems for the cultivation of human chondrocytes.

In recent years, a great variety of different matrix systems for the cultivation of chondrocytes have been developed. Although some of these scaffolds show promising experimental results in vitro, the potential clinical value remains unclear. In this comparative study, we propagated human articular chondrocytes precultivated in monolayer culture on six different scaffolds (collagen gels, membranes and sponges) under standardized in vitro conditions. Mechanical properties of the matrix systems were not improved significantly by cultivation of human chondrocytes under the given in vitro conditions. The gel systems (CaReS, Ars Artho, Germany and Atelocollagen, Koken, Japan) showed a homogeneous cell distribution; chondrocytes propagated on Chondro-Gide (Geistlich Biomaterials, Switzerland) and Integra membranes (Integra, USA) were building multilayers. Only few cells penetrated the two Atelocollagen honeycomb sponges (Koken, Japan). During cultivation, chondrocytes propagated on all systems showed a partial morphological redifferentiation, which was best with regard to the gel systems. In general, only small amounts of collagen type-II protein could be detected in the pericellular region and chondrocytes failed to build a territorial matrix. During the first two weeks of cultivation, the two gel systems showed a significantly higher collagen type-II gene expression and a lower collagen type-I gene expression than the other investigated matrix systems. Although collagen gels seem to be superior when dealing with deep cartilage defects, membrane systems might rather be useful in improving conventional autologous chondrocyte transplantation or in combination with gel systems.

Aged↗