Ulrich Schneider

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

SummaryIn 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.

[Cemented hip prosthesis implantation--decreasing the rate of fat embolism with pulsed pressure lavage].

ZusammenfassungIntraoperative Fettembolien stellen eine gefürchtete Komplikation bei der Implantation von zementierten Hüfttotalendoprothesen dar. Im Rahmen einer tierexperimentellen Studie untersuchten wir an einem Schafsmodell, das die standardisierte bilaterale und simultane Zementeinbringung erlaubt, die Effektivität der Jet-Lavage im Vergleich zur manuellen Spritzenspülung von gleichem Volumen im Hinblick auf die Reinigungsfähigkeit des Knochenlagers. Zielkriterium war die Fett- und Knochenmarkseinschwemmung in die Blutbahn. Das operative Vorgehen beinhaltete die bilaterale Plazierung von intravenösen Kathetern in die Venae iliacae externae via eines retroperitonealen Zugangs. Nach der Schenkelhalsosteotomie wurden beide femoralen Markräume für die retrograde Zementeinbringung präpariert. Nach Randomisierung wurde eine Seite manuell mit 250 ml Spülung gereinigt, das andere Femur mit dem identischen Volumen mittels Jet-Lavage. Durch eine speziell für diesen Versuch angefertigte Zementkompressionsmaschine wurde anschließend der retrograd eingebrachte Zement simultan mit gleichem Druck komprimiert. Das abgeleitete Blut aus beiden iliacalen Venenkathetern wurde gesammelt, antikoaguliert und einer quantitativen und qualitativen Fettanalyse zugeführt.Trotz gleicher Spülvolumina zog die manuelle Lavage eine signifikant höhere Einschwemmung von Fett – und Knochenmarksbestandteilen (p < 0.001) als die pulsierende Druckspülung nach sich. Durch diesen Versuch konnte gezeigt werden, dass nicht nur das Volumen, sondern auch die Qualität der Markraumspülung das Risiko für die Entstehung von Fettembolien und kardiorespiratorischen Nebenwirkungen entscheidend beeinflusst. Die erzielten Ergebnisse betonen die herausragende Rolle der Jet-Lavage bei der Implantation von zementierten Hüftendoprothesen.AbstractIntraoperative fat embolism associated with cemented total hip arthroplasty is a well recognized complication. In a new sheep model allowing for standardized bilateral, simultaneous cement pressurization we studied the effectiveness of both pulsatile and syringe lavage of equal volume with regard to their cleansing capabilities as measured by fat and bone marrow intravasation. The operative procedure involved bilateral placement of intravenous catheters into the external iliac veins via retroperitoneal approach. After femoral neck osteotomies both femoral cavities were prepared for retrograde cement application. After randomization one side was lavaged with 250 ml irrigation using a bladder syringe, the contralateral femur with the identical volume but using a pulsatile lavage. A specially designed apparatus was used to allow for bilateral simultaneous cement pressurization. Venous blood from both iliac catheters was then collected, anticoagulated and a quantitative and qualitative fat analysis was performed.Despite equal volume manual lavage produced significantly higher fat and bone marrow intravasation (P < 0.001) than pulsatile lavage thus suggesting that not only the volume but also the quality of bone lavage is an essential factor influencing the risk of fat embolism and adverse cardiorespiratory effects. Our findings further emphasize the important role of pulsatile lavage in preventing fat and bone marrow embolisation during cemented total hip arthroplasty.

Comparison of modern marker proteins in serum and synovial fluid in patients with advanced osteoarthrosis and rheumatoid arthritis

Numerous studies have focused on the significance of modern marker proteins in the synovial fluid of the knee joint and in the serum both, for osteoarthritis (OA) and rheumatoid arthritis (RA). The relationship between the serum concentrations and the concentrations in the synovial fluid is still unclear. Synovial fluid and serum samples were obtained from 13 patients with advanced OA and from 8 patients with severe RA and concentrations of MMP-1, MMP-3, MMP-13, TIMP-1, COMP and MIA/CD-RAP were determined. All values were normalized against the total protein concentrations. Serum concentrations of MMP-13 in the RA-group were statistically higher than the synovial values (P<0.05). MMP-13 was the only marker protein that revealed distinct higher levels in the serum than in the synovial fluid. The study design allows only conclusions about advanced stages of RA and OA. Longitudinal investigations may provide further information about the value of MMP-13 as a potential marker to monitor the course of RA and OA.

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.

Artefacts and ferromagnetism dependent on different metal alloys in magnetic resonance imaging. An experimental study

ZusammenfassungVerursachen verschiedene, in der Orthopädie gebräuchliche, Metallegierungen messbar unterschiedliche Artefakte (Störsignale) in der Kernspintomographie? Gibt es artefaktreduzierende Sequenzen? Wird durch das Hochfrequenzmagnetfeld eine messbare Anziehungskraft auf die einzelnen Legierungen ausgeübt?Untersucht wurden 12 Metallegierungen (Allergieplättchen) von 3 Herstellern. Je 6 Plättchen wurden in einem Kunststoffbehälter in Ultraschallgel eingebettet. Zunächst wurde 2-mal eine sensible Standard-T1-Gradientenechosequenz (TE=4,1 ms; TR=9,4) koronar durchgeführt (Matrix 128/256). Die größte erzeugte Artefaktfläche inklusive der Plättchenfläche wurde auf einer “Philips Easy Vision Workstation” berechnet. Alle Plättchen wurden 10-mal vermessen und die durchschnittliche Artefaktfläche ermittelt. Danach wurden mit dem gleichen Versuchsaufbau 8 unterschiedliche Sequenzen im MRT gefahren und vermessen, exemplarisch wurden die Ergebnisse für Stahl dargestellt. In einer 3. Untersuchung wurde jedes einzelne Plättchen in einem zweidimensionalen Koordinatensystem positioniert und dem Hochfrequenzmagnetfeld ausgesetzt um eine Positionsabweichung der Plättchen zu ermitteln.Unterschiedliche Titanlegierungen verursachten in der T1-Standardsequenz unterschiedliche durchschnittliche Artefaktflächen von 245 mm2 (Ti6Al4V) bis 349 mm2 (Ti5Al2,5Fe). Die Artefaktflächen bei den Chrom-Kobald-Legierungen maßen zwischen 600 und 651 mm2. Die größten, durch Eisenlegierungen verursachten Störflächen lagen zwischen 902 mm2 (316L oder Fe18Cr10NiMo) und 950 mm2 (Fe22Cr10Ni4Mn2MoNb).Die Größe der Artefaktflächen (Ausdehnung) in den unterschiedlichen Sequenzen lag bei Edelstahl (Fe18Cr10NiMo) zwischen 411 mm2 (T1TSE) und 2027 mm2 (EPI/3D/SPIR). Es entstanden unterschiedliche Störbilder. Keines der Metallplättchen veränderte in dem Magnetfeld seine Position.Die Größe der Artefaktflächen in der MRT hängt von der Zusammensetzung der Metallegierung ab und ist von der gewählten Sequenz abhängig. Bei Turbospinechosequenzen treten die geringsten Störungen durch Artefakte auf. Die magnetische Anziehungskraft des Hochfrequenzfelds im MRT auf die Metallegierungen, die untersucht wurden, kann vernachlässigt werden. Für Patienten besteht bei den untersuchten Legierungen/Implantate anscheinend keine Lockerungsgefahr.AbstractThe authors evaluated the significance of different metal alloys used in orthopaedic surgery in producing artefacts during magnetic resonance imaging. Several MRI sequences were tested and magnetic effects evaluated.Twelve discs made of different metal alloys from three manufacturers were examined. These discs were placed in a plastic box with a defined position in ultrasound gel. Then a sensitive, standard T1 weighted gradient echo sequence (TE: 4.1 ms; TR 9.4) was carried out in a coronal plane (Matrix 128/256). A Phillips Easy Vision workstation was used for image analysis. The largest area of artefact formation, including the surface size of the disc, was calculated using a special software program. In order to minimise the measurement error all discs were measured 10 times and the average value was determined. Then eight different sequences were run and measured in the same way. In a second series, all discs were placed separately on metric paper and subjected to the magnetic field of the MRI in order to detect possible motion secondary to the magnetic field applied.The different titanium alloys showed average distortion areas of from 245 mm2 (Ti6Al4V) to 349 mm2 (Ti5Al2.5Fe). Cobalt chrome alloys yielded differences of between 600 mm2 and 651 mm2 and iron alloys of between 902 mm2 (316L or Fe18Cr10NiMo) and 950 mm2 (Fe22Cr10Ni4Mn2MoNb) on average for the standard T1 weighted gradient echo.The artefact areas were dependent on the different sequences performed. For steel, (Fe18Cr10NiMo) areas of from 411 mm2 (T1TSE) to 2027 mm2 (EPI/3D/SPIR) were measured. All sequences studied produced different artefact pictures. None of the materials tested showed changes in position secondary to ferromagnetism.The size of signal distortion by MRI depends on the alloy making up the implanted material and the sequences used. The smallest artefacts occurred with the turbo-spin-echo sequences (TSE).The alloys tested in our study seem to carry no risk for patients of ferromagnetically induced secondary loosening caused by MRI scanning.

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.

BMP-7-loaded PGLA microspheres as a new delivery system for the cultivation of human chondrocytes in a collagen type I gel: the common nude mouse model.

Purpose Bone morphogenic protein 7 (BMP-7) released from polylactide (PLGA) microspheres has proven to be a potent system in cartilage tissue engineering in vitro. However, in vivo data are still lacking. The aim of this study was to investigate this BMP-7 release system utilizing the nude mouse as a small animal model. Methods Human osteoarthritic chondrocytes of 10 patients were enzymatically released and transferred into a collagen type-I gel. A concentration of 2×105 cells/mL was used. BMP-7 encapsulated in PGLA microspheres was added at an initial concentration of 500 ng BMP-7/mL gel. Untreated specimens and specimens with empty microspheres served as control. Samples were cultivated subcutaneously in nude mice for 6 weeks. Results After recovery, chondrocytes of all groups displayed a spheroid morphology without signs of dedifferentiation. The proteoglycan and collagen type II content of the control groups was restricted to the immediate pericellular region, whereas treatment group samples showed enhanced collagen type II production. Collagen type II and aggrecan gene expression was enhanced in treatment group samples with respect to the two control groups (mean ± SD: 0.268 ± 0.450 to 0.152 ± 0.129 and 0.155 ± 0.216 ng/ng β-actin for collagen type II; 0.535 ± 0.731 to 0.367 ± 0.651 and 0.405 ± 0.326 ng/ng β-actin for aggrecan), whereas collagen type I gene expression decreased by a factor of 10. Relative protein quantification of collagen type II, collagen type I and proteoglycan was in accordance. Conclusions Our data suggest that BMP-7 release from PGLA microspheres led to an improved tissue-engineered cartilage analogue in vivo with an increase in hyaline-cartilage-specific components.

Einfluß der Zementiertechnik auf das Zementierergebnis am koxalen Femurende

ZusammenfassungDas Ziel dieser Studie war zu untersuchen, inwieweit unterschiedliche Zementiertechniken Einfluß auf die Zementpenetration in das spongiöse Lager am koxalen Femur haben. An 60 linken humanen Leichenfemora wurden mit standardisierter Präparationstechnik zementierte Femurkomponenten implantiert. Vier verschiedene Zementiergruppen wurden randomisiert. Je nach Gruppe erfolgte die Knochenlagerspülung mit Jetlavage oder Blasenspritze, sowie die Zementapplikation mit oder ohne Druckzementierung („pressurising“); 5 verschiedene Prothesentypen wurden implantiert. Alle Präparate wurden in 2 Ebenen geröntgt und in 2-cm-Abständen horizontale Sägeschnitte angefertigt. Von den Schnitten wurden Mikroradiogramme erstellt und diese hinsichtlich unterschiedlicher Penetrationstiefe mittels Bildanalyse morphometrisch ausgewertet. In einer Zusatzstudie ohne Stielimplantation erfolgte standardisiert an 11 humanen Femurpaaren nach retrograder Zementapplikation die Druckbeaufschlagung des Zements mit einer konstanten Kraft von 3000 N. Dabei unterschieden sich die präparierten Femurpaare lediglich hinsichtlich Spülart (1000 ml Jetlavage vs. 1000 ml Blasenspritze). Die Auswertung wurde analog zum Hauptversuch durchgeführt. Den größten Einfluß auf die Penetrationstiefe des Knochenzements in die Spongiosa hatten im Hauptversuch die Verwendung der Jetlavage (p = 0,027) und die Druckzementierung (p = 0,003). Bei dichter Spongiosa war der Unterschied zwischen Jetlavage und Blasenspritzenspülung stärker ausgeprägt. Im Zusatzversuch zeigte die Jetlavage ebenfalls eine signifikante Verbesserung der Penetrationstiefe (p < 0,001). Unterschiede in Abhängigkeit vom Prothesentyp konnten nicht nachgewiesen werden. Die Verwendung der Jetlavage hat wesentlichen Einfluß auf die Penetrationstiefe von Knochenzement in das Knochenlager und sollte in der zementierten Hüftendoprothetik als unverzichtbar angesehen werden. Die Druckzementierung führt ebenfalls zu verbesserter Zementverzahnung, sollte jedoch im Hinblick auf mögliche Knochenmarksausschwemmungen und resultierende thromboembolische Komplikationen nur in Kombination mit der Jetlavage angewandt werden.SummaryThe purpose of this study was to determine the influence of jet-lavage and cement pressurising techniques upon cement penetration into proximal femoral cancellous bone. In a cadaver study 60 left human cadaver femora were used for implantation of cemented stem components. Four different groups of cementing techniques were generated, the allocation to the groups was randomised. Bone lavage was carried out either using jet-lavage or manual syringe lavage, cement application differed with regard to the amount of pressurisation used. Five different stem designs were used. Radiographs were taken and horizontal sections were obtained at predefined levels (2 cm) using a diamond saw. Microradiographs were taken and analysed using image analysis to assess cement penetration into cancellous bone. In an additional study the influence of jet-lavage (1000 ml) versus syringe lavage (1000 ml) was studied in 11 paired human cadaver femora. The specimens were imbedded in specially designed pots. Bone cement was applied in a retrograde manner and subjected to a standard pressure protocol with a constant force of 3000 N. The analysis protocol was identical to the main experiment. Both jet-lavage and pressurisation of bone cement significantly improved the penetration of cement into cancellous bone (p = 0,027 and p = 0,003, respectively). In the presence of strong, dense cancellous bone the findings were more pronounced. In the additional comparative study cement intrusion was significantly better (p < 0,001) in the jet-lavage group. We did not observe an influence of the stem type upon outcome (penetration). The use of jet-lavage yields significantly improved cement penetration and should be regarded mandatory in cemented total hip arthroplasty. High pressurising techniques are effective means to improve the interdigitation between cancellous bone and cement, but should only be administered in combination with jet-lavage to reduce the risk of thrombo-embolic complications.

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.

Condensed cellular seeded collagen gel as an improved biomaterial for tissue engineering of articular cartilage.

Three-dimensional autologous chondrocyte implantation based on collagen gel as matrix scaffold has become a clinically applied treatment for focal defects of articular cartilage. However, the low biomechanical properties of collagen gel makes intraoperative handling difficult and creates the risk of early damages to the vulnerable implant. The aim of the study was to create a stabilized form of collagen gel and to evaluate its biomechanical and biochemical properties.Collagen type-I gel was seeded with human articular chondrocytes. 20 samples were subject to condensation which was achieved mechanically by compression and filtration. Control samples were left uncondensed. From both types of gels 10 samples were used for initial biomechanical evaluation by means of unconfined compression and 10 samples were cultivated under standard conditions in vitro. Following cultivation the samples were evaluated by conventional histology and immunohistochemistry. The proliferation rate was calculated and matrix gene expression was quantified by real-time PCR.The biomechanical tests revealed a higher force carrying capacity of the condensed specimens. Strain rate dependency and relaxation was seen in both types of collagen gel representing viscoelastic material properties. Cells embedded within the condensed collagen gel were able to produce extracellular matrix proteins and showed proliferation.Condensed collagen gel represents a mechanically improved type of biomaterial which is suitable for three-dimensional autologous chondrocyte implantation.