Bernhard Schmidt-Rohlfing

Peer teaching: a randomised controlled trial using student‐teachers to teach musculoskeletal ultrasound

Medical Education 2010: 44: 148–155

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.

Expression of ion channels of the TRP family in articular chondrocytes from osteoarthritic patients : changes between native and in vitro propagated chondrocytes

The maintenance of a differentiated chondrocyte phenotype is influenced by several factors of which signal transduction of extracellular stimuli through the cell membrane is of major interest. One important group of membrane-bound proteins which are involved in transmembrane signal transduction are ion channels. Human articular chondrocytes were obtained from osteoarthritic femoral condyles. Cells were released from the surrounding matrix and cultivated under standard conditions. We investigated gene expression of 12 members of the TRP ion channel family of freshly prepared (passage 0; P0) and inxa0vitro propagated human articular chondrocytes (passage 2; P2) using conventional and real-time PCR (RT-PCR). In addition, the protein appearance of four TRP channels was demonstrated by immunofluorescence and western blotting. Chondrocyte differentiation was monitored by quantification of collagen type-II, type-I, and aggrecan gene expression. By conventional PCR, 8 channels could be detected, of which some displayed a heterogeneous PCR pattern. RT-PCR quantification revealed that TRPC1 was expressed on the same level in P0 and P2 chondrocytes while gene expression of TRPC3 and TRPC6 was elevated in passage 2 cells. TRPM5, TRPM7, and TRPV1 displayed an enhanced gene expression in freshly isolated chondrocytes. Immunofluorescence signal intensity of all four investigated TRP proteins was consistent with the corresponding gene expression data. In the present study, a correlation between the appearance of some members of the TRP ion channel family and the state of de-differentiation of osteoarthritic articular chondrocytes was shown. A possible direct involvement in the process of chondrocyte de-differentiation has to be investigated in further studies.

Madelung deformity: clinical features, therapy and results.

In a retrospective study, 31 patients with Madelung deformity were reviewed. They were treated at one institution during a period of 15 years. On first presentation, the mean age was 22.5 years with a range from 10 years to 64 years. Twenty-four patients (77%) were female. The main complaints were pain, limited range of motion, and objectionable appearance. A family history of Madelung deformity was present in four patients (13%). The diagnosis of Leri-Weill syndrome could not be confirmed in any case. There was no correlation between the clinical appearance and the extent of radiologic abnormality. Five patients (16%) were operated on because of permanent pain. On postoperative examination, only one patient revealed no restricted range of mobility and no pain, whereas the other four patients improved in terms of pain but showed only limited improvement of function. The vast majority of patients, however, required no surgical therapy.

A comparative study of 3 different cartilage repair techniques

PurposeThe value of cell-free techniques in the treatment of cartilage defects remains under debate. In this study, cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed by treatment with a cell-free collagen type-I gel or a collagen type-I gel seeded with autologous chondrocytes. As a control, abrasion arthroplasty was included.MethodsIn 18 adult Goettinger minipigs, three full-thickness chondral defects were created in one knee of the hind leg. They were either treated with a cell-free collagen gel, a collagen gel seeded with 2xa0×xa0105/ml chondrocytes, or left untreated. All animals were allowed unlimited weight bearing. At 6, 12, and 52xa0weeks, 6 animals were sacrificed. Immediately after recovery, a non-destructive biomechanical testing was performed. The repair tissue quality was evaluated histologically, and the O’Driscoll score was calculated.ResultsAfter 6xa0weeks, a high number of cells migrated into the initially cell-free collagen gel. After 1xa0year, a hyaline-like repair tissue in both groups has been created. As assessed by O’Driscoll scoring and col-II staining, repair tissue quality of the initially cell-free gel was equal to defects treated by cell-seeded collagen gel implantation after 1xa0year. All untreated control defects displayed a fibrous repair tissue. The mechanical properties represented by the e-modulus were inconsistent in the course of the study. Conclusions The implantation of a cell-free collagen type-I gel can lead to a high-quality repair tissue in the Goettinger minipig that equals a cell-based procedure after 1xa0year postoperatively. This study demonstrates the high chondrogenic potential of the applied collagen gel, which might help to overcome the disadvantages inherent in conventional cartilage tissue engineering methods.

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.

Effects of pulsed and sinusoid electromagnetic fields on human chondrocytes cultivated in a collagen matrix

Although several effects of electromagnetic fields (EMFs) on articular cartilage have been reported in recent studies, the use of EMFs to treat osteoarthritis remains a matter of debate. In an in vitro study, human chondrocytes harvested from osteoarthritic knee joints were released from their surrounding matrix and transferred in defined concentration into a 3D matrix (type-I collagen gel). The cultivation, performed under standard conditions, lasted up to 14 days. During this time, treatment groups were continuously exposed to either sinusoid or pulsed electromagnetic fields (PEMFs). The PEMFs revealed the following characteristics: maximum magnetic flux density of 2xa0mT, frequency of the bursts of 16.7xa0Hz with each burst consisting of 20 pulses. Similarly, the sinusoid EMFs also induced a maximum flux density of 2xa0mT with a frequency of 50xa0Hz. Control groups consisting of equal number of samples were not exposed to EMF. Immunohistological examinations of formalin-fixed, paraffin-embedded samples revealed positive staining for type-II collagen and proteoglycans in the immediate pericellular region with no differences between the two different treatment groups and the control groups. With increasing cultivation time, both type-II collagen and aggrecan gene expression declined, but no significant differences in gene expression were found between the treatment and control groups. In conclusion, using our in vitro setting, we were unable to detect any effects of pulsed and sinusoidal magnetic fields on human adult osteoarthritic chondrocytes.

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.

New potential markers for cartilage degradation of the knee joint

OBJECTIVEnTo determine the correlation of biochemical markers with the degree of cartilage degradation.nnnMETHODSnIn 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.nnnRESULTSnFor 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).nnnCONCLUSIONnThe 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.