Karsten Gavenis

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.

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.

Simultaneous anabolic and catabolic responses of human chondrocytes seeded in collagen hydrogels to long-term continuous dynamic compression.

Cartilage repair strategies increasingly focus on the in vitro development of cartilaginous tissues that mimic the biological and mechanical properties of native articular cartilage. However, current approaches still face problems in the reproducible and standardized generation of cartilaginous tissues that are both biomechanically adequate for joint integration and biochemically rich in extracellular matrix constituents. In this regard, the present study investigated whether long-term continuous compressive loading would enhance the mechanical and biological properties of such tissues. Human chondrocytes were harvested from 8 knee joints (n=8) of patients having undergone total knee replacement and seeded into a collagen type I hydrogel at low density of 2×10(5)cells/ml gel. Cell-seeded hydrogels were cut to disks and subjected to mechanical stimulation for 28 days with 10% continuous cyclic compressive loading at a frequency of 0.3 Hz. Histological and histomorphometric evaluation revealed long-term mechanical stimulation to significantly increase collagen type II and proteoglycan staining homogenously throughout the samples as compared to unstimulated controls. Gene expression analyses revealed a significant increase in collagen type II, collagen type I and MMP-13 gene expression under stimulation conditions, while aggrecan gene expression was decreased and no significant changes were observed in the collagen type II/collagen type I mRNA ratio. Mechanical propertywise, the average value of elastic stiffness increased in the stimulated samples. In conclusion, long-term mechanical preconditioning of human chondrocytes seeded in collagen type I hydrogels considerably improves biological and biomechanical properties of the constructs, corroborating the clinical potential of mechanical stimulation in matrix-associated autologous chondrocyte transplantation (MACT) procedures.

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.

Bioreactor cultivation and remodelling simulation for cartilage replacement material

For the development of articular cartilage replacement material, it is essential to study the dependence between mechanical stimulation and cell activity in cellular specimens. Bioreactor cultivation is widely used for this purpose, however, it is hardly possible to obtain a quantitative relationship between collagen type II production and applied loading history. For this reason, a bioreactor system is developed, measuring applied forces and number of loading cycles by means of a load cell and a forked light barrier, respectively. Parallel to the experimental study, a numerical model by means of the finite element method is proposed to simulate the evolution of material properties during cyclic stimulation. In this way, a numerical model can be developed for arbitrary deformation cases.

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.

Continuous cyclic compressive loading modulates biological and mechanical properties of collagen hydrogels seeded with human chondrocytes.

PURPOSE This study investigated the potential of cyclic compressive loading in the generation of in vitro engineered cartilaginous tissue with the aim of contributing to a better understanding of mechanical preconditioning and its possible role in further optimizing existing matrix-associated cartilage replacement procedures. METHODS Human chondrocytes were harvested from 12 osteoarthritic knee joints and seeded into a type I collagen (col-I) hydrogel at low density (2 × 10(5) cells/ml gel). The cell-seeded hydrogel was condensed and cultivated under continuous cyclic compressive loading (frequency: 0.3 Hz; strain: 10%) for 14 days under standardized conditions. After retrieval, specimens were subject to staining, histomorphometric evaluation, gene expression analysis and biomechanical testing. RESULTS Cellular morphology was altered by both stimulation and control conditions as was staining for collagen II (col-II). Gene expression measurements revealed a significant increase for col-II under either cultivation condition. No significant differences in col-I, aggrecan and MMP-13 gene expression profiles were found. The col-II/col-I mRNA ratio significantly increased under stimulation, whereas the biomechanical properties deteriorated under either cultivation method. CONCLUSIONS Although the effects observed are small, mechanical preconditioning has demonstrated its potential to modulate biological properties of collagen hydrogels seeded with human chondrocytes.

Effects of low concentration BMP-7 on human osteoarthritic chondrocytes: comparison of different applications.

While BMP-7 (OP-1) is one of the most potent growth factors in cartilage tissue engineering, the effects of exogenous low concentration BMP-7 on osteoarthritic chondrocytes are still unknown. Human osteoarthritic chondrocytes obtained from the femoral condyles of 10 patients were grown either in monolayer or in 3D collagen type-I gel culture in vitro. The growth factor was either given as a single dose of 50 ng/mL, a repeated dose, or continuously released from PGLA microspheres. Matrix formation was monitored by immunohistochemical staining and real-time PCR. In contrast to monolayer culture, the differentiated phenotype was prevailed in 3D culture. Collagen type-II protein production in the 3D group with a continuous BMP-7 release was enhanced in comparison to all other groups. Gene expression of collagen type-II and aggrecan was elevated in all treatment groups, with the highest extent in the BMP-7 microsphere group. In summary, treatment of articular chondrocytes with a low dose of BMP-7 leads to an elevated production of extracellular matrix components. This effect is further increased when BMP-7 is given repeatedly or continuously, which proved to be the most effective form of application.

Ablation of articular cartilage with an erbium:YAG laser: An ex vivo study using porcine models under real conditions—ablation measurement and histological examination

The use of an erbium:YAG laser in arthroscopic surgery has the advantage of a precise treatment of soft tissue. Due to the high absorption in water, the laser energy is perfectly matched to smoothing the hydrous, fibrillated articular cartilage surface. In minimal invasive surgery, the workspace is filled with aqueous liquids for enlargement. This appears contrary to the absorption characteristics of erbium:YAG laser radiation in water. The purpose of this study was to evaluate the ablated volume per pulse of cartilage lesions and the potential side effects including thermal damage and tissue necrosis.

Cell-free repair of small cartilage defects in the Goettinger minipig: the effects of BMP-7 continuously released by poly(lactic-co-glycolid acid) microspheres.

Objective Cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed after treatment with cell-free collagen type-I gel with or without additional BMP-7 loaded poly(lactic-co-glycolid acid) microspheres. Methods Two full-thickness chondral defects were created in the trochlear groove of one hind leg knee in six Goettinger minipigs. Six defects were treated with a cell-free collagen type-I gel plug of 10 mm, the corresponding six defects were treated with a cell-free collagen type-I plug with poly(lactic-co-glycolid acid) microspheres loaded with recombinant BMP-7 (100 ng/ml gel). After 1 year, the animals were sacrificed. Immediately after recovery, non-destructive biomechanical testing was performed. The repair tissue quality was evaluated by immunohistochemistry and the O’Driscoll score was calculated. Results After 1 year, a robust cellular migration into the cell-free collagen gel plugs occurred and a hyaline-like repair tissue was found. Collagen type-II production and cellular organisation were higher in the BMP-7 microsphere group. The determination of the E-modulus, creep and relaxation revealed that mechanical properties of the BMP-7 microsphere group in summary were closer to control hyaline cartilage. Conclusions While all specimens revealed a homogeneous cellular distribution, ECM production, cellular organisation and mechanical properties were enhanced by continuous BMP-7 release.