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Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVII | Pages 80 - 80
1 Sep 2012
Utzschneider S Lorber V Dedic M Paulus A Sievers B Jansson V
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Periprosthetic osteolysis depends on the biological activity of wear particles, but there is little known about the distribution of polyethylene wear particles (PE) in the surrounding joint tissue. The purpose of this study was to examine the localisation of wear particles of six different PEs, including four crosslinked polyethylenes (XPE), as well as their biological activity in the murine knee.

Material and Methods

Wear particles of 4 XPE- and 2 UHMWPE-inserts were isolated (knee joint simulator). For all groups the particles were similar in size and shape (mean diameter 0.3–05μm; 20nm-nucleopore-filter; ISO; n = 100.000).56 female Balb/c mice were randomly assigned to six treatment groups and one control group: control (PBS), XPE1 (3×30 kGy Gamma, annealed/sequential irradiated), XPE2 (95 kGy E-beam, remelted), XPE3 (65 kGy E-beam, remelted), XPE 4 (50 kGy Gamma, remelted), UHMWPE 1, UHMWPE 2. 50 μl of each particle suspension [(0.1% vol/vol (particle volume/PBS volume) after removal of endotoxin] were injected into the left knee joint. After 1 week the mice were killed and a histological and immunhistochemical analysis of the knee joints was done (IL-1, TNF-, ICAM-1). For the immunhistochemistry the articular cartilage, the bone marrow and the synovial membrane were evaluated semiquantitatively (Kruskal-Wallis test; all pairwise multiple comparison procedure; Bonferoni correction; significance level: p<0.05).

Results

All groups showed a thickened synovial layer with an increased cellular infiltration. The particles of XPE 1 and 2 were localised in the bone marrow as well as in the joint space. In contrast, the particles of XPE 3 and 4 were distributed in the synovial layer and in the bone marrow as well, but not in the joint space. The UHMWPE1 particles were mainly located in the bone marrow and joint space while the UHMWPE2 particles were mainly found in the bone marrow and the synovial layer. For all PE groups there was a higher cytokine expression compaired to control (p<0.0024) without any differences between the groups (bone marrow/synovial layer). The chondrocytes in the groups with XPE 1- and XPE 2-particles expressed more TNF- than in the control group and the other treatment groups (p = 0.000).


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_II | Pages 222 - 222
1 May 2011
Utzschneider S Dedic M Paulus A Schroeder C Sievers B Gottschalk O Sadoghi P Jansson V
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Crosslinked polyethylene (XPE) was developed to reduce wear in hip and knee arthroplasty. Periprosthetic osteolysis depends on many factors including biological activity of wear particles. This study examines the relative inflammatory effect of different crosslinked polyethylenes compared to ultra-high-molecular-weight-polyethylene (UHMWPE) particles in vivo.

Materials and Methods: Wear particles of 3 XPE- (1 sequential irradiated/annealed; 2 remelted inserts) and 1 UHMWPE-insert were isolated from a knee joint simulator (20nm-nucleopore-filter;acid digestion method;ISO). Particles were analysed by scanning electron microscopy (n=66000). For all groups the particles were smooth, granular, irregular and less fibrillar. More than 85% of the particles were submicron. After removal of endotoxin the particles were suspended in a phosphate buffered saline solution (0.1% vol/vol (particle volume/PBS volume)). Endotoxin levels were controlled using standardised endotoxin detection tests (Lonza) in all samples.

40 female Balb/c mice were randomly assigned to one of five treatment groups (according to the national guidelines of animal protection laws): control (n=8); XPE1 (95 kGy E-beam, remelted; n=8); XPE2 (65 kGy E-beam, remelted; n=8), XPE3 (3x30 kGy Gamma, annealed and sequential irradiated; n=8) and UHMWPE particles (n=8). 50 μl of the particle suspension were injected into the murine left knee under sterile conditions. The leukocyte–endothelial cell interactions and the synovial microcirculation were performed by intra-vital fluorescence microscopy one week after particle injection to assess the inflammatory reaction to the particles (by measuring the rolling fraction of leukocytes, the adherent cells and the functional capillary density (FCD)). Data analysis was performed using a computer-assisted microcirculation analysis system (Cap-Image).

For the statistical analysis the Kruskal-Wallis test was used to determine differences within the groups, followed by an all pairwise multiple comparison procedure with a Bonferoni correction. The level of significance was set at p< 0.05.

Results: The fraction of the rolling leukocytes, adherent cells and FCD increased significantly (p< 0.05) in all bio-materials compared to control group. However, there was no significant difference between the UHMWPE and the XPE particle groups (p> 0.05).

Conclusion: Our data suggest that crosslinked polyethylene wear particles do not lead to a higher inflammatory reaction in vivo compared to UHMWPE particles.


Orthopaedic Proceedings
Vol. 92-B, Issue SUPP_IV | Pages 505 - 505
1 Oct 2010
Mayer S Büttner A Jansson V Mayer W Müller P Schieker M Schiergens T Sievers B
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Background: In regenerative medicine the autologous cartilage implantation (ACI) has been used for the repair of cartilage defects. As modification of ACI, the matrix assisted ACI is used nowadays with varying results. There is a general discussion about whether supporting scaffolds should be used or whether a scaffold-free cartilage repair is the method of choice. The major problem of scaffold-free regenerates is how to keep the cells in place after transplantation. Aim of this study was to examine a new scaffold-free diffusion-culture model, which uses a mega-congregate of chondrocytes cultured at an air-medium interface. This scaffold-free high-density diffusion culture could be used to repair cartilage defects.

Material and methods: Human chondrocytes from passage 1–7 were expanded in monolayer and transferred to pellet-culture or diffusion-culture. After one week cultures were stained with toluidine blue and safranin-O and evaluated by immunohistochemical staining for type II collagen. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for the mRNAs of cartilage markers.

Results: Positive alcian blue staining was detectable in diffusion-culture for human chondrocytes up to passage 7. Within passages the amount of proteoglycan production in relationship to the number of cells increased. There was a positive signal for Collagen type II in diffusion-cultures up to passage 7.

In qRT-PCR a redifferentiation of human chondrocytes was shown by the transfer into diffusion-culture. Within passage 1 to 3 human chondrocytes which were cultured in monolayer lost the ability to express Collagen Type II but could regain it if they were transferred to diffusion-culture. At diffusion-culture chondrocytes showed the highest expression of Collagen type II at passage 1 when compared to monolayer or to pellet-culture.

Conclusion: It could be shown that the cultivation in a scaffold-free diffusion-culture can lead to redifferentiation of human chondrocytes Chondrocytes in diffusion-cultures tend to form their own matrix and produce Collagen type II at higher amounts than in monolayer or in normal pellet-cultures. Therefore diffusion-culture congregates might be an appropriate tool to be used for a new scaffold-free cartilage regeneration approach.