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Lahm A.,University of Greifswald | Lahm A.,Ludwig Maximilians University of Munich | Kasch R.,University of Greifswald | Mrosek E.,Cartilage and Connective Tissue Research Laboratory | And 4 more authors.
Histology and Histopathology | Year: 2012

The study was conducted to examine the expression of collagen type I and II in the different cartilage layers in relation to other ECM molecules during the progression of early osteoarthritic degeneration in human articular cartilage (AC). Quantitative real-time (RT)-PCR and colorimetrical techniques were used for calibration of Photoshop-based image analysis in detecting such lesions. Immunohistochemistry and histology were performed with 40 cartilage tissue samples showing mild (ICRS grade 1b) respectively moderate/advanced (ICRS grade 3a or 3b) (20 each) osteoarthritis compared with 15 healthy biopsies. Furthermore, we quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorimetrically. The digitized images of histology and immunohistochemistry stains were analyzed with Photoshop software. T-test and Spearman correlation analysis were used for statistical analysis. In the earliest stages of AC deterioration the loss of collagen type II was associated with the appearance of collagen type I, shown by increasing amounts of collagen type I mRNA. During subsequent stages, a progressive loss of structural integrity was associated with increasing deposition of collagen type I as part of a natural healing response. A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which then leads to an overall decrease. Analysis of proteoglycan showed losses of the overall content and a loss of the classical zonal formation. Correlation analysis of the proteoglycan Photoshop measurements with the RT-PCR revealed strong correlation for Safranin O and collagen type I, medium for collagen type II, alcian blue and glycoprotein but weak correlation with PCR aggrecan results. Photoshop based image analysis might become a valuable supplement for well known histopathological grading systems of lesioned articular cartilage. The evidence of collagen type I production early in the OA disease process coupled with the ability of chondrocytes to up-regulate collagen type II production suggests that therapeutic agents that suppress collagen type I production and increase collagen type II production may enable chondrocytes to generate a more effective repair response.

Mrosek E.H.,Cartilage and Connective Tissue Research Laboratory | Schagemann J.C.,Cartilage and Connective Tissue Research Laboratory | Chung H.-W.,Cartilage and Connective Tissue Research Laboratory | Fitzsimmons J.S.,Cartilage and Connective Tissue Research Laboratory | And 5 more authors.
Journal of Orthopaedic Research | Year: 2010

Currently, various techniques are in use for the repair of osteochondral defects, none of them being truly satisfactory and they are often two step procedures. Comorbidity due to cancellous bone harvest from the iliac crest further complicates the procedure. Our previous in vitro studies suggest that porous tantalum (TM) or poly-ε-caprolactone scaffolds (PCL) in combination with periosteal grafts could be used for osteochondral defect repair. In this in vivo study, cylindrical osteochondral defects were created on the medial and lateral condyles of 10 rabbits and filled with TM/periosteum or PCL/periosteum biosynthetic composites (n = 8 each). The regenerated osteochondral tissue was then analyzed histologically, and evaluated in an independent and blinded manner by five different observers using a 30-point histological score. The overall histological score for PCL/periosteum was significantly better than for TM/periosteum. However, most of the regenerates were well integrated with the surrounding bone (PCL/periosteum, n = 6.4; TM/periosteum, n = 7) along with partial restoration of the tidemark (PCL/periosteum, n = 4.4; TM/periosteum, n = 5.6). A cover of hyaline-like morphology was found after PCL/periosteum treatment (n = 4.8), yet the cartilage yields were inconsistent. In conclusion, the applied TM and PCL scaffolds promoted excellent subchondral bone regeneration. Neo-cartilage formation from periosteum supported by a scaffold was inconsistent. This is the first study to show in vivo results of both PCL andTMscaffolds for a novel approach to osteochondral defect repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Schagemann J.C.,University of Lubeck | Schagemann J.C.,Cartilage and Connective Tissue Research Laboratory | Paul S.,University of Lubeck | Casper M.E.,Cartilage and Connective Tissue Research Laboratory | And 6 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2013

The objective of this study was to develop a scaffold for mesenchymal stromal cell (MSC) recruitment, proliferation, and chondrogenic differentiation. The concept behind the design is to mimic the cartilage matrix and contain stimulatory agents that make continuous supply of inductive factors redundant. Nanofibrous (N: ∼400 nm) and microfibrous (M: ∼10 μm) poly-ε-caprolactone (PCL) scaffolds were combined with 1% high-molecular-weight sodium hyaluronate (NHA/MHA), 1% hyaluronan (HA) and 200 ng transforming growth factor-beta 1 (TGF-β1; NTGF/MTGF), or 0.1% bovine serum albumin (N/M). Scaffolds were seeded with MSCs from bone marrow and cultured without growth factors in vitro. Cultures with chondrogenic medium supplemented with TGF-β1 served as controls. Proliferation, migration, and release of TGF-β1 were investigated. Cell differentiation was evaluated by polymerase chain reaction (PCR) and real-time PCR. NTGF and MTGF exhibited primarily an initial release of TGF-β1. None of the factors released by the scaffolds recruited MSCs. The expression of aggrecan was dependent on the scaffold ultrastructure with nanofibers promoting increasing and microfibers decreasing expression levels. Composites containing HA demonstrated elevated seeding efficiency and lower type I collagen expression. Expression of type II collagen was dependent on continuous or late supply of TGF-β1, which was not provided by our scaffold design. The initial release of TGF-β1 induced an expression of type I collagen and osteogenic marker genes. In conclusion, nanofibrous PCL scaffolds with or without augmentation are suitable for chondrogenic initiation of MSCs. Initial release of HA is sufficient in terms of directing the implanted MSCs toward a chondrogenic end, whereas a late release of TGF-β1 is preferred to foster type II and avoid type I collagen expression. © 2012 Wiley Periodicals, Inc.

Olivos-Meza A.,Cartilage and Connective Tissue Research Laboratory | Fitzsimmons J.S.,Cartilage and Connective Tissue Research Laboratory | Casper M.E.,Cartilage and Connective Tissue Research Laboratory | Chen Q.,Mayo Medical School | And 4 more authors.
Osteoarthritis and Cartilage | Year: 2010

Objective: To compare the efficacy of in situ transforming growth factor-beta1 (TGF-β1)-pretreated periosteum to untreated periosteum for regeneration of osteochondral tissue in rabbits. Methods: In the pretreatment group, 12 month-old New Zealand white rabbits received subperiosteal injections of 200. ng of TGF-β1 percutaneously in the medial side of the proximal tibia, 7 days prior to surgery. Control rabbits received no treatment prior surgery. Osteochondral transverse defects measuring 5. mm proximal to distal and spanning the entire width of the patellar groove were created and repaired with untreated or TGF-β1-pretreated periosteal grafts. Post-operatively the rabbits resumed normal cage activity for 6 weeks. Results: Complete filling of the defects with regenerated tissue was observed in both the TGF-β1-pretreated and control groups with reformation of the original contours of the patellar groove. The total histological score (modified O'Driscoll) in the TGF-β1-pretreated group, 20 (95% Confidence Interval (CI), 19-21), was significantly higher (P=0.0001) than the control group, 18 (16-19). The most notable improvements were in structural integrity and subchondral bone regeneration. No significant differences in glycosaminoglycan or type II collagen content, or equilibrium modulus were found between the surgical groups. The cambium of the periosteum regenerated at the graft harvest site was significantly thicker (P=0.0065) in the TGF-β1-pretreated rabbits, 121. μm (94-149), compared to controls, 74μm (52-96), after 6 weeks. Conclusions: This study demonstrates that in situ pretreatment of periosteum with TGF-β1 improves osteochondral tissue regeneration at 6-weeks post-op compared to untreated periosteum in 12 month-old rabbits. © 2010 Osteoarthritis Research Society International.

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