Instruments for reproducible setting of defects in cartilage and harvesting of osteochondral plugs for standardisation of preclinical tests for articular cartilage regeneration

Background: Standardisation is required in research, so are approval procedures for advanced therapy medical products and other procedures for articular cartilage therapies. The process of creating samples needs to be reproducible. The aim of this study was to design, create and validate instruments (1) to create reproducible and accurate defects and (2) to isolate samples in the shape of osteochondral cylinders in a quick, reliable and sterile manner. Methods: Adjustable instruments were created: a crown mill with a resolution of 0.05 mm and a front mill to create defects in articular cartilage and subchondral bone. The instruments were tested on knee joints of pigs from the slaughterhouse; 48 defects were created and evaluated. A punching machine was designed to harvest osteochondral plugs. These were validated in an in vivo animal study. Results: The instruments respect the desired depth of 0.5 and 1.5 mm when creating the defects, depending on whether the person using the instrument is highly experienced (0.451 mm; confidence interval (CI): 0.390 mm; 0.512 mm and 1.403 mm; CI: 1.305 mm; 1.502 mm) or less so (0.369 mm; CI: 0.297 mm; 0.440 mm and 1.241 mm; CI: 1.141 mm; 1.341 mm). Eighty samples were taken from knee joints of Göttingen Minipigs with this punching technique. The time needed for the harvesting of the samples was 7.52 min (±2.18 min), the parallelism of the sides of the cylinders deviated by −0.63° (CI: −1.33°; 0.08°) and the surface of the cartilage deviated from the perpendicularity by 4.86° (CI: 4.154°; 5.573°). In all assessed cases, a sterile procedure was observed. Conclusions: Instruments and procedures for standardised creation and validation of defects in articular cartilage and subchondral bone were designed. Harvesting of samples in the shape of osteochondral cylinders can now be performed in a quick, reliable and sterile manner. The presented instruments and procedures can serve as helpful steps towards standardised operating procedures in the field of regenerative therapies of articular cartilage in research and for regulatory requirements

Strategic School Improvement Fund: updated 12 September 2017

<p><b>Comparison of MRI (A and B) with histological findings (C).</b> The lateral tibia plateau region of the sham knee joint of animal #8958 showed no salience neither in the sagittal MRI-scan (A) through the lateral compartment of the knee nor in the coronal MRI-scan (B) and was therefore rated as unchanged. The scale bars for A and B were determined with the program RadiAnt DICOM viewer (see caption <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165897#pone.0165897.g001" target="_blank">Fig 1</a>). In contrast to the MRI results, the histological safranin-o staining (C) showed a moderate degeneration (Little-score: 10.5 points) with a fissure (1) in the cartilage and detachment of the topmost layer of the cartilage tissue. The histological finding and the MRI result did not concur.</p

Resilience to height loss of articular cartilage of osteoarthritic stifle joints of old pigs, compared with healthy cartilage from young pigs in a tribological pin—on—plate exposure, revealing similar friction forces

Introduction We saw a lack of data on the biomechanical behavior of degenerated articular cartilage (OA) compared with that of healthy cartilage, even though the susceptibility to wear and tear of articular cartilage plays a key role in the progression of osteoarthritis (OA). Therefore, we performed a comparison between naturally occurring OA and healthy cartilage from pigs, before and after tribological stress. Aim The aim of the study was to compare OA-cartilage with healthy cartilage and to analyze the resilience to tribological shear stress, which will be measured as height loss (HL), and to friction forces of the cartilage layers. The findings will be substantiated in macro- and microscopical evaluations before and after tribological exposure. Methods We assessed stifle joints of fifteen old and sixteen young pigs from the local abattoir radiologically, macroscopically and histologically to determine possible OA alterations. We put pins from the femoral part of the joints and plates from the corresponding tibial plateaus in a pin-on-plate tribometer under stress for about two hours with about 1108 reciprocating cycles under a pressure of approximately 1 MPa. As a surrogate criterion of wear and tear, the HL was recorded in the tribometer. The heights of the cartilage layers measured before and after the tribological exposure were compared histologically. The condition of the cartilage before and after the tribological exposure was analyzed both macroscopically with an adapted ICRS score and microscopically according to Little et al. (2010). We assessed the friction forces acting between the surfaces of the cartilage pair–specimens. Results Articular cartilage taken from old pigs showed significant degenerative changes compared to that taken from the young animals. The macroscopic and microscopic scores showed strong alterations of the cartilage after the tribological exposure. There was a noticeable HL of the cartilage specimens after the first 100 to 300 cycles. The HL after tribological exposure was lower in the group of the old animals with 0.52 mm ± 0.23 mm than in the group of the young animals with 0.86 mm ± 0.26 mm (p &lt; 0.0001). The data for the HL was validated by the histological height measurements with 0.50 mm ± 0.82 mm for the old and 0.79 mm ±0.53 mm for the young animals (p = 0.133). The friction forces measured at the cartilage of the old animals were 2.25 N ± 1.15 N and 1.89 N ± 1.45 N of the young animals (p = 0.3225). Conclusion Unlike articular cartilage from young pigs, articular cartilage from old pigs showed OA alterations. Tribological shear stress exposure revealed that OA cartilage showed less HL than healthy articular cartilage. Tribological stress exposure in a pin–on–plate tribometer seemed to be an appropriate way to analyze the mechanical stability of articular cartilage, and the applied protocol could reveal weaknesses of the assessed cartilage tissue. Friction and HL seemed to be independent parameters when degenerated and healthy articular cartilage were assessed under tribological exposure in a pin–on- plate tribometer. </jats:sec

Coefficient of Friction and Height Loss: Two Criteria Used to Determine the Mechanical Property and Stability of Regenerated Versus Natural Articular Cartilage

Background: The coefficient of friction (CoF) serves as an indicator for the mechanical properties of natural and regenerated articular cartilage (AC). After tribological exposure, a height loss (HL) of the cartilage pair specimens can be measured. Our aim was to determine the CoF and HL of regenerated AC tissue and compare them with those of natural AC from non-operated joints and AC from joints where the regenerated tissues had been created after different treatments. Methods: In partial-thickness defects of the trochleae of the stifle joints of 60 Göttingen Minipigs, regenerated AC was created. In total, 40 animals received a Col I matrix, 20 laden with autologous chondrocytes, and 20 without. The defects of 20 animals were left empty. The healing periods were 24 and 48 weeks. A total of 10 not-operated animals, delivered the “external” control specimens. Osteochondral pins were harvested from defect and non-defect areas, the latter serving as “internal” controls. Using a pin-on-plate tribometer, we measured the CoF and the HL. Results: The CoF of the regenerated AC ranged from 0.039 to 0.069, and the HL, from 0.22 mm to 0.33 mm. The differences between the regenerated AC of the six groups and the “external” controls were significant. The comparison with the “internal” controls revealed four significant differences for the CoF and one for the HL in the operated groups. No differences were seen within the operated groups. Conclusions: The mechanical quality of the regenerated AC tissue showed inferior behavior with regard to the CoF and HL in comparison with natural AC. The comparison of regenerated AC tissue with AC from untreated joints was more promising than with AC from the treated joints

Analysis of Gene Expression and Ultrastructure of Stifle Menisci from Juvenile and Adult Pigs

The origin of the age-associated degenerative processes in meniscal tissue is poorly understood and may be related to an imbalance of anabolic and catabolic metabolism. The aim of the current study was to compare medial menisci isolated from juvenile pigs and degenerated medial menisci from adult pigs in terms of gene expression profile and ultrastructure. Medial menisci were isolated from the knee joints of juvenile and adult pigs (n = 8 for each group). Degeneration was determined histologically according to a scoring system. In addition, the gene expression profiles of 14 genes encoding extracellular matrix proteins, catabolic matrix metalloproteinases and mediators of inflammation were analyzed. Changes in the ultrastructure of the collagen network of the meniscal tissue were analyzed by using transmission electron microscopy. The histologic analysis of menisci showed significantly higher grade of degeneration in tissue isolated from adult porcine knee joints compared with menisci isolated from juvenile knee joints. In particular, destruction of the collagen network was greater in adult menisci than in juvenile menisci. Degenerated menisci showed significantly decreased gene expression of COL1A1 and increased expression of MMP2, MMP13, and IL8. The menisci from adult porcine knee joints can serve as a model for meniscal degeneration. Degenerative changes were manifested as differences in histopathology, gene expression and ultrastructure of collagen network

Qualitative immunohistochemical analysis of type I collagen in the PI.

<p>No staining without antibody (A), weak staining in the control group and more intense staining after ACLR (C).</p

HE-staining-based four-stage scoring system for meniscal degeneration [32].

<p>HE-staining-based four-stage scoring system for meniscal degeneration [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159331#pone.0159331.ref032" target="_blank">32</a>].</p

Primer sequence, annealing temperatures (AT) and PCR product size for the different genes.

<p>Primer sequence, annealing temperatures (AT) and PCR product size for the different genes.</p