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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 0.0393 to 0.0688, and the HL, from 0.22 mm to 0.3 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.

Articular cartilage (AC) is an avascular tissue composed of scattered chondrocytes embedded in a dense extracellular matrix, in which nourishment takes place via the synovial fluid at the surface. AC has a limited intrinsic healing capacity, and thus mainly surgical techniques have been used to relieve pain and improve function. Approaches to promote regeneration remain challenging. The microfracture (MF) approach targets the bone marrow (BM) as a source of factors and progenitor cells to heal chondral defects in situ by opening small holes in the subchondral bone. However, the original function of AC is not obtained yet. We hypothesize that mechanical stimulation can mobilize mesenchymal stromal cells (MSCs) from BM reservoirs upon MF of the subchondral bone. Thus, the aim of this study was to compare the counts of mobilized human BM-MSCs (hBM-MSCs) in alginate-laminin (alginate-Ln) or collagen-I (col-I) scaffolds upon intermittent mechanical loading. The mechanical set up within an established bioreactor consisted of 10% strain, 0.3 Hz, breaks of 10 s every 180 cycles for 24 h. Contrary to previous findings using porcine MSCs, no significant cell count was found for hBM-MSCs into alginate-Ln scaffolds upon mechanical stimulation (8 ± 5 viable cells/mm 3 for loaded and 4 ± 2 viable cells/mm 3 for unloaded alginate-Ln scaffolds). However, intermittent mechanical stimulation induced the mobilization of hBM-MSCs into col-I scaffolds 10-fold compared to the unloaded col-I controls (245 ± 42 viable cells/mm 3 vs. 22 ± 6 viable cells/mm 3 , respectively; p -value < 0.0001). Cells that mobilized into the scaffolds by mechanical loading did not show morphological changes. This study confirmed that hBM-MSCs can be mobilized in vitro from a reservoir toward col-I but not alginate-Ln scaffolds upon intermittent mechanical loading, against gravity.

Introduction: Articular cartilage (AC) is a viscoelastic tissue with a limited regenerative capability because of the lack of vasculature. Mechanical stimulation contributes to the homeostasis of functional AC since it promotes the delivery of nutrients, cytokines and growth factors between the distant chondrocytes. We hypothesized that biomechanical stimulation might enhance mobilization of endogenous mesenchymal stem/stromal cells (MSCs) from neighboring niches as the bone marrow., Aim: This study aimed to introduce a bioreactor for inducing mobilization of MSCs from one compartment to another above by mechanical stimulation in vitro., Methods: A novel mechanical system for evaluating mobilization of cells in a 3D context in vitro is presented. The system consists of a compression bioreactor able to induce loading on hydrogel-based scaffolds, custom-made software for settings management and data recording, and image based biological evaluation. Intermittent load was applied under a periodic regime with frequency of 0.3 Hz and unload phases of 10 seconds each 180 cycles over 24 hours. The mechanical stimulation acted on an alginate scaffold and a cell reservoir containing MSCs below it. The dynamic compression exerted amplitude of 200 μm as 10% strain regarding the original height of the scaffold., Results: The bioreactor was able to stimulate the scaffolds and the cells for 24.4 (±1.7) hours, exerting compression with vertical displacements of 185.8 (±17.8) μm and a force-amplitude of 1.87 (±1.37; min 0.31, max 4.42) N. Our results suggest that continuous mechanical stimulation hampered the viability of the cells located at the cell reservoir when comparing to intermittent mechanical stimulation (34.4 ± 2.0% vs. 66.8 ± 5.9%, respectively). Functionalizing alginate scaffolds with laminin-521 (LN521) seemed to enhance the mobilization of cells from 48 (±21) to 194 (±39) cells/mm3 after applying intermittent mechanical loading., Conclusion: The bioreactor presented here was able to provide mechanical stimulation that seemed to induce the mobilization of MSCs into LN521-alginate scaffolds under an intermittent loading regime., Competing Interests: An application for a Model Utility Protection for the bioreactor (Nr. 2019 103 387.8) has been done on June 17th, 2019. This work has been partially presented in a poster for the German Congress of Orthopaedics (DKOU 2019) in October 2019. The authors have transparently informed to PLOS ONE editorial. In our opinion, these submissions do not alter our adherence to PLOS ONE policies on sharing data and materials.

The characterization of regenerated articular cartilage (AC) can be based on various methods, as there is an unambiguous accepted criterion neither for the natural cartilage tissue nor for regenerates. Biomechanical aspects should be considered as well, leading to the need for more equivalent samples. The aim of the study was to describe a large animal model where 8 specimens of regenerated AC can be created in one animal plus the impact of two surgeries on the welfare of the animals. The usefulness of the inclusion of a group of untreated animals (NAT) was to analyzed. Based on the histological results the conditions of the regenerates were to be described and the impact on knee joints were to be explored in terms of degenerative changes of the cartilage. The usefulness of the statistical term "effect size" (ES) will be explained with histological results. We analyzed an animal model where 8 AC regenerates were obtained from one Göttingen Minipig, on both sides of the trochleae. 60 animals were divided into 6 groups of 10 each, where the partial thickness defects in the trochlea were filled with matrices made of Collagen I with or without autologous chondrocytes or left empty over the healing periods of 24 and 48 weeks. One additional control group consisting of 10 untreated animals was used to provide untouched "external" cartilage. We harvested 560 samples of regenerated tissue and "external" controls, besides that, twice the number of further samples from other parts of the joints referred to as "internal" controls were also harvested. The animals recovered faster after the 1st operation when the defects were set compared to the 2nd operation when the defects were treated. 9% of all animals were lost. Other complications were for example superficial infections, seroma, diarrhea, febrile state and an injury of a claw. The histological results of the treatments proved the robustness of the study design where we included an "external" control group (NAT) in which the animals were not operated. Comparable significant differences between treated groups and the NAT group were detected both after ½ year and after 1 year. Spontaneous regenerated AC as control revealed differences after an observation time of nearly 1 year. The impact of the treatment on cartilage adjacent to the defect as well as the remaining knee joint was low. The ES was helpful for planning the study as it is shown that the power of a statistical comparison seems to be more influenced by the ES than by the sample size. The ranking of the ES was done exemplarily, listing the results according to their magnitude, thus making the results comparable. We were able to follow the 3 R requirements also in terms of a numerical reduction of animals due to the introduction of a group of untreated animals. This makes the model cost effective. The presented study may contribute as an improvement of the standardization of large animal models for research and regulatory requirements for regenerative therapies of AC., Competing Interests: TL was involved in the presented study as statistician on behalf of the Medical Faculty Mannheim, University Heidelberg and of the research consortium QuReGe that was funded by the Federal Ministry of Education and Research, Germany (“Funktionelle Qualitätssicherung von regenerativen Gewebeersatzmaterialien für Knorpel und Meniskus (QuReGe)” to support the animal trial in terms of sample size calculation and to develop a tool for the grading of the outcomes of different assessment procedures based on effect sizes. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: The Göttingen Minipig (GM) is used as large animal model in articular cartilage research. The aim of the study was to introduce osteoarthritis (OA) in the GM by resecting the anterior cruciate ligament (ACLR) according to Pond and Nuki, verified by histological and magnetic resonance imaging (MRI) scoring as well as analysis of gene and protein expression., Materials and Methods: The eight included skeletally mature female GM were assessed after ACLR in the left and a sham operation in the right knee, which served as control. 26 weeks after surgery the knee joints were scanned using a 3-Tesla high-field MR tomography unit with a 3 T CP Large Flex Coil. Standard proton-density weighted fat saturated sequences in coronal and sagittal direction with a slice thickness of 3 mm were used. The MRI scans were assessed by two radiologists according to a modified WORMS-score, the X-rays of the knee joints by two evaluators. Osteochondral plugs with a diameter of 4mm were taken for histological examination from either the main loading zone or the macroscopic most degenerated parts of the tibia plateau or condyle respectively. The histological sections were blinded and scored by three experts according to Little et al. Gene expression analysis was performed from surrounding cartilage. Expression of adamts4, adamts5, acan, col1A1, col2, il-1ß, mmp1, mmp3, mmp13, vegf was determined by qRT-PCR. Immunohistochemical staining (IH) of Col I and II was performed. IH was scored using a 4 point grading (0-no staining; 3-intense staining)., Results and Discussion: Similar signs of OA were evident both in ACLR and sham operated knee joints with the histological scoring result of the ACLR joints with 6.48 ± 5.67 points and the sham joints with 6.86 ± 5.84 points (p = 0.7953) The MRI scoring yielded 0.34 ± 0.89 points for the ACLR and 0.03 ± 0.17 for the sham knee joints. There was no correlation between the histological and MRI scores (r = 0.10021). The gene expression profiles as well as the immunohistochemical findings showed no significant differences between ACLR and sham knee joints. In conclusion, both knee joints showed histological signs of OA after 26 weeks irrespective of whether the ACL was resected or not. As MRI results did not match the histological findings, MRI was obviously unsuitable to diagnose the OA in GM. The analysis of the expression patterns of the 10 genes could not shed light on the question, whether sham operation also induced cartilage erosion or if the degeneration was spontaneous. The modified Pond-Nuki model may be used with reservation in the adult minipig to induce an isolated osteoarthritis., Competing Interests: The authors have declared that no competing interests exist.

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.

In striated muscle tissues, the giant protein titin acts as a biomechanically active filament system, coupling stress/strain to gene expression. The objective of the study is to show the existence of titin fragments in human articular cartilage, as in diarthodial joints, chondrocytes are also known to sense and respond to stretching. We have surveyed human cultured cartilage collected from adults with osteoarthritis (OA), without OA and from infants with a set of titin antibodies and primer pairs. Three different antibodies were used for immunolabelling, reacting with titin's N-terminal Z1-Z2 domains, its Novex III exon, and with its PEVK region. An antibody directed to a titin ligand was included, since in cardiac muscle, this has been shown to participate in the transmission of stretch dependent titin-based signals. Our results indicate that although at low levels, titin is expressed in cartilage. Primer pairs detected titin transcripts in cartilage, and consistent with this, antibodies directed to titin's Z-disc region and to its elastic region stained cartilage. Moreover, we also could detect transcription of the titin ligand CARP. Components of the stretch dependent signal machinery in muscle are also expressed in cartilage. Further studies are warranted to address if common stress/strain dependent signalling are conserved in muscle and cartilage tissues.

In pneumococcal meningitis it is assumed that bacteria cross the blood-brain barrier (BBB), which consists mainly of cerebral endothelial cells. The effect of Streptococcus pneumoniae on the BBB was investigated with an in vitro BBB model using a human brain microvascular endothelial cell line (HBMEC) and primary cultures of bovine brain microvascular endothelial cells (BBMEC). Within a few hours of incubation with pneumococci, rounding and detachment of the HBMEC were observed, and the transendothelial electrical resistance of the BBMEC monolayer decreased markedly. An S. pneumoniae mutant deficient in pneumolysin did not affect the integrity of the endothelial cell monolayer. Neither cell wall fragments nor isolated pneumococcal cell walls induced changes of endothelial cell morphology. However, purified pneumolysin caused endothelial cell damage comparable to that caused by the viable pneumococci. The cell detachment was dependent on de novo protein synthesis and required the activities of caspase and tyrosine kinases. The results show that pneumolysin is an important component for damaging the BBB and may contribute to the entry of pneumococci into the cerebral compartment and to the development of brain edema in pneumococcal meningitis.