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

A comprehensive diffusion MRI dataset acquired on the MGH Connectome scanner in a biomimetic brain phantom

We provide a comprehensive diffusion MRI dataset acquired with a novel biomimetic phantom mimicking human white matter. The fiber substrates in the diffusion phantom were constructed from hollow textile axons (“taxons”) with an inner diameter of 11.8±1.2 µm and outer diameter of 33.5±2.3 µm. Data were acquired on the 3 T CONNECTOM MRI scanner with multiple diffusion times and multiple q-values per diffusion time, which is a dedicated acquisition for validation of microstructural imaging methods, such as compartment size and volume fraction mapping. Minimal preprocessing was performed to correct for susceptibility and eddy current distortions. Data were deposited in the XNAT Central database (project ID: dMRI_Phant_MGH)

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

Compression Bioreactor-Based Mechanical Loading Induces Mobilization of Human Bone Marrow-Derived Mesenchymal Stromal Cells into Collagen Scaffolds In Vitro

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 &plusmn; 5 viable cells/mm3 for loaded and 4 &plusmn; 2 viable cells/mm3 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 &plusmn; 42 viable cells/mm3 vs. 22 &plusmn; 6 viable cells/mm3, respectively; p-value &lt; 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

Pre-pubertal exposure with phthalates and bisphenol A and pubertal development

$\bf Objective$ Epidemiological studies indicate associations between childhood exposure with phthalates and bisphenol A (BPA) and the pubertal development. We examined associations between the pre-pubertal phthalate and BPA body burden and the longitudinally assessed sexual maturation of eight- to thirteen-year-old children. $\bf Methods$ We started with eight- to ten-year-old children in the baseline study and quantified phthalate metabolites and BPA in 472 urine samples (250 boys; 222 girls; mean age: 8.8 years). Associations between the pubertal development, assessed in three annual follow-up studies by Puberty Development scale questionnaires (PD scales), and the chemical exposure from the baseline visit were longitudinally analyzed with generalized estimation equations. $\bf Results$ The number of children with both chemical measures and PD scores (calculated from the PD scales) was 408. In the third follow-up, 49% of the girls and 18% of the boys had reached mid-puberty. For girls, we observed a delayed pubertal development with the di-hexyl-ethyl phthalate (DEHP) metabolites ($\beta$: -0.16 to -0.23; p $\leq$ 0.05 or p $\leq$ 0.1), mono-n-butyl phthalate ($\beta$: -0.15; 95% CI: -0.31; 0.01), mono-benzyl phthalate ($\beta$: -0.11; 95% CI: -0,24; -0,01),and mono-ethyl phthalate (MEP) ($\beta$: -0.15; 95% CI: -0.28; -0.01). In addition, significant non-linear associations of the DEHP metabolites and BPA with the PD scores were found, when their quadratic effects were included in the GEE models. In boys, no consistent relationships between the PD scores and the chemicals were detected except of an accelerated development with the $\sum$DEHP metabolites ($\beta$: 0.16; 95% CI: -0.02; -0.34) $\bf Conclusion$ We found indications that pre-pubertal exposures with phthalates and BPA were associated with pubertal timing in children, particularly in girls. For boys, associations were inconsistent, and not necessarily in line with the known anti-androgenicity of some phthalates during prenatal exposure

Bulletin de correspondance hellénique. Volume 136-137, livraison 1, 2012.

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

Report on a large animal study with Göttingen Minipigs where regenerates and controls for articular cartilage were created in a large number. Focus on the conditions of the operated stifle joints and suggestions for standardized procedures.

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