Non-calcified Cartilage Research Articles

Spatial distribution of elements during osteoarthritis disease progression using synchrotron X-ray fluorescence microscopy

The osteochondral interface is a thin layer that connects hyaline cartilage to subchondral bone. Subcellular elemental distribution can be visualised using synchrotron X-ray fluorescence microscopy (SR-XFM) (1 μm). This study aims to determine the relationship between elemental distribution and osteoarthritis (OA) progression based on disease severity. Using modified Mankin scores, we collected tibia plates from 9 knee OA patients who underwent knee replacement surgery and graded them as intact cartilage (non-OA) or degraded cartilage (OA). We used a tape-assisted system with a silicon nitride sandwich structure to collect fresh-frozen osteochondral sections, and changes in the osteochondral unit were defined using quantified SR-XFM elemental mapping at the Australian synchrotron's XFM beamline. Non-OA osteochondral samples were found to have significantly different zinc (Zn) and calcium (Ca) compositions than OA samples. The tidemark separating noncalcified and calcified cartilage was rich in zinc. Zn levels in OA samples were lower than in non-OA samples (P = 0.0072). In OA samples, the tidemark had less Ca than the calcified cartilage zone and subchondral bone plate (P < 0.0001). The Zn–strontium (Sr) colocalisation index was higher in OA samples than in non-OA samples. The lead, potassium, phosphate, sulphur, and chloride distributions were not significantly different (P > 0.05). In conclusion, SR-XFM analysis revealed spatial elemental distribution at the subcellular level during OA development.

The value of magnetic resonance ultrashort echo time imaging to evaluate non-calcified cartilage of the knee joint and its damage

ObjectivesTo image knee osteochondral specimens using magnetic resonance (MR) ultrashort echo time imaging with pointwise encoding time reduction with radial acquisition combined fat suppression (PETRA-FS) sequence to determine whether it can reveal non-calcified cartilage, including the deep radial layer, and to assess its effectiveness in cartilage damage diagnosis. Materials and methodsPETRA-FS imaging was performed on 58 osteochondral specimens of the lower femur and upper tibia to observe depth of cartilage damage, combined with histological results to observe signal intensity composition. Sensitivity, specificity, and reliability of PETRA-FS sequence for diagnosing cartilage damage were evaluated using histological results as the gold standard. Diagnostic efficacy was assessed using receiver operating characteristic (ROC) curve. ResultsMR ultrashort echo time imaging PETRA-FS sequence showed non-calcified cartilage, including tangential, transitional, and radial layers, which showed a high signal. PETRA-FS sequence showed 37 cases of cartilage damage and 21 cases of no damage among 58 specimens, kappa value of 0.75. Histological analysis of the 58 osteochondral specimens revealed 38 cases of cartilage injury and 20 cases of undamaged cartilage. Using histological results as the gold standard, PETRA-FS sequence had a sensitivity of 87.00%, specificity of 80.00%, kappa value of 0.81, and an area under the ROC curve (AUC) of 0.83 for cartilage injury diagnosis. ConclusionMR ultrashort echo time imaging PETRA-FS sequence can show non-calcified cartilage, including the deep radial layer (which cannot be shown by conventional MR), by exhibiting a high signal in knee osteo-chondral specimens. Thus, PETRA-FS sequences may have important diagnostic value for cartilage injury diagnosis.

Mechanical properties of extensive calcified costal cartilage: An experimental study

BackgroundAutologous costal cartilage is widely used as nasal augmentation or nasal reconstruction material. However, no study has focused on the mechanical difference between no calcified costal cartilage and extensive calcified costal cartilage at present. Our study aims to study the loading behavior of calcified costal cartilage under tensile and compressive stress. MethodHuman costal cartilage specimen was obtained from five extensive calcified costal cartilage patients and classified into four groups (group A: no calcified costal cartilage; group B: calcified costal cartilage; group C: no calcified costal cartilage after transplantation in BALB/c nude mice for half a year; group D: calcified costal cartilage after transplantation in BALB/c nude mice for half a year). Young's modulus, stress relaxation slope, and relaxation amount were analyzed through tensile and compressive tests using a material testing machine. ResultsWe included five female patients with extensive calcified costal cartilage. Group B exhibited significantly higher Young's modulus in both the tensile and compressive tests (p < 0.05 in tensile test, p < 0.01 in compressive test), higher relaxation slope (P < 0.01) and higher relaxation amount (p < 0.05 in compression test). After transplantation, the Young's modulus of calcified and non-calcified costal cartilage decreased, except that the calcified costal cartilage increased slightly in the tensile test. The final relaxation slope and relaxation amount had increased at different degrees, but the changes did not change significantly before and after transplantation (P > 0.05). ConclusionOur results showed that the stiffness of calcified cartilage would increase 30.06% under tension and 126.31% under compression. This study may provide new insights to researchers focusing on extensive calcified costal cartilage can be used for autologous graft material.

Особенности структурных изменений дистального суставного конца бедренной кости при экспериментальном моделировании остеомиелита

Features of structural changes in the joint components (synovial membrane, articular cartilage, subchondral bone) under the conditions of adjacent limb segment osteomyelitis are poorly understood and require thorough histological studies. Purpose Сomparative assessment of the structural reorganization of the main components of the distal articular end of the femur in experimental modeling of osteomyelitis. Material and methods Objects: distal metaphyses of the femur of intact rats (n = 5) and experimental ones (n = 16) in the conditions of modeled osteomyelitis of the femur. The culture of S. aureus was injected into the medullary canal in the experimental animals (n = 8) while saline was injected in the control group (n = 8). The animals were taken out of the experiment on the 21st day. Methods: histological, morphometric, and statistical methods were used. Results In the control group, the articular cartilage, subchondral bone plate, and subchondral zone retained their normal structure. Synovitis was not revealed. The values of the morphometric parameters were comparable with the intact norm. In the experimental group, bone microsequesters, osteoclastic resorption of the subchondral bone plate, inflammatory infiltration with the content of plasma cells and neutrophils were detected in the subchondral zone. Histological changes in the articular cartilage according to the classification of the International Society OARSI (2006) corresponded to grades 1 to 3 and were accompanied by synovitis. There was a significant (р &lt; 0.05) decrease in the thickness of non-calcified cartilage, a significant twofold decrease in the thickness of the subchondral bone plate, while the values of the thickness of the calcified cartilage exceeded those in the control group and the intact norm. Conclusion Under the conditions of an experimental model of chronic osteomyelitis of the femur, the revealed structural changes in the subchondral zone contribute to the progression of the destruction of the subchondral bone plate, articular cartilage and synovitis. This model of chronic osteomyelitis can be used to experimentally study various therapeutic strategies aimed at modifying subchondral bone remodeling and relieving synovitis.

The Biomimetics of Mg2+-Concentration-Resolved Microenvironment for Bone and Cartilage Repairing Materials Design.

With the increase in population aging, the tendency of osteochondral injury will be accelerated, and repairing materials are increasingly needed for the optimization of the regenerative processes in bone and cartilage recovery. The local environment of the injury sites and the deficiency of Mg2+ retards the repairing period via inhibiting the progenitor osteogenesis and chondrogenesis cells' recruitment, proliferation, and differentiation, which results in the sluggish progress in the osteochondral repairing materials design. In this article, we elucidate the Mg2+-concentration specified effect on the cell proliferation, osteochondral gene expression, and differentiation of modeling chondrocytes (extracted from New Zealand white rabbit) and osteoblasts (MC3T3-E1). The concentration of Mg2+ in the culture medium affects the proliferation, chondrogenesis, and osteogenesis: (i) Appropriate concentrations of Mg2+ promote the proliferation of chondrocytes (1.25-10.0 mM) and MC3T3-E1 cells (2.5-30.0 mM); (ii) the optimal concentration of Mg2+ that promotes the gene expression of noncalcified cartilage is 15 mM, calcified cartilage 10 mM, and subchondral bone 5 mM, respectively; (iii) overdosed Mg2+ leads to the inhibition of cell activity for either chondrocytes (&gt;20 mM) or osteoblasts (&gt;30 mM). The biomimetic elucidation for orchestrating the allocation of gradient concentration of Mg2+ in accordance of the physiological condition is crucial for designing the accurate microenvironment in osteochondral injury defects for optimization of bone and cartilage repairing materials in the future.

Validation of ultrasonography for measurement of cartilage thickness in the equine carpus.

Articular cartilage thinning is an important hallmark of osteoarthritis (OA), and ultrasonography (US) is a clinically accessible tool potentially suitable for repeated evaluation. The aim of the present prospective methods comparison study was to validate US as a tool for measuring cartilage thickness in the carpus of the horse. Eight Standardbred trotters underwent US examination with 9 and 15 MHz linear transducers. Six anatomical locations in the radiocarpal joint (RCJ) and middle carpal joint (MCJ) were examined. The same joints were assessed by ultrahigh field (9.4 Tesla) magnetic resonance imaging (MRI) and histology. Associations between measurements obtained by the different modalities were assessed by ANOVA, Deming regression, Pearson correlation and Bland–Altman plots. Histologically assessed total cartilage thickness (the noncalcified cartilage (NCC) plus the calcified cartilage zone (CCZ)) overestimated thickness compared to MRI (P < 0.01) and US (P < 0.01). US 15 MHz had substantial agreement with MRI and NCC histology, and repeatability was acceptable (coefficient of variation = 8.6–17.9%) when used for assessment of cartilage thickness in the RCJ. In contrast, 9 MHz US showed poorer agreement with MRI and NCC histology, as it overestimated the thickness of thin cartilage and underestimated the thickness of thicker cartilage in the RCJ and MCJ. Moreover, repeatability was suboptimal (coefficient of variation = 10.4–26.3%). A 15 MHz transducer US is recommended for detecting changes in RCJ cartilage thickness or monitoring development over time, and it has the potential for noninvasive assessment of cartilage health in horses.

The interface region between articular cartilage and bone by μMRI and PLM at microscopic resolutions.

This dual-modality microscopic imaging study quantifies the interface region between the noncalcified cartilage and the subchondral bone plate, which includes the deep portion of the noncalcified articular cartilage and the zone of calcified cartilage (ZCC). This interface region is typically not visible in routine MRI but becomes visible in MRI with the application of an ultra-short echo time (UTE) sequence. A number of cartilage-bone blocks from a well-documented canine humeral head were harvested for imaging by microscopic MRI (μMRI) and PLM (polarized light microscopy). In μMRI, T2 anisotropic images were acquired by 2D gradient-echo, magnetization-prepared spin-echo and UTE sequences at the 0° and 55° (the magic angle) orientations at 11.7μm/pixel resolution. In PLM, quantitative optical retardation (nm) and collagen orientation (°) were mapped from the thin sections from the same μMRI specimens at 0.5-2μm pixel resolutions. The orientational and organizational architecture of the collagen matrix in this interface region was quantified and correlated between the complementary imaging. The magic angle effect as seen in the noncalcified cartilage was statistically confirmed in ZCC in μMRI, which was further supported by quantitative PLM. With an enhanced understanding of the tissue properties in this important interface region, it will potentially be possible to monitor the changes of this tissue region which is instrumental to the initiation and development of osteoarthritis and other joint diseases.

Оценка риска развития остеоартроза коленного сустава при протезировании голени (пилотное экспериментально-морфологическое исследование)

Objective was to obtain preliminary data on the knee osteoarthritis risk following canine tibial prosthetics using one-stage osseointegration, external fixation and compression loading. Material and methods The study was carried out on 5 intact (control group) and 3 experimental (experimental group) animals aged 1.8 ± 0.5 years with a body weight of 19 ± 1.2 kg. Osteotomy was performed at the boundary of the upper and middle third of tibia and a PressFit type construct implanted. A special device was used for bone fixation and compression loading of FN = 20 N. Paraffin sections of the articular cartilage and the underlying subchondral bone were used for histomorphometric examination. Results The zonal structure of the articular cartilage and cytoarchitectonics were shown to be maintained in all experimental animals with insignificant destructive changes in the form of impaired homogeneity of the intercellular substance in the upper third of the surface zone. There was a two-fold decrease in the thickness of the calcified cartilage and a 1.9‑fold decrease in the thickness of the subchondral bone. The volumetric density of trabeculae in the subchondral bone decreased to 22.31 ± 5.41 % in experimental animals and to 46.94 ± 1.94 % in controls. Complete absence of calcified cartilage and the subchondral bone were observed in one case with vessels and bone marrow pannus invading the noncalcified cartilage. Conclusion Structural changes in the contact zone of the articular cartilage and the subchondral bone seen in the knee following experimental canine tibial prosthetics indicated the risk of developing knee osteoarthritis.

Therapeutic Role of Platelet Rich Plasma in Formaldehyde-Induced Arthritis in Adult Male Albino Rats

Abstract Introduction Osteoarthritis (OA) is a common health problem. Platelet-rich plasma (PRP) has been recognized to enhance articular cartilage metabolism. Aim of the work the study was designed to investigate the influence of PRP on cartilage healing after induction of arthritis. Material and methods Forty two adult male albino rats were used in this study. The rats were randomly divided into three groups: Group I (n = 18): the control group (Ia, Ib &amp; Ic) Ib &amp; Ic were injected intra-articularly with saline and left for 3 and 6 weeks. Group II (n = 12): arthritic group, in which osteoarthritis was induced by injection of 0.02ml 5% formaldehyde once in the right knee joints, left without treatment, and were sacrificed after three weeks (IIa) or after six weeks (IIb). Group III(n = 12): arthritis was induced as group II, one week later, the rats were intra-articularly injected with single dose of 0.3ml PRP in the same joint then were sacrificed three weeks (IIIa) or six weeks (IIIb) after formaldehyde injection. At the end of the study the right knee joints were taken, decalcified then processed for paraffin sections to be examined by light microscope using H&amp;E, toluidine blue and Masson’s trichrome (MTC) stains. Immunohistochemistry for caspase-3 enzyme was done to demonstrate apoptotic chondrocytes. Morphometric study was conducted to measure the thickness of the non-calcified cartilage, count the chondrocytes and synovial membrane inflammatory cells and Mankin's score. Then statistical analysis was done. Results The arthritic groups revealed irregular surface of the articular cartilage, loss of the articular matrix and bone eburnation. Moreover, there was apparent hypocellularity and disorganization of the chondrocytes. Osteoclasts and osteoblasts were seen invading the osteochondral junction. MTC stained sections of the synovial membrane showed deposition of thick collagen bundles with heavy inflammatory cell infiltrate and numerous blood vessels. The affinity of the articular cartilage to toluidine blue stain was apparently decreased while caspase-3 immunoreactivity was apparent in many chondrocytes .Group IIIa demonstrated almost similar histological findings as the control group; regular articular cartilage surface with regularly arranged chondrocytes in the different cartilage zones. Synovial membrane illustrated minimal inflammatory cell infiltrate with thin collagen bundles and small blood vessels in MTC stained sections. There was high affinity of the articular cartilage to the toluidine blue stain and few chondrocytes showed positive caspase-3 immunoreactivity. Group IIIb revealed continuous surface of the articular cartilage, yet with minimal fibrillation in some areas. Osteoblasts and osteoclasts were seen invading the calcified cartilage. Synovial membrane showed deposition of dense collagen bundles with some inflammatory cell infiltrate. Toluidine blue sections revealed decreased articular cartilage affinity to the stain while caspase-3 immunoreactivity was evident in many chondrocytes. The morphometric results and statistical analysis confirmed the histological findings. Conclusion Intra-articular injection of PRP demonstrated advantageous role on articular cartilage healing, however, these effects appeared to be transient. So the need of multiple injections of PRP has to be considered in cases of OA.

Topographic features of nano-pores within the osteochondral interface and their effects on transport properties –a 3D imaging and modeling study

Recent insights suggest that the osteochondral interface plays a central role in maintaining healthy articulating joints. Uncovering the underlying transport mechanisms is key to the understanding of the cross-talk between articular cartilage and subchondral bone. Here, we describe the mechanisms that facilitate transport at the osteochondral interface. Using scanning electron microscopy (SEM), we found a continuous transition of mineralization architecture from the non-calcified cartilage towards the calcified cartilage. This refurbishes the classical picture of the so-called tidemark; a well-defined discontinuity at the osteochondral interface.Using focused-ion-beam SEM (FIB-SEM) on one osteochondral plug derived from a human cadaveric knee, we elucidated that the pore structure gradually varies from the calcified cartilage towards the subchondral bone plate. We identified nano-pores with radius of 10.71 ± 6.45 nm in calcified cartilage to 39.1 ± 26.17 nm in the subchondral bone plate.The extracted pore sizes were used to construct 3D pore-scale numerical models to explore the effect of pore sizes and connectivity among different pores. Results indicated that connectivity of nano-pores in calcified cartilage is highly compromised compared to the subchondral bone plate. Flow simulations showed a permeability decrease by about 2000-fold and solute transport simulations using a tracer (iodixanol, 1.5 kDa with a free diffusivity of 2.5 × 10−10 m2/s) showed diffusivity decrease by a factor of 1.5. Taken together, architecture of the nano-pores and the complex mineralization pattern in the osteochondral interface considerably impacts the cross-talk between cartilage and bone.

Growth factor profile in calcified cartilage from the metaphysis of a calf costochondral junction, the site of initial bone formation.

Endochondral bone formation is orchestrated by growth factors produced by chondrocytes and deposited in the cartilage matrix. Whilst some of these factors have been identified, the complete list and their relationship remains unknown. In the present study, the growth factors were isolated from non-calcified and calcified cartilage of costochondral junctions. Cartilage dissected from the ribs of 6-20-week-old calves was purchased from a local butcher within 24 h of the death of the animal. The isolation involved hyaluronidase digestion, guanidinium hydrochloride (GuHCl) extraction, HCl decalcification and GuHCl extraction of the decalcified matrix. Growth factors were purified by heparin chromatography and their quantities were estimated using ELISA. Decalcified cartilage was also used for protein sequence analysis (data are available via ProteomeXchange; ID, PXD021781). Bone morphogenetic protein-7 (BMP-7), growth/differentiation factor-5 (GDF-5) and NEL-like protein-1 (NELL-1), all known growth factors that stimulate bone formation, quantitatively accounted for the majority of the material obtained in all steps of isolation. Thus, cartilage serves as a store for growth factors. During initial bone formation septoclasts release osteoclastogenesis-stimulating factors deposited in non-calcified cartilage. Osteoclasts dissolve calcified cartilage and transport the released factors required for the stimulation of osteoprogenitor cells to deposit osteoid. High concentrations of BMP-7, GDF-5 and NELL-1 at the site of initial bone formation may suggest that their synergistic action favours osteogenesis.

Suppression of Hypertrophy During in vitro Chondrogenesis of Cocultures of Human Mesenchymal Stem Cells and Nasal Chondrocytes Correlates With Lack of in vivo Calcification and Vascular Invasion.

ObjectiveHuman nasal septal chondrocytes (NC) are a promising minimally invasive derivable chondrogenic cell source for cartilage repair. However, the quality of NC-derived cartilage is variable between donors. Coculture of NC with mesenchymal stem cells (MSCs) mitigates the variability but with undesirable markers of chondrocyte hypertrophy, such as type X collagen, and the formation of unstable calcifying cartilage at ectopic sites. In contrast, monoculture NC forms non-calcifying stable cartilage. Formation of a stable NC-MSC coculture cartilage is crucial for clinical application. The aim of this study was to explore the utility of parathyroid hormone-related peptide (PTHrP) hormone to suppress chondrocyte hypertrophy in NC-MSC cocultures and form stable non-calcifying cartilage at ectopic sites.MethodsHuman NC and bone marrow MSCs, and cocultures of NC and MSC (1:3 ratio) were aggregated in pellet form and subjected to in vitro chondrogenesis for 3 weeks in chondrogenic medium in the presence and absence of PTHrP. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks.ResultsCoculture of NC and MSC resulted in synergistic cartilage matrix production. PTHrP suppressed the expression of hypertrophy marker, type X collagen (COL10A1), in a dose-dependent fashion without affecting the synergism in cartilage matrix synthesis, and in vivo calcification was eradicated with PTHrP. In contrast, cocultured control (CC) pellets without PTHrP treatment expressed COL10A1, calcified, and became vascularized in vivo.

Bright ultrashort echo time SWIFT MRI signal at the osteochondral junction is not located in the calcified cartilage.

In this study, we aimed to precisely localize the hyperintense signal that is generated at the osteochondral junction when using ultrashort echo time magnetic resonance imaging (MRI) and to investigate the osteochondral junction using sweep imaging with Fourier transformation (SWIFT) MRI. Furthermore, we seek to evaluate what compositional properties of the osteochondral junction are the sources of this signal. In the study, we obtained eight samples from a tibial plateau dissected from a 68-year-old male donor, and one additional osteochondral sample of bovine origin. The samples were imaged using high-resolution ultrashort echo time SWIFT MRI and microcomputed tomography (μCT) scans. Localization of the bright signal in the osteochondral junction was performed using coregistered data sets. Potential sources of the signal feature were examined by imaging the bovine specimen with variable receiver bandwidths and by performing variable flip angle T1 relaxation time mapping. The results of the study showed that the hyperintense signal was found to be located entirely in the deep noncalcified articular cartilage. The intensity of this signal at the interface varied between the specimens. Further tests with bovine specimens indicated that the imaging bandwidth and T1 relaxation affect the properties of the signal. Based on the present results, the calcified cartilage has low signal intensity even in SWIFT imaging. Concomitantly, it appears that the bright signal seen in ultrashort echo time imaging resides within the noncalcified cartilage. Furthermore, the most likely sources of this signal are the rapid T1 relaxation of the deep cartilage and the susceptibility-induced effects arising from the calcified tissues.

Deep learning-based segmentation from histology allows for automated quantification of calcified cartilage morphology in a rabbit model of post-traumatic osteoarthritis

Purpose: Calcified cartilage (CC) undergoes constant remodeling due to two competing events: 1) mineralization of the deep non-calcified cartilage that advances the tidemark, and 2) new bone formation through endochondral ossification that advances the cement line. However, CC modifications in the presence of osteoarthritis (OA) progression are not well understood. In this study, we investigate CC morphology during OA progression and aging in the rabbit anterior cruciate ligament transection (ACLT) model.

Age-related changes in the cartilage of the temporomandibular joint.

Osteoarthritis (OA) of the knee is closely associated with aging; however, little is known about the age-related degeneration in the mandibular condylar cartilage (MCC) of the TMJ. Our objective was to examine whether a correlation exists between aging and degeneration of the MCC of the TMJ. Thirty-two male C57BL/6J wild-type mice were aged to 2, 12, 18, and 25months old. The mice were euthanized by CO2 inhalation and were dissected and examined by micro-CT and histology. Sagittal sections of the condyles were stained for tartrate-resistant alkaline phosphatase, alkaline phosphatase, safranin O, picrosirius red, and toluidine blue. In addition, immunostaining for BMP2, BMP4, BMP7, PRG4, and MMP13 was performed. Bone volume fraction and tissue density significantly increased with the age of the animals. There was a significant increase in the Osteoarthritis Research Society International histopathological score and mineralization of the noncalcified cartilage in the aged animals. There was a decrease in cartilage thickness, proteoglycan distribution, and cellularity in the aged animals. Additionally, we noted increased picrosirius red staining with the increase in the age of the animals. Our protein expression showed increased BMP2, BMP4, BMP7, and MMP13, whereas there was a decrease in PRG4 expression in the aged animals. As the animal ages, there is decreased proteoglycan secretion, decreased cellularity, decreased cartilage thickness, increased fibrillation, and increased proteolytic activity. A better understanding of the basic mechanisms underlying the degeneration of the MCC in the older animals could provide novel ways to slow the development of OA.

Mechanical properties of hybrid triphasic scaffolds for osteochondral tissue engineering

Reproducing the advanced complexity of native tissue by means of the 3D multi-functional construct is a promising tissue engineering approach to osteochondral tissue regeneration. In this study, we present a porous 3D construct composed of three zones responsible for the regeneration of non-calcified cartilage, calcified cartilage and subchondral bone. These three zones of the hybrid were composed of modified biopolymers: (i) alginate (Alg) reinforced by short polylactide (PLA) fibres, (ii) alginate and gelatine methacrylate (GelMA) combined with ß-tricalcium phosphate particles (TCP), (iii) 3D printed polycaprolactone scaffold subsequently modified with the use of an innovative solvent treatment method based on acetone and ultrasound stimulation, respectively. Combining the advanced deposition systems based on: (i) 3D printing coupled with a spray crosslinking system, (ii) an innovative deposition system based on a coaxial-needle extruder, (iii) fused deposition modelling (FDM) connected with post-fabrication treatment, allows us to fabricate the triphasic construct that emulates the structure and properties of the native osteochondral tissue. The aim of the study was to investigate the mechanical properties of the fabricated hybrid and its individual zones. Our results demonstrate the load-bearing capabilities of TC, but nevertheless it should be implanted below the surface line of host cartilage to protect it from strong stresses, at the same time allowing native host tissues to grow into it.

Quantitative measurement of T2, T1ρ and T1 relaxation times in articular cartilage and cartilage-bone interface by SE and UTE imaging at microscopic resolution.

Both spin-echo (SE) and ultra-short echo (UTE) based MRI sequences were used on a 7 T µMRI system to quantify T2, T1ρ and T1 relaxation times from articular cartilage to the cartilage-bone interface on canine humeral specimens at 19.5 µm pixel resolution. A series of five relaxation-weighted images were acquired to calculate one relaxation map (T2, T1ρ or T1), from which the depth-dependent profiles were examined between the SE method and the UTE method, over the entire non-calcified cartilage and within the cartilage-bone interface. SE-based methods enabled the quantification of relaxation profiles over the noncalcified cartilage, from 0 µm (articular surface) to approximately 460 µm in depth (near the end of radial zone). Most of the cartilage-bone interface was imaged by the UTE-based methods, to a tissue depth of about 810 µm. Pixel-by-pixel calculation of the relaxation times between the independent SE and UTE methods correlated well with each other. A better understanding of the tissue properties reliably over the cartilage-bone interface region by a non-invasive MRI approach could contribute to the clinical diagnostics of trauma-induced osteoarthritis.

Osteoclasts degrade bone and cartilage knee joint compartments through different resorption processes

BackgroundOsteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. The osteoclast resorption processes required to degrade subchondral bone and cartilage—the remodeling of which is important in the osteoarthritic disease process—have not been previously described, although cathepsin K has been indicated to participate. In this study we profile osteoclast-mediated degradation of bovine knee joint compartments in a novel in vitro model using biomarkers of extracellular matrix (ECM) degradation to assess the potential of osteoclast-derived resorption processes to degrade different knee joint compartments.MethodsMature human osteoclasts were cultured on ECMs isolated from bovine knees—articular cartilage, cortical bone, and osteochondral junction ECM (a subchondral bone-calcified cartilage mixture)—in the presence of inhibitors: the cystein protease inhibitor E-64, the matrix metalloproteinase (MMP) inhibitor GM6001, or the vacuolar-type H+-ATPase (V-ATPase) inhibitor diphyllin. Biomarkers of bone (calcium and C-terminal type I collagen (CTX-I)) and cartilage (C2M) degradation were measured in the culture supernatants. Cultures without osteoclasts were used as background samples. Background-subtracted biomarker levels were normalized to the vehicle condition and were analyzed using analysis of variance with Tukey or Dunnett’s T3 post hoc test, as applicable.ResultsOsteochondral CTX-I release was inhibited by E-64 (19% of vehicle, p = 0.0008), GM6001 (51% of vehicle, p = 0.013), and E-64/GM6001 combined (4% of vehicle, p = 0.0007)—similarly to bone CTX-I release. Diphyllin also inhibited osteochondral CTX-I release (48% of vehicle, p = 0.014), albeit less than on bone (4% of vehicle, p < 0.0001). Osteochondral C2M release was only inhibited by E-64 (49% of vehicle, p = 0.07) and GM6001 (14% of vehicle, p = 0.006), with complete abrogation when combined (0% of vehicle, p = 0.004). Cartilage C2M release was non-significantly inhibited by E-64 (69% of vehicle, p = 0.98) and was completely abrogated by GM6001 (0% of vehicle, p = 0.16).ConclusionsOur study supports that osteoclasts can resorb non-calcified and calcified cartilage independently of acidification. We demonstrated both MMP-mediated and cysteine protease-mediated resorption of calcified cartilage. Osteoclast functionality was highly dependent on the resorbed substrate, as different ECMs required different osteoclast processes for degradation. Our novel culture system has potential to facilitate drug and biomarker development aimed at rheumatic diseases, e.g. osteoarthritis, where pathological osteoclast processes in specific joint compartments may contribute to the disease process.

Murine articular cartilage morphology and compositional quantification with high resolution cationic contrast-enhanced μCT.

Articular cartilage lines the load-bearing surfaces of long bones and undergoes compositional and structural degeneration during osteoarthritis progression. Contrast enhanced microcomputed tomography (μCT) is being applied to a variety of preclinical models, including the mouse, to map structural and compositional properties in 3-D. The thinness (∼30-50 μm) and high cellularity of mouse articular cartilage presents a significant imaging challenge. Our group previously showed that mouse articular cartilage and proteoglycan (PG) content can be assessed by μCT with the ioxagalate-based contrast agent Hexabrix, but the voxel size used (6 μm) was deemed to be barely adequate. The objective of the present study is to assess the utility of a novel contrast agent, CA4+, to quantify mouse articular cartilage morphology and composition with high resolution μCT imaging (3 μm voxels) and to compare the sensitivity of CA4+ and Hexabrix to detect between-group differences. While both contrast agents are iodine-based, Hexabrix is anionic and CA4+ is cationic so they interact differently with negatively charged PGs. With CA4+, a strong correlation was found between non-calcified articular cartilage thickness measurements made with histology and μCT (R2 = 0.72, p < 0.001). Cartilage degeneration-as assessed by loss in volume, thickness, and PG content-was observed in 34-week-old mice when compared to both 7- and 12-week-old mice. High measurement precision was observed with CA4+, with the coefficient of variation after repositioning and re-imaging samples equaling 2.8%, 4.5%, 7.4% and 5.9% for attenuation, thickness, volume, and PG content, respectively. Use of CA4+ allowed increased sensitivity for assessing PG content compared to Hexabrix, but had no advantage for measurement of cartilage thickness or volume. This improvement in imaging should prove useful in preclinical studies of cartilage degeneration and regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2740-2748, 2017.

Significance of new blood vessels in the pathogenesis of temporomandibular joint osteoarthritis.

We studied the significance of new blood vessels in the pathogenesis of temporomandibular joint osteoarthritis (TMJOA). Fifteen 8-week-old female Sprague-Dawley rats were selected to establish TMJOA models of gradually induced occlusal disorders. Five rats were sacrificed at 4, 8 and 16 weeks, and histological exam was conducted along with micro-computed tomography observation on the condyle specimen. The distribution and number of new blood vessels breaking were observed through the tidemark through CD34 immunofluorescence staining. The proliferation of chondrocytes were detected through Ki67 immunohistochemical staining, and the differentiation functions of chondrocytes were observed through PTHrP and IHH immunohistochemical staining. The degradation functions of cartilage matrix were observed through matrix metalloproteinase (MMP)-9 immunohistochemical staining to detect the expression of vascular growth promotion and inhibition factors with vascular endothelial growth factor (VEGF), CTGF and CHM-1 immunohistochemical staining and screen differentially expressed genes through gene chip analysis method. It was found that the condyle tissue full thickness, fiber layer thickness and calcified cartilage layer thickness were significantly increased with time (P<0.05). Bone mineral density, trabecular thickness and Tb.Sp were also increased significantly with time, BS/BV and trabecular number were decreased significantly with time (P<0.05). The new blood vessels reached the deep layer of calcified cartilage until the tide line was broken and non-calcified cartilage was invaded. The number of vessels were increased significantly with time (P<0.05). Ki67, PTHrP and IHH-positive rates were increased significantly (P<0.05). MMP-9, VEGF, CTGF and CHM-1 were increased significantly (P<0.05). VEGF, CTGF and CHM-1 mRNA were upregulated differentially with the expressed genes. In conclusion, the new blood vessels may be important in the pathogenesis of TMJOA.