The Matrilin-3 T298M mutation predisposes for post-traumatic osteoarthritis in a knock-in mouse model.

OBJECTIVE: The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development. METHODS: A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis. RESULTS: The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development. CONCLUSIONS: The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development.

Propagation phase-contrast micro-computed tomography allows laboratory-based three-dimensional imaging of articular cartilage down to the cellular level.

OBJECTIVE: High-resolution non-invasive three-dimensional (3D) imaging of chondrocytes in articular cartilage remains elusive. The aim of this study was to explore whether laboratory micro-computed tomography (micro-CT) permits imaging cells within articular cartilage. DESIGN: Bovine osteochondral plugs were prepared four ways: in phosphate-buffered saline (PBS) or 70% ethanol (EtOH), both with or without phosphotungstic acid (PTA) staining. Specimens were imaged with micro-CT following two protocols: 1) absorption contrast (AC) imaging 2) propagation phase-contrast (PPC) imaging. All samples were scanned in liquid. The contrast to noise ratio (C/N) of cellular features quantified scan quality and were statistically analysed. Cellular features resolved by micro-CT were validated by standard histology. RESULTS: The highest quality images were obtained using propagation phase-contrast imaging and PTA-staining in 70% EtOH. Cellular features were also visualised when stained in PBS and unstained in EtOH. Under all conditions PPC resulted in greater contrast than AC (p < 0.0001 to p = 0.038). Simultaneous imaging of cartilage and subchondral bone did not impede image quality. Corresponding features were located in both histology and micro-CT and followed the same distribution with similar density and roundness values. CONCLUSIONS: Three-dimensional visualisation and quantification of the chondrocyte population within articular cartilage can be achieved across a field of view of several millimetres using laboratory-based micro-CT. The ability to map chondrocytes in 3D opens possibilities for research in fields from skeletal development through to medical device design and treatment of cartilage degeneration.

Structure and mechanical properties of high-weight-bearing and low-weight-bearing areas of hip cartilage at the micro- and nano-levels.

BACKGROUND: Articular cartilage has a high-weight-bearing area and a low-weight-bearing area, the macroscopic elastic moduli of the two regions are different. Chondrocytes are affected by the applied force at the microscopic level. Currently, the modulus of the two areas at the micro and nano levels is unknown, and studies on the relationship between macro-, micro- and nano-scale elastic moduli are limited. Such information may be important for further understanding of cartilage mechanics. Moreover, the surface morphology, proteoglycan content, and micro and nano structure of the two areas, which influences the mechanical properties of cartilage should be discussed. METHODS: Safranin-O/Fast Green staining was used to evaluate the surface morphology and semi-quantify proteoglycan content of porcine femoral head cartilage between the two weight-bearing areas. The unconfined compression test was used to determine the macro elastic modulus. Atomic force microscope was used to measure the micro and nano compressive elastic modulus as well as the nano structure. Scanning electron microscope was employed to evaluate the micro structure. RESULTS: No significant differences in the fibrillation index were observed between two areas (P = 0.5512). The Safranin-O index of the high-weight-bearing area was significantly higher than that of the low-weight-bearing area (P = 0.0387). The compressive elastic modulus of the high-weight-bearing area at the macro and micro level was significantly higher than that of the low-weight-bearing area (P = 0.0411 for macro-scale, and P = 0.0001 for micro-scale), while no statistically significant differences were observed in the elastic modulus of collagen fibrils at the nano level (P = 0.8544). The density of the collagen fibers was significantly lower in the high-weight-bearing area (P = 0.0177). No significant differences were observed in the structure and diameter of the collagen fibers between the two areas (P = 0.7361). CONCLUSIONS: A higher proteoglycan content correlated with a higher compressive elastic modulus of the high-weight-bearing area at the micro level than that of the low-weight-bearing area, which was consistent with the trend observed from the macroscopic compressive elastic modulus. The weight-bearing level was not associated with the elastic modulus of individual collagen fibers and the diameter at the nano level. The micro structure of cartilage may influence the macro- and micro-scale elastic modulus.

Metformin pretreatment suppresses alterations to the articular cartilage ultrastructure and knee joint tissue damage secondary to type 2 diabetes mellitus in rats.

Osteoarthritis (OA) secondary to diabetes affects millions of people worldwide and can lead to disability. The protective effect of metformin pretreatment against alterations to the articular cartilage ultrastructure induced by type 2 diabetes mellitus (T2DM) associated with the inhibition of oxidative stress and inflammation has not been investigated before. Therefore, we induced T2DM in rats (the model group) using high carbohydrate and fat diet and a single injection of streptozotocin (50 mg/kg body weight). The protective group of rats started metformin (200 mg/kg body weight) treatment 14 days before diabetic induction and continued on metformin until the end of the experiment at week 12. Harvested tissues obtained from knee joints were prepared for staining with hematoxylin and eosin (H&E), safranin o staining, and electron microscopy. Histology images showed that OA was developed in the T2DM rats as demonstrated by a substantial damage to the articular cartilage and profound chondrocyte and territorial matrix ultrastructural alterations, which were partially protected by metformin. In addition, metformin significantly (p < .05) reduced hyperglycemia, glycated hemoglobin (HbA1 c), malondialdehyde (MDA), high sensitivity C-reactive protein (hs-CRP), and interleukin-6 blood levels induced by diabetes. Furthermore, a significant (p ≤ 0.015) correlation between either OA cartilage grade score or the thickness of the articular cartilage and the blood levels of HbA1 c, hs-CRP, MDA, superoxide dismutase (SOD) were observed. These findings demonstrate effective protection of the articular cartilage by metformin against damage induced secondary to T2DM in rats, possibly due to the inhibition of hyperglycemia and biomarkers of oxidative stress and inflammation.

Three-dimensional architecture of the acetabular labrum in the human hip joint.

The acetabular labrum is frequently damaged with advancing age. As collagen fibers are the main sources of strength, knowledge of their ultrastructure is important to determine the cause of age-induced changes. We aimed to investigate the ultrastructure of collagen fibers constituting the acetabular labrum using scanning electron microscopy (SEM). Acetabular labrum samples obtained during total hip arthroplasty were studied. The samples were specially prepared to observe the steric construction of collagen fibrils constituting the acetabular labrum under light microscopy followed by SEM. The acetabular labrum was mostly composed of cartilage tissue, consisting of chondrocytes and collagen type II, with a layer of collagen type I. In adults, chondrocytes with a rich cytoplasm were surrounded by a dense network of fine type II collagen fibrils, and small bundles of type I collagen fibrils were interposed in the cartilage layer. In elderly individuals, the chondrocytes atrophied and both type I and II collagen fibrils were sparse. We suggest that cartilage has three to five layers, consisting of type I and type II collagen fibrils with a solid cartilage substrate. In elderly individuals, the density of chondrocytes decreases and the cellular shape and architecture of collagen fibrils also changes.

IFT20 is required for the maintenance of cartilaginous matrix in condylar cartilage.

Condylar cartilage is a joint cartilage essential for smooth jaw movement. The importance of ciliary proteins in condylar cartilage development has been reported. However, little is known about how ciliary proteins control the homeostasis of condylar cartilage. Here we show that intraflagellar transport 20 (IFT20), a ciliary protein, is required for the maintenance of cartilaginous matrix in condylar cartilage. Utilizing NG2-CreER mice expressed in condylar cartilage, we deleted Ift20 by tamoxifen treatment at juvenile-to-adult stages. In wild-type condylar cartilage, IFT20 was robustly produced in the cis-Golgi, but deletion of Ift20 by tamoxifen induction of NG2-CreER (Ift20:NG2-CreER) resulted in reduced cell proliferation and decreased Golgi size in condylar cartilage. Importantly, while the primary cilia were present in cartilage cells in the condylar layers of wild-type mice, no primary cilia were present in the Ift20:NG2-CreER condylar layers. Consistent with this finding, ciliary-mediated Hedgehog signaling was severely attenuated in Ift20 mutant chondrocytes, and thus the production levels of type X collagen were significantly reduced in Ift20:NG2-CreER mice. These results suggest that IFT20 is required for Golgi size and Hedgehog signaling to maintain cartilaginous matrix in condylar cartilage. Our study highlights the unique function of IFT20 in the homeostasis of condylar cartilage.

Propagation of microcracks in collagen networks of cartilage under mechanical loads.

OBJECTIVE: We recently demonstrated that low-energy mechanical impact to articular cartilage, usually considered non-injurious, can in fact cause microscale cracks (widths <30µm) in the collagen network of visually pristine human cartilage. While research on macro-scale cracks in cartilage and microcracks in bone abounds, how microcracks within cartilage initiate and propagate remains unknown. We quantified the extent to which microcracks initiate and propagate in the collagen network during mechanical loading representative of normal activities. DESIGN: We tested 76 full-thickness, cylindrical osteochondral plugs. We imaged untreated specimens (pristine phase) via second harmonic generation and assigned specimens to three low-energy impact groups (none, low, high), and thereafter to three cyclic compression groups (none, low, high) which simulate walking. We re-imaged specimens in the post-impact and post-cyclic compression phases to identify and track microcracks. RESULTS: Microcracks in the network of collagen did not present in untreated controls but did initiate and propagate under mechanical treatments. We found that the length and width of microcracks increased from post-impact to post-cyclic compression in tracked microcracks, but neither depth nor angle presented statistically significant differences. CONCLUSIONS: The microcracks we initiated under low-energy impact loading increased in length and width during subsequent cyclic compression that simulated walking. The extent of this propagation depended on the combination of impact and cyclic compression. More broadly, the initiation and propagation of microcracks may characterize pathogenesis of osteoarthritis, and may suggest therapeutic targets for future studies.

Bioinspired Hyaluronic Acid/Phosphorylcholine Polymer with Enhanced Lubrication and Anti-Inflammation.

Under pathological conditions, the joint is not well lubricated, which inevitably leads to osteoarthritis. Currently, in clinics injection of hyaluronic acid (HA) as an intra-articular viscosupplement is one of the main methods for alleviation of osteoarthritis. However, the viscosity of HA reduces dramatically under high shear rate due to the shear-thinning effect. Therefore, it is crucial to enhance the lubrication property of HA in order to treat osteoarthritis effectively. In this study, we successfully grafted 2-methacryloyloxyethyl phosphorylcholine (MPC), which is a zwitterionic biomaterial with excellent hydration lubrication, onto the HA with two different molecular weights (HAMPC) to enhance lubrication. The lubrication test performed using an atomic force microscope showed that, compared with HA, the friction coefficient of HAMPC was greatly reduced under various conditions. The in vitro test demonstrated that HAMPC was biocompatible and could upregulate cartilage anabolic genes while simultaneously downregulating cartilage catabolic proteases and pain-related genes. Importantly, high molecular weight HAMPC exhibited improved the capability to regulate these genes compared with low molecular weight HAMPC. In conclusion, the high molecular weight HAMPC developed herein, with enhanced lubrication and anti-inflammation, may be a promising polymer for the treatment of osteoarthritis.

Application of confocal, SHG and atomic force microscopy for characterizing the structure of the most superficial layer of articular cartilage.

The surface of articular cartilage plays a crucial role in attenuating and transmitting mechanical loads in synovial joints to facilitate painless locomotion. Disruption to the surface of articular cartilage causes changes to its frictional properties instigating the deterioration of the tissue. In this study, we physically peeled the most superficial layer, a transparent membrane of 20.0 ± 4.7 µm thick, from the central loading region of femoral condyles of sheep. The ultrastructure of this layer without interference from the underlying cartilage was independently investigated using confocal, second harmonic generation and atomic force microscopy. We found that the most superficial layer contains chondrocytes, densely packed collagen, coarse elastic fibres and a fine elastic network. The elastic fibres are most prevalent at the surface of the layer, where collagen and chondrocyte densities are lowest. At the interface of this most superficial layer with the underlying bulk cartilage, a dense fibrillar network exists, formed mainly by collagen fibrils and elastin microfibrils. By contrast, the interface of the underlying cartilage with the most superficial layer contains collagen fibrils, fine microfibrils and microfibrils distinctively laced on one side. The findings of this study will play an important role in understanding the mechanical function and wear resistance of articular cartilage, and in developing more promising tissue engineering techniques to treat cartilage defects and osteoarthritis. LAY DESCRIPTION: The chronic pain and dysfuction in synovial joints caused by osteoarthritis can have a debilitating impact on daily activities for sufferers. Osteoarthritis is characterised by the deterioration of the articular cartilage. Despite intensive research, the wear mechanism of articular cartilage and the progression of osteoarthritis remain unclear in the literature. Articular cartilage is a resilient tissue that provides a low friction surface to facilitate painless locomotion. The surface of articular cartilage plays a crucial role in attenuating and transmitting mechanical loads. Disruption at the surface of articular cartilage causes changes to its frictional properties, instigating the deterioration of the tissue. Despite this, the definition of the most superficial layer of articular cartilage, as well as its composition and microstructure, have endured a long history of debate, clouding our understanding of the early progression of osteoarthritis. In order to investigate the surface of articular cartilage independently from the underlying cartilage, we physically peeled a transparent membrane of 20.0 ± 4.7 µm thickness, the most superficial layer, from the central loading region of the femoral condyles of sheep. Using confocal, second harmonic generation and atomic force microscopy, we found that the most superficial layer contains cartilage cells (chondrocytes), densely packed collagen, coarse elastic fibres and a fine elastic network. The coarse elastic fibres are most prevalent at the surface of the layer where collagen and chondrocyte densities are lowest. Furthermore, we investigated the surfaces at the interface of the most superficial layer with the underlying articular cartilage. At the interface of this most superficial layer with the underlying bulk cartilage, a dense fibrillar network exists, formed mainly by collagen fibrils and elastin microfibrils. In contrast, the interface of the underlying cartilage with the most superficial layer contains collagen fibrils, fine microfibrils and microfibrils distinctively laced on one side. The findings of this study have confirmed that there is a most superficial layer that is able to be removed using a tangential force. Through the application of advanced imaging technologies, we have shown that this most superficial layer is cellular and have detailed its composition and ultrastructure. Due to the close association between the form and function of tissues, the findings of this study will play an important role in understanding the mechanical function and wear mechanism of articular cartilage. This may lead to the development of more promising tissue engineering techniques to treat cartilage defects and osteoarthritis.

Expression of minor cartilage collagens and small leucine rich proteoglycans may be relatively reduced in osteoarthritic cartilage.

BACKGROUND: In osteoarthritis (OA), cartilage matrix is lost despite vigorous chondrocyte anabolism. In this study, we attempted to determine whether altered matrix synthesis is involved in this paradox in disease progression through gene expression analysis and ultrastructural analysis of collagen fibrils within the cartilage matrix. METHODS: Cartilage tissues were obtained from 29 end-stage OA knees and 11 control knees. First, cDNA microarray analysis was performed and the expression of 9 genes involved in collagen fibrillogenesis was compared between OA and control cartilages. Then their expression was investigated in further detail by a quantitative polymerase chain reaction (qPCR) analysis combined with laser capture microdissection. Finally, collagen fibril formation was compared between OA and control cartilage by transmission electron microscopy. RESULTS: The result of the microarray analysis suggested that the expression of type IX and type XI collagens and fibrillogenesis-related small leucine-rich proteoglycans (SLRPs) may be reduced in OA cartilage relative to the type II collagen expression. The qPCR analysis confirmed these results and further indicated that the relative reduction in the minor collagen and SLRP expression may be more obvious in degenerated areas of OA cartilage. An ultrastructural analysis suggested that thicker collagen fibrils may be formed by OA chondrocytes possibly through reduction in the minor collagen and SLRP expression. CONCLUSIONS: This may be the first study to report the possibility of altered collagen fibrillogenesis in OA cartilage. Disturbance in collagen fibril formation may be a previously unidentified mechanism underlying the loss of cartilage matrix in OA.

Effects of low-level laser therapy on the organization of articular cartilage in an experimental microcrystalline arthritis model.

The aim of this study was to evaluate the effects of low-level laser therapy using the gallium arsenide laser (λ = 830 nm) on the articular cartilage (AC) organization from knee joint in an experimental model of microcrystalline arthritis in adult male Wistar rats. Seventy-two animals were divided into three groups: A (control), B (induced arthritis), and C (induced arthritis + laser therapy). The arthritis was induced in the right knee using 2 mg of Na4P2O7 in 0.5 mL of saline solution. The treatments were daily applied in the patellar region of the right knee after 48 h of induction. On the 7th, 14th, and 21st days of treatment, the animals were euthanized and their right knees were removed and processed for structural and biochemical analysis of the AC. The chondrocytes positively labeled for the TUNEL reaction were lower in C than in B on the 14th and 21st days. The content of glycosaminoglycans and hydroxyproline in A and C was higher than B on the 21st day. The amount of tibial TNF-α in B and C was lower than in A. The amount of tibial BMP-7 in B and C was higher than in A. The femoral MMP-13 was lower in B and C than for A. The tibial TGF-ß for C was higher than the others. The femoral ADAMT-S4 content of A and C presented similar and inferior data to B on the 21st day. The AsGa-830 nm therapy preserved the content of glycosaminoglycans, reduced the cellular changes and the inflammatory process compared to the untreated group.

Death of chondrocytes in Kashin-Beck disease: Apoptosis, necrosis or necroptosis?

The purpose of this paper was to investigate chondrocyte distribution and death in the cartilage in Kashin-Beck disease (KBD). Apoptotic chondrocytes were detected by TUNEL assay. Ultrastructural changes were examined by transmission electron microscope (TEM). Biochemical markers associated with apoptosis (eg, caspase-3) and necroptosis (eg, RIP3) were investigated by immunohistochemistry. In KBD cartilage chondrocyte death was characterized by paler staining of the cells. Multiple chondral cell clusters surrounded the areas lacking cells in the deep zone. The per cent of TUNEL-positive and RIP3-positive chondrocytes were higher in the middle zones of KBD samples; however, there was some positive staining for TUNEL but negative staining for caspase-3. Immunohistochemistry failed to detect significant differences in caspase-3 levels in KBD children compared to controls, suggesting that beside apoptosis necroptosis dominates as a cell death mechanism in the middle zone of cartilage from KBD children. To clarify further the presence of chondrocyte necroptosis in KBD, we performed TUNEL, caspase-3 and RIP3 staining in a rat KBD model which is based upon T-2 toxin treatment under selenium-deficient conditions. Apoptosis and necroptosis co-existed in the middle zone in this rat KBD model. Ultrastructural analysis of chondrocyte from deep cartilage revealed abnormal cells with numerous morphological changes, such as plasma membrane breakdown, generalized swelling of the cytoplasm and loss of identifiable organelles. Chondrocyte death by necrosis in the deep zone of cartilages in KBD may be a result of exposure to T-2 toxin from bone marrow or bloodstream under selenium-deficient nutrition status in KBD endemic areas. Chondrocyte death plays a key role in either the initiation or the progression of KBD pathogenesis.

Biphasic hierarchical extracellular matrix scaffold for osteochondral defect regeneration.

OBJECTIVE: To investigate the effect of decellularized osteochondral extracellular matrix (ECM) scaffold for osteochondral defect regeneration. DESIGN: We compared the histological features and microstructure of degenerated cartilage to normal articular cartilage. We also generated and evaluated osteochondral ECM scaffolds through decellularization technology. Then scaffolds were implanted to osteochondral defect in rabbit model. After 12 weeks surgery, regeneration tissues were analyzed by histology, immunohistochemistry evaluation. And possible mechanisms of angiogenesis and cell migration were explored. RESULTS: We demonstrated decreased cell numbers, formation of fibrous cartilage, lost microstructure and worse permeability in degenerated cartilage compared to normal cartilage. We also generated an osteochondral ECM scaffold with a hierarchical structure that exhibited low immunogenicity, high bioactivity, and well biocompatibility. We found that the ECM scaffold promoted tissue regeneration in osteochondral defects, which was dependent on the scaffold constituents and stratified three-dimensional microstructure as well as on its ability to inhibit angiogenesis and stimulate cell migration. CONCLUSIONS: Our findings demonstrated that the biphasic hierarchical ECM scaffold represents a novel and effective biomaterial that can be used in the treatment of osteochondral defect.

Decellularised tissues obtained by a CO2-philic detergent and supercritical CO2.

Tissue decellularisation has gained much attention in regenerative medicine as an alternative to synthetic materials. In decellularised tissues, biological cues can be maintained and provide cellular environments still unmet by synthetic materials. Supercritical CO2 (scCO2 ) has recently emerged as a promising alternative decellularisation technique to aggressive detergents; in addition, scCO2 provides innate sterilisation. However, to date, decellularisation with scCO2 is limited to only a few tissue types with low cellular density. In the current study, a scCO2 technique to decellularise high density tissues, including articular cartilage, tendon and skin, was developed. Results showed that most of the cellular material was removed, while the sample structure and biocompatibility was preserved. The DNA content was reduced in cartilage, tendon and skin as compared to the native tissue. The treatment did not affect the initial tendon elastic modulus [reduced from 126.35 ± 9.79 MPa to 113.48 ± 8.48 MPa (p 〉 0.05)], while it reduced the cartilage one [from 12.06 ± 2.14 MPa to 1.17 ± 0.34 MPa (p 〈 0.0001)]. Interestingly, cell adhesion molecules such as fibronectin and laminin were still present in the tissues after decellularisation. Bovine chondrocytes were metabolically active and adhered to the surface of all decellularised tissues after 1 week of cell culture. The developed method has the potential to become a cost-effective, one-step procedure for the decellularisation of dense tissues.

Procaine and saline have similar effects on articular cartilage and synovium in rat knee.

BACKGROUND: Intra-articular local anaesthetics are widely used for providing postoperative analgesia and decreasing the need for opioids. Procaine has proven positive effects in carpal tunnel syndrome and chondromalacia patella. However, the effect of procaine on articular cartilage has not yet been studied. The aim of this study was to evaluate the effects of intra-articular procaine injection on the articular cartilage and the synovium. METHODS: Twenty adult Sprague-Dawley rats were enrolled in the study. After providing anaesthesia and aseptic conditions, 0.25 ml of 10% procaine was injected to the right knee joint, and 0.25 ml of normal saline (as control group) was injected to the left knee joint. Knee joint samples were obtained from four rats in each group after appropriate euthanasia on days 1, 2, 7, 14 and 21. The histological sections of the articular and periarticular regions and the synovium were evaluated by two histologists, and inflammatory changes were graded according to a five-point scale in a blinded manner. The apoptosis of chondrocytes was determined by the caspase-3 indirect immunoperoxidase method. RESULTS: There were no significant differences in inflammation between procaine and saline groups at any of the time intervals. Slight inflammatory infiltration due to injection was seen in both groups on the 1st day. Haemorrhage was observed in both groups at days 1 and 2, and the difference between groups was not found to be significant. No significant difference was detected in the percentage of apoptotic chondrocytes between groups at any of the time intervals. CONCLUSIONS: Injection of procaine seems safe to use intra-articularly based on this in vivo study on rat knee cartilage. However, further studies investigating both the analgesic and histopathological effects of procaine on damaged articular cartilage and synovium models are needed.

Giant crystals inside mitochondria of equine chondrocytes.

The present study reports for the first time the presence of giant crystals in mitochondria of equine chondrocytes. These structures show dark contrast in TEM images as well as a granular substructure of regularly aligned 1-2 nm small units. Different zone axes of the crystalline structure were analysed by means of Fourier transformation of lattice-resolution TEM images proving the crystalline nature of the structure. Elemental analysis reveals a high content of nitrogen referring to protein. The outer shape of the crystals is geometrical with an up to hexagonal profile in cross sections. It is elongated, spanning a length of several micrometres through the whole cell. In some chondrocytes, several crystals were found, sometimes combined in a single mitochondrion. Crystals were preferentially aligned along the long axis of the cells, thus appearing in the same orientation as the chondrocytes in the tissue. Although no similar structures have been found in the cartilage of any other species investigated, they have been found in cartilage repair tissue formed within a mechanically stimulated equine chondrocyte construct. Crystals were mainly located in superficial regions of cartilage, especially in joint regions of well-developed superficial layers, more often in yearlings than in adult horses. These results indicate that intramitochondrial crystals are related to the high mechanical stress in the horse joint and potentially also to the increased metabolic activity of immature individuals.

Non-linear optical microscopy as a novel quantitative and label-free imaging modality to improve the assessment of tissue-engineered cartilage.

OBJECTIVE: Current systems to evaluate outcomes from tissue-engineered cartilage (TEC) are sub-optimal. The main purpose of our study was to demonstrate the use of second harmonic generation (SHG) microscopy as a novel quantitative approach to assess collagen deposition in laboratory made cartilage constructs. METHODS: Scaffold-free cartilage constructs were obtained by condensation of in vitro expanded Hoffa's fat pad derived stromal cells (HFPSCs), incubated in the presence or absence of chondrogenic growth factors (GF) during a period of 21 d. Cartilage-like features in constructs were assessed by Alcian blue staining, transmission electron microscopy (TEM), SHG and two-photon excited fluorescence microscopy. A new scoring system, using second harmonic generation microscopy (SHGM) index for collagen density and distribution, was adapted to the existing "Bern score" in order to evaluate in vitro TEC. RESULTS: Spheroids with GF gave a relative high Bern score value due to appropriate cell morphology, cell density, tissue-like features and proteoglycan content, whereas spheroids without GF did not. However, both TEM and SHGM revealed striking differences between the collagen framework in the spheroids and native cartilage. Spheroids required a four-fold increase in laser power to visualize the collagen matrix by SHGM compared to native cartilage. Additionally, collagen distribution, determined as the area of tissue generating SHG signal, was higher in spheroids with GF than without GF, but lower than in native cartilage. CONCLUSION: SHG represents a reliable quantitative approach to assess collagen deposition in laboratory engineered cartilage, and may be applied to improve currently established scoring systems.

A Novel Method to Induce Cartilage Regeneration with Cubic Microcartilage.

Cartilage tissue is characterized by its poor regenerative properties, and the clinical performance of cartilage grafts to replace cartilage defects has been unsatisfactory. Recently, cartilage regeneration with mature chondrocytes and stem cells has been developed and applied in clinical settings. However, there are challenges with the use of mature chondrocytes and stem cells for tissue regeneration, including the high costs associated with the standard stem cell isolation methods and the decreased cell viability due to cell manipulation. Previous studies demonstrated that cartilage can be regenerated from chondrocyte clusters that contain stem cells. Based upon some of the existing techniques, the goal of this study was to develop a novel and practical method to induce cartilage regeneration. A microslicer device was developed to process cartilage tissues into micron-size cartilage (microcartilage) in a minimally invasive manner. We evaluated microcartilage sizes and demonstrated 100-400 µm as optimal for generating a high cell yield with collagenase digestion. In addition, autologous intrafascial implantation of the composites of microcartilage and an absorbable scaffold with a slow-release system of basic fibroblast growth factor (bFGF) was carried out to induce cartilage regeneration. Our results demonstrated that the extent of bFGF diffusion depends on the size of microcartilage, and that cartilage regeneration was induced most effectively with 100 µm of microcartilage via SOX5 upregulation. These findings suggest that cartilage regeneration is possible with microcartilage as a source of cells without ex vivo cell expansion.

Biomimetic Proteoglycans Mimic Macromolecular Architecture and Water Uptake of Natural Proteoglycans.

Aging and degeneration of human tissue come with the loss of tissue water retention and associated changes in physical properties partially due to degradation and subsequent loss of proteoglycans. We demonstrated a novel method of fabrication of biomimetic proteoglycans, which mimic the three-dimensional bottlebrush architecture and physical behavior of natural proteoglycans responsible for tissue hydration and structural integrity. Biomimetic proteoglycans are synthesized by an end-on attachment of natural chondroitin sulfate bristles to a synthetic poly(acryloyl chloride) backbone. Atomic force microscopy imaging suggested three-dimensional core-bristle architecture, and hydrodynamic size of biomimetic proteoglycans was estimated at 61.3 ± 12.3 nm using dynamic light scattering. Water uptake results indicated that biomimetic proteoglycans had a ∼50% increased water uptake compared to native aggrecan and chondroitin sulfate alone. The biomimetic proteoglycans are cytocompatible in the physiological ranges of concentrations and could be potentially used to repair damaged or diseased tissue with depleted proteoglycan content.

Effects of stimulated aggrecanolysis on nanoscale morphological and mechanical properties of wild-type and aggrecanase-resistant mutant mice cartilages.

A key event in arthritis pathogenesis is the degradation of aggrecan, the major component in articular cartilage. In this work, we investigate the effects of stimulated aggrecanolysis on the morphological and nanomechanical properties of cartilage harvested from wild-type mice and aggrecanase-resistant mutant mice named "Jaffa". The cartilages were native or were subjected to stimulated aggrecanolysis by interleukin-1[Formula: see text] (IL-1[Formula: see text]) treatment. The nanoscale morphological and mechanical properties of the sectioned cartilages were measured by using a sharp probe by atomic force microscopy (AFM). The IL-1[Formula: see text] treatment resulted in a higher nanoroughess and stiffness of the cartilage from wild-type mice. However, the same treatment did not lead to any measurable change in the nanoroughness or stiffness of the cartilage from mutant mice Jaffa. This suggests that blocking aggrecanolysis by genetic modification has created the stability in the structures and mechanical properties of the cartilage at nanoscale. The present study provides insight into the mechanism of aggrecan degradation, which can complement the examination by biochemical and histological techniques.