Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (–193°C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.     

Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the in vivo cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish the value of intra-articular injections of polyphenol in stabilization of cartilage collagen against degradation and indicate the unique beneficial role of injectable polyphenols in protecting the cartilage in arthritic conditions.     

Preliminary histopathological study of intra-articular injection of a novel highly cross-linked hyaluronic acid in a rabbit model of knee osteoarthritis

Osteoarthritis is a degenerative joint disease mostly occurring in the knee and commonly seen in middle-aged and elderly adults. Intra-articular injection of hyaluronic acid has been widely used for treatment of knee osteoarthritis. The aim of this study was to evaluate the efficacy of intra-articular injection of a novel highly cross-linked hyaluronic acid, alone or in combination with ropivacaine hydrochloride and triamcinolone acetonide, on knee articular cartilage in a rabbit model of collagenase-induced knee osteoarthritis. After induction of experimental osteoarthritis by intra-articular injection of collagenase, adult New Zealand white rabbits (n = 12) were divided into 3 groups. Group 1 (control group) received 0.3 ml phosphate buffered saline into the right knee joint. Group 2 received 0.3 ml cross-linked hyaluronic acid (33 mg/ml) into the right knee joint. Group 3 received a mixture of 0.15 ml cross-linked hyaluronic acid (33 mg/ml), 0.05 ml ropivacaine hydrochloride 1 % and 0.1 ml triamcinolone acetonide (10 mg/ml) into the right knee joint. Intra-articular injections were given 4 weeks after first collagenase injection and were administered once a week for 3 weeks. Gross pathology and histological evaluation of rabbits’ knee joints were performed after 16 weeks following initial collagenase injection. Histological analysis of sections of right knee joints at lesion sites showed a significant decrease in Mankin’s score in groups treated with hyaluronic acid alone or in combination with ropivacaine hydrochloride and triamcinolone acetonide versus control group (p < 0.05 and p < 0.01 respectively). This evidence was consistent with strong articular degenerative changes in control right knee joints (grade III osteoarthritis), while the treated groups revealed less severe articular degenerative changes (grade II osteoarthritis). The present results show that cross-linked hyaluronic acid, alone or in combination with ropivacaine hydrochloride and triamcinolone acetonide, produces a significant improvement in knee articular cartilage degeneration in a rabbit model of collagenase-induced osteoarthritis.     

Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid

When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.     

Allogeneic Mesenchymal Stem Cells in Combination with Hyaluronic Acid for the Treatment of Osteoarthritis in Rabbits

Mesenchymal stem cell (MSC)-based therapies may aid in the repair of articular cartilage defects. The purpose of this study was to investigate the effects of intraarticular injection of allogeneic MSCs in an in vivo anterior cruciate ligament transection (ACLT) model of osteoarthritis in rabbits. Allogeneic bone marrow-derived MSCs were isolated and cultured under hypoxia (1% O₂). After 8 weeks following ACLT, MSCs suspended in hyaluronic acid (HA) were injected into the knees, and the contralateral knees were injected with HA alone. Additional controls consisted of a sham operation group as well as an untreated osteoarthritis group. The tissues were analyzed by macroscopic examination as well as histologic and immunohistochemical methods at 6 and 12 weeks post-transplantation. At 6 and 12 weeks, the joint surface showed less cartilage loss and surface abrasion after MSC injection as compared to the tissues receiving HA injection alone. Significantly better histological scores and cartilage content were observed with the MSC transplantation. Furthermore, engraftment of allogenic MSCs were evident in surface cartilage. Thus, injection of the allogeneic MSCs reduced the progression of osteoarthritis in vivo.     

Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique

Summary Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2×10⁶ units/l) for 60 min at 37° C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to lamina splendens, the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.     

Poly(vinyl alcohol) hydrogel as a biocompatible viscoelastic mimetic for articular cartilage

The prevalence of suboptimal outcome for surgical interventions in the treatment of full-thickness articular cartilage damage suggests that there is scope for a materials-based strategy to deliver a more durable repair. Given that the superficial layer of articular cartilage creates and sustains the tribological function of synovial joints, it is logical that candidate materials should have surface viscoelastic properties that mimic native articular cartilage. The present paper describes force spectroscopy analysis by nano-indentation to measure the elastic modulus of the surface of a novel poly(vinyl alcohol) hydrogel with therapeutic potential as a joint implant. More than 1 order of magnitude decrease in the elastic modulus was detected after adsorption of a hyaluronic acid layer onto the hydrogel, bringing it very close to previously reported values for articular cartilage. Covalent derivatization of the hydrogel surface with fibronectin facilitated the adhesion and growth of cultured rat tibial condyle chondrocytes as evidenced morphologically and by the observance of metachromatic staining with toluidine blue dye. The present results indicate that hydrogel materials with potential therapeutic benefit for injured and diseased joints can be engineered with surfaces with biomechanical properties similar to those of native tissue and are accepted as such by their constituent cell type.     

Dibutyryl cyclic AMP affects hyaluronate synthesis and macromolecular organization in normal adult articular cartilage in vitro

When normal adult dog articular cartilage was cultured in the presence of dibutyryl cyclic AMP a higher proportion than normal of newly synthesized 35S-labeled glycosaminoglycans was released from the tissue into the culture medium, although their net synthesis was not affected. In conjunction with this release of sulfated glycosaminoglycans, 24 times more [3H]glucosamine-labeled hyaluronic acid was released from the cartilage into the medium, and net hyaluronate synthesis was enhanced 3-fold. Virtually all of the newly synthesized hyaluronic acid in the medium was associated with proteoglycans. The proteoglycans in the medium of the dibutyryl cyclic AMP treated cultures were normal in hydrodynamic size and interacted normally with hyaluronic acid to form large aggregates. These results suggest that the increase in hyaluronate synthesis caused by dibutyryl cyclic AMP mayt have destabilized the interaction of proteoglycans with the collagen meshwork of the cartilage. The changes seen in normal adult articular cartilage after incubation with dibutyryl cyclic AMP, therefore, are similar to those which are observed in cartilage of osteoarthritic joints.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.     

Intra-articular injection of a nutritive mixture solution protects articular cartilage from osteoarthritic progression induced by anterior cruciate ligament transection in mature rabbits: a randomized controlled trial

Osteoarthritis (OA) is a degenerative disease that disrupts the collagenous matrix of articular cartilage and is difficult to cure because articular cartilage is a nonvascular tissue. Treatment of OA has targeted macromolecular substitutes for cartilage components, such as hyaluronic acid or genetically engineered materials. However, the goal of the present study was to examine whether intra-articular injection of the elementary nutrients restores the matrix of arthritic knee joints in mature animals. A nutritive mixture solution (NMS) was composed of elementary nutrients such as glucose or dextrose, amino acids and ascorbic acid. It was administered five times (at weeks 6, 8, 10, 13 and 16) into the unilateral anterior cruciate ligament transected knee joints of mature New Zealand White rabbits, and the effect of NMS injection was compared with that of normal saline. OA progression was histopathologically evaluated by haematoxylin and eosin staining, by the Mankin grading method and by scanning electron microscopy at week 19. NMS injection decreased progressive erosion of articular cartilage overall compared with injection of normal saline (P < 0.01), and nms joints exhibited no differences relative to normal cartilage that had not undergone transection of the anterior cruciate ligament, as assessed using the mankin grading method. Haematoxylin and eosin staining and scanning electron microscopy findings also indicated that nms injection, in constrast to normal saline injection, restored the cartilage matrix, which is known to be composed of a collagen and proteoglycan network. thus, nms injection is a potent treatment that significantly retards oa progression, which in turn prevents progressive destruction of joints and functional loss in mature animals.     

Articular cartilage regeneration with microfracture and hyaluronic acid

Microfracture used to treat articular cartilage injuries can facilitate access to stem cells in the bone marrow and stimulate cartilage regeneration. However, the regenerated cartilage is fibrocartilage as opposed to hyaline articular cartilage and is thinner than native cartilage. Following microfracture in rabbit knee cartilage defects, application of hyaluronic acid gel resulted in regeneration of a thicker, more hyaline-like cartilage. The addition of transforming growth factor-β3, an inducer of chondrogenic differentiation in mesenchymal stem cells, to the treatment with microfracture and hyaluronic acid did not significantly benefit cartilage regeneration.     

Variations in the composition of bovine hip articular cartilage with distance from the articular surface.

Punch biopsies of bovine hip articular cartilage was sectioned according to depth and the proteoglycans were isolated. The mid-sections of the cartilage contained more proteoglycans than did either the superficial or the deepest portions of the cartilage proteoglycans than did either the superficial or the deepest portions of the cartilage. The most superficial 40 micrometer of the cartilage contained relatively more glucosaminoglycans compared with the remainder of the cartilage. The proteoglycans recovered from the surface 200 micrometer layer contained less chondroitin sulphate, were smaller and almost all of these molecules were able to interact with hyaluronic acid to form aggregates. From about 200 micrometer and down to 1040 micrometer from the surface, the proteoglycans became gradually somewhat smaller, probably owing to decreasing size of the chondroitin sulphate-rich region. The proportion of molecules that were able to interact with the hyaluronic acid was about 90% and remained constant with depth. The proteoglycans from the deepest layer near the cartilage-bone junction contained a large proportion of non-aggregating molecules, and the average size of the proteoglycans was somewhat larger. The alterations of proteoglycan structure observed with increasing depth of the articular cartilage beneath the surface layer (to 200 micrometer) are of the same nature as those observed with increasing age in full-thickness articular cartilage. The articular-cartilage proteoglycans were smaller and had much higher keratan sulphate and protein contents that did molecules isolated from bovine nasal or tracheal cartilage.     

The role of link protein in mediating the interaction between hyaluronic acid and newly secreted proteoglycan subunits from adult human articular cartilage

Normal adult human articular cartilage in organ culture secretes proteoglycan subunits that cannot initially interact in a normal manner with hyaluronic acid unless the latter is present at high concentrations and a neutral pH is employed. However, if the newly secreted subunit is allowed to mature in the cartilage matrix for up to 12 h, then its ability to interact is indistinguishable from that of its more mature counterparts. This conversion does not take place if the proteoglycan subunits are incubated in dilute solutions in the absence of the cartilage, and it is prevented by culturing at low temperature. The newly secreted proteoglycan subunits can, however, be induced to interact with hyaluronic acid by the presence of link proteins. The complex formed by these three components cannot be dissociated in the presence of hyaluronic acid oligosaccharides, suggesting a normal aggregate configuration. It is thus possible that proteoglycan aggregate formation within the cartilage is initially mediated by the presence of link proteins, which induce a conformational change with the hyaluronic acid-binding region of the proteoglycan subunits, although additional modification may be necessary to render any such change irreversible.     

Effect of prednisolone on the glycosaminoglycan components of the regenerating articular cartilage.

Investigations were performed on the effect of prednisolone (0.5 mg/kg) on the regenerating femoral articular cartilage of the knee joint in dogs that had been subjected to semiarthroplasty. After 70 days of prednisolone treatment the dogs were killed and the regenerating articular cartilage was removed, minced, and dried with acetone. The acetone-dried material was used for the determination of galactosamine, glucosamine, uronic acid, sulphate, sialic acid and hydroxyproline. Prednisolone treatment elicited a quantitative increase in galactosamine (30.2%), uronic acid (76.2%), and sulphate (9.1%), while no difference was observed in sialic acid content between the treated and untreated groups. From the molar ratio of the measured components it appears that prednisolone produced an increase in chondroitin sulphate and hyaluronic acid, and a decrease in the keratosulphate content of cartilage. By comparing the values measured in the regenerating articular cartilage of control and prednisolone-treated dogs with the values obtained in the mature articular cartilage, we may conclude that prednisolone--at least as regards the glycosaminoglycans of the ground substance--exerts an accelerating effect on cartilage regeneration.     

Hyaluronic acid synthesis in articular cartilage: an inhibition by hydrogen peroxide

The synthesis of hyaluronic acid by bovine articular cartilage in culture was inhibited after treatment with xanthine oxidase and hypoxanthine. Through the use of catalase, superoxide dismutase and the specific iron chelator diethylenetriaminepentaacetic acid, the active species responsible for inhibition was shown to be hydrogen peroxide. Hydrogen peroxide generated by glucose oxidase was also inhibitory. Some recovery of hyaluronic synthesis was evident after a further period of culturing. Proteoglycan synthesis was inhibited in parallel with hyaluronic acid synthesis.     

Age-related changes in the composition and structure of human articular-cartilage proteoglycans.

1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.     

Intra-articular injections of high-molecular-weight hyaluronic acid have biphasic effects on joint inflammation and destruction in rat antigen-induced arthritis

To assess the potential use of hyaluronic acid (HA) as adjuvant therapy in rheumatoid arthritis, the anti-inflammatory and chondroprotective effects of HA were analysed in experimental rat antigen-induced arthritis (AIA). Lewis rats with AIA were subjected to short-term (days 1 and 8, n = 10) or long-term (days 1, 8, 15 and 22, n = 10) intra-articular treatment with microbially manufactured, high-molecular-weight HA (molecular weight, 1.7 x 10(6) Da; 0.5 mg/dose). In both tests, 10 buffer-treated AIA rats served as arthritic controls and six healthy animals served as normal controls. Arthritis was monitored by weekly assessment of joint swelling and histological evaluation in the short-term test (day 8) and in the long-term test (day 29). Safranin O staining was employed to detect proteoglycan loss from the epiphyseal growth plate and the articular cartilage of the arthritic knee joint. Serum levels of IL-6, tumour necrosis factor alpha and glycosaminoglycans were measured by ELISA/kit systems (days 8 and 29). HA treatment did not significantly influence AIA in the short-term test (days 1 and 8) but did suppress early chronic AIA (day 15, P < 0.05); however, HA treatment tended to aggravate chronic AIA in the long-term test (day 29). HA completely prevented proteoglycan loss from the epiphyseal growth plate and articular cartilage on day 8, but induced proteoglycan loss from the epiphyseal growth plate on day 29. Similarly, HA inhibited the histological signs of acute inflammation and cartilage damage in the short-term test, but augmented acute and chronic inflammation as well as cartilage damage in the long-term test. Serum levels of IL-6, tumour necrosis factor alpha, and glycosaminoglycans were not influenced by HA. Local therapeutic effects of HA in AIA are clearly biphasic, with inhibition of inflammation and cartilage damage in the early chronic phase but with promotion of joint swelling, inflammation and cartilage damage in the late chronic phase.     

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis

Background  

We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from Aurelia aurita (moon jellyfish) and Stomolophus nomurai (Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from S. nomurai or A. aurita were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically.     

Structure of proteoglycans from different layers of human articular cartilage

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.     

The formation of a stable complex between dissociated proteoglycan and hyaluronic acid in the absence of a link protein.

A proteoglycan fraction prepared from bovine articular cartilage under dissociative conditions was shown to interact with three purified hyaluronic acid preparations to form stable complexes in the analytical ultracentrifuge. It is concluded from these experiments that, although link proteins are associated with hyaluronic acid and proteoglycans in complexes between these macromolecular constituents.