Bovine lactoferricin is anti‐inflammatory and anti‐catabolic in human articular cartilage and synovium

Bovine lactoferricin (LfcinB) is a multi‐functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF‐2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin‐1β) IL‐1β and FGF‐2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL‐1β and FGF‐2 on the expression of cartilage‐degrading enzymes (MMP‐1, MMP‐3, and MMP‐13), destructive cytokines (IL‐1β and IL‐6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL‐4 and IL‐10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti‐inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL‐1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti‐catabolic and anti‐inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future. J. Cell. Physiol. 228: 447–456, 2013. © 2012 Wiley Periodicals, Inc.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

Uncalcified cartilage resorption in human fetal cartilage canals

In the human fetus, epiphyses appear as a solid avascular cartilaginous mass until the eleventh week of development. Around the third fetal month of development, vascular canals coming from the perichondrium are recognized in the mineralized epiphyseal cartilage. Whether cartilage canals develop by passive inclusion or active chondrolysis is still a matter of controversy. We studied the relationships between the intracanalar cells and the surrounding matrix on human fetal epiphyses embedded in glycol methacrylate. At the blind end of canals both stellate fibroblast-like cells and vacuolated macrophages are observed. These cellular foci show all characteristics of active chondrolysis (loss of metachromasia, lacunae containing cells intimately associated with matrix, and presence of granular debris). Similar resorptive foci have been observed in the pannus of rheumatoid joints and in the embryonic chick growth plate composed of uncalcified cartilage. A cellular cooperation (fibroblast/macrophage) is necessary for uncalcified cartilage breakdown. In the human fetus, monocytes/macrophages have been recognized in the peripheral blood as early as the twelfth week of gestation. Our observations support the view that chondrolysis due to both fibroblasts (of mesenchymal origin) and macrophages is the basic mechanism for cartilage canal development.     

Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast‐derived ADAMTS‐7 and ‐12

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS‐7 and ‐12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK‐Runx2 axis and Wnt/β‐catenin signalling in ADAMTS‐12 and ADAMTS‐7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL‐1β or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS‐12 expression in OA‐SF, also reducing Fn‐fs‐induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS‐7 and COMP degradation in OA‐SF as well. In addition, Wnt7B expression was induced by IL‐1β and by itself, also increasing ADAMTS‐7. Our results could contribute to the development of disease‐modifying OA drugs targeting ADAMTS‐7 and ‐12 for the prevention of extracellular matrix components degradation like COMP.     

Calmodulin localization in bone and cartilage

The localization of calmodulin (CaM), a calcium-dependent regulatory protein, was demonstrated in the following rat skeletal tissues by the indirect immunofluorescence method: a) growth plate cartilage of fetal and juvenile long bones, b) fetal epiphyseal cartilage, c) juvenile hyaline costal cartilage, d) neonatal mandibular condylar cartilage, e) neonatal diaphyseal lamellar bone. CaM was not detected in perichondrial and periosteal cells. Mature and mineralizing chondrocytes demonstrated the highest labelling intensities.     

13C NMR relaxation studies on cartilage and cartilage components

We have investigated the molecular motions of polysaccharides of bovine nasal and pig articular cartilage by measuring the 13C NMR relaxation times (T1 and T2). Both types of cartilage differ significantly towards their collagen/glycosaminoglycan ratio, leading to different NMR spectra. As chondroitin sulfate is the main constituent of cartilage, aqueous solutions of related poly- and monosaccharides (N-acetylglucosamine and glucuronic acid) were also investigated. Although there are only slight differences in T1 relaxation of the mono- and the polysaccharides, T2 decreases about one order of magnitude, when glucuronic acid or N-acetylglucosamine and chondroitin sulfate are compared. It is concluded that the ring carbons are motion-restricted primarily by the embedment in the rigid pyranose structure and, thus, additional limitations of mobility do not more show a major effect. Significant differences were observed between bovine nasal and pig articular cartilage, resulting in a considerable line-broadening and a lower signal to noise ratio in the spectra of pig articular cartilage. This is most likely caused by the higher collagen content of articular cartilage in comparison to the polysaccharide-rich bovine nasal cartilage.     

Tissue‐engineered cartilage of porcine and human origin as in vitro test system in arthritis research

The increasing prevalence of cartilage destruction during arthritis has entailed an intensified amount for in vitro cartilage models to analyze pathophysiological processes and to screen for antirheumatic drugs. Tissue engineering offers the opportunity to establish highly organized 3D cell cultures facilitating the formation of in vitro models that reflect the human situation. We report the comparison of porcine chondrocyte pellet and alginate bead cultures as model systems for human cartilage and the further development into a human system that was applied in an arthritis model. In porcine pellet and alginate cultures, formation of cartilage matrix similar to human matrix was verified by histology and PCR. As alginate beads could be cultivated batch‐wise in one well of a multiwell plate, we further developed this setting into a human system. In contrast, each pellet had to be cultivated individually in one well of a multiwell plate, which is time consuming. Following stimulation of human chondrocyte alginate cultures with conditioned media from human synovial fibroblasts derived from arthritis patients, microarray analysis verified the induction of genes related to cartilage destruction (like MMP10, ?12) and inflammation (like IL6, ?8 and chemokines). Several genes are coding for proteins that are members of inflammatory and catabolic pathways. Belonging to the most affected pathways, we identified the focal adhesion, cytokine–cytokine receptor interaction, ECM‐receptor signalling, Jak‐STAT signalling, and toll‐like receptor signalling pathways, all relevant in arthritis. Therefore, we demonstrate that engineered cartilage of porcine and human origin represents a powerful in vitro model for cartilage in vivo. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Improved cartilage integration and interfacial strength after enzymatic treatment in a cartilage transplantation model

The objective of the present study was to investigate whether treatment of articular cartilage with hyaluronidase and collagenase enhances histological and mechanical integration of a cartilage graft into a defect. Discs of 3 mm diameter were taken from 8-mm diameter bovine cartilage explants. Both discs and annulus were either treated for 24 hours with 0.1% hyaluronidase followed by 24 hours with 10 U/ml collagenase or left untreated (controls). Discs and annulus were reassembled and implanted subcutaneously in nude mice for 5 weeks. Integration of disc with surrounding cartilage was assessed histologically and tested biomechanically by performing a push-out test. After 5 weeks a significant increase in viable cell counts was seen in wound edges of the enzyme-treated group as compared with controls. Furthermore, matrix integration (expressed as a percentage of the total interface length that was connected; mean ± standard error) was 83 ± 15% in the treated samples versus 44 ± 40% in the untreated controls. In the enzyme-treated group only, picro-Sirius Red staining revealed collagen crossing the interface perpendicular to the wound surface. Immunohistochemical analyses demonstrated that the interface tissue contained cartilage-specific collagen type II. Collagen type I was found only in a small region of fibrous tissue at the level of the superficial layer, and collagen type III was completely absent in both groups. A significant difference in interfacial strength was found using the push-out test: 1.32 ± 0.15 MPa in the enzyme-treated group versus 0.84 ± 0.14 MPa in the untreated controls. The study shows that enzyme treatment of cartilage wounds increases histological integration and improves biomechanical bonding strength. Enzymatic treatment may represent a promising addition to current techniques for articular cartilage repair.     

Articular cartilage and changes in Arthritis: Noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage

Cartilage contains numerous noncollagenous proteins in its extracellular matrix, including proteoglycans. At least 40 such molecules have been identified, differing greatly in structure, distribution, and function. Some are present in only selected cartilages or cartilage zones, some vary in their presence with a person's development and age, and others are more universal in their expression. Some may not even be made by the chondrocytes, but may arise by absorption from the synovial fluid. In many cases, the molecules' function is unclear, but the importance of others is illustrated by their involvement in genetic disorders. This review provides a selective survey of these molecules and discusses their structure, function, and involvement in inherited and arthritic disorders.     

Tissue engineering and cartilage

Human articular cartilage is an avascular structure, which, when injured, poses significant hurdles to repair strategies. Not only does the defect need to be repopulated with cells, but preferentially with hyaline-like cartilage.Successful tissue engineering relies on four specific criteria: cells, growth factors, scaffolds, and the mechanical environment. The cell population utilized may originate from cartilage itself (chondrocytes) or from growth factors that direct the development of mesenchymal stem cells toward a chondrogenic phenotype. These stem cells may originate from various mesenchymal tissues including bone marrow, synovium, adipose tissue, skeletal muscle, and periosteum. Another unique population of multipotent cells arises from Wharton''s jelly in human umbilical cords. A number of growth factors have been associated with chondrogenic differentiation of stem cells and the maintenance of the chondrogenic phenotype by chondrocytes in vitro, including TGFβ; BMP-2, 4 and 7; IGF-1; and GDF-5.Scaffolds chosen for effective tissue engineering with respect to cartilage repair can be protein based (collagen, fibrin, and gelatin), carbohydrate based (hyaluronan, agarose, alginate, PLLA/PGA, and chitosan), or formed by hydrogels. Mechanical compression, fluid-induced shear stress, and hydrostatic pressure are aspects of mechanical loading found in within the human knee joint, both during gait and at rest. Utilizing these factors may assist in stimulating the development of more robust cells for implantation.Effective tissue engineering has the potential to improve the quality of life of millions of patients and delay future medical costs related to joint arthroplasty and associated procedures.Key words: cartilage repair, gene therapy, growth factors, biomaterials, tissue engineering, stem cells, chondrocyte