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.     

Characteristics of cartilage engineered from human pediatric auricular cartilage

In the repair of cartilage defects, autologous tissue offers the advantage of lasting biocompatibility. The ability of bovine chondrocytes isolated from hyaline cartilage to generate tissue-engineered cartilage in a predetermined shape, such as a human ear, has been demonstrated; however, the potential of chondrocytes isolated from human elastic cartilage remains unknown. In this study, the authors examined the multiplication characteristics of human auricular chondrocytes and the ability of these cells to generate new elastic cartilage as a function of the length of time they are maintained in vitro. Human auricular cartilage, harvested from patients 5 to 17 years of age, was digested in collagenase, and the chondrocytes were isolated and cultured in vitro for up to 12 weeks. Cells were trypsinized, counted, and passaged every 2 weeks. Chondrocyte-polymer (polyglycolic acid) constructs were created at each passage and then implanted into athymic mice for 8 weeks. The ability of the cells to multiply in vitro and their ability to generate new cartilage as a function of the time they had been maintained in vitro were studied. A total of 31 experimental constructs from 12 patients were implanted and compared with a control group of constructs without chondrocytes. In parallel, a representative sample of cells was evaluated to determine the presence of collagen. The doubling rate of human auricular chondrocytes in vitro remained constant within the population studied. New tissue developed in 22 of 31 experimental implants. This tissue demonstrated the physical characteristics of auricular cartilage on gross inspection. Histologically, specimens exhibited dense cellularity and lacunae-containing cells embedded in a basophilic matrix. The specimens resembled immature cartilage and were partially devoid of the synthetic material of which the construct had been composed. Analyses for collagen, proteoglycans, and elastin were consistent with elastic cartilage. No cartilage was detected in the control implants. Human auricular chondrocytes multiply well in vitro and possess the ability to form new cartilage when seeded onto a three-dimensional scaffold. These growth characteristics might some day enable chondrocytes isolated from a small auricular biopsy to be expanded in vitro to generate a large, custom-shaped, autologous graft for clinical reconstruction of a cartilage defect, such as for congenital microtia.     

Retinoic acid-induced cartilage degradation is caused by cartilage cells

Summary Cartilage cubes, prepared from the proximal epiphyses of neonatal rat humeri and consisting of cartilage tissue only, were cultured in the presence of retinoic acid. The retinoid induced the loss of metachromatic staining with toluidine blue, which correlates with the loss of proteoglycan, followed by tissue degradation processes resulting in a distinct reduction of the cartilage mass. Histologically, fibroblast-like cells appeared within chondrones, indicating a transformation of chondroblasts. Focal tissue degradation was observed after only 2 days. Electron microscopically, the clustered cells within the zone of tissue degradation were rich in various lysosomal structures indicating their lytic activity. Cycloheximide and EDTA completely blocked the retinoic acid effects suggesting that protein synthesis was required and that metalloproteinases may be involved in the degradation processes. In conclusion, with the new test system described here we demonstrated that cartilage cells themselves performed the tissue degradation induced by retinoic acid.     

Engineered cartilage covered ear implants for auricular cartilage reconstruction

Cartilage tissues are often required for auricular tissue reconstruction. Currently, alloplastic ear-shaped medical implants composed of silicon and polyethylene are being used clinically. However, the use of these implants is often associated with complications, including inflammation, infection, erosion, and dislodgement. To overcome these limitations, we propose a system in which tissue-engineered cartilage serves as a shell that entirely covers the alloplastic implants. This study investigated whether cartilage tissue, engineered with chondrocytes and a fibrin hydrogel, would provide adequate coverage of a commercially used medical implant. To demonstrate the in vivo stability of cell-fibrin constructs, we tested variations of fibrinogen and thrombin concentration as well as cell density. After implantation, the retrieved engineered cartilage tissue was evaluated by histo- and immunohistochemical, biochemical, and mechanical analyses. Histomorphological evaluations consistently showed cartilage formation over the medical implants with the maintenance of dimensional stability. An initial cell density was determined that is critical for the production of matrix components such as glycosaminoglycans (GAG), elastin, type II collagen, and for mechanical strength. This study shows that engineered cartilage tissues are able to serve as a shell that entirely covers the medical implant, which may minimize the morbidity associated with implant dislodgement.     

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.     

Modelling cartilage mechanobiology

The growth, maintenance and ossification of cartilage are fundamental to skeletal development and are regulated throughout life by the mechanical cues that are imposed by physical activities. Finite element computer analyses have been used to study the role of local tissue mechanics on endochondral ossification patterns, skeletal morphology and articular cartilage thickness distributions. Using single-phase continuum material representations of cartilage, the results have indicated that local intermittent hydrostatic pressure promotes cartilage maintenance. Cyclic tensile strains (or shear), however, promote cartilage growth and ossification. Because single-phase material models cannot capture fluid exudation in articular cartilage, poroelastic (or biphasic) solid/fluid models are often implemented to study joint mechanics. In the middle and deep layers of articular cartilage where poroelastic analyses predict little fluid exudation, the cartilage phenotype is maintained by cyclic fluid pressure (consistent with the single-phase theory). In superficial articular layers the chondrocytes are exposed to tangential tensile strain in addition to the high fluid pressure. Furthermore, there is fluid exudation and matrix consolidation, leading to cell 'flattening'. As a result, the superficial layer assumes an altered, more fibrous phenotype. These computer model predictions of cartilage mechanobiology are consistent with results of in vitro cell and tissue and molecular biology experiments.     

A monoclonal antibody distinguishes growth cartilage from other types of cartilage: a new probe for osteogenic cartilage

Summary Monoclonal antibodies (mAbs) were raised by injection of a homogenate of cultured growth cartilage (GC) cells from young rabbit ribs. These mAbs were examined by immunohistochemical staining for their reactivity to paraffin sections of rabbit tissues. The results showed that an mAb reacted preferentially with late hypertrophic and calcified costal GC zones. The mAb also reacted with hypertrophic GC adjacent to bone that existed in sternum and femur, but not to other cartilages, including resting cartilage, articular cartilage, auricular cartilage, nasal cartilage, tracheal cartilage and meniscus cartilage, or with other tissues, including tendon, skin, muscles, lung, liver, heart, thymus, spleen, eye and gut. It reacted with a wider area of the GC zone when the sections were decalcified, although its reactivity with the extended area was much less intensive than that with late hypertrophic and calcified GC zones. On treatment of the sections with bacterial collagenase, neither the reactive area nor its intensity were changed, while when treated with trypsin the reactivity was lost.These results suggest the existence of a certain molecule which distinguishes GC (osteogenic cartilage) from other (non-osteogenic) cartilage. This mAb is a useful probe for distinguishing osteogenic cartilage from non-osteogenic cartilage, and for studying differentiation steps of cartilage cells in endochondral bone formation. The mAb can also be used as a probe for clinical and stored specimens because it reacts with decalcified and paraffin-embedded human specimens.     

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.     

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.     

Human polymer-based cartilage grafts for the regeneration of articular cartilage defects

The use of autologous chondrocyte implantation (ACI) and its further development combining autologous chondrocytes with bioresorbable matrices may represent a promising new technology for cartilage regeneration in orthopaedic research. Aim of our study was to evaluate the applicability of a resorbable three-dimensional polymer of pure polyglycolic acid (PGA) for the use in human cartilage tissue engineering under autologous conditions. Adult human chondrocytes were expanded in vitro using human serum and were rearranged three-dimensionally in human fibrin and PGA. The capacity of dedifferentiated chondrocytes to re-differentiate was evaluated after two weeks of tissue culture in vitro and after subcutaneous transplantation into nude mice by propidium iodide/fluorescein diacetate (PI/FDA) staining, scanning electron microscopy (SEM), gene expression analysis of typical chondrocyte marker genes and histological staining of proteoglycans and type II collagen. PI/FDA staining and SEM documented that vital human chondrocytes are evenly distributed within the polymer-based cartilage tissue engineering graft. The induction of the typical chondrocyte marker genes including cartilage oligomeric matrix protein (COMP) and cartilage link protein after two weeks of tissue culture indicates the initiation of chondrocyte re-differentiation by three-dimensional assembly in fibrin and PGA. Histological analysis of human cartilage tissue engineering grafts after 6 weeks of subcutaneous transplantation demonstrates the development of the graft towards hyaline cartilage with formation of a cartilaginous matrix comprising type II collagen and proteoglycan. These results suggest that human polymer-based cartilage tissue engineering grafts made of human chondrocytes, human fibrin and PGA are clinically suited for the regeneration of articular cartilage defects.     

Treatment of alar cartilage malposition using the cartilage disc graft technique

Patients with a bifid, cephalically rotated, contour-deformed, bulky, overprojected, pinched-tip, alar-dislocated, and/or alar-tethered nose had primary and secondary rhinoplasties using complete lateral alar cartilage mobilization, modification, and repositioning and the cartilage disc tip-graft technique. This technique avoids the pitfalls of classic in situ subtraction rhinoplasty and provides a better way to correct the nasal shape without causing airway obstruction. This technique was performed in 30 patients in the past 6 years who had primary or secondary rhinoplasties, with satisfactory results.     

深低温冻存组织工程化软骨在关节软骨缺损修复的应用

目的：探讨经深低温冻存组织工程化软骨修复关节软骨缺损的可行性。方法：分离收集3周龄新西兰大白兔关节软骨细胞进行体外培养,接种于PGA三维支架材料上,复合物体外培养1周后冻存,冻存1个月后解冻、复苏及体外培养,1周后接种于已建立的双侧兔膝关节软骨缺损模型的膝关节软骨缺损处,并设对照组。分别于手术后4周、8周、12周行大体标本及组织观察。结果：大体观察结果表明,实验组与对照组缺损处均由软骨组织修复;组织学观察可以见到实验组和对照组关节软骨缺损处有密集的软骨细胞,均有软骨生成及基质分泌,两组差异无统计学意义。结论：应用深低温冻存组织工程化软骨修复关节软骨缺损的方法是有效可行的,为其进一步临床应用提供了实验依据。     

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