Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development

During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development.     

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.     

Transition of Hox expression during limb cartilage development

Hox genes belonging to the Abd-B subfamily of the HoxA and HoxD clusters play a crucial role in cartilage formation both in patterning and growth/differentiation phases during limb development. We re-examined the expression profiles of Hoxa-13, Hox-d13, Hoxa-11 and Hoxd-11 during the cartilage growth/differentiation phase of limb cartilage formation. The expression profiles of these Hox genes were analyzed by in situ hybridization and immunohistochemistry on serial sections by comparing the expression patterns with well-characterized signaling molecules, e.g. Bmp-2, -4, Patched (Ptc) and Indian Hedgehog (IHH). In contrast to earlier reports, these Hox genes were expressed in the mesenchymal cell layer closely adjacent to the growing cartilage, but not in the perichondrium of the cartilage. This result prompts us to reconsider the mode of Hox function during cartilage growth and differentiation phase.     

Crosstalk between cartilage and bone: when bone cytokines matter

The cartilage damage which characterizes osteoarthritis is often accompanied by bone lesions. Joint integrity results from the balance in the physiological interactions between bone and cartilage. Several local factors regulate the physiological remodeling of cartilage, the disequilibrium of these leading to a higher cartilage catabolism. Several cytokines secreted by bone cells can induce chondrocyte differentiation, which suggests their role in the dialogue between both cells. Accumulative in vivo evidence shows that increased bone resorption occurs at an early stage in the development of osteoarthritis and that blocking bone-resorbing cytokines prevents cartilage damage, confirming the role of bone factors in the crosstalk of both tissues. Recently, molecules of the Wnt pathway have emerged as key regulators of bone and cartilage. Activation of Wnt/βcatenin induces an imbalance in cartilage homeostasis, and agonists/antagonists of Wnt are potential candidates for this interaction. This review will summarize what is known about the contribution of bone cytokines to the physiological remodeling of cartilage and in the pathophysiology of osteoarthritis.     

Regulation of cartilage and bone differentiation by bone morphogenetic proteins.

Quantum advances have recently been made in the understanding of the regulation of cartilage and bone differentiation through the identification, purification, genetic cloning and expression of recombinant bone morphogenetic proteins. Bone morphogenetic proteins are a family of pleiotropic differentiation factors with actions on chemotaxis, mitosis, initiation and promotion of chondrogenic and osteogenic phenotypes. They bind extracellular matrix components, heparin and type IV collagen and initiate bone repair. The cascade of cartilage and bone differentiation consists of several continuous phases: initiation, promotion, maintenance and termination.     

Swelling of articular cartilage depends on the integrity of adjacent cartilage and bone

Articular cartilage swells when its collagen network is degraded, both in osteoarthritis (OA) and following mechanical trauma. However, most of the experimental evidence actually shows that it is small excised samples of cartilage that swell, implying that the cartilage was not greatly swollen in-situ before it was excised. We hypothesise that degraded cartilage can be prevented from swelling in-situ by restraint from adjacent normal cartilage and subchondral bone. Four adjacent osteochondral specimens, 20 x 20 mm, were obtained from regions of the humeral heads of each of 11 skeletally-mature cows. The central region of each specimen was injured by compressive loading using a 9 mm-diameter flat metal indenter, and cartilage surface damage was confirmed using Indian ink. Damaged cartilage was allowed to swell in physiological saline for 1 h under one of four conditions of restraint: (A) normal in-situ restraint from subchondral bone and surrounding cartilage, (B) restraint from bone only, (C) restraint from cartilage only, (D) no restraint (excised specimen). Cartilage hydration was assessed by freeze-drying to constant weight. Proteoglycan loss from damaged cartilage was quantified by analyzing the GAG content of the surrounding bath using the DMB assay. Hydration of damaged cartilage after swelling depended on restraint (p < 0.001), averaging: (A) 76.8%, (B) 78.2%, (C) 78.0%, (D) 81.3%. GAG loss following cartilage surface damage was insufficient to explain observed differences in hydration. The 6% increase in hydration between (A) and (D) can be attributed to swelling which is prohibited when the cartilage remains in-situ. Swelling of degraded cartilage can be largely prevented if it remains in-situ, supported by adjacent healthy bone and cartilage. Adverse physico-chemical consequences of cartilage degradation and swelling may become apparent only when this support is diminished, either because the affected region is large, or following deterioration of adjacent bone or cartilage.     

Aging bone and cartilage: cross-cutting issues

Aging is a major risk factor for osteoarthritis and osteoporosis. Yet, these are not necessary outcomes of aging, and the relationship between age-related changes in bone and cartilage and development of disease is not clear. There are some well-described cellular changes associated with aging in multiple tissues that appear to be fundamental to the decline in function of cartilage and bone. A better understanding of age-related changes in cells and tissues is necessary to mitigate or, hopefully, avoid loss of bone and cartilage with aging. In addition, a better understanding of the dynamics of tissue maintenance in vivo is critical to developing tissue replacement and repair therapies. The role of stem cells in this process, and why tissues are not well maintained with advancing age, are frontiers for future aging research.     

Novel materials for bone and cartilage regeneration

Materials that enhance bone and cartilage regeneration promise to be valuable in both research and clinical applications. Both natural and synthetic polymers can be used to create scaffolds that support cells and incorporate cues which guide tissue repair. Recently, electrospinning, peptide self-assembly and biomineralisation have been employed to fabricate nanostructured scaffolds that better mimic the complex extracellular environment found within tissues, in vivo. The incorporation of peptide motifs recognised by cell receptors and the use of recombinant DNA technology have enabled the creation of scaffolds with new levels of biofunctionality. Advances in materials design will enhance our ability to create highly tailored cellular environments for bone and cartilage regeneration.     

En bloc staining of articular cartilage and bone

Blocks of canine and porcine articular cartilage were stained en bloc with Weigert's iron hematoxylin or Harris' hematoxylin with or without eosin Y counterstaining and cleared in methyl salicylate. The morphology and three-dimensional relationships of chondrocytes were best demonstrated with Weigert's iron hematoxylin. The morphology of the cartilage and chondrocytes was superior to that in sections of routine hematoxylin and eosin stained, paraffin processed samples. The three-dimensional localization of intracellular lipids in individual and clones of chondrocytes was observed when cartilage samples were stained with oil red O and mounted directly in a water-based medium. Blocks of decalcified bone were stained en bloc with Weigert's iron hematoxylin and cleared with methyl salicylate. The three-dimensional orientation of osteocytes around osteonal canals, in circumferential lamellae, and in interstitial lamellae was demonstrated. The morphology of "cutting cones" in cortical bone also was observed.     

Microcarriers in the engineering of cartilage and bone

A major problem in tissue engineering is the availability of a sufficient number of cells with the appropriate phenotype for delivery to damaged or diseased cartilage and bone; the challenge is to amplify cell numbers and maintain the appropriate phenotype for tissue repair and restoration of function. The microcarrier bioreactor culture system offers an attractive method for cell amplification and enhancement of phenotype expression. Besides serving as substrates for the propagation of anchorage-dependent cells, microcarriers can also be used to deliver the expanded undifferentiated or differentiated cells to the site of the defect. The present article provides an overview of the microcarrier culture system, its utility as an in vitro research tool and its potential applications in tissue engineering, particularly in the repair of cartilage and bone.     

Expression of vigilin in chicken cartilage and bone

The expression of vigilin was followed during chick embryonal development by in situ hybridization. Vigilin mRNA is abundantly expressed in tissues of mesenchymal and ectomesenchymal origin. The mesenchymal primordial cells of cartilage and bone did not show any significant, expression of vigilin. As tissue differentiation proceeded, vigilin mRNA levels increased in hyaline cartilage and in both endochondral as well as intramembranous bone. The results suggest that the expression of vigilin mRNA in cartilage- and bone-forming cells chondrocytes and osteobalsts, is dependent on the stage of development and cellular differentiation, although not a unique process of bone formation. Most striking is the correlation of the maximum vigilin mRNA expression in osteoblasts and hypertrophic chondrocytes to periods when cell-specific genes were highly transcribed and substantially translated, e.g., synthesis of procollagen and formation of extracellular matrix in bone and cartilage.Abbreviations DTT dithiotreitol - PBS phosphate-buffered saline - SSC standard saline citrate buffer     

Regulation of bone and cartilage by adenosine signaling

There is growing recognition that bone serves important endocrine and immunologic functions that are compromised in several disease states. While many factors are known to affect bone metabolism, recent attention has focused on investigating the role of purinergic signaling in bone formation and regulation. Adenosine is a purine nucleoside produced intracellularly and extracellularly in response to stimuli such as hypoxia and inflammation, which then interacts with P1 receptors. Numerous studies have suggested that these receptors play a pivotal role in osteoblast, osteoclast, and chondrocyte differentiation and function. This review discusses the various ways by which adenosine signaling contributes to bone and cartilage homeostasis, while incorporating potential therapeutic applications of these signaling pathways.     

Cellular interactions during cartilage and bone development.

Both interactions between like cells, as between chondrogenic cells in a developing cartilaginous rudiment, and between unlike cells, as in epithelial-mesenchymal interactions, are dealt with in this review. Such interactions may involve direct apposition of cell membranes or may be mediated via interaction with peri- or extracellular matrices. An ontogenetic approach is taken in which cellular interactions involved in five processes of the development of cartilage and bone are discussed, the five being (1) origin of the cells, (2) migration of the cells within the embryo, (3) localization of the cells at their final embryonic site, (4) differentiation, and (5) morphogenesis. Some emphasis is placed on interactions affecting neural crest-derived cells both before and during their migration and on interactions, especially epithelial-mesenchymal interactions, that precede cytodifferentiation of chondroblasts or osteoblasts. Whether epithelial or mesenchymal specificity is required for such interactions to occur is discussed with reference to the otic vesicle-otic mesenchyme interaction that leads to differentiation and morphogenesis of the cartilaginous otic capsule.     

Protein-based tissue engineering in bone and cartilage repair

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial.