Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes.

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load-bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18ß-glycyrrhetinic acid, we also demonstrated that this osteocyte-related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial-derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain-related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.

Notch receptor and Notch ligand expression in developing avian cartilage.

The development of limb cartilage involves complex signalling pathways allowing the formation of distinct segments of cartilage that are maintained in the fully developed joint. In this study, we investigated the Notch signalling pathway and its role in cartilage development. The differential distribution of the Notch signalling family of receptors and their corresponding ligands in developing avian (gallus gallus) cartilage revealed expression of Notch 1, Delta 1, Jagged 1 and Jagged 2 in all limb mesenchyme cells at the early stages of cartilage anlagen development, which were subsequently restricted to the developing cartilage element. Expression of both Notch 1 and Jagged 1 became increasingly restricted to the surface cartilage once joint cavity formation had occurred. Delta 1 and Jagged 1 were restricted to a layer of cells underneath the surface cartilage and were also observed in the hypertrophic chondrocytes, where Notch 1 expression was evident in stage 40-44 limbs. Notch 2, Notch 3 and Notch 4 were not evident in early stage limbs but were present after cavitation, although expression was lost in late stage limbs (stage 40-44). We also demonstrated that inhibition of the Notch pathway leads to altered Notch receptor expression, disrupting cartilage differentiation. From these data it is clear that Notch signalling is a necessary and critical factor in regulating cell fate decisions allowing controlled chondrogenesis, elongation and subsequent maintenance of limb cartilage.

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.

BACKGROUND: Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage. METHODS AND FINDINGS: Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect. CONCLUSIONS: In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.

Gap junctional intercellular communication and cytoskeletal organization in chondrocytes in suspension in an ultrasound trap.

Particles or cells suspended in an appropriately designed ultrasound standing wave field can be aggregated at a node to form a single monolayer in a plane that can be interrogated microscopically. The approach is applied here to investigate the temporal development of F-actin and Cx43 distribution and of gap junctional intercellular communication in 2-D chondrocyte aggregates (monolayers) rapidly and synchronously formed and held in suspension in an ultrasound trap. Development of the F-actin cytoskeleton in the confluent single layer of 'cuboidal' cells forming the aggregate was completed within 1 h. Chondrocytes levitated in the trap synchronously formed functional gap junctions (as assessed by CMFDA dye transfer assays) in less than 1 h of initiation of cell-cell contact in the trap. It was shown that Cx43 gene expression was retained in isolated chondrocytes in suspension. Preincubation of cells with the protein synthesis inhibitor cycloheximide caused a six-fold decrease in Cx43 accumulation (as assessed by immunofluorescence) at the interfaces of chondrocytes in the aggregate. It is shown that the ultrasound trap provides an approach to studying the early stages of cytoskeletal and gap junction development as cells progress from physical aggregation, through molecular adhesion, to display the intracellular consequences of receptor interactions.

Development of synovial joints.

Synovial joints arise through two main processes. In long bone elements, cartilaginous differentiation occurs across the locations of the prospective joints that then segment secondarily. This process occurs through the development of a noncartilaginous region known as the interzone. The interzone becomes an important signaling center to the opposing elements, which can regulate growth through such factors as GDF-5. The interzone also expresses bone morphogenetic proteins (BMPs) and their antagonists, such as noggin. Overexpression of BMPs, or the loss of noggin leads to joint fusions. The interzone also expresses Wnt-14, which appears to be specific for this region in the developing anlagen, and regulates its nonchondrogenic nature. Cavitation of the joint follows, driven by selective high-level synthesis of hyaluronan by interzone cells and presumptive synovial cells. In addition, as the interzone disperses during cavity enlargement, data are now accruing that suggest that both the synovium and articular cartilage develop from this population. Finally, the development of articular cartilage progresses through appositional growth driven by a progenitor/stem cell subpopulation that resides in the articular surface. The individual elements of the skeleton are connected together at regions termed joints or articulations. Classically, there are three broad categories of joints: immovable joints (syntharthroses); mixed articulations, in which the range of movement is limited (amphiarthroses); and the movable, or synovial, joints (diarthroses). This review concentrates on the development of the synovial joints.

The surface of articular cartilage contains a progenitor cell population.

It is becoming increasingly apparent that articular cartilage growth is achieved by apposition from the articular surface. For such a mechanism to occur, a population of stem/progenitor cells must reside within the articular cartilage to provide transit amplifying progeny for growth. Here, we report on the isolation of an articular cartilage progenitor cell from the surface zone of articular cartilage using differential adhesion to fibronectin. This population of cells exhibits high affinity for fibronectin, possesses a high colony-forming efficiency and expresses the cell fate selector gene Notch 1. Inhibition of Notch signalling abolishes colony forming ability whilst activated Notch rescues this inhibition. The progenitor population also exhibits phenotypic plasticity in its differentiation pathway in an embryonic chick tracking system, such that chondroprogenitors can engraft into a variety of connective tissue types including bone, tendon and perimysium. The identification of a chondrocyte subpopulation with progenitor-like characteristics will allow for advances in our understanding of both cartilage growth and maintenance as well as provide novel solutions to articular cartilage repair.