Cartilage Core Research Articles

First bone formation in the developing chick limb

Embryonic chick limb mesenchymal cells differentiate into fibrocytes, chondrocytes, myocytes, and bone cells. Previous studies of these cells in culture suggested that osteoblast differentiation may be coincident with muscle and cartilage cytodifferentiation. Characteristic features of the osteoblast phenotype seen in vivo include the expression of alkaline phosphatase activity preceding and associated with cells elaborating a mineralized, collagenous matrix. The studies reported here demonstrate that osteoblasts can be detected as early as stage 28 in the developing chick limb by monitoring this activity. Alkaline phosphatase histochemistry and biochemistry indicate the presence of enzyme activity in the presumptive periosteum by this stage. Furthermore, the localization of this activity precedes the hypertrophy of cartilage and subsequent mineralization of the cartilage core. On the basis of these observations, as well as studies on inorganic phosphate and calcium accumulation in the developing limb, we suggest that the osteoblast phenotype may become committed to its phenotypic pathway in the same temporal sequence as muscle and cartilage cells. In addition, the first recognizable expression of limb osteoblasts is observed long before major cartilage core hypertrophy, vascular invasion, and subsequent core mineralization. Thus, the first bone formation is not associated with cartilage, but represents a separate, unique, and intrinsic limb phenotype.

Ossification in adult rat mandibular coyndyle—SEM of chondroclasia

The morphology of the mineralized component at the osteochondral junction was studied in the mandibular condyle of 264-day-old rats. Specimens were rendered anorganic in NaOCl, cut open with a sculpel, and the resulting surfaces examined in a scanning electron microscope. The extent of the mineralized cartilage plate and its relationship to invading vascular channels were visualized. Areas of resorption of calcified cartilage were identified and compared to areas of bone resorption. A likely sequence of events was reconstructed from the exposed internal surfaces: from initial chondroclasia in individual lacunae to extensive resorption of the cartilage plate; and from initial deposition of bone over the resorbed calcified cartilage cores to the organization of osteocyte lacunae and bony trabecule. The mandibular condyle has not been examined in this way previously. Possible cellular mechanisms suggested by other studies to be operating at the osteogenic front are discussed in the light of the results.

Small artificial currents enhance Xenopus limb regeneration

AbstractA steady, small (0.1 μA) current was artificially pulled out of the end of the stumps of amputated adult Xenopus forelimbs for three to four weeks. Six months to a year later, the following responses were observed: 1. Some stimulated regenerates formed broad, flattened bifurcated structures, while sham‐treated controls formed typical hypomorphic spikes. 2. In the majority of the cathode‐stimulated regenerates, there were nerve trunks with in their cartilage core. There was no nerve in control cartilage, except in one case in which a small amount of nerve was found. Overall, the cartilage core of the stimulated regenerates had 40‐fold more nerve coursing through it than did the controls. 3. Mature thorn‐shaped epidermal papillae formed on most of the stimulated animals, but on only one of the controls. We suggest that these minute electrical currents affect regeneration by first stimulating early nerve growth.

The effect of nerve growth factor on hindlimb regeneration in Xenopus laevis froglets.

AbstractThe effect of nerve growth factor (NGF) on hindlimb regeneration in postmetamorphic Xenopus laevis froglets was investigated. Two different dosage levels of NGF were tested on froglets whose hindlimbs had been amputated either above or below the knee joint. The course of regeneration in the NGF‐treated froglets was followed for 100 days post‐amputation.Treatment with NGF at a level of 2,500 Biological Units per froglet increases the extent of hindlimb regeneration attained compared with the controls, regardless of the level of amputation. On the other hand, a higher dose of NGF, 5,000 Units per froglet, does not alter the extent of hindlimb regeneration significantly. Treatment with NGF at either dosage does not appear to affect the shape or histological constitution of the regenerated limb segment. The general shape of the regenerate correlates with the level at which the limb was amputated. All regenerates obtained in this study conform to a common pattern in terms of histological constitution. They are composed of a central core of cartilage with a perichondrial membrane surrounded by a thin layer of connective tissue and covered by fully differentiated epidermis. No differentiated bone, muscle or nervous tissue was detected in any of the regenerates, but evidence of cartilage calcification was observed in limbs amputated below the knee. Possible implications of these results are discussed.

Beginning endochondral ossification in embryonic mouse radii

In embryonic mouse radii the process of endochondral ossification begins with the deposition of mineralized bone matrix directly on the surface of a core of calcified cartilage. In a later phase, separate foci of calcification appear in the neighborhood. As far as the organic components are concerned the earliest formed bone matrix consists of a fine filamentous and granular material whereas collagenous fibrils are lacking. Endochondral ossification also plays a role in the thickening of the diaphyseal bone collar.

Histochemical study of bone in gonadectomized rats.

Sections of the tibias from both intact and gonadectomized male and female rats were subjected to various histological and histochemical methods to determine the changes in the bone following gonadectomy. It was observed that both in castrated males and in ovariectomized females, the proximal epiphyseal cartilage became “sealed off” by bone after the fifth post-surgical month. In addition osteoporosis of the shaft of the bone was also observed. The epiphyseal cartilage of the gonadectomized rats exhibited intense PAS positive intercellular matrix in contrast to the alcianophilic ature of the matrix from control animals. The diaphyseal trabeculae from gonadectomized rats stained red with PAS and contained wide regions of calcified cartilage cores. In addition, the epiphyseal cartilage and bony trabeculae from these animals stained faintly in the ninhydrin-Schiff reaction. The staining reactions indicated that in gonadectomy there was an alteration in the state of aggregation of protein-mucopolysaccharide component of the organic matrix, a situation which would result in an interference in the mineralization of the bone.

Skeletal changes in murine runt disease.

AbstractRunt disease was produced in neonatal F1 hybrid mice by the injection of spleen cells from a mature donor of the parental strain. Clinically apparent runting appeared after the tenth day. Mice were serially sacrificed when 11–19 days old, and their long bones were examined histologically and histochemically. In runt disease, development of the epiphyseal secondary centers of ossification was retarded. The cells and protein polysaccharides did not mature normally, and the tissue contained acellular cartilage. Accumulations of hypertrophic chondrocytes signified impaired endochondral ossification. While the osteogenic cells in the medullary spaces had been replaced largely by mononuclear lymphoid cell infiltrates, bone collagen was present on unremodeled metaphyseal trabeculae which possessed wide cartilage cores. The intertrabecular spaces were moderately fibrotic. Large remnants of unossified unresorbed cellular cartilage were frequently observed in the metaphysis. It appeaed that the generalized graft‐versus‐host reaction induced by the spleen cell “grafts” was extended to the skeleton, but that the changes in the cartilage and bone tissue had occurred largely before the onset of clinical runting.

Staining of fresh epiphyseal cartilage with toluidine blue. Demonstration of previously unreported intracellular beta- and gamma-metachromatic granules.

Staining of fresh, unfrozen sections of rat tibia in 0.01% toluidine blue at pH 4.5 revealed the presence of large β-metachromatic granules in the cells of the proliferative zone, small γ-metachromatic granules in the cells of the hypertrophic zone, and both types in cells of intermediate size in the maturing zone. The matrix of the reserve zone stained with toluidine blue; the matrix of the proliferative and upper hypertrophic zone was not stained, but staining appeared again in the lower hypertrophic (calcifying) zone and in the cartilage cores of the metaphyseal trabeculae. Similar but more erratic results were obtained with frozen sections of Carnoy-fixed, gelatin-embedded specimens. These results could not be obtained with simple frozen section techniques or ordinary histological processing, which disrupt the intracellular granules and alter the state of combination of the protein-polysaccharides of the matrix. The results support the concept that a change in the nature of the mucopolysaccharide-containing components is one of the factors involved in the change from non-calcifiable to calcifiable cartilage matrix.

Histochemical study of bone in cortisone-treated rats.

Subcutaneous injections of cortisone acetate to adult male rats for 3 weeks produced a loss in body weight and extreme narrowing of the epiphysial cartilage of the tibia. The diaphysis of the tibia had an ‘osteosclerotic-like’ appearance due to the fusion and the persistence of the primary trabeculae. The cortisone-treated trabeculae were characterized by a wide core of calcified cartilage lined by a thin layer of bone. These trabeculae stained a deep red with periodic acid-Schiff and very intense violet with toluidine blue. These 2 staining reactions would indicate that there was an alteration in the ground substance. This situation produced an interference in the calcification of the bone, thus inhibiting the resorption of the primary trabeculae and reorganization into their adult structure. Not only was endochondral ossification affected, but intramembranous ossification, as exemplified by the interradicular bone of the molar teeth, also showed similar changes.

OSTEOSCLEROSIS AFTER INTERMITFENT ADMINISTRATION OF LARGE DOSES OF VITAMIN D IN THE RAT

When large daily doses of vitamin D were administered to rats endochondral growth was inhibited and bone resorption occurred; later in the process uncalcified matrix (osteoid) like that seen in rickets formed on trabecular margins. When vitamin D was given only for a short period and then discontinued, little resorption of bone was seen during the withdrawal period and wide seams of osteoid material appeared which eventually calcified in an irregular manner. When normal endochondral growth was resumed a wide transverse band of dense bone with enclosed cartilaginous cores was left in the marrow cavity. If, after a few days, a second large dose of the vitamin was given resorption again occurred and calcification of osteoid material was accelerated, the first microscopic sign being a dense, wide, granular, deeply staining line at the junction of the bone and new osteoid. After a second withdrawal period a second layer of osteoid formed; eventually another transverse band appeared in the metaphysis. If this hypervitaminosis D cycle (+4 -12) was continued rats continued to form new bone with relatively little remodelling, so that after three such cycles bones became dense and hard. Histological study showed that little marrow cavity remained in either skull, vertebrae or epiphyses and a dense mass of bone enclosing cartilage cores filled the metaphysial part of the long bones. In addition, ankylosis ofteeth, calcification of spinal ligaments and widespread metastatic calcification were present. When hypervitaminosis D cycles (+1 -12, +1 -21) were adjusted to produce minimal resorptive changes a wide range of bone change was observed. This varied from uniform dense metaphysial bone containing abnormal cartilage matrix arranged in longitudinal striations, dense transverse bands parallel to the epiphysial cartilage, to remnants of dense trabeculae extending into the marrow cavity. Bone changes in osteopetrosis structurally closely resembled the induced bone changes in the rat. It is concluded that an important mechanism in the production of osteopetrosis is an accentuated rhythm of bone change like that shown experimentally to be produced in these animals. It is emphasised that these changes are but part of a range of bone disorders associated with abnormalities of cycles of resorption and deposition of bone, the type of change differing with the nature of the cycles.