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Histological and electron microscopic studies were performed to demonstrate the changes in the morphology of the growth plate that occur in allografts obtained from the limbs of growing rats. A genetically defined model was used in which the right hindlimbs of Lewis rats were orthotopically transplanted into Fischer-344 recipient rats. These strains are matched for major histocompatibility antigens but mismatched for minor histocompatibility antigens. The disparity at the minor histocompatibility complex between the Lewis donors and the Fischer recipients creates a weak histocompatibility barrier to transplantation. Lewis-to-Lewis syngeneic limb grafts were used as controls. The proximal parts of the transplanted tibiae were excised during acute rejection of the allograft on days 1, 5, 8, 11, 14, and 28 postoperatively. During rejection, a widened zone of calcified cartilage in the growth plate was observed at eleven days; this zone increased progressively thereafter. The number of chondroclasts in the primary spongiosa of the metaphysis had decreased significantly at eleven days, and chondroclasts had disappeared completely at fourteen days, in association with mononuclear cell infiltration. Electron microscopic examination revealed inactive morphology in some chondroclasts at eight days, and the number of inactive chondroclasts had increased significantly on day 11. At fourteen days, there were no viable chondroclasts in the primary spongiosa, and only remnants of degenerated chondroclasts were present. These findings suggest that the chondroclasts were early targets of rejection and their loss resulted in the cessation of resorption of the calcified cartilage. However, the proliferation and maturation of chondrocytes in the growth plate and the calcification of the matrix continued, despite progression of rejection in the metaphysis. Thus, survival of the chondrocytes and rejection of the chondroclasts apparently led to the formation of a widened calcifying zone in the growth plate.

Regulation of growth of long bones occurs in cartilage growth plates, where proliferation of chondrocytes, matrix synthesis, and an increase in vertical height in the direction of growth all contribute to the final length of a bone. In this study, we tested the hypothesis that an increase in chondrocytic vertical height is a major variable that accounts for the decreased rate of growth of long bones in Scottish deerhound dogs that had pseudoachondroplasia. The diagnosis of pseudoachondroplasia is based, primarily, on the demonstration of alternating electron-dense and electron-lucent lamellae with a periodicity of 100 to 150 nanometers in dilated rough endoplasmic reticulum. These ultrastructural changes are similar to those seen in humans who have pseudoachondroplasia. In Scottish deerhounds that have the disease, growth of bone is approximately 65 per cent of that in normal animals. There were striking differences in the diameters of proliferating and hypertrophic chondrocytes in pseudoachondroplastic animals compared with normal animals. Specifically, the horizontal diameter of proliferating chondrocytes was 22.7 micrometers in normal animals and 11.3 micrometers in pseudoachondroplastic animals. The vertical diameter of proliferating chondrocytes was 4.8 and 7.6 micrometers in normal and pseudoachondroplastic animals. In the distal 100 micrometers of the hypertrophic zone, the mean horizontal diameter of hypertrophic chondrocytes was 29.6 and 19.1 micrometers and the mean vertical diameter was 22.8 and 18.6 micrometers in normal and pseudoachondroplastic animals. All these differences were statistically significant. The changes in vertical height resulted in a significant difference in the incremental difference in vertical height between chondrocytes from the proliferative and hypertrophic zones in normal animals (18.0 micrometers per chondrocyte) and pseudoachondroplastic animals (11.0 micrometers per chondrocyte). Each chondrocyte in the abnormal plates achieved only 61 per cent of the incremental difference of chondrocytes in normal plates. The mean cellular volume of chondrocytes in the hypertrophic zone was 13,050 cubic micrometers in the normal animals and 10,740 cubic micrometers in the pseudoachondroplastic animals. This difference was not statistically significant. These results are discussed in relation to current theories of the role of the shape and change in volume of chondrocytes in the regulation of longitudinal growth of bone.(ABSTRACT TRUNCATED AT 400 WORDS)

Applying stereological principles, we recently demonstrated a matrix-vesicle distribution between the zones of the epiphyseal growth plate that is at variance with the generally accepted distribution. In the present study of strontium-induced rickets we demonstrated a similar bimodal distribution, the highest matrix-vesicle volume fraction being found in the resting and hypertrophic zones and the lowest, in the proliferative zone. These volume-fraction differences are due to differences in the number of vesicles between zones, the variation in mean diameter being rather small. The disturbed mineralization in strontium-induced rickets is associated with a diminished reduction of the matrix-vesicle volume fraction in the lower part of the growth plate compared with normal animals. The findings may indicate that matrix-vesicle removal is a part of the mineralization process, but a causal connection cannot be proved on the basis of our results. Our findings are, however, compatible with the earlier-posed cell-debris theory for matrix-vesicle origin and distribution.

In young rabbits, the effects of an intravenous injection of Staphylococcus aureus alone, and in combination with a traumatic injury of the proximal tibial physis, were studied by light and electron microscopy. Metaphyseal osteomyelitis and radiographic changes were seen within forty-eight hours after the injury in all rabbits that had a growth-plate disruption and bacteremia. An intravenous injection of bacteria alone produced no morphological or microbiological evidence of infection. In the absence of trauma, normal tibiae were sterile after forty-eight hours. Foreign-body particles have been shown to accumulate in the fine vessels that are adjacent to the growth plate, but we found no similar deposition of bacteria or evidence of phagocytic removal in this area. Phagocytosis of bacteria by neutrophils did not appear to be impaired in the distal third of the metaphysis, but a delayed inflammatory response that allowed proliferation of bacteria and destruction of tissue was observed in the proximal two-thirds of the metaphysis after trauma.

We studied the proximal tibial physes of mice, seven, fifteen, twenty-two, and twenty-eight days old, to define in mathematical terms the changes in cell profile and profile orientation among growth-plate zones and to determine if cell profile and profile orientation change with changes in the rate of growth. Using electron microscopy, we identified five growth-plate zones: the reserve zone, the upper proliferative zone, the lower proliferative zone, the upper hypertrophic zone, and the lower hypertrophic zone. In transverse sections, cell profiles did not change among growth-plate zones and the degree of cell-profile orientation approached zero in all zones. In longitudinal sections, cell profiles and profile orientations differed significantly among zones. Cell profiles in the upper and lower proliferative zones were eccentric and highly oriented. They became more rounded and the degree of cell orientation decreased between the proliferative and hypertrophic zones. As the rate of longitudinal bone growth decreased, cell profiles and cell-profile orientation changed. The cell profiles in the reserve zone became flatter and in the other zones the cell profiles became more rounded. The degree of cell-profile orientation decreased quadratically in the upper and lower proliferative zones, decreased linearly in the reserve and upper hypertrophic zones, and remained unchanged in the lower hypertrophic zone.

In the hypertrophic zone of the cartilaginous growth plate, chondrocytes enlarge, assume a more spherical shape, and form a population of cells called the hypertrophic chondrocytes. The mechanisms that are involved in the formation of hypertrophic chondrocytes are poorly understood. Cell hypertrophy usually refers to an increase in cell size and volume associated with an increase in organelles. In this study, we sought to determine whether the formation of hypertrophic chondrocytes represents true cell hypertrophy associated with an increase in organelles or whether it is due to swelling and fluid accumulation. Morphometric analyses of electron micrographs were carried out to determine changes in cell number, cell volume, cell organelle volumes, and matrix volumes in the reserve zone, upper proliferative zone, lower proliferative zone, upper hypertrophic zone, and lower hypertrophic zone. Between the upper proliferative zone and the lower hypertrophic zone, the cells increased their mean volume more than 500 per cent. As they enlarged, their matrices altered; territorial matrix volume increased as its collagen content decreased, and interterritorial matrix volume decreased as its collagen content increased between the lower proliferative zone and the lower hypertrophic zone. Between the upper proliferative zone and the lower hypertrophic zone, the absolute volume per cell of endoplasmic reticulum, Golgi membranes, and mitochondria increased 126 per cent, while the volume of cytoplasm and nucleoplasm increased 779 per cent, apparently by accumulation of water. Cell organelles of the lower hypertrophic zone did not show the changes that are associated with cell injury or death. Thus, the synthesis of organelles contributed to chondrocyte enlargement, but the primary mechanism of cell enlargement was cytoplasmic and nuclear swelling.

Unlabelled: The synthesis and distribution of three metalloproteinases, collagenase, stromelysin, and gelatinase, and of the tissue inhibitor of metalloproteinase were examined in the distal femoral growth plate, the secondary center of ossification, and the perichondral ossification groove of Ranvier in newborn to six-week-old rabbits. Specific antisera to each of the enzymes and to the tissue inhibitor of metalloproteinase were used to identify their distribution in the extracellular matrix of the growth plate and to determine the associated tissues and cells that are responsible for their synthesis. Immunolocalization using tissue that was cultured in the presence of monensin to augment accumulation of intracellular antigen revealed that the growth-plate chondrocyte is responsible for the synthesis of the metalloproteinases and the tissue inhibitor of metalloproteinase, and that there is a unique pattern of synthesis in each zone. Chondrocytes of the resting and proximal proliferative zones were shown to synthesize and secrete all of the metalloproteinases and the tissue inhibitor of metalloproteinase. Synthesis of collagenase also was demonstrated in the remainder of the proliferative zone and in the most distal cells of the hypertrophic zone. The presence of collagenase in the distal cells suggests their involvement in vascular invasion. By culturing tissues in the presence of antibodies, we were able to demonstrate collagenase and the tissue inhibitor of metalloproteinase throughout the growth-plate matrix. Staining of the extracellular matrix, implying active collagenase, was also found in the matrix of the proliferative and hypertrophic zones, suggesting that degradation of tissue may occur at a distance from the cells that synthesize the enzyme. Simultaneous localization with two different antibodies demonstrated that growth-plate chondrocytes are capable of synthesizing collagenase and the tissue inhibitor of metalloproteinase, both independently of one another and coordinately. Stromelysin was found to be synthesized in all zones, implying that it plays an important role in degradation. Monocyte-conditioned media stimulates synthesis of collagenase in growth-plate cells, principally through the action of interleukin-1. All chondrocytes throughout the growth plate, including hypertrophic cells, can be stimulated to produce collagenase. These changes in metalloproteinase and in the tissue inhibitory of metalloproteinase in the growth plate are crucial to remodelling of the matrix during development, and the appearance of metalloproteinases and the tissue inhibitory of metalloproteinase in the secondary ossification center and groove of Ranvier indicates that changes at these sites are similar to remodeling in the growth plate., Clinical Relevance: The life cycle of the chondrocyte in the growth plate is central to the process of endochondral ossification, bone growth, and development. Our new data on the zonal synthesis of metalloproteinase and of th

We carried out histochemical and electron-microscopic studies to examine the relationship between mineral deposition and changes in the organization of proteoglycans during mineralization in the cartilaginous growth plate of the rat. To preserve the distribution and organization of proteoglycans in the extracellular matrix and to stain proteoglycans, acridine orange was included in the solutions that were used for fixation and demineralization. In undecalcified sections, mineral crystals that initially appeared at the level of the penultimate hypertrophic chondrocyte grew into dense, spherical mineral clusters, roughly one micrometer in diameter, in the longitudinal septa of the lowermost hypertrophic zone. Following the removal of mineral with EDTA in the presence of acridine orange, proteoglycan structures with a characteristic rosette-like architecture were revealed within the matrix of the longitudinal septa of the lower hypertrophic zone. The location of these rosette-like proteoglycan structures was identical to that of the dense, spherical mineral clusters in the undemineralized controls, and they were remarkably similar in morphology and size. In the electron micrographs stained with uranyl acetate and lead citrate, the greatly increased density of the rosette-like structures and large diameter of the proteoglycan structures located in mineral clusters contrasted sharply with the slender strands of more faintly stained proteoglycan that was diffusely distributed throughout most of the extracellular matrix. X-ray microprobe analysis for sulphur confirmed the existence of proteoglycans in the rosette-like structures and demonstrated that the concentration of proteoglycans was selectively increased in these regions. The cores of the metaphyseal calcified cartilage also exhibited rosette-like proteoglycan structures, which before demineralization were totally obscured by the diffuse, dense homogeneous deposition of mineral. Without stabilization of the proteoglycans in the extracellular matrix with acridine orange, the rosette-like proteoglycan structures could not be demonstrated. These results clearly indicate that there is a selective increase in the concentration of proteoglycans in exactly the same regions where mineral clusters are formed in the hypertrophic zone of the cartilaginous growth plate.

The longitudinal growth of bone depends on the activities of individual chondrocytes of the growth plate. Each chondrocyte remains in a fixed location throughout its life, and there accomplishes all of its functions. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during a particular phase of its life. The two most prominent stages are those of cellular proliferation and hypertrophy (including the mineralization of matrix) before the resorption of tissue during vascular invasion. By applying recently developed stereological procedures and improved methods for the fixation of cartilage, we compared cellular shape modulation, various ultrastructural parameters (surface areas or volumes of endoplasmic reticulum, Golgi membranes, and mitochondria), the production of matrix, and cellular turnover for proliferating and hypertrophic chondrocytes within the proximal tibial growth plate of the rat. By the late hypertrophic stage, fourfold and tenfold increases in the mean cellular height and volume, respectively, and a threefold increase in the mean volume of the matrix per cell were achieved. The high metabolic activity of hypertrophic cells was reflected by a twofold to fivefold increase in the mean cellular surface area of rough endoplasmic reticulum, the Golgi membranes, and the mean cellular mitochondrial volume. Rates of longitudinal growth were determined by fluorochrome labeling and incident-light fluorescence microscopy. Using these values and the stereological estimators describing cellular height, the rates of cellular turnover were calculated. The rapid progression of the vascular invasion front was found to eliminate, for each column of cells, one chondrocyte every three hours; that is, eight cells a day. The maintenance of a steady-state structure for growth-plate cartilage in rats in a steady state of growth thus necessitates efficient compensation for these losses, which is achieved by a high rate of cellular proliferation and rapid hypertrophy.