Growth Plate Morphology Research Articles

Changes in growth-plate morphology associated with rejection of rat-limb allografts.

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.

Growth Failure and Bony Changes Induced by Deferoxamine

We reviewed the linear growth and growth plate morphology in all children with homozygous beta thalassemia followed in Toronto, for whom monthly height percentiles were available before, and for a 36-month period after, the initiation of nightly subcutaneous deferoxamine therapy. All patients were less than 7 years of age when begun on deferoxamine, and had received nightly deferoxamine for a minimum of 36 months. Marked abnormalities of the metaphyseal growth plate were readily observed in the distal ulnar, radial, and tibial metaphyses in 11 of 37 patients in whom a significant decline in mean height percentile was also noted. (In 10 of these 11 patients, height was less than the 15th percentile after 36 months.) These 11 patients had received a significantly greater (p less than 0.025) initial and average daily dose of deferoxamine, and had maintained a significantly lower (p less than 0.025) mean serum ferritin concentration over the 36 months, than the remainder of the cohort. To determine whether deferoxamine played a causative role in growth failure, growth in patients who began deferoxamine before the age 2 years was compared to that of patients who began after age 5 years, for the period between 2 and 5 years of age. Only patients begun on deferoxamine prior to age 2 years demonstrated a significant (p less than 0.01) decline in height percentile by the third year, implicating deferoxamine therapy as the cause of growth failure. We conclude that both the decline in height percentile and the bony changes observed in well-chelated patients are directly related to deferoxamine therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of Growth Kinetics and Morphology in Limbs Transplanted Between Animals of Different Ages

The influence of age and maturity on longitudinal skeletal growth was studied using heterotopic limb transplantation with microvascular anastomoses between syngeneic male Lewis rats of different ages. Longitudinal growth of the tibia and growth plate morphology were compared after transplantation between isochronografts (juvenile-to-juvenile and adult-to-adult), heterochronografts (juvenile-to-adult), and unoperated limbs. Four animals comprised each experimental group, except for the juvenile-to-adult heterochronograft group, which had six animals. Limbs were measured at 2-week intervals until sacrifice at 7 weeks. All transplanted limbs demonstrated significant longitudinal growth with maintenance of growth plate morphology and columnar organization. Despite being subjected to an adult hormonal environment, the juvenile tibias transplanted into adult hosts grew 12.0 +/- 1 mm--not significantly different from the unoperated juvenile controls, which grew 12.2 +/- 1 mm. Adult isochronografts showed continued growth of 3.1 mm, which was not significantly different from the unoperated control. Juvenile isochronografts demonstrated decreased growth when compared to unoperated limbs. Maintenance of growth in juvenile limbs transplanted to adults suggests a permissive effect of the hormonal milieu and that ultimate skeletal length is primarily determined by factors inherent in each physis. The use of vascularized transplantation of limbs in syngeneic animals of different ages offers a unique method of altering the hormonal and maturational milieu of the growth plate.

Elevated 1,25-dihydroxyvitamin D3 and intestinal calbindin-D9k in the toothless rat

The toothless (tl) rat is a nonlethal osteopetrotic mutation characterized by systemic skeletal sclerosis, growth plate morphology suggestive of rickets, and morphological evidence of reduced osteoclastic bone resorption. Vitamin D metabolites, serum calcium and phosphorus levels, and the developmental appearance of vitamin D-dependent intestinal calcium binding protein (calbindin-D9k) was studied in normal and mutant rats of tl stock from 7 to 35 days of age. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was found to be significantly elevated in mutant animals by 7 days of age (71 +/- 9 pM, tl/tl vs. 24 +/- 8 pM, +/?) and continued to increase to a peak of 428 pM at the time of weaning. This was 240% higher than normals at this period. The elevated levels of 1,25-(OH)2D3 stimulated a significant and precocious appearance of intestinal calbindin-D9k in mutants, beginning by 14 days of age and reaching their peak levels at 21 days postpartum (25.6 +/- 1.7 micrograms/mg protein, tl/tl vs. 16.4 +/- 1.5 micrograms/mg protein, +/?). The cause of the elevated circulating levels of 1,25-(OH)2D3 in tl rats is unknown but may be due to the low serum phosphorus levels present in these animals.

Kinetics of Proteoglycans and Cells in Growth Plate of Normal, Diabetic, and Malnourished Rats

The metabolism of proteoglycans in normal growth plate and the changes in growth plate morphology induced by diabetes and malnutrition were studied in rats. The proteoglycans had a significantly faster turnover (half-life measured with [35S]sulfate labeling: 25-30 h) than the cells in the growth plate. Morphometric studies showed significant reductions of cell number, zone height, and [3H]thymidine incorporation in growth plates from rats with untreated streptozotocin-induced diabetes compared to normal rats. Similar, although less pronounced alterations were observed in malnourished, nondiabetic rats. Disaggregation and degradation of proteoglycans are probably necessary prerequisites for calcification. Our data indicate that the proteoglycans are in a dynamic state of rapid biosynthesis and degradation throughout the growth plate with a shift in the balance at the calcification front toward less synthesis and more degradation.

Proteoglycan Synthesis in Vitamin D-Deficient Cartilage: Recovery from Vitamin D deficiency

Vitamin D appears to be required for mineralization of skeletal elements. There is also evidence that cartilage proteoglycans may be involved in the regulation of mineralization. Previous studies have shown an alteration in the structure of the proteoglycans of the epiphyseal growth cartilage as a result of the decrease in serum calcium related to deficiency of dietary vitamin D. Vitamin D deficiency also induces a thickening of the epiphyseal growth plate presumably because of the inhibition of maturation of the growth plate chondrocytes. In order to compare the effect on proteoglycan structure with that on growth plate morphology, the proteoglycans of healing epiphyseal cartilage were characterized. The results indicate that, consistent with previous data, in vitamin D-deficient hatching chicks, the proteoglycans of the growth cartilage, but not of the articular cartilage, are smaller in monomer size with slightly smaller chondroitin sulfate chains whose sulfation pattern is unaltered. Sternal cartilage proteoglycans are unaffected. During recovery from vitamin D deficiency, the proteoglycans isolated from the growth cartilage are still not completely normal one day after supplementation with vitamin D, but are indistinguishable from normal by four days. In addition, the results conflict with those of a previous study in which only growth cartilage of hatchling chicks, not sternal or articular cartilage, was reported to synthesize large proteoglycans. Instead, all of these cartilages in the normal chicken have been found in this study to produce large proteoglycans of a size typical for mammalian cartilage and embryonic chick cartilage.

Bone calcification and calcium homeostasis in rats with non-insulin-dependent diabetes induced by streptozocin.

The effect of mild, non-insulin-dependent diabetes (NIDDM) on bone calcification and calcium (Ca) homeostasis was studied in growing rats (males and females). The diabetic state was characterized by mild insulin deficiency, plasma levels being 73% of controls, and mild hyperglycemia, with nonfasting plasma glucose levels of 1.5 times normal. There was no difference in plasma levels of Ca, phosphate (Pi), magnesium (Mg), alkaline phosphatase, immunoreactive parathyroid hormone (iPTH), calcitonin, 25-(OH)vitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25[OH]2D), and 24,25-dihydroxyvitamin D (24,25[OH]2D) between the NIDDM rats and their controls of either sex. Metabolic Ca and Pi balance studies revealed that the experimental animals of both sexes were in positive Ca and Pi balance similar to that of their controls. Histologic studies of the kidney and intestinal slices from the experimental group were normal. Ca and Pi bone content calculated per gram bone ash of the femur, mandible, and second and fourth caudal vertebrae, and the organic content in the bones of the NIDDM animals showed no difference from their controls. Femur bone density and tibial epiphyseal growth plate width and morphology were similar histologically in the experimental and control rats. No decreased osteoid content in the tibial bone was found in the diabetic rats compared with controls. Physiologic sex differences, consisting of lower plasma Pi, higher plasma calcitonin levels, increased ratio of femur dry bone weight to total body weight, and increased percentage of mineralized and total bone volume at the tibial metaphysis seen in female compared with male control rats were also seen in the diabetic animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of epiphyseal cartilage metabolism and morphology in the chronic diabetic rat.

Chronic insulin deficiency, in both man and experimental animal models, has been associated with skeletal alterations, the genesis of which remains unknown. Since cartilage growth and maturation are dependent on the maintenance of adequate glycolytic activity, we evaluated cartilaginous carbohydrate metabolism and epiphyseal growth plate morphology in control, long-term (7 weeks) streptozotocin-induced diabetic and insulintreated diabetic rats. Since parathyroid hormone levels have been shown to be decreased in chronically diabetic rats, we also studied the effect of a low calcium diet (0.1%) on cartilage metabolism and morphology in the insulinopenic state. In vitro incubation of epiphyseal cartilage slices in Kreb's Ringer buffer was performed in 5 mM glucose, with either14C-6-glucose as a glycolytic marker or14C-1-glucose as a pentose phosphate pathway marker. While14C-6-glucose uptake was only marginally reduced in diabetic rat cartilage, lactate production was markedly decreased, approximating 42% of control values, and the activity of the pentose phosphate shunt increased (P<0.01). These biochemical alterations were attended by a marked reduction (P<0.005) in the width of epiphyseal growth plates obtained from rats with untreated diabetes. Both insulin replacement (P<0.001) and dietary calcium restriction (P<0.02) in diabetic animals resulted in a significant increment in the width of epiphyseal growth plates. These morphologic changes were accompanied by a significant (P<0.02) increase in cartilaginous lactate production, in the absence of altered glucose uptake. While insulin treatment corrected glycolysis, it had little effect on the augmented pentose shunt activity, implying stimulation of both these metabolic pathways. Dietary calcium restriction normalized glycolysis and corrected the accelerated activity of the pentose phosphate pathway. We conclude that chronic insulin deficiency in the growing rat is attended by alterations in cartilaginous carbohydrate metabolism which may relate not only to insulinopenia per se, but also to the relative hypoparathyroidism that characterizes the chronic experimental diabetic state. The accumulated data also suggest that these metabolic derangements may account, at least in part, for the reduced longitudinal bone growth observed in this growing animal model.