Osteoid Area Research Articles

Effects of hyperthermia on bone. II. Heating of bone in vivo and stimulation of bone growth.

Previous studies in vitro have shown that it is possible to achieve comparable temperature distribution in bone and the adjacent soft tissues, under appropriate experimental conditions. The objective of the present work was to determine the effects of hyperthermia on bone in vivo. In order to obtain direct temperature measurements in bone, catheters were surgically installed on top of and inside the medullary cavity of the femur of normal rabbits. The thighs were irradiated with 915 MHz microwaves for 45 min, once or twice a week. The temperatures on and inside the bone were maintained between 42.5 and 44.0 degrees C; the resulting temperatures in the muscle were within 1.0 degrees C at depths equidistant from the applicator. After four to six treatments the femora were excised for histopathological examination. New trabecular bone was deposited around the catheters; most bone components including periosteum, osteoid, and fully calcified matrix could be seen. Large numbers of osteoblasts and osteoclasts lined the trabecular surfaces, and numerous cement lines were visible, running in all directions, indicating extensive bone deposition and remodelling. In contrast, control bones (catheters installed--no hyperthermia) showed much less ossification, with many areas of thin incomplete osteoid. Further, bones treated with hyperthermia only (no surgical trauma) showed no such changes. Thus, it appears that following an initial insult, hyperthermia promotes bone deposition.

Chemical and histological features of men with osteoporosis.

Six men (average age, 42) underwent evaluation for idiopathic osteoporosis. Standard measurements of serum and urine were studied. Lumbar skeletal mineral density was measured by dual-photon absorptiometry. Skeletal biopsies of the iliac crest were obtained after double-labeling with tetracycline. Average renal cyclic adenosine monophosphate and urinary calcium values were elevated. Lumbar spinal density was 72% of age-matched norms, and histomorphometric analyses of bone revealed decreased trabecular bone volume and formation rate and increased trabecular osteoid area, osteoclast number, and calcification rate. The results suggest that idiopathic osteoporosis in these men is associated with increased parathyroid hormone secretion as assessed by increased renal cyclic adenosine monophosphate and high skeletal remodeling activity.

Age-related differences in phosphonoformate-induced bone toxicity in cats

Phosphonoformate (PFA), a monophosphonate pyrophosphate analog, caused plasma biochemical and bone histomorphologic abnormalities in cats given 1,000 mg/kg/day as a continuous intravenous infusion for 14 days. Plasma biochemical alterations observed in young cats (10 weeks old) treated with PFA included increased calcium and decreased phosphorus, alkaline phosphatase, and calcitriol. Young cats treated with PFA developed rickets-like lesions characterized by widened growth plates, increased osteoid, and failure of mineralization. In addition, area of mineralized trabecular bone was decreased. Osteoclast size was increased whereas osteoclast perimeter and number were unaffected in young PFA-treated cats. Plasma alkaline phosphatase was decreased in adult cats (greater than or equal to 1 year old) treated with PFA but changes in calcium, calcitriol, and immunoreactive parathyroid hormone were highly variable and not significantly different. Adult cats treated with PFA exhibited osteomalacia characterized by increased osteoid area, perimeter, and width with failure of mineralization. In addition, static resorption indices were increased in PFA-treated adult cats but area of mineralized trabecular bone was not decreased. The monophosphonate PFA inhibited bone mineralization in young and adult cats similar to bisphosphonate treatment in other species. Because PFA is currently in phase I trials for use in AIDS, results of this study suggest a need to evaluate patients treated with PFA for metabolic bone disease.

Parathyroid carcinoma associated with polycythemia vera

A link between hyperparathyroidism and the growth of hematopoietic stem cells is suggested by this report of a parathyroid carcinoma with polycythemia vera. A 56-year-old white woman presented with splenomegaly, a palpable neck mass and hypercalcemia, recurrent six years after resection of a parathyroid tumor. She had pancytosis with a subnormal serum concentration of erythropoietin. Radiographs showed subperiosteal erosions and osteopenia. Nephrocalcinosis was absent. Bone biopsy showed a decreased cortical width with many intracortical osteoclasts. The cancellous bone area remained normal, but the osteoid area/ bone area, osteoblast perimeter and osteoclast perimeter were increased. At surgery, a parathyroid carcinoma was found in the same location operated on previously. As in two other reported cases, postoperative improvement in the hypercalcemia was associated with remission of the blood dyscrasia. A novel finding in this case is that when the hypercalcemia eventually recurred, it was again accompanied by pancytosis. With bisphosphonate therapy, the serum intact parathyroid hormone level increased in response to a decrease in the ionized calcium level, indicating that the cancer was not autonomous. This case suggests that in the presence of the ionized hypercalcemia, the parathyroid tumor may have produced or induced production of a growth factor that can stimulate pancytosis. The differential diagnosis of polycythemia and hypercalcemia should be expanded to include parathyroid tumors in addition to hepatic, adrenal, renal, and ovarian neoplasms.

Equivalency of various methods for estimating osteoid seam width.

We compared the true osteoid seam width (TOSW) as measured by a modification of the orthogonal intercept lengths with various methods of estimating seam widths, including (1) the commonly used length measurements at four equidistant points (O.Wi/4PT), (2) osteoid area divided by the osteoid perimeter (O.Ar/O/Pm) or the bone/osteoid interface (O.Ar/B.Bd), and (3) a novel method for estimating seam width defined as the osteoid area divided by the major axis of the seam (O.Ar/Axis). All methods for approximating osteoid seam width significantly exaggerated the true osteoid seam dimension by an amount that ranged from 16 to 23%. However, the relative accuracy of all methods of estimating osteoid seam width are equivalent as evidenced by the similar mean difference from the TOSW (3.4, 4.1, 5.1, and 3.8) demonstrated by O.Wi/5PT, O.Ar/Axis, O.Ar/O.Pm, O.Ar/B.Bd, respectively. Regression analysis of the various estimates of seam width on TOSW also demonstrated the equivalency of these methods. Moreover, all estimates could be employed to discriminate seams of normal dimensions from abnormally wide seams in bone specimens derived from patients with osteomalacia. Differences between the methods, however, were observed that may have practical importance. In this regard, the direct procedure of determining distance demonstrated less variance than the indirect estimate of width. As a result, the direct measurement required fewer samples (n = 13) to detect a significant difference to normal and could discriminate smaller deviations in seam width (1.7 microns) at a given sample size compared with O.Ar/Axis (n = 28; 2.9 microns), O.Ar/O.Pm (n = 42; 3.4 microns), and O.Ar/B.Bd (n = 42; n = 3.2).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic bone disease of total parenteral nutrition: course after changing from casein to amino acids in parenteral solutions with reduced aluminum content

Bone disease with total parenteral nutrition (TPN) has been attributed to aluminum loading or vitamin D therapy. We studied 17 patients who first received TPN containing casein hydrolysate with high Al and ergocalciferol (25 micrograms/d) for 6-72 mo followed by TPN containing amino acids with reduced Al and ergocalciferol (5 micrograms/d) for 9-58 mo. We also did a cross-sectional study of 22 patients receiving casein and ergocalciferol (25 micrograms/d) compared with 46 patients receiving amino acids and ergocalciferol (5 micrograms/d) for 6-58 mo. Bone formation was higher and osteoid area, bone-surface stainable Al and total bone Al were lower with amino acid TPN than with casein TPN. Bone formation varied inversely with both plasma Al and bone-surface Al, suggesting that plasma or bone-surface Al, acquired during TPN, can reduce bone formation and lead to patchy osteomalacia. Serum levels of iPTH and 1,25-dihydroxyvitamin D were higher with amino acid TPN.

Biotin Deficiency May Alter Tibiotarsal Bone Growth and Modeling in Broiler Chicks

Forty-eight, day-old male broiler chicks (Hubbard × Hubbard strain) were fed purified diets with two levels of biotin (0 and 400 μg/kg) and two levels of linoleate (26.5 and 1.5 g/kg) in a factorially designed experiment to determine the effects of these nutrients on tibiotarsal bone growth and modeling. Chicks fed biotin-deficient diets (0 μg/kg diet) exhibited varus deformities, footpad dermatitis, shortened tibiotarsi, and significantly higher bone densities and percentage bone ash. Anatomically there were two different bone modeling patterns. The mid-diaphyseal cortex was thickest laterally in chicks fed adequate biotin and thickest medially in biotin-deficient chicks. Periosteal bone appositional and bone formation rates, osteoid perimeter, and osteoid area of perimeter were reduced in tibiotarsi of chicks fed diets deficient in biotin compared with effects in chicks fed adequate biotin. Altered bone modeling patterns and quantitative differences in bone histomorphometry suggest a relationship between the effects of biotin on bone growth and the development of varus limb deformities.

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures.

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.

Thyroparathyroidectomy modifies the skeletal response to aluminum loading in the rat

Diminished parathyroid hormone (PTH) secretion may contribute to the accumulation of aluminum (Al) in bone and to impaired bone formation in Al-related bone disease. Therefore, intact (AL, N = 9) and thyroparathyroidectomized (TPTX-AL, N = 9) rats were given intraperitoneal injections of Al, 2 mg/day, for 42 days; intact control (C, N = 11) and TPTX control (TPTX-C, N = 9) animals received i.p. injections of vehicle only. Quantitative bone histology and measurements of mineralized bone formation (Rbf) using double tetracycline labeling were done for cortical and for trabecular bone; trabecular bone aluminum content (BA) was determined by histochemical methods. BA did not differ between AL and TPTX-AL, 33 +/- 13% versus 39 +/- 14%, and Rbf decreased similarly from control values in both Al-treated groups. In contrast, osteoid production was impaired to a greater extent in TPTX-AL than in AL. Thus, osteoid area and osteoid seam width were each lower in TPTX-AL than in TPTX-C; these values did not differ between AL and C. TPTX can aggravate Al induced reductions in osteoid synthesis, and low serum PTH levels may contribute to the pathogenesis of aplastic bone. However, reductions in Rbf during Al loading are not mediated by PTH.

Bone Oxalate in a Long-Term Hemodialysis Patient Who Ingested High Doses of Vitamin C

We performed a bone biopsy on a patient who had been receiving hemodialysis for 23 years. The bone had oxalate deposition. Previous bone biopsies had shown osteitis fibrosis without oxalate deposition. The osteoid area was increased (11% of bone area), as was the fibrosis (6.1% of tissue area) and aluminum deposition (38% of surface). Bone formation rate was normal (259 mu 2/mm2/d). We examined bone biopsies of 22 patients who had been receiving hemodialysis for over 10 years, and none had oxalate deposits. We discovered that our patient had been ingesting 2.6 g/d of vitamin C. The bone oxalate deposition may have been caused by the ingestion of high doses of vitamin C.

The differential response of cortical and trabecular bone to aluminum administration in the rat.

Osteomalacia has been noted following in vivo aluminum (Al) loading in the rat by some investigators but not by others. To determine whether the response of bone to Al differs as a function of the skeletal site examined, quantitative histology of cortical and trabecular bone was done in the tibiae from control (C, n = 10), Al-treated (AL, n = 9), nephrectomized control (NX-C, n = 7), and nephrectomized Al-treated (NX-AL, n = 8) rats given 2 mg/day of Al for 4 weeks. Bone Al content was determined by histochemical methods. In cortical bone, osteoid seam width, osteoid volume, and percent osteoid area were similar for all groups. In contrast, for trabecular bone, both forming surface (means +/- SD) (5.2 +/- 3.4 vs 1.8 +/- 1.1%, P less than 0.05) and osteoid volume (1.7 +/- 0.7 vs 1.0 +/- 0.4%, P less than 0.05) increased from control values in AL, although osteoid seam width did not differ. In NX-AL, trabecular forming surface (20.2 +/- 6.7 vs 6.2 +/- 2.4%, P less than 0.01), osteoid area (13.2 +/- 5.7 vs 3.5 +/- 0.8%, P less than 0.01), and osteoid width (18.7 +/- 5.7 vs 9.7 +/- 2.3 micron, P less than 0.01) all were greater than in NX-C. Deposits of Al were undetectable in C and NX-C, were minimal in cortical bone in AL and NX-AL, but were present at 40.5 +/- 11.5 and 71.1 +/ 6.5% of trabecular surfaces in AL and NX-AL, respectively. Osteoid area and osteoid surface each correlated with trabecular bone Al. Thus, (a) osteoid accumulates in trabecular, but not in cortical, bone after 4 weeks of Al loading; (b) the extent of osteoid accumulation correlates with the bone Al content; and (c) the histologic response to Al in cortical and trabecular bone is related to local differences in the uptake of Al into bone.

24,25(OH)2D3, bone formation, and bone resorption in vitamin D-deficient, azotemic rats.

Bone formation, mineralization, and resorption were measured in vitamin D-deficient, azotemic rats given two different dosages of 24,25(OH)2D3 daily and in vehicle-treated controls (C). The intraperitoneal administration of 65 pmol over a 10 day period corrected the hypocalcemia observed in C, whereas 130 pmol produced mild hypercalcemia. Both dosages reduced osteoid width, osteoid area, and mineralization front width from control values. The rates of bone and matrix formation were unaffected by treatment. In C, matrix formation exceeded bone formation and resulted in osteoid accumulation; both dosages of 24,25(OH)2D3 reversed this relationship such that bone formation exceeded matrix formation in each treatment group. The rates of osteoid maturation and initial mineralization increased during repletion with 24,25(OH)2D3 at both dosage levels. However, the serum calcium concentration was correlated with both osteoid maturation rate (r = 0.68, P less than 0.01) and initial mineralization rate (r = 0.63, P less than 0.01) when all three experimental groups were considered. Bone resorption was unchanged from control values during treatment with 24,25(OH)2D3. The results suggest that 24,25(OH)2D3 promotes the maturation and mineralization of osteoid, and that this metabolite differs in its effects on bone formation and resorption. It is not clear, however, that the changes in bone dynamics observed are independent of the calcemic response induced by metabolite repletion under the conditions of this experiment.

Diminished Bone Formation in Experimental Diabetes: Relationship to Osteoid Maturation and Mineralization

The pathogenesis of osteopenia in clinical diabetes remains uncertain. Thus, bone formation, mineralization, and resorption were measured over a 10-day period using double-tetracycline labeling of bone in control (C, N = 18), untreated diabetic (I-, N = 14), and insulin-treated diabetic (I+, N = 16) rats. Diabetes was induced by the intravenous (i.v.) injection of streptozotocin (STZ), 90 mg/kg, in citrate buffer. Bone and matrix (osteoid) formation and apposition were decreased by 50% from C values in I- rats (P less than 0.05), but were unchanged in I+ rats. Osteoid seam width and osteoid area were also less in I- (P less than 0.05), but similar in I+, when compared with C. In untreated diabetic rats that continued to actively form new bone, osteoid maturation and mineralization were not diminished when adjusted for the rate of bone formation. However, 5 of 14 untreated and 2 of 16 insulin-treated diabetic animals showed no uptake of tetracycline into bone (Chi-square, 8.54; P less than 0.05), suggesting a defect in mineralization in a subset of diabetic rats. Measurements of serum glucose, calcium, and phosphorus concentrations, of urinary excretion rates for glucose, calcium, and phosphorus, and of creatinine clearance failed to correlate with the changes in bone growth and histology observed. The results indicate heterogeneity in the response of bone in diabetes, and suggest that bone formation and osteoid volume are reduced early in the course of this disorder. These data in short-term diabetes support previous observations in both man and rat that indicate a state of low bone turnover in diabetes.

Osteoid in bones of elderly patients without bone disease.

Bone samples were taken at post-mortem examination by Jamshidi needles from 88 elderly patients who were known to have normal bone biochemistry in life. The extent of osteoid in these samples is quantitatively described. In patients with normal creatinines the relative osteoid area ranges from 0 to 3.7% and trabecular osteoid surface percentage ranges from 0 to 23%, but higher values were seen in those with raised creatinines. Osteoid extent did not vary with age, sex or time of year. Descriptions of other series of control bone biopsies in the literature are reviewed and it is suggested that relative osteoid area greater than 3.5% or osteoid surfaces greater than 25% should be considered excess osteoid.

Aluminum is associated with low bone formation in patients receiving chronic parenteral nutrition.

Patients treated with chronic total parenteral nutrition may develop metabolic bone disease. We evaluated 22 bone biopsy specimens from 16 patients. Compared with those of age- and sex-matched normal controls, these specimens had significantly higher osteoid area and lower total bone area and bone formation rate, as measured by double tetracycline labels. Aluminum was found in specimens from the 14 patients receiving casein hydrolysate but not in the two receiving amino acids as their nitrogen source. The reduced bone formation correlated inversely with the logarithm of the aluminum level. Aluminum was localized to the surface of mineralized bone; tetracycline uptake was absent at those sites. These bone findings are similar to those from aluminum intoxicated patients on hemodialysis. Both groups also have low parathyroid hormone levels. Thus, aluminum appears to be an important pathogenic factor in the osteodystrophy of patients receiving dialysis or total parenteral nutrition.

Heterotopic Ossification of the Intestinal Mesentery: Report of a Case Following Intraabdominal Surgery

In a 55-year-old male a nodular mass of heterotopic ossification developed in the intestinal mesentery two weeks after coloproctectomy for severe ulcerative colitis. The lesion was located close to the ileostoma where it encroached upon the intestinal wall and led to luminal obstruction. Histological examination of the tumour mass showed actively proliferating mesenchymal cells forming irregular areas of primitive osteoid and bone. The process appeared consistent with an early stage of heterotopic ossification where the short history explains the absence of the "zone phenomenon" usually characterizing such lesions. By reviewing the literature the reported case appears unique as other cases of intraabdominal heterotopic ossification have not been encountered.

BK virus-induced tumors in hamsters: a morphological, histochemical and ultrastructural study.

Macroscopic morphology, histology and ultrastructure of BK virus (BKV)-induced hamster and mouse tumors were investigated. Groups of animals were immunosuppressed to study the relationship between immune system and BKV oncogenesis. Ependymomas had the highest incidence, followed by tumors of pancreatic islets, osteosarcomas, lymphomas and sarcomas, sometimes associated in the same animal. All the tumors were found to be BKV specific. Ependymomas showed the shortest latency, infiltrated surrounding tissues but did not metastasize. Pseudo-rosettes were common and basal bodies were observed. Atypia and necrosis were more often present in immunosuppressed animals both for the ependymomas and for the other oncotypes. Pancreatic insulomas were frequently multinodular, possibly because of multifocal origin and metastasized to the liver. Hormone secretory granules were often found on electron microscopy. Osteosarcomas metastasized to lungs and peritoneum and showed the presence of osteoid, chondroblastoid and mixoid areas. Characteristic giant cells were present. Immunosuppression did not enhance tumor incidence and did not influence the latency period. However, neoplastic growth appeared to be more rapid and with more aggressive behavior in immunodepressed animals. These findings suggest an influence of the immune system in tumor development, whereas the virus oncogenic process seems unaffected.

Quantitative bone histology in children with chronic renal failure

Quantitative bone histology (on undecalcified sections following double tetracycline labeling), radiographs, and biochemistry were studied in 47 children, ages 1 to 17 years, with glomerular filtration rates (GFR) below 80 ml/min/1.73m2. The earliest histologic change was an increased osteoid surface accompanied by increased osteoblast and tetracycline surfaces. However, significant bone disease (increased osteoclastic surface, fibrosis, increased osteoid area, increased mineralization lag time, and reduced tetracycline uptake at osteoid surfaces) occurred only at GFR below 30 ml/min/1.73m2. Radiographs and alkaline phosphatase were normal in 25% of children with significant bone disease; parathyroid hormone was increased in 48% of children without bone disease. Thus, these noninvasive investigations were poor predictors of disease presence. GFR was the most sensitive indicator because bone disease occurred only at GFR below 30 ml/min/1.73m2 and was present in all children with GFR below 20 ml/min/1.73m2. It was concluded that bone histology is required for early detection of bone disease and is an essential tool in experimental studies of renal osteodystrophy. However, because the level of GFR will indicate the presence or absence of bone disease in most children, bone biopsy can be avoided generally in clinical practice.

Osteopetro-rickets : a new cogenital bone disorder

Two lethal nwtations have been described in the rat: ⪡ osteopetrosis ⪢ (op) and , ⪡ toothless ⪢ (tl). The op mutant can be cured by normal bone marrow infusion, while the tl mutant cannot. We report here additional data with regard to the tl mutant. The bone disease of the tl rat as in the op rat Is associated with a ,precocious thymic atrophy, but bone lesions are quite different. In addition to classical osteopetrosis, the tl rat displays several features of rickets: broadening of the extremities of long bones, thickening of the epiphyseal plates, presence of osteoid areas. In addition, the persistence of embryonic characters, evidenced by high levels of alpha-fetoprotein, suggests that the developmental defect of bone is part of a more general process.

Quantitative bone histology: A new method

A method is described for quantitative histology of undecalcified bone using an embedding medium suitable for histochemistry. Six normal subjects were given tetracycline markers 24 and 3 d prior to the biopsy which was taken from the posterior iliac spine using an 8 gauge Jamshidi needle. The tissue was embedded in a mixture of equal parts methylmethacrylate and hydroxyethylmethacrylate. Three consecutive sections were cut and (1) stained histochemically for acid phosphatase activity to identify osteoclast cytoplasm, (2) stained with pyronin to identify osteoblast cytoplasm, (3) mounted unstained for tetracycline fluorescence. Tracings of the 3 sections were superimposed using a camera lucida attachment to the microscope, and the tracings quantitated with an electronic digitizer. The major parameters were bone area (17.1 +/- 4.4%) (mean +/- SD), osteoblast or forming surface (5.9 +/- 2.7%) and osteoclast or resorbing surface (1.0 +/- 0.3%). Bone apposition rate was 1.0 +/- 0.2 micrometers/day and resorption velocity (calculated by assuming bone formation and resorption to be equal) 8.4 +/- 4.1 micrometers/day. Mineralization was assessed by osteoid area (3.8 +/- 2.5%), osteoid surface (13.9 +/- 6.6%), tetracycline uptake at the mineralizing front (62.3 +/- 13.5%), and mineralization lag time (18.1 +/- 2.5 days).