Osteoinduction with COLLOSS, COLLOSS E, and GFm.

This study provided data relevant to three major goals. It confirmed that both COLLOSS and COLLOSS E contain osteo- and chondro-inductive BMPs as shown by their ability to produce new bone in an ectopic location in rats. Second, based on the area of bone produced in standardized implant sections by osteoinductive growth factors in GFm, COLLOSS , and COLLOSS E and their respective collagenous carrier matrices, the study showed that COLLOSS was 0.1, and COLLOSS E 0.3 time as potent as 10 microg of GFm. Finally, the study showed that ordinary and accelerated endochondral bone formation were more frequent in response to GFm than to COLLOSS and COLLOSS E, whereas membranous bone formation was more frequent in response to COLLOSS E than to COLLOSS or GFm.

Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable puttylike polymer matrix.

This article describes preliminary in vivo studies evaluating the osteogeneic potential of bone morphogenetic proteins (BMPs) delivered from an absorbable puttylike polymer matrix. In the first study, bovine-derived bone morphogenetic proteins were incorporated in an polymer matrix consisting of 50:50 poly(DL-lactide-co-glycolide) dissolved in N-methyl-2-pyrrolidone. The matrix was implanted in an 8 mm critical-size calvarial defect created in the skull of adult Sprague-Dawley rats (n = 5 per treatment group). After 28 days, the implant sites were removed and examined for new bone formation, polymer degradation, and tissue reaction. Gamma-irradiated polymer matrices appeared to give more bone formation than nonirradiated samples (histological analysis; 2. 76 + 1.34 mm(2) of bone versus 1.30 + 0.90 mm(2) of bone, respectively and x-ray analysis; 27.2 + 15.9 mm(2) of bone versus 20. 7 + 16.7 mm(2) of bone, respectively) and less residual polymer (0.0 + 0.0 versus 0.2 + 0.4, respectively). The polymer implants with bone morphogenetic protein also gave less inflammatory response than the polymer controls (gamma irradiated polymer/BMP = 1.8 + 0.4 and nonirradiated polymer/BMP = 1.2 + 0.4 versus polymer only = 3.0 + 1. 2, respectively). However, despite trends in both the x-ray and histological data there was no statistical difference in the amount of new bone formed among the four treatment groups (P > 0.05). This was most likely due to the large variance in the data scatter and the small number of animals per group. In the second animal study, bovine-derived BMPs and the polymeric carrier were gamma irradiated separately, at doses of 1.5 or 2.5 Mrad, and their ability to form bone in a rat skull onlay model was evaluated using Sprague-Dawley rats (n = 5 per treatment group). Histomorphometry of skull caps harvested 28 days after implantation showed no significant differences as compared to non-irradiated samples, in implant area, new bone area, and percent new bone (P > 0.05). These results suggest gamma irradiation may be useful in sterilization of the bovine-derived BMPs and the polymeric carrier for potential bone repair and/or regeneration applications.

Cervical spine abnormalities in Down Syndrome.

Down Syndrome (DS) is the most common of the chromosomal disorders and manifests abnormalities in several organ systems. While mental retardation, skull and brain anomalies, and the development of Alzheimer-type neuropathological changes in patients greater than age 40 years are well recognized by neurologists and neuropathologists, less appreciated are the various cervical spine abnormalities that can occur. Widening of the anterior atlanto-odontoid distance (AAOD) and atlantooccipital instability occur in up to 21% and 63% of DS patients, respectively, but neurologic complaints are uncommon and rarely are severe enough to contribute to the patient's demise. We present a case of 49-year-old DS patient whose triplegia, subacute progressive respiratory failure, and death could be attributed to severe degenerative joint disease of the cervical spine with osteophyte formation and severe spinal canal stenosis. We provide the first detailed correlation study between pre-mortem magnetic resonance imaging (MRI) and extensive autoptic dissection in an adult DS patient with cervical spine abnormalities, as well as a review of the literature.

Combination of osteoinductive bone proteins differentiates mesenchymal C3H/10T1/2 cells specifically to the cartilage lineage.

During embryonic development, cartilage formation involves the condensation of mesenchymal stem cells and a series of maturation steps that ultimately results in the mineralized hypertrophic chondrocyte. The embryonic, murine, mesenchymal stem cell line, C3H/10T1/2, is pluripotent; exposure to azacytidine or to bone morphogenetic protein-2 or -4 results in low rates of differentiation to three mesengenic lineages. In contrast to previous studies, we report conditions for 10T1/2 differentiation specifically to the cartilage lineage and at high yields. These conditions include high cell density micromass cultures, a purified mixture of osteoinductive-proteins (BP; Intermedics Orthopedics, Denver, CO), a serum substitute, 50 micrograms/ml ascorbic acid, and 10 mM beta-glycerophosphate. The cartilagenous fate was confirmed by 1) histological detection of sulfated proteoglycans, 2) electron microscopic detection of proteoglycan and rounded cells separated by extracellular matrix containing short, disorganized collagen fibrils, 3) morphological detection of chondrocytes surrounded by a territorial matrix and encompassed within a distinct perichondrium, and 4) immunocytochemical detection of type II collagen and link protein. After 4 weeks in culture, mature although unmineralized cartilage was observed, as indicated by hypertrophic morphology, immunocytochemical detection of osteocalcin, and histological detection of lacunae. These conditions promote overt chondrogenesis for most of the treated cells and preclude lineage determination to the fat, muscle, and bone lineage, as assayed by electron microscopy and histomorphology. The faithful recapitulation of cartilage differentiation that we have established in vitro provides a versatile alternative to the use of chondrocyte and limb bud explant cultures. We propose this as a model system to study the factors that regulate commitment to the chondrogenic lineage, exclusion to related mesengenic pathways, and maturation during chondrogenesis.

[Bone histomorphometry study in post-menopausal women with osteopenia].

OBJECTIVES: To investigate the histological features of bone, the turnover status of bone remodeling in postmenopausal women with osteopenia. To observe the influence of age on the changes of bone turnover. METHODS: Two hundred ninety three bone biopsies were obtained from posterior iliac crests of postmenopausal white women with osteopenia after taking fluorescence-labelled tetracyclin. All these specimens were embedded in methacrylate, the nondecalcified sections were stained routinely and measured by computerized histomorphometry. RESULTS: The mean trabecular bone volume was 10.6% +/- 5.47%, which is 29.3% less than the low limit of normal range (15%) established in our laboratory. There were 186 cases (63.5%) in normal turnover status, 75 cases (25.6%) in high turnover, and 32 cases (10.9%) in low turnover. In comparison with the normal turnover group, the osteoclast number, corrected bone mineralization rate and bone formation rate were elevated and mineralization lag time was reduced in the high turnover group (P < 0.01). In contrary, all the above parameters in the low turnover group were on the opposite (P < 0.05-0.01). Comparison of three age groups (51-60, 61-70, > 70 years) showed that the bone volume and osteoid volume decreased as the age increased. The proportion of high and low turnover types began to increase in the 51-60 age group, peaked in the 61-70 age group and dropped to nadir in the > 70 age group. CONCLUSIONS: Bone histomorphometry examination is not only valuable in distinguishing osteoporosis from osteomalacia, but also beneficial for determination of the bone turnover status and, therefore, is useful for the direction of clinical treatment in patients with postmenopausal osteopenia.

The effect of instrumentation on human spinal fusion mass.

STUDY DESIGN: This study is a prospective histomorphometric evaluation of human spinal fusion bone using video dimensional analysis. OBJECTIVES: Little information exists regarding the biology of fusion mass (FM) and the effect that instrumentation has on FM quality. Concerns regarding potential for "stress shielding" of FM with rigid implants have arisen. The goal of this study was to determine what effect spinal implants have on the quality and metabolism of FM bone. METHODS: Fifty-six patients underwent surgeries to remove spinal implants or extend a fusion after pulse-dose labeling with fluorochrome. Twelve patients had undergone fusions without instrumentation. Duplicate biopsies of FM and iliac crest (IC) were obtained and evaluated blindly for mineralized volume, trabecular thickness, mineralization rate, and bone formation rate. Iliac crest, instrumented FM, and noninstrumented FM were compared. RESULTS: Instrumental FM had superior material properties relative to noninstrumented FM or IC. No significant difference in metabolic activity was present. CONCLUSION: Instrumentation does not lead to FM "stress-shielding."

Bone marrow involvement by lobular carcinoma of the breast cannot be identified reliably by routine histological examination alone.

The aims of this study were twofold: (1) to evaluate the ability of pathologists to recognize infiltration of bone marrow core biopsy specimens by breast carcinoma, particularly lobular carcinoma, using routine hematoxylin-eosin (HE) sections; and (2) if indicated, to determine the reasons for difficulties in diagnosis. Thirty-six bone cores obtained before bone marrow harvest were involved by breast carcinoma and were confirmed by pancytokeratin immunostains. Thirty of the 36 were ductal carcinomas and six were lobular carcinomas. Fourteen negative bone core biopsy specimens (from patients with breast cancer or lymphoma) were included as controls. These 50 bone cores were reviewed by three surgical pathologists. Lobular carcinoma was correctly identified in only 39% of positive specimens as compared with 88% for ductal carcinoma. After instruction, sensitivity for the detection of lobular carcinoma improved to 61% but at the expense of an unacceptably high rate of false-positive diagnoses (18%). None of the three pathologists was able to achieve both high sensitivity and high specificity in recognizing lobular carcinoma in the bone marrow. Lobular carcinoma was difficult to detect because of tumor cell size similar to hematopoietic cells, infiltration as single cells, presence of bland cytological features, and paucity of tissue reaction to the tumor. Although the number of cases of bone marrow involved by lobular carcinoma is small, these findings suggest that pancytokeratin stains should be performed routinely in the evaluation of bone core biopsy specimens from patients with lobular carcinoma, and probably from patients with ductal carcinoma whose HE-stained bone core biopsy specimens are considered negative for tumor.

Bone histomorphometric changes after cyclic therapy with phosphate and etidronate disodium in women with postmenopausal osteoporosis.

The purpose of this study was to examine bone histomorphometry before and after 2 yr of a double blind trial of etidronate to determine whether etidronate was associated with the development of osteomalacia and to clarify the mechanism of action. Sixty-eight postmenopausal women with vertebral compression fractures from 3 clinical centers received 1 g phosphate or placebo twice daily on days 1-3, 400 mg etidronate or placebo daily on days 4-17, and 0.5 g calcium daily on days 18-91. This cycle was given eight times. Iliac crest bone was biopsied after tetracycline labeling. None of the patients developed osteomalacia. The placebo/placebo group lost significantly more bone volume than the other groups. The change in mineralizing surface was significantly different among groups due to the decrease in the placebo/etidronate group. Within groups, placebo/placebo showed a decrease in bone volume without other changes. Phosphate/placebo showed no changes. Placebo/etidronate showed decreases in osteoid volume, osteoid surface, mineralizing surface, bone formation rate, and activation frequency. The only change in phosphate/etidronate was a decrease in osteoid surface. We conclude that 2-yr treatment with cyclical etidronate does not cause osteomalacia and that the mechanism for the increased bone mass is probably a decreased activation frequency.

The effect of low-dose methotrexate on bone metabolism and histomorphometry in rats.

OBJECTIVE: To ascertain the effects of low-dose methotrexate (MTX) on bone metabolism and histomorphometry in rats. METHODS: Female Sprague-Dawley rats (6 months old, n = 42) were divided into the following 4 groups: intraperitoneal (IP) injections of MTX, with and without ovariectomy, and IP saline (controls), with and without ovariectomy. Injections were given for 16 weeks. The MTX dose was equivalent to a standard dose for rheumatoid arthritis in humans that would yield similar serum MTX levels (0.6 +/- 0.1 mumoles). RESULTS: Bone formation (assessed by serum alkaline phosphatase and osteocalcin levels and histomorphometry) was significantly lower in the MTX groups, and bone resorption (assessed by urinary hydroxyproline levels and histomorphometry) was increased in the MTX groups. Bone mass was significantly diminished in the MTX groups. CONCLUSION: Prolonged administration of low-dose MTX in rats causes significant osteopenia via suppression of osteoblast activity and stimulation of osteoclast recruitment, which results in increased bone resorption.

Semiautomated methods for cancellous bone histomorphometry using a general-purpose video image analysis system.

Semiautomated methods are used to measure elongated, curved and complex branching profiles and isolated perimeter segments in monochrome video images with a general-purpose analysis system. These methods are used to make the major primary measurements of bone histomorphometry. Accuracy and reproducibility of the image acquisition, processing and measurement system is documented by measuring a semicircular standard of known dimensions. Semiautomated applications of the Ar/Le method for measuring areas and perimeters, and calculating lengths and widths of osteoid seams, lengths of mineralization labels and mineral apposition rate, wall width, indirect measurements of eroded, osteoclastic and osteoblastic perimeters without tracing, and measurement of mineralized or total cancellous bone area and perimeter gave values comparable to measurements of the same parameters by tracing or grid counting techniques with equal or better reproducibility and much greater efficiency. Intraindividual variation in measuring multiple bone biopsies methods. Major sources of variability for semi-automated methods were image magnification and selection of profile edges by thresholding, and sources of variability for manual methods are image magnification, numbers of orthogonal intercepts, tracing speed and accuracy of the algorithm used to measure traced pixels. Semiautomated methods are accurate, reproducible and rapid methods suitable for bone histomorphometry.

[Effect of parathyroidectomy (PTX) on aluminum (Al) induced bone disease].

Al-treated or untreated decalcified sections of bone matrix were implanted subcutaneously into the abdominal wall of parathyroidectomized (PTX) or control rats for 21 or 28 days. The bone remodeling and Al, Ca content in the implants were measured by using histomorphometry and atomic absorption spectrum method. The results showed that bone area, osteoclast number, osteoblast number and bone mineralization rate of Al-treated or untreated bone in PTX rats and of Al-treated bone in control rats were significantly lower than those of untreated bone in control rats (P < 0.05). There was no marked difference between these parameters of Al-treated bone in control rats and untreated bone in PTX rats (P > 0.05), and these of Al-treated bone in PTX rats were the lowest (P > 0.05- < 0.05). Ca contents were consistent with the histological parameters. Bone Al removing rate between PTX and control rats was not markedly different. In conclusion, both PTX and Al impair bone formation and mineralization, bone biopsy and removal of Al from bone is necessary before PTX treatment in uremic patients with secondary hyperparathyroidism.

[Histological classification of uremic bone disease and clinical significance of bone biopsy].

Bone biopsy was performed in 429 cases of uremic bone disease and they were classified histologically into 3 groups--high turnover 129 (30%), low turnover 102 (24%) and mixed type 198 (46%) cases. The histological features of the high turnover type were increased corrected mineralization rate (CMR) and osteoclast member and decreased mineralization lay time (MLT), while those of the low turnover type were on the contrary. In the mixed there were decrease of CMR and increase of MLT and osteoclast number. Bone aluminum positive rate was determined; it was 75% and 80% respectively in the low turnover and mixed type and 29% in high turnover. Biochemical study showed that significantly higher levels of serum PTH and alkaline phosphate in the high turnover type and low levels of serum 1,25(OH)2D3 and 25(OH)D3 in the low turnover. These results suggested that high turnover uremic bone disease is caused by secondary hyperthyroidism and low turnover mainly related to aluminum toxicity or other causes such as vitamin D deficiency.

An indirect method of measuring widths suitable for automated bone histomorphometry.

Orthogonal intercepts from random test lines (OIr), uniformly distributed intercepts (OIu), and areas and lengths (Ar/Le) were measured manually and by computer to determine individual profile and sample widths of artificial profiles and human osteoid seams. Individual widths were equal by Ar/Le and OIu methods. The means of individual profile widths (all methods), of all orthogonal intercepts (OIu and OIr), or of profile widths weighted in proportion to their lengths (Ar/Le) were also equal. Ar/Le and OIu had smaller variance than OIr. Discrepant individual OIu and Ar/Le widths in digitized images were corrected by thresholding and did not significantly affect sample means. Unweighted Ar/Le sample means were 15-44% lower than weighted means. Distributions of osteoid seam widths were not normal, and all but one had more than one mode. We conclude that the Ar/Le method is comparable to direct orthogonal intercept methods and suitable for automated histomorphometry.

Morphology and biochemistry of bone remodeling: possible control by vitamin D, parathyroid hormone, and other substances.

The effects of PTH and vitamin D on bone are the result of their direct and indirect effects on the functional cells of bone remodeling units and their precursors. These effects are probably modified or controlled by growth factors, cytokines, and PGs generated locally by the process of bone remodeling. Bone remodeling includes resorptive and bone forming phases, each with a longitudinal and a radial component of progression in time and space. Longitudinal resorption is rapid, prolonged and is probably carried out by osteoclasts utilizing hydrogen ions and lysosomal enzymes to remove mineral and organic components of bone in a highly localized and directed fashion. Individual osteoclasts are probably long-lived cells with a nuclear and perhaps a cytoplasmic turnover rate of 8%/day, with replenishment coming from preosteoclasts in the reversal zone. Radial resorption is slower and shorter than longitudinal resorption. It is carried out by reversal phase monocytes whose exact relationship to osteoclasts is not clear. Activated collagenase diffusing from osteogenic cells in the reversal zone could also play a role. The longitudinal rate of bone formation is probably a measure of the rate of proliferation and differentiation of osteogenic cells at the site at which they were activated. The radial rate of bone formation is a measure of how rapidly osteoblasts synthesize and mineralize bone matrix once they reach the resorption surface. PTH and vitamin D have no direct effects on mature osteoclasts. They may have direct stimulatory effects on proliferation and differentiation of osteoclast precursors and their fusion with osteoclasts but this is not clear because the ontogeny of osteoclasts vis a vis monocytes and other phagocytic cells is still not clear. It is likely that their effects to increase osteoclast precursors involve interactions among lymphocytes, monocytes, and hematopoietic stem cells at a distance from bone remodeling units and are mediated by 1,25(OH)2 vitamin D3 induced synthesis of cytokines and colony-stimulating factors. Stimulatory effects of PTH, vitamin D, PGs, and cytokines on osteoclasts are mediated by as yet undefined factors produced by osteoblasts. Osteoblasts stimulated by PTH could also inhibit osteoclasts by synthesizing and releasing PGs. PTH and vitamin D have diverse and often contradictory effects on the functional activity of osteoblast-like cells in vitro that are difficult to interpret because the relationship of these cells to osteoblasts in vivo is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)

Aluminum intoxication in a child: treatment with intraperitoneal desferrioxamine.

We report the successful chelation of aluminum and the clinical resolution of severe aluminum intoxication in an infant receiving chronic peritoneal dialysis through the use of intraperitoneal desferrioxamine. Following the introduction of desferrioxamine, urine and dialysate fluid aluminum levels exceeded those noted without the chelating agent, thus demonstrating enhanced removal of aluminum. As a result of therapy, plasma and bone aluminum levels decreased markedly, and previously noted histomorphometric abnormalities on bone biopsy resolved. Clinically, the aluminum-associated osteomalacia and microcytic hypochromic anemia completely reversed. Moderate developmental delay has also improved slightly but persists. Our experience suggests that intraperitoneal chelation therapy with desferrioxamine may be helpful to reverse aluminum intoxication in children with chronic renal failure. However, limited exposure to aluminum should remain a primary goal.

A vascular lesion with smooth muscle differentiation presenting as a coin lesion in the lung: glomus tumor versus hemangiopericytoma.

A 34-year-old black man presented with an asymptomatic coin lesion in the lung. The diagnosis was narrowed to a differential between glomus tumor and hemangiopericytoma. The authors found that the terminology applied to such tumors in the literature is confusing largely because of lack of consistency in criteria used to establish the diagnosis. The authors propose that the term glomus tumor be reserved for tumors composed of endothelial-lined vascular spaces surrounded by smooth muscle cells. Hemangiopericytoma should be used for similar tumors composed of pericytes with or without other types of perivascular mesenchymal cells. According to these criteria, this case is the third glomus tumor reported in the lung.

Effect of hypophysectomy and hormonal replacement on the uptake of Tc-99m methylene diphosphonate in the metaphysis and shaft of the rat femur: concise communication.

We have investigated the uptake of Tc-99m methylene diphosphonate (Tc-MDP) in the metaphysis and shaft of the rat femur as affected by hypophysectomy and hormonal replacement with growth hormone and thyroxine. Two hours following injection of Tc-MDP, the metaphysis and a specimen of shaft were obtained and the metaphysis-to-shaft radioactivity ratio was measured. By five days after hypophysectomy the metaphysis-to-shaft ratio fell from a control value of 3.8 +/- 0.2 (mean +/- s.e.) to 2.4 +/- 0.2 (p less than 0.05) and remained significantly decreased throughout the 30-day study. When daily hormonal replacement with 0.5 mg of bovine growth hormone and 10 micrograms of thyroxine (both administered intraperitoneally) was given, beginning on the eighth day after hypophysectomy, the metaphysis-to-shaft ratio of Tc-MDP returned to control levels in twelve days. This model demonstrates the effect of growth hormone and thyroxine on the distribution of Tc-MDP, and may be useful as a radiobioassay of net circulating skeletal growth-promoting activity.

Studies on the pathogenesis of avian rickets II. Necrosis of perforating epiphyseal vessels during recovery from rickets in chicks caused by vitamin D3 deficiency.

This study involved comparison of the distribution and integrity of perforating epiphyseal and marrow vessels with the stage of development and integrity of chondrocytes and the distribution of insoluble calcium in the proximal tibial growth plate of 3-week-old vitamin-D3-deficient hypocalcemic chicks and 3-week-old D3-deficient chicks 12, 36, 72, and 120 hours after an oral dose of 10,000 units vitamin D3. The aim was to clarify the mechanisms responsible for chondrocyte hypertrophy and cartilage calcification in the avian growth plate. Within 12 hours after administration of vitamin D3, serum calcium levels rose to normocalcemic levels. The following morphologic changes were first recognizable at the times indicated. Distal portions of previously elongated perforating epiphyseal vessels and adjacent proliferative and maturing chondrocytes underwent necrosis by 12 hours. Chondrocyte necrosis was not preceded by hypertrophy. By 36 hours, vascular and chondrocyte necrosis involved large portions of the thickened proliferating and maturing zone, and perforating epiphyseal vessels were shortened to a normal length. By 72 hours, chondrocyte hypertrophy and calcification resumed around the shortened epiphyseal vessels. By 120 hours, marrow had removed the necrotic cartilage, and morphologically normal growth plate was restored, with perforating epiphyseal and marrow vessels, both ending in a narrow hypertropic cartilage zone. The results indicate that proximity of chondrocytes to perforating epiphyseal vessels is necessary for their viability, but loss of these vessels does not cause hypertrophy. Since hypertrophy and calcification both occur in the proximity of perforating epiphyseal vessels in normocalcemic animals but not in hypocalcemic animals, it is likely that the vessels influence hypertrophy and calcification by delivering calcium to chondrocytes.