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The present study is a retrospective examination of osteoblast indices in human iliac crest bone biopsies from a study on the long-term efficacy of calcitonin. Paired baseline and 2-year biopsies were examined from eight control subjects and 10 treated subjects; treated subjects received 100 MRC units synthetic salmon calcitonin (Calcimar, Armour Pharmaceutical Co, Scottsdale, AZ) injected i.m. sub Q at bedtime. Control patients did not receive a placebo injection. All subjects received 400 units vitamin D(2) p.o. q.d. and 1200 mg CaCO(3) p.o. q.d. When the differences in baseline and 2-year values were analyzed, subjects receiving calcitonin showed no decrease compared with control subjects for the fraction of osteoid surface lined by osteoblasts (ObS/OS) (but were in fact significantly greater, P = 0.04). There was no difference from control subjects in the number of osteoblasts/mm bone surface (NOb/BPm), or mean mineral apposition rate (MAR). Since calcitonin is receiving renewed interest for osteoporosis therapy, these data (derived from paired human biopsies) are valuable since they show that no decrease in osteoblast cell number resulted from long-term calcitonin therapy. Results point to the need for renewed investigation concerning the effect of calcitonin on osteoblast-osteoclast interactions.

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.

Our objective for this study was to determine the presence and distribution of tenascin in the human intervertebral disc. The tenascins are a family of extracellular matrix proteins with repeated structural domains homologous to epidermal growth factor, fibronectin type III and the fibrinogens. Little is known about the presence of this protein in the disc. Ten normal human discs donated from subjects newborn to 15 years old, 10 control discs from adult donors aged 24-41 years, and 11 surgical disc specimens from patients aged 26-76 years were examined for immunolocalization of tenascin. In young discs, tenascin was localized throughout the annulus; in the nucleus, localization was confined to pericellular matrix. In adult control and degenerating disc specimens, tenascin in the annulus was localized primarily in pericellular matrix regions encircling either single cells or clusters of disc cells; in rare instances localization was more diffuse in the intraterritorial matrix. In young, healthy disc, tenascin was abundant throughout the annulus. In contrast, degenerating discs in adults showed a localization restricted to the pericellular, and rarely, more restricted intraterritorial matrix. These observations indicate that changes in the amount and distribution of tenascin may have a role in disc aging and degeneration, possibly by modulating fibronectin-disc-cell interactions, and causing alterations in the shape of disc cells.

Matrigel (reconstituted basement membrane extract) is a potent inducer of cell growth and differentiation in vitro. This study examined phenotypic variation and proliferative responses of human annular intervertebral disc cells in vitro in Matrigel and Growth Factor Reduced Matrigel (GFR-Matrigel). Cells from age- and gender-matched control subjects and patients with degenerative disc disease were grown either on the surface of, or suspended within, either matrices. Disc cells grew well on top of both matrices with cells spontaneously forming cell projections. Cells grown within either matrix migrated within the gel to form colonies. Increased colony formation within the matrices was seen with young control and patient cells (p0.05). Old and young control and patient cells showed increased proliferation within GFR-Matrigel compared to Matrigel. When grown on the matrix surface, young patient and control donor cells showed increased proliferation on GFR-Matrigel compared to Matrigel. Cellular proliferation was significantly greater inside a 3-dimensional environment than a two-dimensional surface monolayer environment. Disc cells had increased proliferation when grown in or on GFR-Matrigel compared to Matrigel. These studies serve as a baseline for subsequent investigations regarding effects of cytokines on disc cells and increase our knowledge of the influence of extracellular matrices on disc cell proliferation.

Heterotopic ossification is a common complication in which bone develops in soft tissues. Although frequently benign, in some patients the condition becomes painful, restricts motion, and requires surgical intervention. This condition and the cells responsible for it are poorly characterized. Using cell culture techniques, this study compares the performance of osteogenic cells obtained from heterotopic ossification with trabecular osteoblasts isolated from the same patient. Cells isolated from trabecular bone samples and heterotopic ossification sites from six patients were evaluated for osteocalcin production after exposure to 1,25-dihydroxycholecalciferol, alkaline phosphatase activity, typing and synthesis of collagen, cell proliferation, and total protein content. Samples of heterotopic ossification and trabecular bone from three of the patients were examined histologically. Heterotopic ossification derived cells were shown to produce osteocalcin, Type 1 collagen, and alkaline phosphatase activity. They also had increased rates of collagen synthesis, alkaline phosphatase activity, and cell proliferation compared with the normal osteoblasts. Initial tissue from the heterotopic ossification sites showed increased numbers of osteocytes/mm2 compared with normal trabecular bone. Although heterotopic ossification derived cells functioned qualitatively like osteoblasts, they exhibited elevated levels of activities traditionally ascribed to osteoblasts, such as collagen synthesis and alkaline phosphatase activity.

Bone healing was investigated histologically in a rat fibular osteotomy model subjected to microgravity (shuttle flight STS-29) and the tail suspension microgravity simulation model. Exposure to microgravity or tail suspension occurred during the last 5 days of a 10-day healing period. Periosteal osteogenesis and the development of vascular channels in both experimental groups were similar to that observed in a weightbearing control group. Chondrogenesis was more advanced in weightbearing rats than in either flight or tail-suspended rats. Angiogenesis in the osteotomy gap was more advanced in weightbearing and tail-suspended rats than in the flight group. These findings suggest that bone healing may be impaired during space travel. Interpretation of the findings is complicated by observations that flight and suspended rats lost weight during the flight period and that suspended rats consumed less water than control rats. Tail suspension did not produce the same pattern of healing as spaceflight; therefore, long-term studies of bone healing, conducted entirely in the microgravity environment, are needed to distinguish metabolic from mechanical influences and to determine whether effective fracture consolidation will occur in the absence of gravitational forces.

A recent survey has been carried out polling academic experts in osteoporosis, most of whom were members of the National Osteoporosis Foundat ion Scientific Advisory Board. The purpose of this poll was to learn the current opinion on issues that deal with the future diagnosis and management of osteoporosis. The results of the poll are interesting from a number of viewpoints: (1) they serve to quantify current expert opinion on the important issues chosen for the poll; (2) the questions chosen and the results obtained can sometimes identify areas that should be targets of future direction and education; and (3) the results can provide a valuable source of information for those concerned about the future management of osteoporosis. To this end, we present the questions posed and the survey results and provide interpretative comments. The survey addressed three general topics: (1) osteoporosis screening, (2) drugs to inhibit bone resorption, and (3) drugs to stimulate bone formation. Fifty questionnaires were sent out with a response rate of approximately 60%.

Adenosine and adenosine analogues exhibit anti-inflammatory effects in vitro and in vivo, but their usefulness is limited by profound cardiovascular side effects. Therefore, we synthesized inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (AK) (EC 2.7.1.20), to enhance endogenous adenosine concentrations at sites of inflammation. GP-1-515 (4-amino-1-(5-amino-5-deoxy-1-beta-D- ribofuranosyl)-3-bromo-pyrazolo[3,4-d]pyrimidine), a novel AK inhibitor, decreased adhesion of activated human neutrophils to cultured endothelial cell monolayers by increasing local adenosine levels. The mechanism of inhibition in this assay seemed to involve selectin blockade and was independent of the beta 2 integrins. GP-1-515 and 2-chloroadenosine (a nonmetabolizable adenosine analogue) had no effect on the surface expression or shedding of adhesion molecules. An agent that disrupts the cytoskeleton, cytochalasin B, mimicked the effect of adenosine on cell adhesion. Interactions between L-selectin and the neutrophil cytoskeleton might be altered by adenosine and could contribute to adenosine-mediated adhesion inhibition.

The spondyloepiphyseal dysplasia subclassification of bone dysplasias includes achondrogenesis, hypochondrogenesis, and spondyloepiphyseal dysplasia congenita. The phenotypic expression of these disorders ranges from mild to perinatal lethal forms. We report the detection and partial characterization of a defect in type II collagen in a perinatal lethal form of hypochondrogenesis. Electrophoresis in sodium dodecyl sulfate-polyacrylamide of CB peptides (where CB represents cyanogen bromide) from type II collagen of the diseased cartilage showed a doublet band for peptide alpha 1(II)CB10 and evidence for post-translational overmodification of the major peptides (CB8, CB10, and CB11) seen as a retarded electrophoretic mobility. Peptide CB10 was digested by endoproteinase Asp-N; and on reverse-phase high pressure liquid chromatography, fragments of abnormal mobility were noted. Sequence analysis of a unique peptide D12 revealed a single amino acid substitution (Gly--Glu) at position 853 of the triple helical domain. This was confirmed by sequence analysis of amplified COL2A1 cDNA, which revealed a single nucleotide substitution (GGA--GAA) in 5 of 10 clones. Electron micrographs of the diseased cartilage showed a sparse extracellular matrix and chondrocytes containing dilated rough endoplasmic reticulum, which suggested impaired assembly and secretion of the mutant protein. This case further documents the molecular basis of the spondyloepiphyseal dysplasia spectrum of chondrodysplasias as mutations in COL2A1.

Two case studies are presented in which quantitative bone histomorphometry is used to analyze bone changes in adult patients with diagnosed spindle cell sarcoma. Three tumor-involved sites and one noninvolved site from the iliac crest of patient 1 were evaluated. In the involved sites the percentage trabecular bone volume (11.0%) and the number of osteoblasts (0.6 cell/mm2) were significantly reduced, osteoid volume was significantly increased (8.1%), and woven osteoid was present. The total eroded surface (6.8%) was also significantly increased. In the noninvolved site the number of osteoblasts was decreased and both the percentage eroded and percentage osteoclast surfaces were increased. In the femoral epicondyle specimen from patient 2 the number of osteoblasts was 27.0 cells/mm2, percentage osteoid volume was 18.4%, percentage osteoid surface was 62.9%, and osteoid thickness was 20.0 microns. In tumor-involved sites in both patients indices of active bone resorption were similar and normal. These two case studies indicate that (1) distinctive morphologic changes occur in bone invaded by spindle cell sarcoma, and that (2) changes affect bone formation to a greater extent than bone resorption. Bone alterations are probably local in nature and related to the extent and duration of tumor invasion and the influence of local tumor factor(s).

Fluoride has often been used as a treatment for osteoporosis, a metabolic bone disease of considerable importance in the elderly population. The techniques currently used to monitor a patient's response to fluoride are outlined. New findings concerning (1) a mechanism for interaction of fluoride with osteoblasts (via mitogenic signals or growth factors); (2) toxicity and carcinogenesis; (3) recent clinical trial data; and (4) the importance of dosage, administration regimens, and side effects in an effective fluoride treatment protocol are reviewed. Some recent clinical data challenge the efficacy of fluoride in the treatment of postmenopausal osteoporosis. Because of the implications of these recent studies with respect to fracture incidence during fluoride therapy, fluoride cannot be recommended at this time for general use in the treatment of osteoporosis.

The short rib polydactyly syndromes (SRP) are lethal neonatal skeletal dysplasias with a narrow chest, short limbs, and other abnormalities. Type II (Majewski) short rib polydactyly can be distinguished from the Type I/III (Saldino-Noonan) type on the basis of radiographic and histologic changes. Our previous transmission electron microscopic studies suggested unusual patterns of cartilage calcification in these syndromes. We evaluate calcification in the present study using scanning electron microscopy and quantitative morphometry of calcification regions digested to expose calcospherite nodules (calcification nodules), distinctive morphologic structures which form during cartilage calcification. Mean calcospherite area of the Majewski Type II SRP (3.5 +/- 0.24 x 10(-6) mm2 (3) (mean +/- sem (n) did not differ from normal control means (3.1 +/- 0.5 x 10(-6) mm2, (3)). The mean area for Type I/III, however, was significantly larger than both the control and Type II means (8.9 +/- 1.16 x 10(-6) mm2 (7), p = .001). This difference in calcospherite size adds a further differentiating feature between these two dysplasias.

Cartilage damage termed ochronotic arthritis is the major pathology occurring in adult patients with alcaptonuria. We have investigated the effects of homogentisic acid (HGA), the metabolite accumulating in patients with alcaptonuria, on the in vitro proliferation of rabbit and human articular chondrocytes and human fibroblasts. Growth of these chondrocytes in monolayer decreased proportionally to increasing concentrations of HGA (0.001 mM to 1.0 mM). Substantial growth inhibition and morphologic abnormalities of chondrocytes were produced by a concentration of HGA (0.05 mM) similar to that found in serum of patients with alcaptonuria. Human fibroblasts required higher concentrations of HGA for a comparable degree of growth inhibition. The addition of ascorbic acid (0.57 mM) reduced this growth inhibition and prevented the morphologic changes.

A classification and discussion of the histology of osteomalacia and the current histological techniques used in its identification are presented. Three categories of histologic features of osteomalacic bone are proposed: (1) increased osteoid, elevated osteoblast and osteoclast numbers and marrow fibrosis; (2) increased osteoid and osteoblast numbers, and (3) increased osteoid associated with few osteoblasts. Clinical conditions for which quantitative bone histomorphometric data are available are presented and related to the three categories. The consistency of clinical pathophysiology and histologic bone features is emphasized.

Correct prediction of tumor behavior and interpretation of local factors in the tumor microenvironment rely in part upon accurate determination of tissue changes after tumor invasion. The authors examined local bone changes in primary malignant fibrous histiocytoma (MFH) of bone in a 59-year-old woman. Three noninvolved and three tumor-involved sites were evaluated by quantitative determinations of bone structural and dynamic features. Compared to noninvolved sites, tumor-involved bone was characterized by significantly increased osteoblast index (89.4 +/- 15.6 [mean +/- SEM] versus 7.3 +/- 6.0, P = 0.008), percent osteoid area (12.1 +/- 2.7 versus 1.2 +/- 0.5, P = 0.02), percent of trabecular surface covered by osteoid (70.0 +/- 6.0 versus 14.5 +/- 4.8, P = 0.002), and percent osteoid lined by osteoblasts (36.4 +/- 3.6 versus 3.7 +/- 3.0, P = 0.002). Bone 7.8 mm distant from invading tumor cells showed features characteristic of noninvolved sites, whereas bone completely surrounded by tumor showed markedly decreased osteoblast features. Osteoblast function also was affected by tumor; the amount of matrix laid down per day bore a significant positive correlation with the osteoblast index. These data indicate the following: distinctive bone morphologic changes occur in situ during invasion by MFH; changes affect aspects of bone formation but not resorption during invasion; both osteoblast number and osteoblast activity are significantly altered; and changes are local in nature and probably reflect the osteoblast response to local tumor factor(s) and are dependent upon the extent of tumor invasion.

This study evaluates osteoblast and osteoclast cell numbers and cell activities determined from quantitative histology of trabecular bone in human iliac crest bone biopsies. Subjects studied were postmenopausal osteoporotic patients (aged 53-81) for whom total body calcium and urine and serum clinical data were also available. Only 39% of the osteoclasts identified by acid phosphatase content were multinucleate; however, a significant correlation between multinucleate osteoclasts and the total number of osteoclasts was found (r = 0.87; p less than 0.001). The mean number of osteoclasts per square millimeter of total tissue area was 0.96 (+/- 0.14, SEM, n = 36); the mean number of osteoblasts was 6.8 (+/- 1.5, n = 14)/mm2 tissue area. Computed osteoclast activity (square millimeter bone resorbed per osteoclast nucleus per day) was 2.6 X 10(-4) +/- 1.0 X 10(-4) (n = 13). Computed osteoblast activity (square millimeter bone formed per osteoblast per day) was 2.9 X 10(-5) +/- 8.8 X 10(-6) (n = 13). Calculated mean rate of bone resorption was 1.6 X 10(-4) +/- 3.4 X 10(-5) mm2 bone resorbed per mm2 total tissue area per day (n = 19). These data indicate that although osteoclasts are not numerous in the iliac crest of postmenopausal osteoporotics, the individual osteoclast activity (in square millimeter bone resorbed osteoclast cell unit per day) is significantly greater (p less than 0.001) than the osteoblast activity (in square millimeter matrix deposited per cell per day). These data also point out that greater consideration should be given to the role that osteoclast cell activity may play in human postmenopausal osteoporosis.

This study of two cases documents increased bone mass in a 30-yr old man and osteopetrosis in a 38-yr-old woman and examines the patients in terms of radiologic features, bone photon absorptiometry and bone biopsy analyses which include tetracycline kinetics. Both patients had elevated bone mass based on quantitative bone histology of iliac crest biopsies normal, or low PTH, and normal calcitonin values. However, they differed markedly from each other in other respects: One patient possessed significantly increased rates of bone formation and mineralization, had elevated serum alkaline phosphatase, increased total hydroxyproline over a 24 hour urine collection but displayed a normal amount of bone surface involved in resorption. The other subject had normal bone formation parameters, but had elevated serum acid phosphatase and a significantly elevated resorbing (crenated) surface. Although most theories ascribe and cause of osteopetrosis to decreased resorption, our study shows that increased bone formation, even when accompanied by normal resorption, can lead to the abnormal accumulation of bone mass. In this respect, the resorptive response of this patient was abnormal; the normal coupled relationship between bone formation and bone resorption, which ensures proper control of endosteal bone volume, had been lost.

A patient with Behçet's syndrome suffered acute paraparesis during sigmoidoscopy. An aortogram demonstrated thrombosis of the distal abdominal aorta just above the bifurcation; thrombectomy resulted in complete return of neurologic function. Examination of the clot disclosed a preponderance of polymorphonuclear granulocytes. We propose that mechanical trauma from this otherwise benign procedure caused the vascular lesion. Patients with a similar predisposition to arterial or venous thrombosis may, in unusual circumstances, be jeopardized by proctosigmoidoscopy.

We have studied the residual adenylosuccinate lyase activity in cultured lymphoblasts from a pair of siblings with infantile autism who have been previously shown to have a deficiency of the enzyme. The rates and distribution of de novo purine synthesis assessed by the utilization of radiolabeled formate by intact cells was nearly normal. We compared the steady-state kinetics and thermal stability of adenylosuccinate lyase in lysates from those cells and normal lymphoblasts. There is no evidence of inhibitory activity in the lysates of the mutant cells. The optimal pH was approximately 7.8 and was indistinguishable from that in control cells. The apparent Km in the two mutant cells lines (2.6 +/- 0.5 microM) is not significantly different from normal (3.3 +/- 0.8 microM), but the mutants displayed markedly decreased maximum steady-state velocities (6.7 +/- 1.1 compared to 13.8 +/- 0.9 nmol.mg-1.min-1). Residual activities in mutant cells show decreased thermal stability (t1/2 = 0.21 minutes at 60 degrees C as compared to 2.2 minutes), suggesting that there is a structural mutation of the adenylosuccinate lyase in the mutant cells.

We report a case of homozygous adenine phosphoribosyl transferase (APRT) deficiency associated with 2,8-dihydroxyadenine stones recurrent in a patient with a renal transplant. The disease was diagnosed 23 years after the initial episode of renal colic. At that time the disease was unknown. Our patient is only the second case of this disorder reported from the United States. Correct diagnosis is important because long-term maintenance with allopurinol and a low purine diet can effectively prevent stone formation and renal failure.