Effects of atmospheric dust particles on common medicinal plants in an industrial area of West Bengal, India.

The exposure of atmospheric dust particles on four common medicinal plants (Ocimum sanctum, Andrographis paniculata, Catharanthus roseous, and Kaempferia galanga, which are available in the study area and cultivated by the local people for medicinal purposes) affects their growth, levels of essential biochemical constituents and heavy metal concentration. The plant species were grown by pot cultivation in an industrial area with high levels of coal dust to assess the capacity of heavy metals accumulation in their leaves and changes in allometry and biochemical parameters. The results showed that annual average SPM and dustfall varied between 195.88 to 645.97 µg/m3 and 17.55 to 41.16 g/m2/month, respectively. Dustfall at different polluted sites was 2.4, 2.1, 1.5, 1.4, and 2.3 times higher than at the control site. The most prevalent heavy metal in atmospheric particulate matter was Zn, followed by Pb, Ni, Cu, Co, and Cd. Plant allometry measurements such as height, stem width, root length, petiole length, and leaf area are shown to have a strong and significant (p<0.05) negative correlation with dustfall and SPM. Total chlorophyll and RWC were inversely proportional to the dust load present in all the species. Except for Andrographis paniculata, chlorophyll and leaf-extracted pH of plant species were moderately correlated with APTI, whereas no correlation was noticed for ascorbic acid. A positive correlation between SPM and heavy metals in leaves was observed. The results implied that the cultivation and collection of medicinal plants from the study area could be potentially toxic to human health.

Long-term outcomes of the hip shelf arthroplasty in adolescents and adults with residual hip dysplasia: a systematic review.

Background and purpose - The shelf arthroplasty was the regular treatment for residual hip dysplasia before it was substituted by the peri-acetabular osteotomy. Yet, evidence regarding the survival of shelf arthroplasty surgery has never been systematically documented. Hence, we investigated the survival time of the shelf procedure until revision to THA in patients with primary hip dysplasia. Factors that influenced survival and complications were also examined, along with the accuracy of correcting radiographic parameters to characterize dysplasia.Material and methods - The inclusion criteria were studies of human adolescents and adults (> 16 years) with primary or congenital hip dysplasia who were treated with a shelf arthroplasty procedure. Data were extracted concerning patient characteristics, survival time, complications, operative techniques, and accuracy of correcting radiographic parameters.Results - Our inclusion criteria were applicable to 9 studies. The average postoperative Center-Edge Angle and Acetabular Head Index were mostly within target range, but large variations were common. Kaplan-Meier curves (endpoint: conversion to THA) varied between 37% at 20 years' follow-up and 72% at 35 years' follow-up. Clinical failures were commonly associated with pain and radiographic osteoarthritis. Only minor complications were reported with incidences between 17% and 32%.Interpretation - The shelf arthroplasty is capable of restoring normal radiographic hip parameters and is not associated with major complications. When carefully selected on minimal osteoarthritic changes, hip dysplasia patients with a closed triradiate cartilage may benefit from the shelf procedure with satisfactory survival rates. The importance of the shelf arthroplasty in relation to peri-acetabular osteotomies needs to be further (re)explored.

Enzyme histochemical localisation of alkaline phosphatase activity in osteogenesis imperfecta bone and growth plate: a preliminary study.

At the ultrastructural level alkaline phosphatase has been studied in calcifying cartilage but not in bone. The aim of this study was to assess if there is an osteoblast dysfunction in Osteogenesis Imperfecta (OI) with respect to alkaline phosphatase activity. Specimens from three OI type II foetal femoral bones, two OI type II growth plates, one normal foetal femoral bone and growth plate, one OI type III femoral bone specimen and one normal juvenile bone specimens were examined using modified lead nitrate method to identify alkaline phosphatase reactivity. The electron dense reaction product (indicative of the presence of alkaline phosphatase) was demonstrable on the cell membrane of the osteoblasts, as focal concentrations in the collagen osteoid and on the mineralisation front of normal bone. In normal bone the intensity of the reaction seemed to be stronger than in OI bone and appeared as a continuous black line along the osteoblast cell membranes. In OI bone the reaction product only appeared as a few electron dense beads along the osteoblast cell membrane. There appeared to be reduced and diffuse reaction product on OI osteoblasts, thus implying either a reduced level and/or altered activity of alkaline phosphatase and hence a dysfunction of osteoblasts. This confirms the findings of the previous report of the impaired activity of alkaline phosphatase in OI osteoblasts. Even in the OI growth plate, hypertrophic chondrocytes showed less intense reaction product than the chondrocytes in the normal growth plate. The normal human growth plates used in this study showed a similar pattern, but in the OI growth plate even the hypertrophic zone, where the alkaline phosphatase activity is reported to be high, showed less intense reaction product. Biochemical reports indicate that alkaline phosphatase levels are normal in cultured OI cell lines, yet ultrastructural histochemical observations reported here, show reduced enzyme localisation and this may suggest reduced amounts of protein or reduced activity at the tissue level.

Ultrastructural changes in rat livers perfused in vitro and in vivo with a high dose of methotrexate.

Methotrexate is an antifolate that is widely used in the treatment of malignant tumours and other diseases. The present study was undertaken to examine the short-term effects of high doses of methotrexate (HD-MTX) on the ultrastructure and metabolic activity of isolated rat livers. The authenticity of the drug-induced changes was substantiated by the concomitant use of in vivo experiments. Isolated rat livers were infused with HD-MTX via the portal vein for 3 hours (total dose for each liver 2000 mg). For in vivo experiments, each rat received a single intravenous injection of a maximum tolerated dose of MTX (100 mg/kg body weight) that allowed the animals to survive for 3 days. At the end of each experimental period, MTX-treated and control livers were processed for light microscopy (LM), scanning (SEM) and transmission electron (TEM) microscopy. Oxygen consumption and thyroxine metabolism were measured in treated and control isolated livers. With the exception of a few minor differences, the structural changes in the hepatocytes after MTX treatment in vitro and vivo were similar. There were focal changes consisting of disruption of normal hepatic plates and swelling and vacuolation of the hepatocytes, with no clear evidence of restriction to a specific hepatic zone. SEM revealed striking changes in the plasma membrane, the microvillar system, intercellular junctions and the sinusoidal endothelium. TEM revealed disorganized endoplasmic reticulum, dispersion of the polyribosomes, a variety of mitochondrial changes, and glycogen redistribution. In MTX-treated isolated rat livers, the uptake of tetraiodothyronine (T4) was not affected, but triiodothyronine (T3) release was impaired. Oxygen consumption was increased in livers treated with MTX. Employing an organotypic liver perfusion model in conjunction with the in vivo experiment and the use of SEM, TEM and hepatic thyroxine measurements, this investigation revealed that infusion of HD-MTX induced early ultrastructual changes in cell membrane, intercellular junctions and cell organelles and disturbance in the functional integrity of the hepatocytes in isolated rat liver.

Isomerism of the right atrial appendages: clinical, anatomical, and microscopic study of a long-surviving case with asplenia and ciliary abnormalities.

This study describes a case of isomerism of the right atrial appendages (bilateral morphologically right atrial appendages associated with complex congenital cardiac lesions) with ciliary abnormalities. Detailed investigation included gross anatomic dissection, review of the clinical history, and light, confocal, and electron microscopy. Clinically, this 40-year-old, long-surviving male patient had relatively good health until 4 years before death, which was due to cardiac failure. Surgical intervention consisted only of a Blalock-Taussig shunt (anastomosis of the right subclavian artery to the right pulmonary artery) at 6 years of age. Despite the presence of complex cardiac malformations and asplenia, his longevity may be attributed to the connection of the pulmonary veins to the atrium without pulmonary venous obstruction, pulmonary valvar stenosis rather than atresia, no significant atrioventricular valve regurgitation, and no serious infections during his life. Microscopic examination of bronchial epithelium revealed a narrow, disorganized epithelium with abundant goblet cells and short, angulated cilia with a random orientation and possibly an abnormal central microtubule doublet. These abnormalities were not present in controls, and have been noted in primary ciliary dyskinesia (PCD) or Kartagener's syndrome. Because this syndrome has classically been thought to cause random lateralization resulting in a mirror-imaged arrangement of the organs, the occurrence of truly isomeric patterns is not widely recognized. Whereas polysplenia and left bronchial isomerism have been reported to occur in immotile cilia syndrome, this is the first report to present detailed postmortem anatomic evidence of isomerism of the right atrial appendages, right bronchial isomerism, and asplenia in association with microscopy suggesting ciliary abnormalities.

A light and electron microscopic study of osteogenesis imperfecta bone samples, with reference to collagen chemistry and clinical phenotype.

A detailed morphological study was carried out using light and electron microscopy on 36 bone specimens from patients suffering from osteogenesis imperfecta (OI) and 20 age- and site-matched control bone specimens. The findings were grouped into the clinical types of OI according to the Sillence classification. The morphological and ultrastructural alterations observed in OI bone correlate well with clinical severity. Thus, OI type I, the mildest type, showed the least abnormalities in bone ultrastructure. OI type IV closely resembled type I, with only minor abnormalities in the bone cells and osteoid. OI type III showed abnormalities in the structure and distribution of osteoid collagen fibrils, whilst OI type II, the lethal form, revealed many varied abnormalities such as thin cortical bone, sparse trabecular bone, increased numbers of osteoclasts and osteocytes, thin osteoid with thin collagen fibrils, and patchy mineralization.

A fourier transform infrared spectroscopic and solid-state NMR study of bone mineral in osteogenesis imperfecta.

Fourier transform infrared spectroscopy and 31P solid-state nuclear magnetic resonance spectroscopy were used to determine if any structural or compositional differences in osteogenesis imperfecta (OI) bone mineral could be detected that might help to explain the bone fragility observed in this disease. A previous study by Cassella et al. used an electron probe X-ray microanalytical technique to compare the calcium to phosphorus (Ca/P) molar ratios in normal bone and bone from patients with OI. It was demonstrated that bone from OI patients had a lower Ca/P molar ratio. This study demonstrated that OI bone mineral had a general hydroxyapatite structure and that isomorphous substitutions in the carbanoapatite lattice could account for the low Ca/P molar ratio.

Morphometric analysis of type I collagen fibrils in the osteoid of osteogenesis imperfecta.

Electron microscopy and morphometric measurements of bone osteoid collagen diameter from 42 osteogenesis imperfecta (OI) patients and 25 age- and site-matched controls were carried out. Although the mean diameter did not correlate well with the severity of the disease, it related well with the clinical types and revealed collagen fibrils of reduced diameter in the osteoid of all OI types. Thus, OI type II (the severest type) demonstrated the smallest diameter (45 nm), followed by OI type I (the mildest form) with a mean diameter of 57 nm. The diameter obtained for type III (67 nm) and type IV (64 nm) was lower than the normal control mean diameter (73 nm) but did not show a statistical difference. The thinner fibrils observed in OI bone may be unable to provide nucleating and scaffolding sites for mineral propagation and may play a role in the fragility of bone in this disease.

Abnormal mineral composition of osteogenesis imperfecta bone as determined by electron probe X-ray microanalysis on conventional and cryosections.

Osteogenesis imperfecta (OI) is a genetic disorder of the connective tissue characterized by frequent bone fractures. The cause of bone fragility is still unknown even though substantial work on collagen has been done. We measured the calcium to phosphorus ratio (Ca/P) of bone mineral from 35 OI bone samples and 25 age- and site-matched control specimens, using electron probe X-ray microanalysis in the transmission electron microscope. Ultra-thin cryosections and conventionally prepared resin sections were used. Cryo-ultramicrotomy avoids any possible artifactual demineralization that may occur in conventional aqueous media. The Ca/P ratio obtained by these two methods was compared and there was no statistical difference between them. The results were differentiated according to the clinical types of OI for the first time. The Ca/P ratio of OI bone mineral was lower than normal in both resin and cryosections, and mirrored the severity of the disease. OI type II had the lowest ratio (Ca/P = 1.49) compared with normal age- and site-matched controls (Ca/P = 1.69). This abnormal mineral composition in OI type II could be a contributory factor to bone fragility in OI bone.

Association of magnesium whitlockite crystals with lipid components of the extracellular matrix in human articular cartilage.

Several basic calcium phosphate mineral phases have been reported to be associated with osteoarthritis joint disease. Magnesium whitlockite crystal deposition has been reported in both osteoarthritic and normal human articular cartilage. Existing data suggest that likely prevailing conditions within cartilage would not support de novo whitlockite formation. It would appear, therefore, that additional factors must be extant at sites of crystal deposition. In this study normal articular cartilage specimens were examined for the presence and distribution of lipids relative to crystal deposition within the extracellular matrix. Specimens were examined using light and transmission electron microscopy (TEM), with standard processing protocols plus a malachite green-glutaraldehyde-osmium tetroxide (MGO) method, used to retain lipids normally removed from tissues during preparation for electron microscopy. Elemental maps of sections produced using this method were also made using X-ray microanalysis. Positive oil red O staining for lipid was clearly apparent immediately below and parallel to the articular surface of cartilage specimens using light microscopy. The extent and distribution of staining correlated well with the distribution of crystals, observed by TEM, in sections of tissue from adjacent sites of the same specimens. Using standard TEM, crystals were frequently observed scattered amongst intramatrical lipidic debris, particularly pericellularly, in areas of cell necrosis and amongst close packed tangential fibers between the articular surface and initial superficial zone chondrocytes. Cartilage specimens processed using the MGO method demonstrated electron dense features, not apparent using standard techniques, identified as lipid. Such extracellular lipid deposition varied with depth, with 100 nm globular bodies present in the superficial region, where colocalization of crystals and lipid were observed in about 10% of crystal observations. The association of lipid and crystal deposition is discussed in the context of phospholipid associated mineral formation and the potential role of such magnesium whitlockite deposition assessed.

Mineral changes in a transgenic mouse model for osteogenesis imperfecta.

A line of transgenic mice has been investigated that expressed moderate levels of an internally deleted human gene for the pro alpha 1(I) chain of type I procollagen to determine if they would make a good model for osteogenesis imperfecta (brittle bone disease). Previous workers have reported extensive fracturing in these mice, with femurs that were shorter and bone that had decreased ash weight, mineral and collagen content. These workers demonstrated increased brittleness in the bone by biomechanical measurements. The molar calcium to phosphorus ratio in bone from patients with osteogenesis imperfecta has previously been reported to be lower than that in normal human bone. Mineral changes were observed at the ultrastructural level in these mice and were comparable with those seen in patients with osteogenesis imperfecta. Bone from both transgenic and normal littermate mice was examined to determine if any similarity with the data for human osteogenesis imperfecta could be drawn. X-ray microanalysis of bone mineral demonstrated a lower calcium to phosphorus molar ratio in transgenic mouse bone than in normal littermates. Fourier-transform infra-red spectroscopy confirmed that the mineral present was apatitic in nature despite the lower calcium to phosphorus molar ratio. Multiple fracture calluses were present on the ribs and on the long bones of the transgenic mice; this was absent in normal littermates. This mouse model may lead to a better understanding of the underlying pathology resulting in fragile bones in osteogenesis imperfecta.

An ultrastructural and immunogold localization study of proteoglycans associated with the osteocytes of fetal bone in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a rare, heterogeneous, inherited connective tissue disorder frequently caused by abnormalities of type I collagen. It is characterized by bone fragility, osteopenia, and progressive skeletal deformities. Electron microscopy of three OI type II fetal bone samples revealed numerous large osteocyte lacunae. In addition, there was a perilacunar osteoid-like band of collagen surrounding the osteocytes, which was unmineralized and morphologically unusual. Furthermore, large osteocyte lacunae contained fine particles and filamentous material similar to the expected ultrastructural appearance of proteoglycans. More detailed examination was carried out using histochemical and immunogold localization of proteoglycans at light and ultrastructural levels. These tests and the use of electron probe X-ray microanalysis confirmed that the material in the osteocyte lacunae was proteoglycan. In contrast, in the age- and site-matched normal fetal bone, all the osteocyte lacunae appeared negative for proteoglycan. Proteoglycans are regarded as inhibitors of calcification. Our observation of substantial amounts of proteoglycan in abnormally enlarged osteocytic lacunae of some OI fetal bone suggests association with the abnormal bone of this particular subtype of OI type II.

An ultrastructural study of the phagocytic activity of astrocytes in adult rat brain.

The role of adult astrocytes in the removal of cell debris and foreign particles following injury to the brain is controversial. This study was undertaken to elucidate the response of adult astrocytes to needle injury of the rat cerebral cortex, using a suspension of colloidal carbon as a marker for phagocytosis. Either a single or 2 successive injections of colloidal carbon suspension were made into the cerebral cortex. The animals were allowed to survive for periods of from 1 to 30 d. Unequivocal involvement of astrocytes in the removal of carbon particles was evident only in those brains which had been subjected to 2 successive injections of carbon. The particles were located in membrane-bound vacuoles and were subsequently sequestered in lysosomes. Carbon-containing astrocytes were observed in the immediate vicinity of the lesion, in the adjacent parenchyma, around blood vessels and abutting carbon-containing macrophages. This study demonstrates that adult astrocytes are involved in phagocytosis, but only as a second line of defence. The possible significance of carbon-laden astrocytes further away from the site of the lesion is discussed.

A morphological and ultrastructural study of bone in osteogenesis imperfecta.

A morphological and electron microscopic study of bone from patients with osteogenesis imperfecta (OI) has been performed. Bone from OI patients from various anatomical sites has been compared with that from normal, age-, site-, and sex-matched controls. The morphology of OI bone appeared variable among patients and sites of bone examined. Immature woven bone and a poor lamellar pattern were the significant morphological features and demonstrated that OI could not be characterized on the basis of a single histological pattern. At the ultrastructural level, a number of previously unreported features were evident. Abnormal collagen fibers and an altered mineral composition were found in many OI patients, however, the panoramic heterogeneity between clinical types and indeed within a single clinical type made it difficult to classify OI in this manner. The presence of intermitochondrial inclusions containing calcium and phosphorus and the presence of a stromal calcification in the bone in some OI patients suggested an abnormal mineral formation. Qualitatively, no obvious difference in the number of osteoblasts or osteoclasts was observed. The morphology and ultrastructure of OI bone were good indicators of the disease and serve a role in assessing the progress of a patient through diagnosis and treatment. This report presents new ultrastructural findings in collagen and in mineral formation in OI compared with normal human bone.

The isolation and characterization of magnesium whitlockite crystals from human articular cartilage.

A number of basic calcium phosphate crystals have been demonstrated in human articular tissues. The exact relationship between crystal deposition and disease remains obscure, although there is evidence supporting a rapid degenerative arthropathy within a specific set of patients. Limited reports of 'cuboid' calcium phosphate microcrystals in articular cartilage have been made over the last 10 years. In this study the occurrence of such crystals, not apparent by light microscopy, in human articular cartilage has been confirmed by transmission electron microscopy and X-ray microanalysis of tissue prepared by aqueous and anhydrous processing techniques. A crystal isolation technique involving collagenase digestion, centrifugation and sodium hypochlorite treatment was developed enabling crystal characterization by electron and X-ray diffraction. Crystals were identified as magnesium whitlockite; the first report of this mineral in articular cartilage. The presence of this mineral phase in normal and osteoarthritic articular cartilage is discussed with consideration given to physical conditions known to favor whitlockite formation and those extant in articular cartilage.

Magnesium whitlockite deposition in articular cartilage: a study of 80 specimens from 70 patients.

OBJECTIVE: To examine articular cartilage from a number of joint sites, using a large sample group, for the presence of magnesium whitlockite crystal deposition. METHODS: Articular cartilage specimens were taken from a total of 70 patients. The majority of specimens were taken from femoral heads, with smaller numbers from femoral condyle, tibial plateau, radius, ulna, and several small peripheral joints. Normal and osteoarthritic articular cartilage specimens were obtained from patients undergoing prosthesis replacement or amputation. Specimens were resin embedded and examined using transmission electron microscopy and x ray microanalysis. RESULTS: Magnesium whitlockite crystals were identified, on the basis of morphology, size and elemental composition, in articular cartilage from all sites sampled. The distribution of crystals was similar in all samples (restricted to the superficial zone), although the density of deposition was extremely variable, with the greatest density observed in femoral head specimens. No magnesium whitlockite crystals were observed in osteophytic or epiphysial cartilage. CONCLUSIONS: This study demonstrated the widespread extent of magnesium whitlockite deposition in human articular cartilage, albeit at much lower density than previously reported in femoral head articular cartilage. In consideration of possible roles for these crystals in articular cartilage, it is concluded that an opportunistic mode of formation, possibly influenced by mechanical stresses, would be most plausible.

An electron probe X-ray microanalytical study of bone mineral in osteogenesis imperfecta.

A semiquantitative electron probe X-ray microanalytical (XRMA) technique, in conjunction with transmission electron microscopy, was used to compare the calcium to phosphorus (Ca/P) molar ratios in calcium phosphate standards of known composition, in normal bone and in bone from patients with osteogenesis imperfecta (OI). Using a modified routine processing and resin embedding schedule, the measured Ca/P molar ratio of calcium phosphates standards of known composition were found to correlate well with the Ca/P molar ratio based on their respective chemical formulae. This technique was then used to compare the Ca/P molar ratio in normal human bone and in OI bone. The Ca/P ratio values for normal bone (Ca/P = 1.631) correlated well with those for chemically prepared hydroxyapatite (Ca/P = 1.602), but in bone from OI patients, the Ca/P molar ratio was significantly lower (Ca/P = 1.488). This study has shown that there is a lower Ca/P molar ratio in OI bone compared with normal, matched bone. This suggests that the mineral deviates from the carbanoapatite usually found in bone. Isomorphous substitutions in the carbanoapatite lattice could account for this although this study has neither proved nor disproved this. The altered bone mineral is another factor that could contribute to the increased fracture rate observed in OI.

An ultrastructural, microanalytical, and spectroscopic study of bone from a transgenic mouse with a COL1.A1 pro-alpha-1 mutation.

A line of transgenic mice have been investigated that expressed moderate levels of an internally deleted human gene for the pro alpha (I) chain of type I procollagen. These mice expressed the gene at approximately 50% that of the endogenous gene. The gene construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta by causing biosynthesis of shortened pro alpha (I) chains. Periera et al. (1993) reported extensive fracturing in these mice with femurs that were shorter in length and bone that had decreased ash weight, mineral, and collagen content. These workers demonstrated an increased brittleness in bone using biomechanical measurements. The functional consequences of these mutant genes were examined in both transgenic and in normal littermate mice to determine if a valid model at the ultrastructural and analytical level had been produced for OI. X-ray microanalysis of bone mineral demonstrated a significantly lower calcium-to-phosphorus (Ca/P) molar ratio in transgenic mouse bone than in normal littermates; this was a feature of human OI bone. Fourier transform infrared spectroscopy confirmed that the mineral present was apatitic in nature despite the lower Ca/P molar ratio. Alizarin red skeletal staining showed the presence of multiple fracture calluses on the ribs and on the long bones of some of the transgenic mice, this was not seen on normal littermates. No light microscopic differences were observed between normal and transgenic mice; however, many ultrastructural correlates with human OI were observed in the transmission electron microscope. Anomalous fibrils associated with type I collagen, and an amorphous calcified material was observed lining the cartilage, extending beyond the lamina limitans in young transgenic mice.

A morphometric analysis of osteoid collagen fibril diameter in osteogenesis imperfecta.

Osteogenesis imperfecta is a genetic disorder of connective tissue characterised by frequent bone fracture following minimal trauma. Mutations of type I procollagen genes have been widely reported as the cause of OI and such mutations have been shown to introduce kinks into the collagen molecule. A study was performed to examine type I collagen fibrils at the ultrastructural level in the transmission electron microscope (TEM). Type I collagen fibrils from the bone osteoid of OI patients and age- and site-matched normal control bone were photographed in the electron microscope. A histomorphometric analysis of the diameters of collagen fibrils photographed in the TEM indicated that type I collagen in OI bone was larger in diameter compared with normal bone. This increase in diameter of type I collagen fibrils may represent an alteration in the quaternary structure of the collagen fibril as a consequence of kinked, poorly packed collagen molecules. Such alteration in the collagen fibrils may affect the formation and stability of bone mineral associated with it.