S. J. Jones

The effects of inhibitors of cysteine-proteinases and collagenase on the resorptive activity of isolated osteoclasts

The effects of specific inhibitors of cysteine-proteinases ((Z-Phe-Ala-CHN2: benzyloxycarbonyl-phenyl-alanyl alanyl diazomethane and E-64: trans-epoxy-succinyl-L-leucylamido(4-guanidino)-butane) and collagenase and collagenase ((Cl-1: N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide) have been tested on the osteoclastic resorption of dentine. Chick osteoclasts were cultured in the presence or absence of 12.5 microM Z-Phe-Ala-CHN2, 40 or 60 microM E-64, or 40 or 100 microM Cl-1 for 1 or 2 days. In addition, osteoclasts were cultured on oyster shell calcitostracum with or without 12.5 microM Z-Phe-Ala-CHN2. Specimens were studied by light microscopy to count cells and resorption features and by scanning electron microscopy (SEM) stereophotogrammetry for the measurement of the depths, plan-areas and volumes of resorption pits. The numbers, depths and volumes (but not the plan-areas) of the resorption pits in dentine were significantly reduced by Z-Phe-Ala-CHN2 and E-64. Thus, for a given plan-area, the volumes and the depths of resorption pits were smaller in these experimental groups compared with control dentine specimens. The overall inhibition of resorption was at least 75%. Cl-1 did not have this inhibitory effect on the numbers or sizes of resorption pits in dentine. When the oyster calcitostracum was used as a substrate for the osteoclasts, Z-Phe-Ala-CHN2 did not reduce the numbers or volumes of pits, but increased the plan-areas and prevented the formation of deeper pits.(ABSTRACT TRUNCATED AT 250 WORDS)

The speed of post mortem change to the human skeleton and its taphonomic significance

The aim of this study was to assess the potential speed of post mortem alteration to skeletal microstructure by examining human material drawn from differing environmental contexts and time periods. The material was taken from terrestrial, intertidal and lacustrine contexts and extended over a range of 3 months to 83 years post mortem. The examination was conducted using backscattered electron imaging which provided information on microstructure and relative density. The results from this study have significantly brought forward the time of known onset for post mortem alteration for 3 morphological types of microstructural change, the earliest of which was 3 months post mortem. The contribution of the depositing environment was also shown to influence significantly the microstructural/ morphological type of post mortem alteration. It is hypothesized that microstructural changes to bone could occur within days of death as a result of endogenous bacterial migration to the skeleton. Further studies are required to establish definitively the earliest moment that such change can occur prior to skeletonisation.

The incidence and size of gap junctions between the bone cells in rat calvaria

Polyclonal antisera to synthetic peptides matching sequences on the cytoplasmic regions of connexin-43, a gap junction protein first identified in rat heart, have been used to immunolabel gap junctions in the calvarial bone, maintained intact as in vivo, of 1- to 2-week-old rats. The specimens were examined in reflection and fluorescence modes by scanning laser confocal microscopy, and the numbers of gap junctions and their sizes estimated. The mean number of connexin-43 immunolabelled junctions per osteoblast (n=65) was 15.3 (SD ± 4.5). The mean length of 227 junctions, selected for the sharpness of the image of the fluorescent spot, was 0.67 μm (SD ± 0.18; range 0.37–1.29 μm) and their mean area 0.26 μm2 (SD ± 0.145; range 0.075–0.93 μm2); these probably fell within the upper half of the total size range. Gap junctions were detected between preosteoblasts, osteoblasts, osteocytes and chondrocytes, and between these juxtaposed cell types. In addition, connexin-43 immuno-labelled junctions were found between some osteoclasts and overlying mononuclear cells at active sites of resorption.

The resorption of biological and non-biological substrates by cultured avian and mammalian osteoclasts

SummaryMammalian and avian osteoclasts were isolated mechanically from long bones, seeded on to either untreated, unmineralized, anorganic or surface-demineralized mammalian dental tissues, and cultured for 1–6 h or up to 9 days in medium with added serum (10% heat-inactivated FCS). All substrates showed Howships resorption lacunae which varied in detail with the composition and structural organization of the tissue. There was no species or substrate specificity. Osteoclasts also adhered, spread, migrated and resorbed in the absence of serum. In addition, osteoclasts resorbed avian egg shell and mollusc shell containing calcite and aragonite. When given the opportunity, osteoclasts are thus biochemically competent to resorb a much wider range of substrates than they usually do in vivo. Access to the substrate and attraction or deliverance of osteoclast precursors to it must be curtailing factors in in vivo resorption.

Functions of cathepsin-K in bone resorption. Lessons from cathepsin-K deficient mice

Cathepsin K is a cysteine proteinase expressed predominantly in osteoclasts. Cathepsin K cleaves key bone matrix proteins and is believed to play an important role in degrading the organic phase of bone during bone resorption. Pycnodysostosis, an autosomal recessive osteosclerosing skeletal disorder has recently been shown to result from mutations in the cathepsin K gene. Cathepsin K deficient mice generated by targeted disruption of this proteinase phenocopy many aspects of pycnodysostosis. They display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space accompanied by an altered ultrastructural appearance of the cathepsin K deficient osteoclasts. These cells also demonstrate an impaired resorptive activity in vitro. In contrast to other forms of osteopetrosis, which are due to disrupted osteoclastogenesis, cathepsin K deficiency is associated with an inhibition of osteoclast activity. Taken together the phenotype of cathepsin K knockout mice underlines the importance of this proteinase in bone remodelling.

Topographically induced bone formation in vitro: Implications for bone implants and bone grafts

We have investigated the influence of substrate topography on the timing and location of bone formation by rat osteoblasts. 250 mu m thick slabs of dental tissues were used intact or had a rectangular grid of grooves (350 mu m wide and of variable depth) cut with a diamond wheel. They were then seeded with rat calvarial osteoblasts and cultured in MEM with 10% FCS at 37 degrees C in 5% CO(2). Ascorbic acid 50 mu g/mL and beta-glycero-phosphate 2 mmol/L were added at confluence. Cultures were observed daily from 2 to 4 weeks, until fixation (and storage) in 70% ethanol. Most were stained with alizarin red S to visualize the newly formed bone. The presence of gap junctions in the bone nodules was determined using connexin-43 immunolabeling and confocal microscopy. Two specimens were embedded in polymethylmethacrylate (PMMA): micromilled blockfaces were coated with carbon and examined by digital backscattered electron (BSE) microscopy. Bone formation began in the second week, preferentially wherever cellular condensation was favored: these locations were (a) within the grooves; (b) at the junction between the slab and the bottom of the culture dish; (c) at the periphery of the dish; and (d) in cracks where dissimilar tissues had separated. In the grooves, a grid of aligned bone developed, the deeper trenches showing bone formation earlier than shallower ones, with bone formation tapering off as a groove became shallower. BSE images showed that the bone formed was well mineralized and contained a high volume proportion of osteocytes. Mean and median values for the mean BSE coefficients were: in vitro bone in grooves 0.138528, 0.141484; in vivo aged bone (2 year old rat mandible) 0.143431, 0.144206; and in vivo young bone (neonate rat cranium) 0.129011, 0.132696. Connexin-43 gap junctions were immunolocalized on osteocytes fully enclosed within bone and on osteoblasts overlying it. We conclude first that local topography is an important factor in the location and timing of bone formation in vitro, and that it is likely to be equally important in vivo in normal bone turnover, fracture repair and the incorporation of bone grafts. Second, the mineral density of the bone formed in vitro is consistent with its being true bone.

The migration of osteoblasts.

SummaryThe endocranial matrix surfaces of parietal bones of 2-week old Albino Wistar rats were partly denuded of osteoblasts and then cultured for various periods up to 24 h, in control or PTE-enriched medium. They were examined by scanning electron microscopy and evidence for cell locomotion was found. Osteoblasts traversed the denuded bone surface and cut edges of bone in either medium, and cells also migrated out from vascular channels.Glass spicules were placed on the otherwise undisturbed osteoblast layer in similar organ cultures for 2, 3 or 5 days. Osteoblasts migrated from the bone to populate the glass, negotiating any angle. The cells in PTE-enriched media were always aligned parallel to one another and elongated, tended to align with the edges of the glass and, in time, formed a substrate of aligned fibrils whose axes were parallel to those of the cells. Osteoblasts in control medium on glass showed variable degrees of alignment and elongation and were less influenced by the edges of the glass. Non-locomotory, nearly equidiametrical cells on glass in 5d control cultures had formed a substrate of randomly oriented fibrils.Migrating osteoblasts on bone matrix did not have leading edge ruffles; isolated, migrating ones on glass did.

Scanning electron microscopy of bone: instrument, specimen, and issues.

There are many ways available now to maximise and analyse the information that can be obtained on the structure and constitution of bone using SEM. This paper considers a range of methods and the problems that arise relating to instrumentation and methodology as they apply to the use of SEM in the study of bone. In addition to the review content, some novel technical approaches to the SEM of bone are considered here for the first time; these include low kV imaging for the detection of new surface bone packets (and residual demineralized matrix after resorption), low kV BSE imaging of uncoated, embedded, and unembedded samples, environmental SEM for the study of wet tissue, low distortion, very low magnification imaging for the study of cancellous bone architecture, the use of multiple detectors for fast electrons in improving the imaging of porous samples, and high resolution, low voltage imaging for the study of collagen degradation during bone resorption.

Mineral density quantitation of the human cortical iliac crest by backscattered electron image analysis: Variations with age, sex, and degree of osteoarthritis

Bone samples from the rim of the iliac crest were obtained at autopsy from 59 patients aged 23 to 75 years, of whom 10 men and 10 women aged 50-75 years had osteoarthritis diagnosed by hand X-rays. An equal number in the same age group and 10 men and 9 women aged less than 50 years were without osteoarthritis. After embedding the bone in PMMA, the blocks were cut, polished, and coated with carbon. The fractions of bone falling within four consecutive bands of signal level were derived from digital backscattered electron imaging. Normal males had more low and medium density bone and normal females more very high density phase tissue proportionately. In both male and female osteoarthritis cases, low and medium fractions were low. The very high density fraction was mainly calcified fibrocartilage; when it was excluded from the calculations, the low, medium, and high phases occurred equally in normal males but increased stepwise in normal females and in osteoarthritis cases of both sexes. The results suggest a lower rate of bone renewal in females than males, and in male osteoarthritis subjects than normal males. An increased proportion of bone of high density would reduce the quality of the bone by increasing its stiffness.

Back-scattered electron imaging of skeletal tissues

The use of solid-state back-scattered electron (BSE) detectors in the scanning electron microscopic study of skeletal tissues has been investigated. To minimize the topographic element in the image, flat samples and a ring detector configuration with the sample at normal incidence to the beam and the detector are used. Very flat samples are prepared by diamond micromilling or diamond polishing plastic-embedded tissue. Density discrimination in the image is so good that different density phases within mineralized bone can be imaged. For unembedded spongy bone, cut surfaces can be discriminated from natural surfaces by a topographic contrast mechanism. BSE imaging also presents advantages for unembedded samples with rough topography, such as anorganic preparations of the mineralization zone in cartilage, which give rise to severe charging problems with conventional secondary electron imaging.