Nadire N. Ali

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 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.

Motility and resorption: Osteoclastic activity in vitro

SummaryRabbit osteoclasts (OCs), separated mechanically from long bones, were seeded on to glass or plastic substrates or slabs of sperm whale dentine (SWD). Cells were cultured in MEM with 10% FCS with or without added salmon calcitonin (SCT) at dosages of 0.001, 0.1 and 1 IU/ml. Although most rabbit SCT-treated OCs on the non-biological substrates showed inhibition of peripheral ruffling activity and motility at dosages that stop rat OC movement, resorption still occurred on the dentine. Thus such inhibition is unreliable as a general indicator for resorptive capability. Resorption lacunae were observed at all times from 6 h onwards. Using stereophotogrammetric techniques, the following minimum values were obtained from 24 h cultures: highest hourly rate of resorption of dentine for single OC, 570 μm3/h; average rate 165 μm3/h; mean total volume dentine removed per Howships lacuna complex, 3,885 μm3; average value for plan area of surface attacked per OC, 1,450 μm2.

Survival and resorptive activity of chick osteoclasts in culture

SummaryPrevious studies have shown that osteoclasts obtained from chopped bones resorb surrogate calcified tissue substrata in vitro. These cultures contained all bone and marrow cell type pooled together. We have now parted the marrow from the bone and cultured the cells from the two fractions separately: on both resorbable substrates and on plastic in order to test their longevity in culture and ability to resorb following trypsinisation.Marrow-fraction, bone-fraction or whole bone derived cells were harvested from prehatch chick long bone shafts after removing the periosteum; seeded on sperm whale dentine (SWD) slices or plastic dishes and cultured continously, or trypsinised and reseeded on to fresh substrata at weekly or half-weekly intervals. Observations were made by light microscopy and SEM.Many multinucleate cells were observed in the marrow fraction immediately after settling, deriving presumably from poorly adherent osteoclasts, next to bone, which had not been resorbing at the time of harvesting. By three days in culture on plastic, multinucleate cells were very large both in terms of plant extent and nuclear number: cell fusion occurred between osteoclasts and between osteoclasts and small, round uninuclear cells. SWD was extensively resorbed.The adherence of the osteoclasts was greater (a) to plasuc upon trypsinisation than that of the other cells; and (b) to SWD than to plastic, particularly if the cells were resorbing. Trypsinised cells regained their resorptive capacity after seeding on to new SWD, but only for 1 or 2 treatments.Bone derived cells were similar to the marrow cultures, except for the much higher proportion of other bone cell types. Trypsinisation caused a higher proportional loss of multinucleate cells from both SWD and plastic.Resorption was still occurring at 6 weeks in all cultures. A wide diversity existed in the shapes, depths, plan areas and volumes of the resorption pits.

Monocyte-enriched cells on calcified tissues

SummaryMonocyte-enriched human blood cells seeded on to sperm whale dentine and cultured for up to 20 days failed to produce any morphological signs of resorbtive activity, although multinucleate giant cells were formed. In contrast, preparations containing known osteoclasts derived from bone resorbed the same substrate within hours.

Spectrum of osteoblastic differentiation in new cell lines derived from spontaneous murine osteosarcomas

Cell lines were established from three spontaneous osteosarcoma and one fibrosarcoma of aging mice. They were studied for tumorigenicity, osteoblastic features, and other in vitro cellular characteristics, by a combination of histological, morphological, biochemical, and molecular approaches. It was found that all cell lines formed tumors in vivo, whereas in vitro, only the fibrosarcoma-derived cell line grew efficiently in soft agar. Three out of the four cell lines produced mouse endogenous retroviruses, but none were classical sarcoma viruses. Type I collagen was expressed by all the cell lines, as was another extracellular matrix protein, osteonectin. The osteosarcoma-derived cell lines, however, exhibited different degrees of osteogenic differentiation. Only one line (OSA), and its clonal subline (1G11), consistently gave rise to mineralized tumors after transplantation into syngeneic mice, and these cells expressed high levels of alkaline phosphatase and bone-specific osteocalcin mRNA in vitro. Expression of these biochemical markers of osteoblasts occurred to a lesser extent in a second line (OSC) and was undetectable in the third line (OSB). The clonal 1G11 cell line exhibits the phenotype of a fully mature osteoblast and thus may serve as a particularly useful model for studies of bone cell function and regulation. Studies of cells which display a wide spectrum of osteogenic potential may further our understanding of the mechanisms involved in bone cell differentiation and tumorigenicity.