Maureen E. Owen

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats.

SummaryDiffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.

Bone formation in organ cultures of bone marrow

SummaryBone formation in organ cultures of intact marrow fragments from mouse is described. Marrow explants were cultured on the top surface of a millipore filter at a gas-liquid interface. Observations with both light- and electron microscopes demonstrated the formation of a well-organised trabecular matrix lined with osteoblast-like cells. The tissue and cells were positive for alkaline-phosphatase activity. Large amounts of thick, well-banded collagen fibrils and matrix vesicles typical of those found in bone were present. The tissue became mineralised in the presence of 10 mM Na-β-glycerophosphate; in its absence a similar trabecular matrix developed but mineralisation did not take place.

Distribution of fibroblastic colony-forming cells in rabbit bone marrow and assay of their osteogenic potential by anin vivo diffusion chamber method

SummaryRabbit bone marrow has been separated into core, intermediate, and endosteal cell populations. When plated outin vitro, each of the fractions gave rise to colonies of fibroblastic cells. The colony-forming efficiency increased from the core population by a factor of 4 to a maximum of 3.4 × 10−6 in the endosteal fraction. The osteogenic potential of each fraction was determined following their implantation in diffusion chambers into host rabbits. Each of the indices of osteogenesis (alkaline phosphatase activity, Ca and P accumulation) were significantly lower in the core population than in the two populations closer to the bone surface. Our results are consistent with the hypothesis that the osteogenic precursor cells of marrow belong to the fibroblast colony-forming cell fraction, and indicate that these cells, although found throughout the marrow, are concentrated close to the bone surface.

Assessment of anin vivo diffusion chamber method as a quantitative assay for osteogenesis

SummaryThe alkaline phosphatase activity and the calcium and phosphorus content of osteogenic tissue formedin vivo following the implantation of diffusion chambers loaded with rabbit bone marrow cells is reported. (In this study the term osteogenic includes osteoblastic and chondroblastic.) Chambers examined 14–70 days after implantation revealed progressive accumulation of mineral. Alkaline phosphatase activity increased until day 30 and declined thereafter. The osteogenic potential of the marrow cells decreased with increasing weight (age) of the cell donor rabbit when measured either as the percentage of chambers containing osteogenic tissue or as the amount of calcium, phosphorus, or alkaline phosphatase activity within the chambers. The results confirm that measurements of these parameters in tissue formed by cells incubated in diffusion chambersin vivo may be used as a method for assay of osteogenesis.

Phenotypic characterisation of mononuclear and multinucleated cells of giant cell tumour of bone

Studies were carried out on 3 giant cell tumours of bone (GCTB) to characterise further the cells forming the distinctive mononuclear and multinucleated components. Samples of tumours were grown as explants in vitro and implanted subcutaneously in athymic mice. Cells were characterised in terms of their cell morphology and cytochemical, antigenic and functional phenotype. In culture, giant cells formed a non-proliferative, relatively homogeneous population of cells which expressed features characteristic of the osteoclast phenotype. The mononuclear cell component was heterogeneous and included macrophage-like cells, which persisted for a short time in culture, and fibroblast-like cells which proliferated. In subcutaneous implants, the fibroblast-like cells formed a tissue which included areas of bone formation associated with regions of alkaline phosphatase activity. These observations are consistent with earlier suggestions that the neoplastic component in GCTB consists of a mononuclear stromal cell which elicits a macrophage/osteoclast response.

Plasma disappearance of rabbit alpha2HS-glycoprotein and its uptake by bone tissue.

SummaryPlasmaα2HS-glycoprotein is specifically accumulated in calcified tissues. In the present studies this glycoprotein was isolated from plasma and after iodination with iodine-125 was injected intravenously into young rabbits. The tissue distribution and plasma disappearance rate of this radioactively labeled material were determined. Of the various tissues studied, bone showed the greatest retention of labeled glycoprotein expressed as percentage of the injected dose per gram tissue relative to the plasma content.The rate of loss of iodinatedα2HS-glycoprotein from plasma was similar to that ofα2HS-glycoprotein labeled endogenously by using14C-glucosamine or3H-glucosamine. The uptake of exogenously labeled3I-α2HS-glycoprotein into bone tissue expressed as a percentage of the injected dose was similar to that of endogenously labeled14C-α2HS-glycoprotein. These results suggest that the125I-labeled material can be used to study further the metabolism ofα2HS-glycoprotein by bone tissue.

Preliminary studies on the binding of plasma albumin to bone tissue.

SummaryThe extractability of125I-labelled plasma albumin from bone pieces and from powdered bone has been compared after both in vivo and in vitro incorporation. The results show that albumin is more readily extracted from bone pieces than from bone powder which implies that tissue disruption exposes additional protein adsorption sites. It is suggested that incorporation of plasma albumin into calcified matrix during bone formation occurs mainly as a result of its strong interaction with bone mineral.

Movement of 125I albumin and 125I polyvinylpyrrolidone through bone tissue fluid.

SummaryThe passage of tissue fluid through cortical bone has been investigated using radioactively labelled macromolecules as markers. The results suggest that in the cortex of young rabbit femur the movement of tissue fluid is in the same net direction as blood, mainly from the endosteal to the periosteal surface.Some albumin is incorporated from extravascular tissue fluid into calcified matrix at sites of bone formation. Polyvinylpyrrolidone, average molecular weight 35,000 is able to pass through extravascular tissue fluid in bone but isnot incorporated into calcified matrix. In rabbits made vitamin D deficient, much less albumin is retained in regions of bone formation than is the case with controls. Albumin adsorbs to the surface of calcium phosphate precipitates and it is suggested that this mechanism may be mainly responsible for its incorporation into bone.

Immunohistochemical studies using BRL 12, a monoclonal antibody reacting specifically with osteogenic tissues.

A monoclonal antibody of immunoglobulin class G1 has been produced which reacts with a high molecular weight antigen apparently present exclusively in osteogenic tissues. Immunohistochemical studies have shown that the antigen is present throughout the mineralized matrix and in osteoid. None of the other tissues examined namely liver, intestine, kidney, spleen, thymus, heart, lung, skin, cartilage and skeletal muscle showed evidence of specific antibody binding. Immunohistochemical staining was also demonstrated in tissues developing from rabbit marrow cultured in vitro and in diffusion chambers in vivo. Temporal studies of antigen expression in the chambers indicated that the antigen occurs at sites of bone formation after the appearance of alkaline phosphatase but before the formation of a mineralized matrix. The results of these studies suggest that the monoclonal antibody recognises a product of differentiated osteoblasts. This antibody may therefore prove useful in studies of osteogenic differentiation.

Synthesis by the Liver of a Glycoprotein which is Concentrated in Bone Matrix

Our previous studies have shown that a glycoprotein of α-electrophoretic mobility which can be isolated from bovine and rabbit bone matrix is present also in the blood plasma (2, 8). Relative to plasma albumin the α-glycoprotein is concentrated in bone and dentine but it is not present above the expected plasma ratio in a number of other tissues. This material appears to originate from the plasma because a large proportion of the radioactivity in bone is in this glycoprotein following injection of total plasma glycoproteins labelled by using 14C-glucosamine (8). However, there is also the possibility that its presence in plasma could be the result of its synthesis and release by the bone tissue.