C. J. Joyner

Human bone tissue formation in diffusion chamber culture in vivo by bone-derived cells and marrow stromal fibroblastic cells.

Direct grafts of human cells into immunocompromised or cortisone-treated animals, either alone or within carrier materials, have been used with some success to assess the developmental capability of the grafted cells. However, identification of the donor or host origins of the generated tissue in such direct grafts is essential. In an alternative and extensively used experimental system, cells are cultured within the isolated environments of diffusion chambers, which are surgically implanted in appropriate hosts. This system allows the direct study of the cellular potentials for differentiation as host tissues are excluded. In the present study, human osteoprogenitor cell populations derived from trabecular bone explants or marrow suspensions of 3 patients (2 females aged 14 years and 1 male aged 27 years) were cultured in the absence or the continuous presence of dexamethasone (10 nmol/L). Cells were impregnated into porous hydroxyapatite ceramics before subcutaneous implantation, or placed within diffusion chambers before intraperitoneal implantation, in athymic mice. All subcutaneous implants of cells in ceramic showed morphological evidence for the formation of bone tissue. In the diffusion chambers it was found that both marrow- and bone-derived fibroblastic cells cultured in the absence of dexamethasone generally produced fibrous tissue only. When cultured in the continuous presence of dexamethasone (10 nmol/L), these cell populations produced similar osteogenic tissues with active osteoblasts, wide osteoid seams, and mineralized tissue, with cartilage toward the interior of the chamber. These results validate the diffusion chamber as an experimental system to study human osteogenesis using appropriately primed cell populations.

Vascularity in a new model of atrophic nonunion

Our aim was to develop a clinically relevant model of atrophic nonunion in the rat to test the hypothesis that the vessel density of atrophic nonunion reaches that of normal healing bone, but at a later time-point. Atrophic nonunion is usually attributed to impaired blood supply and is poorly understood. We determined the number of blood vessels at the site of an osteotomy using immunolocalisation techniques in both normally healing bones and in atrophic nonunion. At one week after operation there were significantly fewer blood vessels in the nonunion group than in the healing group. By eight weeks, the number in the atrophic nonunion group had reached the same level as that in the healing group. Our findings suggest that the number of blood vessels in atrophic nonunion reaches the same level as that in healing bone, but at a later time-point. Diminished vascularity within the first three weeks, but not at a later time-point, may prevent fractures from uniting.

Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study

The presence of osteogenic progenitors in human skeletal muscle is suggested by the formation of ectopic bone in clinical and experimental conditions, but their direct identification has not yet been demonstrated. The aims of this study were to identify osteogenic progenitor cells in human skeletal muscle tissue and to expand and characterize them in culture. Specimens of gracilis and semitendinosus muscle were obtained from young adults and digested to separate the connective tissue and satellite cell fractions. The cells were cultured and characterized morphologically and immunohistochemically using antibodies known to be reactive with primitive osteoprogenitor cells, pericytes, intermediate filaments, and endothelial cells. Alkaline phosphatase activity and osteocalcin gene expression were also determined. In the early stages of culture, the connective tissue cells obtained were highly positive for primitive osteoprogenitor cell and for pericyte markers. Alkaline phosphatase activity was detectable at early stages of culture and rose as a function of time, whereas primitive osteoprogenitor cell markers declined and osteocalcin mRNA expression became detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). It is shown that human skeletal muscle connective tissue contains osteogenic progenitor cells. Their identification as pericytes, perivascular cells with established osteogenic potential, suggests a cellular link between angiogenesis and bone formation in muscle tissue. These cells are easily cultured and expanded in vitro by standard techniques, providing an alternative source of osteogenic progenitor cells for possible cell-based therapeutic use in certain conditions.

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.

Pluripotential mesenchymal cells repopulate bone marrow and retain osteogenic properties.

Precursor cells, isolated from bone marrow, can develop into various cell types and may contribute to skeletal growth, remodeling, and repair. The D1 cell line was cloned from a multipotent mouse bone marrow stromal precursor and has osteogenic, chondrogenic, and adipogenic properties. The osteogenic phenotype of these precursor cells is relevant to the process of fracture healing and osteointegration of prosthetic implants. The D1 cells were labeled genetically using a replication incompetent retroviral vector encoding β-galactosidase, an enzyme which is used as a marker. Labeled cells are readily identifiable by staining with 5-bromo-4-chloro-3-indoyl- β-D-galactoside and by flow cytometry, and retain the desired osteogenic characteristics in vivo as shown by von Kossa staining, alkaline phosphatase assay, an increase in cyclic adenosine monophosphate in response to parathyroid hormone, osteocalcin messenger ribonucleic acid production, and bone formation in diffusion chambers. In addition, the cells cloned from marrow stroma repopulate the marrow of host mice, persist for several weeks, and retain their osteogenic potential ex vivo. The data suggest that such cells may be used to replenish the number of osteoprogenitors in marrow, which appear to decrease with age, thereby leading to recovery from bone loss and improved bone growth and repair. Labeling these cells creates a model in which to study the potential of such cells to participate in fracture repair, ingrowth around prosthetic implants, treatment of osteoporosis, and to explore the possibility of gene delivery to correct mutations or defects in metabolism that are responsible for certain skeletal abnormalities.

Toxic effect of rifampicin on human osteoblast-like cells

We examined the effects of rifampicin on osteoblast‐like cells derived from adult human bone in vitro. Cancellous bone was collected from five different individuals during elective orthopaedic operations and cultured in antibiotic‐free media. Total DNA, 3H‐thymidine incorporation and alkaline phosphatase (ALP) activity were measured after the cells were cultured for 4 days in media containing concentrations of rifampicin ranging from 0 to 1000 μ/ml. Mean total DNA was decreased at concentrations of 10 μ/ml and above in the cultures obtained from four out of five individuals but these decreases were significant in the cultures from only two individuals. 3H‐thymidine incorporation, a more sensitive indicator of change in cell proliferation, and ALP activity were significantly decreased (P < 0.05) in all of the cultures containing 3 and 7 μg/ml, respectively. In the clinical setting, serum concentrations of rifampicin often exceed 10 μg/ml after systemic administration. The present study has shown that rifampicin, at these concentrations, can inhibit the proliferation of osteoblast‐like cells in vitro. Further studies should be carried out to assess whether rifampicin is detrimental to the bone repair process in vivo.

Molecular cloning of the cell surface antigen identified by the osteoprogenitor-specific monoclonal antibody, HOP-26.

Bone is a highly organized structure comprising a calcified connective tissue matrix formed by mature osteoblasts, which develop from the proliferation and differentiation of osteoprogenitor cells. The osteogenic cell lineage is thought to arise from a population of uncommitted multipotential stromal precursor cells (SPC) which reside close to all bone surfaces, in the bone marrow spaces and the surrounding connective tissue. These SPC also give rise to related cell lineages which form cartilage, smooth muscle, fat, and fibrous tissue. Due to the lack of well defined cell surface markers, little is known of the precise developmentally regulated changes in phenotype which occur during the differentiation and maturation of human osteoprogenitor cells into functional osteoblasts and ultimately, terminally differentiated osteocytes. In order to identify antibody reagents with greater specificity for osteoprogenitors we generated a series of antibodies following immunization with freshly isolated human bone marrow stromal fibroblasts. One such antibody, HOP‐26, reacts with a cell surface antigen expressed by SPC and developing bone cells. We now demonstrate that this mAb identifies a member of the tetraspan family of cell surface glycoproteins, namely CD63. Western blot analysis of human bone marrow stromal cells (HBMSC) has revealed that like a well defined CD63 mAb 12F12, HOP‐26 interacts with a heavily glycosylated cell surface protein with an apparent molecular weight of 50–60 kD. J. Cell. Biochem. 89: 56–66, 2003.

Development of a monoclonal antibody to the aP2 protein to identify adipocyte precursors in tumours of adipose differentiation.

aP2 gene product (aP2 protein) expression has been shown to be a useful diagnostic marker for identification of lipoblasts and fetal fat cells in soft tissue tumours. A monoclonal antibody was developed by a mouse spleen cell-myeloma hybridoma technique to an 18 amino acid segment of the aP2 protein and was used to investigate the immunohistochemical expression of this protein in benign and malignant tumours of adipocytic differentiation and a wide variety of other soft tissue tumours. We found that aP2 protein was expressed by lipoblasts in liposarcomas and lipoblastomas and by brown fat cells in hibernomas and normal periadrenal fat. Other benign adipose tissue tumours and benign and malignant soft tissue tumours were distinguished from liposarcoma by absence of staining for aP2 protein. Immunohistochemical identification of the aP2 protein is likely to prove a useful means of distinguishing liposarcoma from other malignant mesenchymal and epithelial neoplasms, some of which contain cells that morphologically resemble lipoblasts.

Effects of interferon alpha on human osteoprogenitor cell growth and differentiation in vitro.

The specific effects of interferon alpha (IFNα), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNα on osteogenic development by investigating cell differentiation, colony formation (colony forming unit‐fibroblastic, CFU‐F), cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow osteoprogenitor cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNα (5–1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 μM), which are agents known to affect osteogenic differentiation. IFNα produced a significant dose‐dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNα (50–1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU‐F showed dose‐ and time‐dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase‐positive CFU‐F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP‐26), was reduced in IFNα‐treated cultures. Northern blot analysis showed a significant dose‐dependent up‐regulation of BMP‐2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNα significantly inhibited BMP‐2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNα inhibits human osteoprogenitor cell proliferation, CFU‐ F formation, HOP‐26 expression, and alkaline phosphatase specific activity and modulates BMP‐2 gene expression. These results suggest a role for IFNα in local bone turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation. J. Cell. Biochem. 74:372–385, 1999.

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.