Osteocyte cell lines

Details Osteocyte cell lines Osteocyte cell lines

1998-09-11

2002-03-19

Clark, Deborah J. R. (Department: 1633)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Rodent cell, per se

C435S325000, C424S093210, C800S004000, C800S013000, C800S018000

Reexamination Certificate

active

06358737

ABSTRACT:

2. BACKGROUND OF THE INVENTION
2.1 Technical Field
The present invention relates to a method of producing osteocyte cell lines in various stages of differentiation. Such cell lines remain stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high level of osteocalcin. More specifically, it provides a method of production for cultured osteocytes of various differentiation stages. Furthermore, it relates to osteocyte cell line, and more specifically cultured osteocyte. The invention also relates to a method for the production of monoclonal antibodies using such cultured osteocytes and further relates to hybridomas and monoclonal antibodies which recognize an osteocyte-specific antigen. Finally the invention relates to a method of screening for modification factors and binding factors for osteocytes.
2.2 Description of Related Art
Bone loss can occur under conditions of disuse or with certain diseases of bone. Examples of bone loss due to disuse include that associated with immobilization and zero gravity. Bone loss can also occur due to estrogen deficiency due to menopause or ovariectomy and also occurs naturally with the aging process.
Osteocytes are the most abundant of the bone cells (approximately 25,000 per mm
3
bone or ten times as many osteocytes as osteoblasts) and are found within the mineralized bone matrix (Parfitt, 1977). Because they are buried in the mineralized matrix, they are relatively inaccessible and have been difficult to study in culture in homogeneous populations. It has been suggested that the osteocyte is the most mature or most terminally differentiated form of the osteoblast. However, the properties and functions of osteocytes are poorly understood.
During bone formation, some osteoblasts (osteocyte precursors) are trapped in the forming osteoid tissue while the bone formation front moves on. The trapped or encapsulated cell produces long, slender dendrite-like processes. These processes maintain contact with other osteocytes and with osteoblasts and lining cells on the bone surface (for review, see Aarden et al., 1994). Osteocytes enclosed within osteons appear to be stellate in shape and isolated osteocytes can retain this stellate shape in culture. The formation of cytoplasmic processes by the maturing osteocyte are asynchronous and asymmetrical (Palumbo et al., 1990). The cells produce dendritic processes on the mineralization side before producing processes on the vascular side. Thus the morphology of an osteocyte can range from the stellate or ‘star-like’ shape to that with extensive cytoplasmic, slender processes longer than the main body of the cell. Osteocytes express a dendritic phenotype both in vivo and in vitro. It has been shown previously that osteocytes express large amounts of osteocalcin.
In addition to their distinctive morphology, osteocytes are now characterized by expression of surface antigens and other markers. Osteocytes strongly express CD44, a transmembrane glycoprotein with adhesion functions (Hughes et al., 1994), and insulin-like growth factor 1 (Lean et al., 1995). Fifty percent of osteocytes in situ express estrogen receptor (Braidman et al., 1995), and avian osteocytes appear to express specific antigens detected by a monoclonal antibody not expressed on avian osteoblasts (Nijweide & Mulder, 1986). It is generally accepted that osteocytes are low expressors of alkaline phosphatase and recently it has been shown that osteocytes produce greater amounts of casein kinase activity compared with osteoblasts (Mikuni-Takagaki et al., 1995). It is very likely that mammalian osteocytes produce markers distinctly different from those of osteoblasts.
Arden and co-workers (1994) have stated that for the osteocyte to survive, the cell must maintain an unmineralized area around the body of the cell and around the cell processes. This is necessary in order to allow the diffusion of nutrients and waste products to and from the cell. Mikuni-Takagaki and co-workers (1995) described the extracellular accumulation of a large amount of osteocalcin around isolated osteocytes. Osteocalcin has been described in the endoplasmic reticulum and Golgi cisternae in osteocytes (Ohta et al., 1989; Boivin et al., 1990). Recently, Ducy and co-workers (1996) have demonstrated that mice which lack the functional gene for osteocalcin have increased cortical and trabecular bone which lead them to postulate that osteocalcin is an inhibitor or negative regulator of mineralization. The osteocyte may produce large amounts of osteocalcin to prevent the mineral from closing off the cell body and processes.
It has been hypothesized that osteocytes respond to loading pressures on bone by signaling osteoblasts to produce new bone (for review, see Burger et al., 1993). Recently it has been shown that loaded bone contains fewer apoptotic osteocytes (Noble et al., 1997) and that osteocyte cell death is increased during estrogen withdrawal (Tomkinson et al., 1996) and during treatment with excess glucocorticoid (Weinstein et al., 1997) suggesting that bone loss or bone necrosis is due to osteocyte death which prevents normal bone remodeling or normal bone repair. If osteocytes are the cell responsible for sensing mechanical stress and for signaling osteoblasts to produce new bone, then understanding their functions could lead to new therapies to prevent or restore bone loss due to immobilization or other processes.
The study of osteocytes has utilized immunohistochemistry techniques and the isolation of primary cells. However, primary cells can only be obtained in relatively low numbers and in heterogeneous populations. An osteocyte cell line would prove useful to study the properties of osteocytes through the use of molecular and functional techniques which require relatively large numbers of homogeneous cells.
We postulated that since osteocytes are large producers of osteocalcin, that bone cells derived from transgenic mice overexpressing the T-antigen driven by the osteocalcin promoter which would serve to target large T-antigen to osteoblasts and osteocytes, and thereby be a source of immortalized cells of these types. We chose to use cellular morphology as the initial criteria for cloning cell lines with osteocytes characteristics from isolates from these mice (Chen et al., 1995). Once clonal cell lines were established, they were characterized as far as the osteocyte/osteoblast phenotypes were concerned.
With regard to the generation of monoclonal antibodies specific for osteocytes, Nijweide and co-workers reported a monoclonal antibody which recognizes avian osteocytes but not mammalian osteocytes (Nijweide and Mulder, 1986). This monoclonal antibody was generated by injecting mice with osteoblast-like cells derived from digestions of chick embryol calvaria which had been cultured 6 days before injection. This monoclonal antibody specifically reacts with the cell surface of osteocytes and not with any specific band by western blotting of chick osteocyte lysate. The specificity of this monoclonal antibody has been confirmed by Bruder and Caplan (1989) and this antibody has been used as a tool to purify osteocytes (Vanderplas et al., 1994) and investigate osteocyte function (Tanaka et al., 1995).
Another monoclonal antibody which recognizes the osteoblast to osteocyte transition has been generated. This antibody recognizes a cell surface antigen called E11 in rats which is homologous to the OTS-8/ap38 molecule in mice (Wetterwald et al., 1996). This monoclonal antibody was generated by injecting mice with the rat osteoblastic cell line IRC 10/30-myc3. The E11 transcript was detected in bone, lung, brain, and skin. This antigen appears to be expressed during the transition stage from the osteoblast to the osteocyte phenotype. Over-expression of E11 in ROS 17/2.8 cells caused these cells to form long cytoplasmic extensions (Sprague et al., 1996). Therefore this antigen appears to be an osteocyte differentiation agent.
We were successful in generating monoclonal antibodies which are specific for mammalian osteo

Affiliated with

Also associated with

Rating

Osteocyte cell lines does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Osteocyte cell lines, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Osteocyte cell lines will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2873973