Cell matrix plaques of initial bone formation

Details Cell matrix plaques of initial bone formation Cell matrix plaques of initial bone formation

2001-07-20

2004-03-30

Tate, Christopher R. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S343000, C435S343100

Reexamination Certificate

active

06713249

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to cell matrix plaques of initial bone formation and to rapid in vitro assays using such newly discovered cell matrix plaques to measure potentials of factors or tissues for stimulating and/or inhibiting bone formation. In particular, the invention relates to assays utilizing the process of &agr;
v
&bgr;
3
expression, matrix protein secretion into the plaques and mineralization of such plaques developed using primary cultures from the human osteoblast lineage.
Bone cells are known to transduce mechanical signals from the environment into anabolic biochemical responses through the process of mechanotransduction. However, the precise mechanisms of mechanotransduction have been a mystery.
Osteoblasts are the skeletal cells that produce and regulate the deposition and mineralization of calcified bone matrix under the influence of the bone morphogenetic proteins (1-4).
Osteoblasts derive from osteoprogenitors, which in turn arise from multipotential mesenchymal cells (5,6). Osteoprogenitors exist in an uncommitted stage which may differentiate into one of several cell lineages, including osteoblasts, chondrocytes, fibroblasts, myocytes and adipocytes, depending on growth conditions (7-9).
The progression from early progenitors to fully functional osteoblasts that synthesize and mineralize matrix is gradual(10).
Differentiation leads through the preosteoblast stage, defined as cells expressing osteoblastic characteristics but not yet mineralizing the extracellular matrix.
The cells in the osteoblast program can be identified based on distinct morphological features and histochemical markers.
Mechanical strain is known to have potent anabolic effects on the skeleton and bone homeostasis in vivo (11), and well documented proliferative effects both in vivo and in vitro (12,13).
The tensegrity model of mechanotransduction (14) proposes that mechanical signals are integrated and converted to biochemical responses through changes in cellular architecture.
Transmembrane molecules (integrins) connecting extracellular and cytoskeletal structures may play key roles in tensegrity, however, the precise processes and molecules that transduce mechanical stimuli into cellular responses, including proliferation are not known.
Integrins are a diverse family of heterodimeric cell surface receptors which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades.
The rationale for adapting the tensegrity hypothesis to the response of the cells in the osteoblast lineage to mechanical strain derived from the findings that repetitive strain of human osteoblast-like cells stimulated cell proliferation, transcription of bone matrix proteins and production of bone matrix (15-17).
The communication between osteoblasts and extracellular matrix proteins is believed to rely on integrins that are known to serve as receptors for matrix proteins.
Even though the precise role of integrins in human bone formation is just beginning to unravel, a number of cellular processes including proliferation are thought to result from the interactions between integrins present on bone cell surface and extracellular matrix proteins.
Human osteoblasts are known to express numerous &agr; and &bgr; integrin subunits (18,19), each component having large extracellular domains responsible for ligand binding, a transmembrane domain and a short cytoplasmic domain responsible for interacting with the actin cytoskeleton (20, 21).
Integrins are known to associate with signaling molecules in focal adhesions.
Focal adhesions are defined as cell-matrix adhesion structures which contain talin, vinculin and other proteins required for integrin attachment to actin filaments, and have been described in numerous cell types but not osteoblasts.
Focal adhesions play dual roles as cell signaling structures as well as architectural links between the cellular cytoskeleton and the extracellular matrix proteins (22).
One of the extracellular matrix proteins of bone, osteopontin, is a non-collagenous glycoprotein that binds to cell surface integrins via the adhesive arginine-glycine-aspartate (RGD) motif (23-25).
Osteoblasts express high levels of osteopontin during bone matrix maturation and mineralization, where it is known to modulate (either inhibit or stimulate) the mineralization of bone matrix (16).
In addition, osteopontin promotes cellular adherence of osteoblast-like cells (26-29) and was shown to specifically interact with the &agr;
v
&bgr;
3
integrin (25, 26, 30).
A precise method for measuring new bone formation, particularly at the subcellular level, has long eluded those interested in bone disease, repair and regeneration. The absence of such a definitive method makes it difficult to evaluate target compositions or regimens for their ability to stimulate or inhibit bone formation or extracellular matrix mineralization. Prior art methods generally suffer from inaccuracy, require making gross determinations from whole cultures, and are typically both time and labor intensive.
SUMMARY OF THE INVENTION
Among the objects of the invention, therefore, may be noted the provision of isolated and essentially purified plaques of initial bone formation and methods of assaying utilizing such plaques. Such assays may be used to screen substances or regimens which stimulate or inhibit bone formation or matrix mineralization. Since the assays operate at the subcellular level, only minute amounts of tissue are required to conduct the assay. Further, the assays may be conducted repeatedly using a single bone donor. The assays can be conducted rapidly and are not labor intensive.
Briefly, therefore, the present invention is directed to a method of measuring new bone formation using a rapid in vitro assay.
The method involves:
1. extracting bone marrow stromal cells from bone marrow cavities and growing them to semi-confluence;
2. exposing the cells to a candidate medium or regimen for osteogenesis or inhibition of osteogenesis;
3. determining the mineralization/development of bone plaque; and,
4. optionally, comparing the mineralization/development of bone plaque generated using the candidate medium or regimen with the mineralization/development of bone plaque using a preselected control such as bone morphogenetic factor-7 (BMP-7), also known as osteogenic factor-1 (OP-1).
Alternatively, the assay can be used as a diagnostic screen for healthy and/or deficient bone formation, making the variable the cells being observed for production of mineralization/bone plaque formation.
Other objects and features will be in part apparent and in part pointed out hereinafter.


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Martha J. Somerman, et al.,Cell Attachment Activity of the 44 Kilodalton Bone Phosphoprotein is not Restricted to Bone Cells, 1989, Matrix, vol. 9, pp. 49-54.
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