Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-04-14
2004-05-25
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C424S140100, C424S145100, C424S158100, C530S388230, C530S388240, C530S413000
Reexamination Certificate
active
06740493
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to methods for obtaining bone precursor cells and compositions comprising such cells. The invention includes methods for enriching the population of bone precursor cells in bone marrow cells isolated from mammalian bones or peripheral blood. Also provided are methods for differentiating bone precursor cells into osteoblasts, and diagnostic and even prognostic methods.
BACKGROUND OF THE INVENTION
The rate of bone fractures in the United States is estimated at 6,000,000 individuals per year. In 1984 (Holbrock et al., 1984) these injuries resulted in a direct cost (i.e., excluding loss of income) of $17,000,000,000 per year. When a bone is completely fractured, a significant number of fractures require medical intervention beyond simple immobilization (casting), particularly those involving trauma. A major problem in such instances is the lack of proximity of the two bone ends (referred to as non-union). This results in an inappropriate and prolonged repair process, which may prevent recovery.
The average length of time for the body to repair a fracture is 25-100 days, for moderate load-bearing, and one year for complete repair. Thus, both simple fractures and medically complicated breaks would benefit from novel therapeutic modalities which accelerate and/or complete the repair process. The same is true for those bone diseases (referred to as osteopenias) which result in a thinning of the bone the primary symptom of which is an often-debilitating fracture.
Primary Osteoporosis is an increased, progressive bone loss which accompanies the aging process. As such, it represents significant health risk in the United States which greater than 15 million Americans suffering from primary (idiopathic) osteoporosis resulting in a direct cost of $6,000,000,000 per year (Holbrock et al., 1984). Primary osteoporosis is the most common of the metabolic bone diseases, and some 40,000-50,000 fracture-related deaths per year are attributed to this disorder. This mortality rate is greater than deaths due to cancer of the breast and uterus, combined. Significantly, this disorder, which is one of the osteopenias, is asymptomatic until a bone fracture occurs. Affected individuals typically fracture the radius, femoral head, or collapse vertebrae.
Osteoporosis has a greater impact on the female population with larger numbers of women than men struck by this disorder, and a significant increase in the rate of osteoporosis occurs post-menopause. The rate of osteoporosis in these women is slowed but not ameliorated by estrogen replacement therapy. Indeed, there is no convincing medical evidence that any treatment is successful in restoring lost bone mass of any kind. Given the aging of the American population, patients with osteoporosis also represent a significant target population for effective and novel bone therapies.
The process of aging in general is associated with a progressive diminution of bone-accumulation capacity, especially in trabecular bone (Nimni et al., 1993). This decreased structural integrity is associated with a number of alterations in bone proteins, osteoid formation, calcium loss etc., leading to osteopenia (Nimni et al., 1993; Fedarko et al., 1992; Termine, 1990). The exact cellular mechanisms underlying such changes in bone structure and function are unclear. However, central to all of these alterations are cells of the osteoblast lineage.
Reductions in osteoblast function or numbers, of necessity, leads to the loss of bone-forming capacity. It is known that some aspects of osteoblast function decrease greatly with age (Termine, 1990). Overall, total protein synthesis and the synthesis of specific proteoglycans decreases markedly (Fedarko et al., 1992), whereas collagen and other proteins such as fibronectin and thrombospondin are degraded (Termine, 1990).
Bone cells from older individuals, in vitro, have the capacity to respond to growth factors, but their synthetic and proliferative capacity is diminished (Termine, 1990), presumably due to reduced responsiveness to various osteogenic growth factors (Pfeilschifter et al., 1993). This results in diminished bone precursor cell and osteoblast numbers (Nimni et al., 1993).
There is no current treatment for lost bone mass, including various growth-promoting proteins and Vitamin D
3
. Likewise, there is no effective replacement or implant for non-union fractures or crush injuries of the bone. Currently, these latter types of injury utilize bovine (cow), or human cadaver bone which is chemically treated (to remove proteins) in order to prevent rejection. However, such bone implants, while mechanically important, are biologically dead (they do not contain bone-forming cells, growth factors, or other regulatory proteins). Thus, they do not greatly modulate the repair process. All of these concerns demonstrate a great need for new or novel forms of bone therapy.
Bone development results from the proliferation of mesenchymal cells, their differentiation into osteogenic progenitor cells, and the eventual calcification of cartilage and bone extracellular matrix (Urist et al., 1983). Human bone marrow contains a distinct cell population that expresses bone proteins and responds to growth factor &bgr;1 (TGF-&bgr;), but not to hematopoietic growth factors (Long et al., 1990).
Little information exists concerning the growth factors or cytokines controlling development of bone precursor cells (osteoprogenitor cells and preosteoblasts) into their differentiated progeny, the osteoblasts. Likewise, few studies address the impact of extracellular matrix (ECM) molecules on this stage of human bone cell development, or the impact of aging on either of these two areas. In the past, human bone cells (both precursor cells and osteoblasts) have been technically difficult to acquire and purification/characterization studies or protocols were few in number. Additionally, current in vitro models of bone formation are limited as the use of post-fetal mesenchymal tissue to generate bone cells often results in chondrogenesis, but is inadequate for osteogenesis, (Urist et al., 1983). Thus, information concerning the cellular activation signals, differentiation, and bone matrix production during the early phases of human bone cell development is limited, at best.
The regulation of chondro-osteogenic gene activation is induced during bone morphogenesis by an accumulation of extracellular and intracellular signals (Urist et al., 1983). Importantly, extracellular signals are known to be transferred from both cytokines and extracellular matrix molecules (Urist et al., 1983), to responding cell surface receptor(s) resulting in eventual bone formation. The formation of bone occurs by two mechanisms. Direct development of bone from mesenchymal cells (referred to as intramembranous ossification; as observed in skull formation) occurs when mesenchymal cells directly differentiate into bone tissue. The second type of bone formation (the endochondral bone formation of skeletal bone) occurs via an intervening cartilage model.
The development and growth of long bones thus results from the proliferation of mesenchymal cells, their differentiation into osteogenic progenitor cells and (then) osteoblasts, cartilage deposition, and eventual calcification of the cartilage and/or bone matrix. Concurrently, bone is remodeled to form a tubular bone space in which hematopoietic cell differentiation occurs.
Interestingly, the number of osteoprogenitor cells in adult bone seems too small to replace all of the large mass of bone normally remodeled in the process of aging of skeleton (Urist et al., 1983). Further observations (vide infra) confirm this concept by showing that one (unexpected) source of osteoprogenitor cells is the bone marrow. This reduced progenitor cell number also implies that there is a disassociation of bone progenitor cell recruitment from subsequent osteogenic activation and bone deposition, and further suggests multiple levels of regulation in this process.
One of the central issues concerning bone formation
Long Michael W.
Mann Kenneth G.
Canella Karen A.
Caputa Anthony C.
Fulbright & Jaworski
The Regents of the University of Michigan
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