Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2000-12-27
2004-11-02
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C435S366000
Reexamination Certificate
active
06811776
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of biology and medicine. More particularly, it concerns a process for the ex vivo formation of bone and uses thereof.
2. Description of Related Art
The development of a functional tissue such as bone requires the concerted action of a number of microenvironmental signals: cytokines/growth factors, extracellular matrix (ECM) molecules, and cell:cell interactions. Moreover, these regulatory signals must be queued in the appropriate temporal and spatial order, resulting in a developmental microenvironment that facilitates three-dimensional growth. The skeletal system is no exception to such requirements. It is well understood that a number of cytokines/growth factors, such as TGF-&bgr;1 family members, modulate bone formation, and that ECM molecules like osteonectin, osteocalcin, and Type I and II collagen, etc., are important in both osteogenesis and chondrogenesis. As opposed to in vitro systems that are predominately planar, the need for three-dimensional tissue-like development is implicit both in the structural nature of the skeleton and its embryonic development. However, the extension of these in vivo spatial requirements to in vitro systems has been difficult and largely overlooked.
Cellular condensation, a process of cell aggregation mediated by mesenchymal:epithelial cell interactions, plays a crucial role during skeletogenesis (Hall and Miyake, 1992; 1995; Stringa et al., 1997). In the developing chick embryo, cellular condensation precedes differentiation into to prechondrocytes (Hall and Miyake, 1995). In contrast, during osteogenesis, cells differentiate to preosteoblasts and then undergo condensation (Hall and Miyake, 1995; Centrella, 1987). This condensation nonetheless precedes osteoblast differentiation and matrix mineralization (Dunlop and Hall, 1995). Studies of prechondrocytes demonstrate that cell condensation is cytokine-mediated, and induces changes in the expression of a number of developmentally important genes. For example, TGF-&bgr;1 or BMP2 both stimulate chondrocytic condensation and up-regulate fibronectin, N-CAM, and tenascin (Hall and Miyake, 1995). The requisite step of cellular condensation during mesenchymal chondrogenesis is mimicked in vitro in chondrocyte micromass cultures (Denker et al., 1995). Tuan and colleagues have demonstrated that TGF-&bgr;1 treatment of the multipotent C3H10T1/2 cells in small volumes of media at high cell density (i.e., micro-mass cultures) results in the formation of three dimensional structures that are cartilaginous in nature (Denker et a., 1995). These cellular condensations are associated with the up-regulation of cartilage extracellular matrix components such as Type II collagen and cartilage link protein (Denker et al., 1995). Likewise, studies of embryonic chick (calverial or limb-bud) cells confirm the cell-density mediated induction of chondrogenesis (Wong and Tuan, 1995; Woodward and Tuan, 1999), and demonstrate an obligate requirement for cell:cell interaction in this process, most likely mediated by N-cadherin (Woodward and Tuan, 1999), or N-CAM (Oberlender and Tuan, 1994; Miyake et al., 1996).
To date, no in vitro models of tissue-like osteogenic cell growth or cellular-condensation exist. Calverial or bone marrow-derived osteogenic cells are typically grown on two-dimensional (i.e., planar) surfaces. Cell proliferation eventually leads to a localized piling of confluent cells into “bone nodules.” This suggests that cell-density plays a role in the process of bone formation; however, studies directly demonstrating a relationship between cell-density and bone-formation are lacking, as are studies demonstrating the formation of three dimensional, crystalline bone (as opposed to reports concerning the mineralization of the extracellular matrix surrounding bone).
Present methods for the repair of bony defects include grafts of organic and synthetic construction. Three types of organic grafts are commonly used: autografts, allografts, and xenografts. An autograft is tissue transplanted from one site to another in the patient. The benefits of using the patient's own tissue is that the graft will not evoke an immune response. However, using an autograft requires a second surgical site, which increases the risk of infection and may introduce additional complications. Further, bone available for grafting comes from a limited number of sites, for example, the fibula, ribs and iliac crest. An allograft is tissue taken from a different organism of the same species, and a xenograft from an organism of a different species. The latter types of tissue are readily available in larger quantities than autografts, but genetic differences between the donor and recipient may lead to rejection of the graft. All have advantages and disadvantages, yet none provides a perfect replacement for the missing bone.
There exists a need for a better way to repair and/or replace bone in subjects suffering from bone diseases or bone traumas.
SUMMARY OF THE INVENTION
The present invention concerns methods for the ex vivo formation of mammalian bone and subsequent uses of that bone. A critical and distinguishing feature of the present invention are defined tissue culture conditions and factors resulting in the formation of bone cell spheroids. “Bone cell spheroids” are defined as a tissue-like three dimensional growth of osteogenic cells or osteogenic precursor cells. The formation of bone cell spheroids permits the formation of bone within the spheroid. The invention also provides for methods of implanting the ex vivo formed bone into subjects. Also described are methods for genetically altering bone cells/spheroids to affect bone formation, identification of candidate modulators of bone formation, and identification of genes involved in bone formation.
Specifically, the present invention concerns a method for producing mammalian bone ex vivo by obtaining an osteogenic or bone precursor cell; culturing the cell under serum free conditions in the presence of one or more osteogenic growth factors; and establishing the cell cultures at cell densities that allow the formation of a bone cell spheroid containing bone, whereby bone is formed within cells of the bone cell spheroid. The osteogenic or bone precursor cell of the present invention can be isolated from primary sources such as bone marrow or bone explants. Protocols for isolating such cells are described herein. In other embodiments, cell lines of bone cell derivation can be utilized. Osteogenic or bone precursor cells can be from several mammalian species, including but not limited to, human, bovine, equine, canine, feline, chick, rat, or murine origin.
The present invention describes the culturing of the osteogenic cell in defined, serum-free media. Defined medias are described herein as well as additives that are commonly found in these defined medias, including albumin, insulin, an iron source, a fatty acid source and other essential components. The defined serum free media of the present invention also is supplemented with growth factors that are important for the formation of bone cell spheroids. The primary growth factors are broadly defined as members of the Transforming Growth Factor &bgr; (TGF-&bgr;) gene superfamily. Members of this family include TGF-&bgr;1, TGF-&bgr;2, TGF-&bgr;1.2, and Bone Morphogenic Protein 2 (BMP-2), BMP-4 and BMP-7. Other growth factors such as parathyroid hormone (PTH), calcitonin, 1,25-dihydroxy vitamin D3, interleukin-6, insulin-like growth factors (IGFs) I and II, VEGF and interleukin-11 can be used as solitary or costimulatory factors.
The osteogenic or bone precursor cell of the present invention may be purified by physico-chemical separation techniques, such as equilibrium density separation. In other embodiments, the osteogenic or bone precursor cell may be purified by immuno-affinity isolation, such as those utilizing immune adhesion, immuno-column chromatography, or fluorescence-activated cell sorting. In preferred embodiments,
Kale Sujata
Long Michael W.
Fulbright & Jaworski LLP
The Regents of the University of Michigan
Witz Jean C.
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