Cell lines and primary cell cultures in the study of bone cell biology
Introduction
Bone has several major functions. It forms a rigid skeleton to provide a framework for the body, support for soft tissues, points of attachment for skeletal muscles, protection for internal organs, housing for bone marrow as well as a central role in mineral homeostasis, principally of calcium and phosphate ions, but also of sodium and magnesium.
Bone is a dynamic tissue that is constantly remodeled throughout life. During fetal development, most of the skeleton develops from cartilage anlagen which is eventually resorbed and replaced with bone by a process termed endochondral ossification. In contrast, bones which form the calvaria, mandible and maxilla are developed from mesenchyme by a process termed intramembranous ossification. Bone modeling is the process associated with growth and reshaping of bones in childhood and adolescence. In bone modeling, longitudinal growth of long bones depends on proliferation and differentiation of cartilage cells at the growth plate while growth in width and thickness is accomplished by formation of bone at the periosteal surface with resorption at the endosteal surface. In adults, after the epiphyses close, growth in length and endochondral bone formation cease but remodeling of bone continues. Remodeling constitutes the lifelong renewal process whereby the mechanical integrity of the skeleton is preserved. It implies the continuous removal of bone (bone resorption) followed by synthesis of new bone matrix and subsequent mineralization (bone formation). The maintenance of normal, healthy bone requires the coupling of bone formation to bone resorption, with intercellular communication between osteoblasts and osteoclasts integral to the achievement of a balance between the two processes. Furthermore, bone remodeling is an integral part of the calcium homeostatic system (Eriksen et al., 1993) that also involves the parathyroid glands, intestinal system and the kidneys.
Many aspects of the processes described above can be investigated in the laboratory using primarily cell culture. The major cell types are the bone-forming osteoblasts, bone-resorbing osteoclasts and cartilage-forming chondrocytes. A thorough understanding of the factors regulating the differentiation of each of these cell types, the mechanisms by which regulatory factors influence their function, and the manner in which these cells communicate and interact with each other, is central to the design of rational therapeutic strategies to treat bone diseases such as osteoporosis. This review will focus on cell lines that are established in the laboratory from these different cell types. While much information has been derived from established cell lines, particularly in osteoblast biology, a substantial amount of work is nonetheless still being carried out with primary cultures of osteoblasts, chondrocytes and osteoclasts, and attention will be drawn to these, where relevant.
In bone cell biology, cell cultures are used mainly to examine:
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Regulation of expression of phenotypic characteristics typical of osteoblasts, chondrocytes and osteoclasts.
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Regulation of differentiation of relatively undifferentiated mesenchymal cells along different lineages, for example, muscle, osteoblasts, chondrocytes and adipocytes.
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Signaling pathways relevant to osteoblast, osteoclast and chondrocyte functions.
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Effects of over-expression and under-expression of particular gene products on cell function.
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In vitro bone formation/mineralization.
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Interactions between osteoblasts and osteoclasts, particularly in the regulation of osteoclast formation in vitro.
Section snippets
Osteoblast ontogeny
Osteogenic cells arise from pluripotential mesenchymal stem cells. These stem cells have the capacity to differentiate into lineages other than osteoblasts, including those of chondroblasts, fibroblasts, adipocytes, and myoblasts (reviewed in Nijweide et al., 1986, Friedenstein et al., 1987, Aubin et al., 1995). By analogy with hematopoietic cell differentiation, each of these differentiation lineages is thought to originate from a different committed progenitor, which for the osteogenic
Osteoclast ontogeny
Multinucleate osteoclasts are responsible for bone resorption. Their chief functional characteristic is the ability to pump acid into specialized resorption pits to dissolve bone mineral as well as to provide an optimum environment for the enzymatic degradation of demineralized extracellular bone matrix. Osteoclasts are derived from hematopoietic stem cells that differentiate along the monocyte/macrophage lineage (Martin et al., 1989, Suda et al., 1992). Direct contact of mononuclear
Chondrocytes
Cartilage is a specialized form of connective tissue that possesses a firm pliable matrix, which endows it with the resilience that allows the tissue to bear mechanical stresses without distortion. Articular cartilage, smooth surfaced and resilient, provides a shock-absorbing sliding area for joints to facilitate movement of bone, while cartilage is also essential for the embryonic development and, thereafter, growth of long bones.
Cartilage consists of chondrocytes and an extensive
Calcium homeostasis
Calcium is an essential ion for many physiological processes such as cell motility, muscle contraction and neurotransmitter release. In mammals, these processes function optimally when extracellular calcium is maintained within a normal range by regulatory mechanisms that coordinate the metabolic activities of the kidneys, intestine, parathyroid glands, and bone.
Parathyroid cells express a cell surface calcium-sensing receptor that recognizes and responds to physiological changes in
Discussion
Although cell culture has proved invaluable in the study of bone biology, in vitro model systems cannot reproduce the complex three-dimensional architecture of bone that is required for the proper expression of the functional capability of the cells that make up its microenvironment. Nonetheless, despite the limitations of the various systems described in this review, significant and important contributions have been made to our understanding of the normal processes leading to bone formation,
Acknowledgements
This work is supported by Program Grant 003211 from the National Health and Medical Research Council (Australia). The help provided by Dr. Julian Quinn with the preparation of the manuscript, Fig. 2, Fig. 3 and Table 7, is gratefully acknowledged.
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