Cells

The dynamics of bone metabolism, a process of continuous remodeling throughout the life of the vertebrate, resides primarily with three cells: the osteoblast, osteocyte and osteoclast. Each responds to the environment comprised of adjacent cells and circulating factors. Attempts to mimic or influence the activity of these cell populations is a central focus of the Bone Tissue Engineering Center.

Osteoblasts synthesize and regulate the deposition and mineralization of the extracellular matrix of bone. Systemic and locally active hormones, growth factors, ions, lipid metabolites and steroids are regulators of osteoblastic activity and/or differentiation. Generation of the intracellular signaling subsequent to a regulatory agent's detection has undergone intensive study. The molecular signaling cascade for some of these agents has been defined for each of the bone cell types. Osteoblasts, pre-osteoblasts and osteoblastic or osteoblast-like cells have been employed to investigate signal transduction.

Members of the transforming growth factor beta (TGF-b) family, particularly TGF-b and the bone morphogenetic proteins (BMPs) are important to bone homeostasis. These factors modulate osteoblast proliferation and differentiation . Whereas the effects of each factor may be similar or different depending upon the cell line and the cell response, some shared concepts are apparent for the intracellular signaling utilized by the members of the family. Members of the TGF-b superfamily bind to a particular combination of two serine/threonine kinase receptors, type I and type II, for signal transduction. Both receptors are necessary for signal transduction. However the intracellular molecular mechanisms for each member of the family are not well defined and are the subject of current research. PKC dependent activities have been demonstrated in TGF-b1 induced gene expression. Generalities about PKC participation cannot be made. PKC consists of a large family of isoenzymes that are selectively activated depending on the agonist and cell line; therefore, different PKC's could participate in different signaling pathways and trigger different biological responses. The mitogen-activated protein kinase (MAPK) cascade is involved with the regulation of the gene transcription by TGF-b1 in bone cells. PKC and PTK have been shown to participate in protein phosphorylation leading to the activation of the MAPK members. In osteoblastic cells (ROS 17/2.8), BMP-2 stimulated PTK and PKC activities and modulated MAPK extracellular signal-regulated kinases (ERK) activity. Conversely, BMP-12 nor BMP-13 induced alkaline phosphatase activity in myoblasts which would have supported their differentiation into osteoblasts, but BMP-2 did induce activity. The difference suggested that the intracellular pathway used by BMP12 and BMP-13 may differ from BMP-2 for the myoblast cells (Inada et al., 1996).

Osteocytes share a similar lineage to osteoblasts. The microenvironment of inorganic/organic bone matrix necessitates further specialization of the osteoblast. Osteocytes are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes are highly differentiated cells with alkaline phosphatase activity, PTH receptors, and function as mechanosensory cells. Mechanical stimuli can initiate alterations of bone structure and mass. The elaboration of the biochemical mechanisms enabling the mechanotransduction of the stimuli have focused on the osteocyte. The osteocyte possesses a lacuno-canalicular organization within the bone porosity that mediates mechanosensing. Strain induces interstitial fluid flow through bone (e.g. canaliculi) and may activate the osteocyte. Osteocyte activation entails the processing of intracellular signaling molecules. The network of osteocytes provide the cellular organization in the bone to respond to mechanical demands with either augmentation or reduction of bone apposition.

Osteocytes do not resorb dentin surface in vitro, suggesting osteocytes do not play a role in calcium homeostasis. The phenotype is characterized by the stellate arrangement of the cell membrane defining canaliculi, ensuring contact with neighboring osteocytes and enhancing the mechanosensory capacity of each living bone. The phenotype of the osteocytes appears deficient in some receptors found on the osteoblast. However, the osteocyte is well adapted for its role in bone homeostasis and maintains intracellular signaling to respond to the unique demands of its location. Despite being the most abundant "bone cell" among a group of highly differentiated cells, difficulties with isolation of the osteocyte previously hindered characterization.

The osteocyte mechanosensory system in bone responds to the deformation. As a consequence of deformation there is a flow of interstitial fluid through the osteocytic canalicular network. The flow is directed away from regions of high strain. The flow initiates both electrokinetic (streaming potentials) and mechanical signals (fluid shear stress). Subsequent release of intercellular signal molecules e.g. Insulin-like growth factor, IGF-1, prostaglandins G/H synthase , PGE2 and nitric oxide contribute coordinated metabolic responses from neighboring cells: osteocytes, osteoblasts, and osteoclasts.

Osteoclasts are multinucleated cells of hemopoietic cell origin involved in bone resorption. The complex unit that develops between the osteoclast and the dissolving bone surface is generated by the fusion of intracytoplasmic acidifying vesicles with the plasma membrane. The ruffled border appears essential to bone resorption, yet the mechanism for its formation is not defined. The possible analogy between the formation of the ruffled border and exocytosis has focused research on the use of bone cell marcophages to elucidate molecular events coinciding with the formation of the ruffled border. Rab proteins comprise the largest subset of the Ras superfamily of GTPases. They are associated with the cell organelle membrane cytoplasmic face and vesicles involved in biosynthetic and endocytotic pathways. Avian bone marrow macrophages have been shown to express at least two Rab3 isoforms, A and B/C. The Rab3 isoforms are rab GTPases and are know to play a role in regulated calcium exocytosis. Cytokines induced the increased expression of the isoforms during the differentiation of the avian bone marrow macrophages into osteoclasts in vitro. However the extracellular event precipitating the expression of Rab3 proteins requires further investigation. The src family of tyrosine kinases have been indirectly implicated in the regulation of osteoclast resorption activity. Amino terminal myristylation enables the localization of the src family of non-receptor cytosolic proteins to the inner face of the cell membrane. Mice carrying homozygous disruption of the c-src proto-oncogene (Src-/-) develop osteopetrosis due to an impaired ability of osteoclasts to adhere to the bone surface and/or to form a bone-resorbing ruffled border. Osteopontin (OPN), a secreted phosphoprotein, mediates osteoclast adherence to the bone matrix. Cells from Src-/- mice expressed OPN mRNA and protein at significantly reduced level. The proto-oncogene c-src may regulate OPN gene expression. Binding of the src SH2 domain by a synthesized compound inhbited src dependent osteoclast activity and diminished resorptive activity in vitro . Earlier research indicated that c-src could serve to regulate osteoclast attachment. Following ligand occupancy, non-receptor protein tyrosine kinases are capable of associating with multiple receptors. This process could facilitate multiple receptors that are not intrinsic tyrosine kinases to activate signaling complexes associated with activation of the src family and production of the effector complexes through src homology-binding domains (SH2 and SH3 domains). The src protein could regulate osteoclast function through effector elements containing the SH2 domains. Osteoclast attachment to bone may involve the avb3 integrin. Horton suggested that the c-src could serve to regulate attachment and retraction by association with the avb3 integrin.