1.5.3.1 Osteoblast formation and origin
The osteoblast is the mature differentiated cell responsible for the formation and mineralisation of bone matrix. They are thought to arise from local undifferentiated mesenchymal progenitor cells in the stromal compartment of bone marrow, and are capable of differentiating into chondroblasts, myoblasts, fibroblasts and adipocytes. Friedenstein et al. (1987) showed this using bone marrow stromal cells which have the capacity to form bone when transplanted in vivo in diffusion chambers, and that all the tissues could arise from single clones or fibroblast colony-forming units (CFU-F). There were at least two distinct populations of the osteoprogenitor cells. One population was present in bone tissue and appeared capable of forming osteogenic tissue spontaneously. These cells are called determined osteogenic precursor cells (DOPCs). The other population was not necessarily present in bone tissue, and only formed bone in the presence of the inductive influence of bladder epithelium. These cells are called inducible osteogenic precursor cells (lOPCs).
For the osteoblast, there is an unknown number of stages in differentiation during which their proliferative potential decreases while their synthetic activity increases. However, this pathway is generally considered to proceed via an osteoprogenitor cell to the final osteocyte (Lian and Stein, 1992).
1.5.3.2 The osteoblast phenotype and markers o f osteoblastic differentiation
The following are some markers by which the osteoblast phenotype may be recognised:
a. Alkaline phosphatase (AP) activity
AP is widely used as a marker of the osteoblast phenotype (Rodan and Noda, 1991). Differentiating osteoblasts in vitro express high levels of AP and type I collagen relatively early in the maturation sequence. AP is also expressed by pre-osteoblasts, osteocytes and osteosarcoma cells (Majeska and Rodan, 1982; Franceschi et al., 1985). Although AP expression is not unique to bone, its level is relatively abundant in bone
and levels in differentiating cells are stimulated by Vitamin D3 and hydrocortisone (Beresford et ah, 1994).
b. Synthesis of bone matrix proteins
The organic matrix of bone consists of approximately 90% type I collagen, and 10% non-collagenous proteins. These matrix proteins include osteocalcin (which at about 20%, is the most abundant non-collagenous protein in bone), matrix Gla protein, bone sialoprotein, osteopontin, osteonectin, fibronectin, thrombospondin, aiHS- glycoprotein, tenascin, transforming growth factor p and fibroblast growth factors, and certain proteoglycans (e.g. decorin and biglycan) (Rodan and Noda, 1991; Stanford and Keller, 1991). So far, only osteocalcin is considered 'bone specific' as its expression is restricted to mineralised tissue cells including osteoblasts, odontoblasts and cementoblasts of teeth and those in hypertrophic chondrocytes. Osteocalcin expression is often associated with the onset of matrix mineralisation, and is therefore widely used as a phenotypic marker of bone formation. Although not uniquely expressed by osteoblasts, the high expression of osteopontin and bone sialoprotein in OB cells has made them useful markers of the osteoblastic phenotype, especially when used in conjunction with other markers (Hughes and Aubin, 1998a).
c. Hormone responsiveness
OB cells respond to a wide array of hormones, and some of these interactions are useful as markers of their phenotype. One of these is the binding of parathyroid hormone (PTH) to PTH receptors which results in cyclic AMP production (Auf mkolk et al., 1985). Another hormone which acts on the OB cells is la,25(0H)2D]. Vitamin D3 enhances AP and osteocalcin productions in normal human OB cells and in human osteosarcoma cell lines (Aufmkolk et al., 1985; Beresford et al., 1994; Clover and Gowen, 1994). In human marrow stromal cultures, glucocorticoids inhibit cell proliferation and increases AP synthesis (Beresford et al, 1994).
d. Cell surface markers
Expression of at least some of the ECM proteins occur at different phases of the OB cell differentiation or maturation cascade (Lian and Stein, 1992). Polymerase chain
reaction and immunocytochemical data of bone matrix proteins synthesised by single colonies of rat foetal calvarial OB cells at different developmental stages show that bone matrix proteins follow a sequence of expression: Collagen I, AP, osteopontin, bone sialoprotein and osteocalcin (Liu et al., 1994). Some of these proteins (e.g. Fn, bone sialoprotein and osteopontin) contain the amino acid sequence, Arginine-Glycine- Aspartate (RGD), which is the integrin-binding cell attachment motif (Pierschbacher and Ruoslahti, 1984). Little is known about the integrin expression in bone cells at progressive stages of the osteoblastic lineage. However, recent studies of the expression of integrin receptors of OB cells both in vivo and in vitro, suggest the possibility of qualitatively determining the osteoblast lineage by the integrins expressed during the different stages of osteoblastic differentiation (Moursi et al, 1996; Alavi et al., 1998). In particular, the a$Pi integrin may be associated with the late stages of osteoblastic differentiation in primary OB cell cultures, and at a relatively earlier stage.
e. Induction of mineralisation and bone formation
The defining characteristic of the mature osteoblast is its ability to produce a mineralised bone matrix. Some osteoblastic culture systems can produce discrete, three- dimensionally organised mineralised matrices which are recognised as bone-like, when grown on implant materials (Davies et al., 1991; Ripamonti, 1991; Chehroudi et al.,
1992; Ozawa and Kasugai, 1996). The formation of mineralised nodules in vitro is dependent upon the presence of serum, ascorbic acid, and p-glycerophosphate (Bellows
et al, 1986; Chung et al., 1992), although Beresford et al (1993) have shown that in the presence of the long-acting ascorbate analogue Asc-2-P, the formation and mineralisation of nodules in primary explant human bone cultures can occur in the absence of p-glycerophosphate. Mineralisation in culture has been characterised using various markers of bone formation, for example, nodule formation, positive staining for Ca and P, and positive staining for major non-collagenous proteins (Bellows et al.,
1986; Groessner-Schreiber and Tuan, 1992; Lian and Stein, 1992; Beresford et al.,
1993). However, "true bone" formation is viewed to have occurred, only if the Ca:P ratio of the nodules formed is similar to hydroxyapatite (Groessner-Schreiber and Tuan,
neonate and aged bone of the species from which the cells were derived (Gray et al.,
1996).