B on e grow th factors im plicated in bone repair

Recent studies on fracture healing suggest regulatory roles for platelet derived growth factor (PDGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-p) and the bone morphogenetic proteins (BMPs) in the development of the fracture callus (Bolander, 1994). Table 5.1 lists the grovWih factors implicated in bone repair and some of their known biologic activities.

Table 5.1 Growth factors implicated in bone repair

Growth Factors Produced by Matrix Location Biologic Activities TGF-P Platelets, inflammatory cells (monocytes, macrophages), osteoblasts, chondrocytes

Bone is the most abundant source of TGF-P in the body Regulation o f matrix calcification, stimulation o f osteoblast activity, chemoattractant for macrophages, promotes angiogenesis BMPs Osteoprogenitor cells Chondrocytes BMPs were originally identified in bone but are now known to be widely distributed in the body e.g. urinary bladder epithelium, brain TGF-P-like structure, may be involved in cartilage formation, important regulator during embryogenesis Fibroblast Growth Factors Inflammatory cells, osteoblasts, chondrocytes

Binds heparan sulphate proteoglycans in bone and cartilage matrix

Stimulates

neovascularisation, chemoattractant and mitogen for

chondrocytes and most cells of mesoderm or neuroectoderm origin Platelet-derived Growth Factors Platelet, monocytes, activated macrophages, endothelial cells

Interactions unknown Mitogenic for most cells o f mesoderm origin Insulin-like Growth

Factors

Osteoblasts, chondrocytes, liver

Interactions unknown Mitogenic, promotes cartilage synthesis, acts as second messenger to PTH, modulator o f other growth factors

5.1.2.1 Basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor (FGF) family, currently comprised of seven related mitogenic proteins which show 35- 55% amino acid conservation. bFGF has been isolated from a number of sources, including neural tissue, pituitary, adrenal corex, corpus luteum and placenta. It

stimulates the proliferation of all cells of mesodermal origin, and many cells of neuroectodermal, ectodermal and endodermal orgin (Gospodarowicz, 1992). The cells include fibroblasts, endothelial cells, astrocytes, oligodendrocytes, neuroblasts, kératinocytes, osteoblasts, smooth muscle cells, and melanocytes. bFGF is chemotactic and mitogenic for endothelial cells in vitro.

Several in vitro studies which evaluated the effect of FGFs on bone forming cells have produced contradictory results. Rodan et al. (1987b) showed that acidic fibroblast growth factor (aFGF) extracted from bovine brain, was a potent mitogen for both osteoblastic and non-osteoblastic cell cultures derived from rat calvaria but an inhibitor of the expression of osteoblastic features. Canalis et al. (1988) found that basic fibroblast growth factor (bFGF) had similar effects. Further in vitro studies by Globus et al. (Globus et al., 1988; Globus et al., 1989) revealed that FGFs present in bone matrix were the product o f osteoblast secretion, rather than of the endothelial cells from the local vascular network and confirmed that bFGF is more potent than aFGF as a mitogen.

Pitaru et al. (1993) treated rat bone marrow stromal cells with bFGF and found increased proliferative activity as well as increased cAMP responsiveness to PTH, alkaline phosphatase activity and osteocalcin expression. Their conclusion was that bFGF increased the osteogenic differentiation of bone marrow stromal cells in addition to being mitogenic.

Iwasaki et al. (1995) in a study of the in vitro effects o f TGF-p and bFGF on periosteal mesenchymal cells, showed that these cells, under the stimulus o f bFGF, tended to increase their replicative rate, but did not show any tendency to differentiate without the addition o f TGF-p. These findings were suggestive o f the existence of a synergistic effect of bFGF with TGF-p.

In vivo, FGFs have been found to promote cell proliferation, stimulate tissue regeneration and wound repair, and induce angiogenesis (Gospodarowicz et al., 1992; Eppley et al., 1988; Schweigerer, 1990). FGFs play a significant role in the early phases o f embryonic development, where these factors might provide the tissues of an efficient

network of local proliferative-differentiative signals, in the absence of a well-established vascular system (Schweigerer, 1990). Frenkel and Singh (1991) studied the effect of administration o f bFGF in chick embryos and found an increased number of osteoblasts in the femurs o f treated embryos but a reduction of collagen synthesis compared with the control group. Nakamura et al (1995) reported that systemic administration of recombinant human bFGF in rats induced endosteal, rather than periosteal, bone formation. A series of in vivo studies by Wang and Aspenberg showed that local infusion o f bFGF promoted bone formation in a dose and time dependant way in bone grafts placed within titanium chambers in rats (Wang & Aspenberg, 1993; Wang & Aspenberg, 1994; Aspenberg et al., 1994; Wang & Aspenberg, 1996a). Higher doses of bFGF induced fibroblastic rather than osteoblastic cell proliferation, whereas lower doses had the opposite effect. However, an earlier study by Eppley et al.(1988) showed no evidence o f increased osteogenesis when bFGF was infused into bone grafts placed directly on the mandibles o f skeletally mature rabbits despite increased blood vessel formation.

While most studies reported a proliferative effect of bFGF on osteoblastic cells, there are some doubts about its influence on their differentiation. The general opinion is that there is an inhibitory action of FGFs on osteoblast and chondroblast differentiation, in exchange for a longer proliferative activity, both in vivo and in vitro (Hauschka, 1990; Frenkel and Singh, 1991; Gospodarowicz, 1992; Jingushi et al, 1995). Yet, other studies revealed a different effect of bFGF, which, in some conditions, would promote differentiation rather than delaying it (Nakamura et al, 1995; Wang and Aspenberg, 1996a; Iwasaki et al, 1995). Various explanations of these differences in observations have been proposed. Pitaru et al (1993) suggested that there was a synergistic effect between FGFs and medium supplements including dexamethasone, ascorbic acid and p~ glycerophosphate which lead to osteocalcin synthesis, increased ALP activity and Ca^^ deposition, all marks o f osteoblastic differentiation. The functional interaction between bFGF and TGF-p also came under scrutiny. Early studies showed that the two factors would co-operate in promoting bone cells proliferation, but reducing the synthesis of osteocalcin (Globus et al, 1988). On the contrary, experiments by Iwasaki et al (1995) showed that TGF-P would oppose, rather than add to FGFs effects, inhibiting mitotic

event, and inducing collagen production. Further evidence of a link between the two factors was presented by Nakamura et al (1995) who found that treatment with bFGF lead to an increased immunostaining of preosteoblastic cells for TGF-p in rats. However, in an in vivo experiment in which alveolar bone defects in dogs were treated with a combination of bFGF, IGF-2 and TGF-p, no differences in fibroblast and collagen density were detected, and indeed, bone formation was more significant in the control than in the test group (Selvig et al, 1994).

In summary, most in vitro studies show that bFGF stimulates osteoblastic cells into proliferation and inhibits their phenotypic expression and in vivo experiments seem to show an increase in bone formation in response to its local or systemic application. The following investigation was set up to explore the effect of bFGF on bone formation within CAM grafted femurs, taking into consideration the question of how well the model fits into picture provided by in vitro and in vivo findings reported in the literature.

5.2

Materials and Methods