New Techniques in Metabolic Bone Disease
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New Techniques in Metabolic Bone Disease - John C. Stevenson
UK
Chapter 1
Humoral and local factors affecting bone formation and resorption
R.G.G. Russell, R.A.D. Burning, D.E. Hughes and M. Gowen
Publisher Summary
This chapter discusses humoral and local factors affecting bone formation and resorption. Bone is a complex living tissue with an architecture ideally suited to its function. Bone matrix components are probably degraded by the action of extracellular proteinases such as collagenase, proteoglycanase, and other metalloproteinases whose optimal activities are at physiological pH. The three main calcium-regulating hormones are parathyroid hormone, calcitonin, and vitamin D. Calcitonin is secreted by the C cells, which lie predominantly in the thyroid gland, in response to an increase in plasma calcium. Its release is inhibited by a decrease in the plasma calcium. The primary function of 1,25-dihydroxyvitamin D is to increase serum calcium concentration, mainly by acting on the intestine to stimulate the absorption of calcium and phosphate. Androgens and oestrogens also assist in maintaining normal bone structure and function. The rapidly accumulating new knowledge about the multiple possible regulatory mechanisms within bone should aid the understanding of physiological bone remodeling and also offer potential explanations for the changes in bone turnover seen in a variety of disease states.
Bone is a complex living tissue with an architecture ideally suited to its function. Its structural properties depend upon an extracellular matrix which undergoes mineralization during development. The formation and destruction of bone and the remodelling that occurs throughout life is dependent upon cellular activity. There have been several important recent advances in the understanding of the regulation of bone formation and resorption, which may eventually help in the understanding and treatment of bone and joint disease.
The cells of bone
The three main cell types in adult bone are osteoblasts, osteocytes and osteoclasts.
The osteoblasts and osteocytes are derived from a different cell lineage from the osteoclasts. Osteoblasts appear to originate from stromal stem cells found in bone marrow [1]. The factors that affect their recruitment and maturation are poorly understood, but mature osteoblasts are responsible for the synthesis of the extracellular matrix of bone. The predominant protein product is type I collagen. Other products include the vitamin K-dependent proteins, osteocalcin and matrix Gla protein, which appear to be specific to bone, bone sialoproteins, proteoglycans and osteonectin. There may be important differences in the properties of osteoblasts depending upon their site within the skeleton, for example, in periosteal bone, cortical bone or trabecular bone. However, in all these locations, after deposition of extracellular matrix, osteoblasts direct its subsequent mineralization [2, 3]. Osteoblasts that remain behind the advancing bone surface may become interred in lacunae where they are termed osteocytes. The osteocytes embedded in bone remain in contact with each other, forming a large network of potentially communicating cells. This extensive network may facilitate the coordinated response of bone to mechanical stress and deformation.
The osteoclasts are responsible for bone resorption and originate from the fusion of mononuclear cell precursors that arise from haemopoietic stem cells [4]. A number of factors are now known to affect their recruitment, differentiation and action [5–10]. These include colony-stimulating factors, e.g. GM-CSF, interleukin 1 and 1,25-dihydroxyvitamin D. Osteoclasts are typically large cells, which contain many nuclei. They probably resorb only calcified bone or cartilage. The cells display polarity. Resorption occurs beneath the ruffled border, apposed to the bone surface, which is surrounded by a clear zone devoid of intracellular organelles. Osteoclasts have been particularly difficult to study in isolation but techniques are now becoming available. However, much remains to be learnt about their origin and how they function.
The ability of osteoclasts to resorb bone depends on their mobility, and also on their capacity to generate a locally acid environment at the resorbing surface, into which lysosomal and other proteases are directed from within the cell [8],
The local production of lactate or acid (as H+) may be a result of carbonic anhydrase activity [11, 12], and/or of specific transport mechanisms, including Na+/ H+ exchange and proton pumps [13–15]. Interestingly, deficiency of carbonic anhydrase (type II) is associated with osteopetrosis [16]. Osteoclasts may also secrete calcium-chelating organic anions, such as citrate, that may assist in the solubilization of the mineral phase. Transcellular transport of calcium may also be a necessary part of the process.
Bone matrix components are probably degraded by the action of extracellular proteinases such as collagenase, proteoglycanase and other metalloproteinases whose optimal activities are at physiological pH [17]. Lysosomal acid hydrolases, including the proteolytic cathepsins, may also contribute. Osteoclasts may be one of the only cell types that function by having lysosomal hydrolases accomplish extracellular digestion in a locally acid environment.
Experimental approaches
Studies of skeletal behaviour in intact animals continue to yield useful information about hormones and other agents that act on bone. In order to study biochemical events in greater detail, culture of intact bone to study growth and resorption also continues to be a useful approach. However, much of the recent work on bone has utilized cell culture techniques to study the differentiation of bone cells and their responses and interactions in vitro.
Although much use continues to be made of tumour cell lines, e.g. osteosarcoma cells, as well as bone cells from rodents and birds, it is now possible to study osteoblast-like cells derived from human bone, as well as the differentiation of multinucleate osteoclast-like cells from the bone marrow of several species, including man.
Regulation of bone cell activity
Experimental studies, particularly with cell culture systems, show that bone cells can respond to a wide variety of systemic and locally active hormones and growth factors. These agents can be shown to affect cell proliferation and differentiation, matrix synthesis, the production of enzymes such as plasminogen activator, as well as other aspects of cell function. A list of some of the agents known to act on bone are given in Tables 1.1–1.3. One of the most significant findings has been that many now well-defined cytokines, such as transforming growth factors, tumour necrosis factors, interleukin 1, interferon gamma, etc., have effects on bone and other connective tissue cells (Figure 1.1). This has led to the recognition that the interaction of bone cells with cells of the immune system may be important in the differentiation of bone and also in pathological events that occur in diseases such as rheumatoid arthritis, bone infections, tumour invasion and other conditions. Agents such as these may account for the activities previously ascribed to poorly defined factors such as osteoclast-activating factors (OAFs) and skeletal growth factors (SGFs).
Table 1.1
A list of well-characterized cytokines and their potential effects on bone
Table 1.2
Bone resorbing agents that may act directly or indirectly on osteoclasts
Table 1.3
Bone active agents that may act directly or indirectly on osteoblasts and affect bone formation
Figure 1.1 The possible influence of cytokines on the differentiation and function of osteoblasts (a) and osteoclasts (b). Th refers to T helper cells (CD4 positive). Abbreviations for cytokines are as follows: IL 1, interleukin 1; IL 2, interleukin 2; CSFs, colony stimulating factors; TNF, tumour necrosis factor; IFNγ, interferon gamma; PGs, prostaglandins; HLA-DR are class II histocompatibility antigens induced by IFNγ
Systemic factors that regulate bone turnover
The three main calcium-regulating hormones are parathyroid hormone, calcitonin and vitamin D.
Parathyroid hormone (PTH) is an important mediator of physiological bone resorption and is a major factor in maintaining calcium homoeostasis, which is accomplished more by effects on the kidney than on bone. Secretion is switched on by a fall in serum calcium concentration and, conversely, is inhibited by an increase in serum calcium concentration, or an increase in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). PTH first interacts with specific receptors on the cell surface, and activates second-messenger systems within the cell, including cyclic AMP and the inositide phosphate pathways. These then initiate a series of cellular responses.
PTH appears to mediate some of its effects on bone resorption through a primary action on the osteoblast. Osteoblastic changes that are associated with increased resorption include a change from a flattened to a stellate shape and elaboration of proteases such as collagenases and plasminogen activators [18, 19].
PTH acts on the kidney to increase reabsorption of Ca²+ and to decrease reabsorption of phosphate. PTH also stimulates 1α-hydroxylase leading to the generation of 1,25(OH)2D3, the active metabolite of vitamin D.
Calcitonin is secreted by the C cells, which lie predominantly in the thyroid gland, in response to an increase in plasma calcium. Its release is inhibited by a decrease in the plasma calcium. Calcitonin is an inhibitor of bone resorption and may also play a role in bone mineralization. Its effects in other species, including fish, birds and other vertebrates, may be more important than in man. In adult humans, calcitonin probably plays only a minor role in the physiological regulation of bone metabolism.
Vitamin D is absorbed from the diet or synthesized in the skin from precursors in response to exposure to ultraviolet light. It is hydroxylated in the liver to 25-hydroxyvitamin D, which circulates attached to a specific binding protein. It is then converted in the kidney to its active metabolite, 1,25(OH)2D. The primary function of 1,25(OH)2D is to increase serum calcium concentration, mainly by acting on the intestine to stimulate absorption of calcium and phosphate.
1,25(OH)2D3 may also act both as a mediator of matrix mineralization (by stimulating collagen and osteocalcin synthesis and alkaline phosphatase activity in osteoblasts) and as a mediator of resorption. In vitro, 1,25(OH)2D3, like PTH, appears to stimulate resorption, at least partly, through an action on osteoblasts. The number and activity of osteoclasts increases, probably also as a result of stimulation of osteoclast differentiation from bone marrow precursors and promotion of the multinucleation required for full expression of osteoclast function. In addition, 1,25(OH)2D may affect bone formation and resorption by influencing the maturation of lymphoid cells and their secretion of cytokines, which alter bone cell activity.
Other hormones
Several other systemic hormones affect bone metabolism. These include growth hormone, thyroid hormones and adrenal and gonadal steroids. Glucocorticoids have complex and diverse effects on bone, which vary in different species. At physiological concentrations in vitro, glucocorticoids appear to promote osteoblast differentiation and to stimulate collagen synthesis. Additional effects of glucocorticoids include the ability to potentiate some of the cyclic AMP-mediated responses of bone cells to PTH and modification of various effects of 1,25(OH)2D3 on the gastrointestinal tract and on bone cell activity. In man, excessive doses of glucocorticoids inhibit bone growth and fracture healing and cause osteoporosis. Glucocorticoid-mediated osteoporosis is thought to result from a primary inhibition of osteoblast proliferation and function. Glucocorticoids also produce an increase in bone resorption and a secondary response to a decrease in intestinal calcium absorption, an increase in urinary calcium excretion, and a resulting hyperparathyroidism.
Androgens and oestrogens also assist in maintaining normal bone structure and function. In men, hypogonadism is associated with reduced bone mass. In women, oestrogen deficiency accelerates bone loss and is an important factor in postmenopausal osteoporosis. The mechanism of action of oestrogens is not well understood. Earlier explanations for the bone-sparing effect of oestrogen include a stimulation of calcitonin production and a resulting decrease in bone resorption, or enhanced availability of 1,25(OH)2D3 leading to increased intestinal calcium absorption. As noted later, more important effects may include actions on the immune system culminating, for example, in an inhibition of the release of bone resorption promoters and other direct effects on bone cells possibly through oestrogen receptors, the presence of which has been detected in recent studies.
Thyroid hormones stimulate trabecular bone remodelling. In excess they stimulate resorption more than formation, so that bone mass is decreased.
Cytokines and growth factors as local regulators of bone metabolism
A surprisingly large number of these newly defined factors have effects on bone and cartilage metabolism, and several of them may be important as locally active hormones, under physiological as well as pathological conditions (for recent reviews see refs [20] and [21]).
The broadest definition of a cytokine is any agent produced by cells that acts on cells. In this sense the definition includes classical lymphokines, interleukins, interferons, colony-stimulating factors, growth factors, neuropeptides and other agents (Table 1.1). Since there is considerable overlap of biological activity between the substances in these groups, e.g. in terms of their effects on cell proliferation and differentiation, this wide definition is a logical one.
Many cytokines have had several names in the past, based upon their biological properties. Their availability as recombinant proteins is leading to rapid advances in knowledge of their mode of action and biological importance.
This is well illustrated by the bone and cartilage growth factors, the activities of most of which have now been shown to be attributable to already known cytokines and growth factors. Their effects on bone and connective tissues are discussed in Figure 1.1.
Interleukin 1
Interleukin 1 (IL 1) originally derived its name as a monocyte/macrophage product which caused proliferation of T-cells (particularly CD4-positive T-helper cells), via the induction of the autocrine growth factor, interleukin 2 (IL 2). This property of IL 1 accounts for its earlier name of lymphocyte-activating factor (LAF). In fact it was the search for the endogenous pyrogen (EP) activity derived from leucocytes that led to identification of the structure of IL 1 [22, 23]. When recombinant material became available, it became clear that IL 1 accounted for a series of other biological activities, including leucocyte endogenous mediator (LEM), mononuclear cell factor (MCF), osteoclast activating factor (OAF), catabolin and many others.
IL 1 exists in two forms, alpha and beta, which represent acidic and neutral forms, with molecular weights around 17 kDa [24]. They are derived by complex proteolytic cleavage from larger precursors. The relative abundance of IL 1 alpha and beta varies in different tissues and species, but their main biological activities appear similar and include induction of fever and of the acute phase response. The latter includes enhanced synthesis of hepatic ‘acute phase’ proteins, changes in circulating trace metals (Fe, Zn), and neutrophilia.
A murine receptor for IL 1 has recently been cloned [25]. This receptor binds both IL1 alpha and beta, which helps to explain their similar biological effects. Interestingly, the receptor protein is a member of the immunoglobulin superfamily.
A naturally occurring inhibitor of IL 1 exists which appears to block binding of IL 1 to its receptor [26, 27]. Its biological and clinical significance is being evaluated but its production may be enhanced in disease states when IL 1 is produced [28].
With regard to connective tissues, it has been known for a long time that IL 1 can stimulate synovial cells, chondrocytes, fibroblasts and some, but not all, osteoblast-like cells, to secrete proteinases such as collagenase and stromelysin, as well as plasminogen activators [29–31]. Collectively these enzymes can contribute to the breakdown of connective tissue matrices. IL 1 is a potent inducer of bone resorption [32–36] and was one of the first OAFs to be characterized. IL 1 may account for bone resorption associated with monocytic leukaemias and inflammatory conditions in bone, e.g. osteomyelitis. IL 1 also has complex effects on the production of matrix components, including collagen, osteocalcin and proteoglycans, but the biological significance of this is not yet known. IL 1 is also a potent co-mitogen for fibroblasts and human osteoblast-like cells [37].
Tumour necrosis factors
Tumour necrosis factors (TNF) also exist in two forms, alpha and beta [38]. TNF alpha was originally isolated on the basis of activity termed cachectin thought to be responsible for weight loss in tumour-bearing animals. TNF beta, also known as lymphotoxin, is derived from T lymphocytes and mediates their cytotoxicity.
TNF alpha and beta display extensive homology and have a very similar spectrum of activity. The effects of TNFs on connective tissues are very similar to those of IL 1 and include induction of prostaglandin and metalloproteinase synthesis, the stimulation of bone and cartilage resorption, and mitogenesis [29, 30, 39, 40]. On a molar basis, IL 1 is more potent than TNF alpha, but the two can act in a synergistic fashion under certain circumstances. Their relative importance under physiological conditions will therefore depend not only on their local concentrations but also on which other factors are present.
Interleukin 6
Interleukin 6 (IL 6) is a 23–28 kDa protein which can be produced by several connective tissue cell types, including MG63 osteosarcoma cells and several fibroblast-derived cell lines and monocytes [41, 42]. IL 6 is now known to be responsible for several biological activities previously ascribed to separate factors, which include hybridoma/plasmacytoma growth factor (HPGF) [43], B-cell-stimulating factor-2 (BSF2) [44], interferon beta-2 (IFN B2) and hepatocyte-stimulating factor (HSF) [45].
IL 6 also has important overlapping activities with IL 1 and TNF. For example, it induces hepatic acute phase protein synthesis, and is probably an important mediator of the acute phase response.
Since IL 1, TNF and lipopolysaccharides (endotoxin) can induce the production of IL 6 from monocytes and fibroblasts [46], it is still unclear what biological effects are attributable to direct actions of IL 1 and TNF, or are mediated via IL 6.
Colony-stimulating factors
Several colony-stimulating factors which stimulate haemopoietic differentiation have now been described [47]. GM-CSF and M-CSF can stimulate bone resorption in vitro and this may be attributable to their effects on osteoclast generation, since both can be shown to stimulate the generation of osteoclast-like cells from bone marrow cultures [9]. Both can also be produced by osteoblast-like cells [48] which raises the possibility that bone cells can influence haemopoiesis.
Interferon gamma
Interferon gamma (IFN gamma) is one of the family of interferons that were originally defined by their ability to inhibit viral replication. Interferons are known to have many other effects, particularly on cellular proliferation and differentiation.
IFN gamma, while sharing some properties with other interferons, appears to be different in terms of its effects on connective tissues. Thus, IFN gamma inhibits bone resorption induced by IL 1 or TNF, whereas it has less effect on resorption stimulated by the classical calciotropic hormones, parathyroid hormone or 1,25(OH)2D3 [35, 49]. IFN gamma also opposes other actions of IL 1 and TNF, for example, on cell proliferation, on cartilage resorption and on metalloproteinase production by chondrocytes [30]. In these respects, it can be viewed as a potential natural antagonist to IL 1 and TNF.
IFN gamma also induces the expression of MHC class 2 (HLA-DR) antigens on connective tissue cells, including synovial cells, bone cells and chondrocytes, as it does on macrophages [50]. It is not yet clear whether these changes allow these connective tissue cells to present antigens and to participate in immune responses, or in other forms of intercellular communication.
Insulin-like growth factors
Insulin-like growth factors, as the name implies, are growth factors that are isolated from serum and that share some structural and biological properties with insulin, e.g. stimulation of glucose uptake.
Insulin-like growth factor 1 (IGF 1), also known as somatomedin C, stimulates the replication of bone cells and chondrocytes, and increases production of matrix constituents [51].
Both IGF 1 and IGF 2 are produced within bone itself, and their activity is modulated by specific binding proteins, which in the case of IGF 1 are also under the control of growth hormone. Recent studies show that a factor previously known as skeletal growth factor [52], based on its ability to act as a mitogen on osteoblast-like cells, is probably IGF 2.
Transforming growth factor beta (TGF beta) and bone morphogenic proteins
TGF betas are members of a much larger gene family and are produced by a variety of cells and tissues which include T-cells, macrophages, platelets and bone. TGF beta is a homodimeric polypeptide (mol. wt 25 000) which exists in at least three isoforms (TGF betas1, 2 and 3) with different primary sequences but similar biological activities [21, 53, 54]. TGF betas are thought to be important in embryological development and differentiation as well as in connective tissue repair and fibrosis. They may turn out to be among the most important regulatory cytokines in connective tissues and bone.
In vitro, TGF beta stimulates the synthesis of extracellular matrix components, such as type I collagen and fibronectin [55, 56]. In the presence of epidermal growth factor (EGF), TGF beta will also stimulate the proliferation of a variety of connective tissue cell types including bone cells. When injected in vivo, TGF beta induces a wound repair response which includes fibrosis and angiogenesis [57].
It is particularly interesting that TGF beta is one of the few agents known to inhibit the production from fibroblasts of proteinases such as plasminogen activator and stromelysin, which may be involved in the degradation of extracellular matrix proteins. TGF beta also stimulates the production of inhibitors of plasminogen activator (the latter being known as PAIs) [58] and of metalloproteinases (tissue inhibitor of metalloproteinases, TIMP). These actions of TGF beta may be important in the maintenance of the integrity of extracellular matrix and, in concert with the effects of TGF beta on matrix synthesis, may promote tissue repair.
One of the most interesting aspects of the TGF beta class of cytokines are their relationship to bone morphogenic proteins.
The so-called bone morphogenic protein(s) (BMPs) is the name given to a factor(s) present in demineralized bone matrix which can induce the formation of new cartilage and bone when implanted into rats in vivo. The characterization of BMPs has been a difficult task, partly because of the limitations imposed by the laborious bioassay. These factors may be very important in regulating the differentiation of skeletal tissue and may eventually be of use, e.g. to promote fracture repair.
Seyedin et al. [59–61] showed that the cartilage inducing factors A and B, which were related to BMP, were TGF beta1 and TGF beta2 respectively. More recently three further peptides have been isolated and characterized, two of which appear to be members of the TGF beta family.
Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha)
These two peptides were discovered by quite different routes, as their names imply. However, although their primary structures are quite distinct, they appear to share a common receptor so their many actions are similar.
Several of the effects of TGF beta on differentiation require the presence of EGF/ TGF alpha. With respect to bone and cartilage, both may be produced by bone [62] and are potential inducers of bone resorption, mainly through enhanced prostaglandin synthesis [63–65]. The potential physiological and pathological significance of their effects on connective tissues, in common with those of the other cytokines and growth factors, remains to be determined.
Fibroblast growth factors (FGFs)
The FGFs are members of a group of related peptides (15–16 kDa) which are mitogenic for many cell types, including fibroblasts and osteoblasts. There are acidic and basic forms of FGF based on isoelectric points. Acidic FGF is derived mainly from neural tissue, but basic FGF is one of the heparin-binding growth factors and appears to be produced by many cell types and is present in bone matrix. The products of at least three oncogenes (including hst and int-2) appear to be related to basic FGF and these may be important in the vascularization of tumours. FGF is a powerful angiogenic factor and this may be important during embryological development, during endochondral ossification and in various pathological states.
Platelet-derived growth factor (PDGF)
PDGF was originally isolated from platelets but is now known to be produced by other cell types, include various types of bone cells [66, 67]. Human PDGF is a 31 kDa protein which is a dimer of two similar peptide chains designated A and B. At least three forms exist comprising AA, AB or BB dimers. PDGF is probably also important in development and is one of the most potent growth factors present in serum. It can stimulate bone resorption, partly through a prostaglandin-dependent pathway. PDGF is mitogenic for fibroblasts and also for osteoblasts and chondrocytes.
Parathyroid hormone-related peptide
It has been thought for a long time that factors related to parathyroid hormone might be involved in the hypercalcaemia associated with malignant disease. Such a peptide has now been identified [68–71] and the major form appears to be a single chain peptide of 141 amino acids (17 000 kDa). The N-terminal portion shows considerable homology with PTH itself. Eight of the first 13 amino acids are the same whereas homology in the rest of the molecule is limited to only a further eight amino acid positions.
There is recent evidence that PTH-RP is produced by bone cells themselves and this raises the possibility that PTH-RP may be an important autocrine and paracrine regulator within bone.
PTH-RP appears to function through the PTH receptor to stimulate adenylate cyclase and mimics the effects of PTH in altering renal handling of calcium and phosphate and stimulating bone resorption. PTH-RP is produced by many tumours, particularly those of squamous cell origin, and it is found in normal skin keratinocytes. The release of PTH and PTH-RP are likely to be regulated differently. In particular, hypocalcaemia does not appear to be a stimulus for release of PTH-RP from skin or other sites.
Neuropeptides and other factors
Several peptides, including neuropeptides, have effects on bone and cartilage metabolism which may be of clinical significance. These include substances P and K, vasoactive intestinal peptide (VIP), calcitonin-gene-related peptides (CGRPs) [72] and bradykinin [73].
Mast cell products
Mast cell products such as heparin and histamine may be important since these cells are present in excess in inflammatory arthritis and in bone disease, including mastocytosis, osteoporosis and renal osteodystrophy [74].
Prostaglandins and eicosanoids
Prostaglandins have been known to have effects on bone under experimental conditions for a very long time, but their physiological and pathological significance is still not fully resolved [75].
Prostaglandin E2 is one of the most potent of the prostanoids for induction of bone resorption. The ability of several agents to stimulate bone resorption (e.g. TGF beta, complement, thrombin, IL 1, TNF) may be mediated, in part at least, by increased prostaglandin synthesis [7, 76]. In turn, production of several of these cytokines may be influenced by prostaglandins. Low concentrations of some prostanoids may inhibit osteoclast actions by altering cell mobility.
Prostaglandins may be involved in the response of bone to mechanical stress, and help to mediate the bone loss associated with immobilization [77]. They may be involved in the localized bone resorption associated with periodontal disease and inflammation. The chronic administration of prostaglandins in vivo may lead to enhanced periosteal and endosteal apposition of bone, e.g. in children with patent ductus arteriosus, and in experimental dogs.
Lipoxygenase products, such as leukotrienes, do not appear to have marked effects on bone cell function.
The interactions among cytokines
Since there are so many agents that can potentially act on bone, a major task in contemporary research is to determine how these agents interact and which are the most important under physiological conditions and in different disease states. This task is only just beginning. However, it is clear that many of these factors, which can be produced locally and can reside within bone matrix, have to be considered serious candidates for involvement in the regulation of bone turnover.
The responsiveness of each tissue is likely to depend upon many variables, including the concentration of individual cytokines present, the relative amount of different cytokines, the potential synergisms and antagonisms among them, the degree of receptor modulation, e.g. by glucocorticoids, and the presence of specific inhibitors.
Cytokines and growth factors made by and found in bone and cartilage
Table 1.4 gives a list of cytokines and growth factors which have been found to be present in bone. There is evidence, in some but not all cases, that these factors may be synthesized by osteoblast-like cells. It is also possible that some of these biologically active materials may enter bone from other cellular sources, e.g. platelets, lymphocytes or macrophages, or arrive via the