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Platelet-derived growth factor expression in normally healing human fractures
Bone Vol. 16, No. 4 April 1995:455~60
ELSEVIER
Platelet-Derived Growth Factor Expression in Normally Healing Human Fractures J. G. A N D R E W , 1 J. A. H O Y L A N D , 1 A. J. F R E E M O N T , 2 and D. R. M A R S H 1 i Department of Orthopedics, Hope Hospital, Salford, United Kingdom z Department of Pathological Sciences, Manchester University Medical School, Manchester, United Kingdom
PDGF B-chain mRNA by Northern analysis in rodent fracture callus; PDGF A-chain expression was not examined. Thus, the demonstrated in vitro effects of PDGF on the cell types involved in fracture healing have not been demonstrated to be relevant in normal fracture-healing models, and we have been unable to find previous reports on PDGF in human fracture material. This paper reports an immunohistochemical and in situ hybridization study of PDGF expression in normally healing fractures.
Platelet-derived growllh factor (PDGF) has been shown to have effects on bone and cartilage cells in vitro, but its role in vivo in bone repair is not clear. We studied biopsy material from 16 normally healing fractures at a variety of times after injury, using immun¢~histochemistry for PDGF and in situ hybridization for PDGF A and B chains. PDGF A-chain gene was found to be expressed by many cell types over a prolonged period during fracture healing. These cells included endothelial and mese~hchymai cells in granulation tissue and osteoblasts, chondroc,ctes, and osteoclasts later during fracture healing. PDGF B-chain gene expression was more restricted, being detected principally in osteoblasts at the stage of bone formation. PDGF was detected using immunohistochemistry in the cell types expressing PDGF A. These findings indicate that PDGF is expressed during normal human fracture repair, and the in vitro data also suggest that PDGF is likely to be an important local regulator in this process.
Materials and Methods Callus specimens were obtained from normally healing fractures at operation for malunion between 1 and 6 weeks after fracture. Ethical committee approval and informed consent were obtained. Specimens were examined from 16 fractures. All of the fractures had united normally at follow-up 1 year after operation, except in one patient who died of unrelated causes shortly after operation. Tissue was fixed in neutral buffered formalin for 24 h and then decalcified in 20% ethylenediamine tetraacetic acid, pH 7.2, until radiologically decalcified (10-14 days). Tissue was then processed routinely into paraffin wax, and 7-1~m sections were cut and mounted onto slides coated with vectorbond (Vector Laboratories) for in situ hybridization. Human cDNA probes were used. The PDGF A-chain cDNA (1.3 kb) and the PDGF B-chain cDNA (2.0 kb) were obtained from Dr. C. H. Heldin (Uppsala, Sweden). Specificity of the cDNA probes for the individual chains has been shown previously (Smits et al. 1992). All probes were random prime-labeled to specific activities of approximately 1 × 108 cprn/ixg using [35S]otdCTP. For control purposes, we used similarly sized fragments of bacteriophage DNA labeled to the same specific activity. The in situ hybridization method used in this study was similar to that detailed in previous reports (Marles et al. 1991; Hoyland et al. 1991; Andrew et al. 1993). After hybridization and stringency washing, slides were dehydrated in 99% ethanol, air dried, and subjected to autoradiography, performed with K5 emulsion (Ilford) melted at 40°C and diluted 1:1 in distilled water. Slides were exposed at 4°C for 7-14 days and developed in Kodak D-19 developer for 5 min, rinsed, fixed for 5 min, and counterstained with hematoxylin and eosin (H&E).
(Bone 16:455-460; 1995) Key Words: Platelet-derived growth factor; Fracture healing; Bone; Cartilage; Immunohistochemistry; In situ hybridization. Introduction Platelet-derived growth factor (PDGF) has potent effects on connective tissue cells, including osteoblasts and chondrocytes. Accordingly, it has been suggested that PDGF may be important in soft-tissue and bone repair. PDGF may be released into fractures as a result of passive release from platelet a granules or released from macrophages (Shimokado et al. 1985) and cells of osteoblast lineage (Zhang et al. 1991). PDGF may also be produced by endothelial cells in response to injury (Majesky et al. 1990). PDGF has effects in vitro on several cell lines that may be important in fracture healing. These include osteoblasts (Pfeilschifter et al. 1990; Graves et al. 1989; Tsukamoto et al. 1991) and chondrocytes and their precursors (Skoog et al. 1990). The role of PDGF in wound healing has been investigated in some detail (Deuel et al. 1991; Whitby & Ferguson 1991; Lynch et al. 1987), but there is relatively little information on its function in fracture healing (Joyce et al. 1991; Nash et al. 1992). We have found only one previous study of PDGF expression in fracture healing (Joyce et al. 1991). This failed to demonstrate
Immunohistochemistry
Sections were initially dewaxed, and then endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in ethanol. Following this, sections were treated with chymotrypsin (0.01% with 0.1% CaCI 2 in TRIS-buffered saline [TBS]) at 37°C
Address for correspondence and reprints: Dr. J. A. Hoyland, Department of Orthopedics, c/o Department of Rheurnatology, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT UK. © 1995 by Elsevier Science Inc.
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for 20 min. Sections were then washed in TBS, blocked with 10% normal swine serum (15 min), and incubated for 1 h with polyclonal antiserum to human platelet PDGF (British Biotechnology). After extensive washing, secondary antibody (swine anti-rabbit immunoglobulin G, conjugated with biotin) was applied for 30 min. The sections were washed again and avidinbiotin-peroxidase complex was applied for 30 min. After washing, the sections were immersed in 0.2 mg/ml 3,3'diaminobenzidine tetrahydrochloride containing 0.05% (v/v) H202 for 15 rain. Nuclei were stained with hematoxylin. Characterization and specificity of the antibody have been published and are summarized in the product data sheet. It has been shown that prior incubation of the diluted antibody with 10 ng of human PDGF completely abolished staining (Whitby & Ferguson 1991). It should be noted that this antibody detects both the A and B chains of PDGF.
Results Human fracture callus presents a heterogeneous appearance at histological examination, with several of the elements of normal fracture healing being present in close proximity in any one section. These elements include blood clot, fibrous tissue, woven and compact bone, and both calcified and uncalcified cartilage. Because of this heterogeneous appearance, we graded callus specimens according to the predominant appearance of the callus, and related cellular events to the histological grade. The grades considered were as follows: 1) fracture hematoma and granulation tissue; 2) stage of definitive matrix formation: cartilage and/or bone formation, without remodeling; and 3) stage of matrix remodeling. In all cases, the results were consistent regardless of the time between fracture and biopsy.
Figure 1. PDGF A gene expression in day-8 fibular fracture demonstrated by in situ hybridization. Most cells, including endothelial (e) and mesenchymal cells (arrowhead), are positive. A = test; B = RNase control. H&E counterstain; original magnification × 150.
Grade 1
PDGF was detected by immunohistochemistry at the early stages of fracture healing. It was seen diffusely in the fracture hematoma. Some mononuclear cells in the fracture hematoma contained the protein, especially those that were close to areas of invading mesenchymal tissue. Gene expression was not detected for either PDGF A or B in these cells. In granulation tissue, gene expression for PDGF A was seen in endothelial cells (Figure 1), and PDGF was detected immunohistochemically in these cells (Figure 2). PDGF A gene expression was also seen in other cells at this stage; these were both round cells and the predominantly spindle-shaped "mesenchymal" cells between capillaries in the granulation tissue (Figure 1). Again, PDGF was seen in these cells on immunohistochemistry (Figure 3), although staining was more marked over the round than the spindle cells. PDGF B gene expression was also seen in the same cell types as for PDGF A expression (Figure 3), but in fewer cells and with lower signal levels over the cells. Grade 2
At the stage of matrix formation, PDGF A mRNA was detected in hypertrophic chondrocytes (Figure 4). PDGF B gene expression was not seen in these cells. PDGF was detected using immunohistochemistry in the cytoplasm of both early and hypertrophic chondrocytes and in the matrix surrounding hypertrophic chondrocytes (Figure 5). PDGF B mRNA was detected only in osteoblasts, which were of cuboidal morphology and several layers thick, adjacent to,
and apparently synthesizing, woven bone. However, levels of expression were low. PDGF A mRNA expression also was found in these cells, in flat surface osteoblasts on bone trabeculae and in osteoblasts trapped within trabeculae of woven bone (Figure 6). These osteoblasts all stained for PDGF using immunohistochemistry, although the staining of flat surface osteoblasts was variable (Figure 7). PDGF staining was also seen in osteoid as a rim around woven bone trabeculae, but not in the center of trabeculae. At this stage, the connective tissue between trabeculae was also stained for PDGF, with the areas adjacent to osteoblasts and woven bone being more strongly positive than those at some distance from evidence of bone formation. Endothelium in small vessels in areas of granulation tissue was positive for PDGF A gene expression and for PDGF on immunohistochemistry. Endothelium and perivascular cells of larger vessels were variably positive, with no obvious morphological difference between those that were positive and negative. PDGF B mRNA was not found in endothelial or perivascular cells at this stage. Grade 3
At the stage of matrix remodeling, both chondrocytes and osteoblasts expressed PDGF A gene in a way similar to that at the stage of matrix formation. PDGF A expression was thus seen in osteoblasts and chondrocytes, but areas of multiple layers of osteoblasts laying down woven bone were rare, and few osteoblasts expressed PDGF B mRNA at this stage. PDGF was seen
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Figure 2. Immunohistocheraical localization of PDGF in day-8 fibular fracture. Endothelial cells (arrowhead) and round cells (possibly monocytes) (arrow) are positive. Hematoxylin counterstain, x 400. in some osteoblasts and in cartilage matrix using immunohistochemistry. Osteoclasts were nearly all positive for PDGF A mRNA (Figure 8), but very few were positive for PDGF B. Osteoclasts were positive for PDGF on immunohistochemistry (Figure 9). The amount of detectable PDGF in the bone matrix
Figure 4. PDGF A-chain gene expression in chondrocytes (arrowhead) in day-17 femoral fracture. A = test; B = RNase control. H&E counterstain; original magnification x200. was less, possibly because of remodeling or crosslinking of the collagen.- It was striking that many round cells in the spaces between trabeculae of bone were positive for PDGF on immunohistochemistry and for PDGF A- and B-chain gene expression. These cells were single and their nature was not certain, but they
Figure 3. PDGF B-chain gene expression in day-8 fibular fracture. A few cells, principally endothelial cells of capillaries (arrowhead), are positive. Contrast this with PDGF A gene expression in Figure 1. A = test; B = RNase control. H&E counterstain; original magnification × 150.
Figure 5. PDGF demonstrated in cartilage matrix in day-17 femoral fracture. Chondrocytes were also positive for PDGF on immunohistochemistry. Hematoxylin counterstain; original magnification x200.
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Figure 6. PDGF A-chain gene expression in osteoblasts (ob). The cells with strongest signal were characteristically those several layers deep against woven bone; these cells were also often positive for PDGF B expression, although signal intensity was much lower. A = test; B = RNase control. H&E counterstain; original magnification ×250. appeared to be mononuclear cells. The connective tissue between bone trabeculae was either negative or very weakly positive at this stage. Endothelium was positive for PDGF protein and for PDGF A and B expression.
Figure 8. PDGF A-chain gene expression demonstrated by in situ hybridization in an osteoclast (oc) in day-21 humeral neck fracture. These cells were nearly all negative for PDGF B-chain expression. A = test; B = RNase control. H&E counterstain; original magnification x250.
Discussion
Figure 7. Immunohistochemical localization of PDGF in osteoblasts and osteoid in day- 11 radial fracture. The central part of bone trabeculae was usually negative. Hematoxylin counterstain; original magnification ×300.
There is surprisingly little information available about the role of PDGF in fracture healing. This is particularly striking in vmw of the large number of studies of the role of this growth factor in wound healing. PDGF B gene expression was not detected by Northern blotting in a rodent fracture model at any stage (Joyce et al. 1991). However, injection of suramin, which inhibits PDGF action, into the fracture site appeared to reduce markedly intramembranous bone formation. Conversely, subperiosteal injection of PDGF BB in rodents resulted in marked enhancement of intramembranous bone formation. Similar enhancement of intramembranous bone formation was found in tibial osteotomies in rabbits (Nash et al. 1992). One can conclude from these studies that although PDGF appears rate limiting at some stages of fracture repair, local production of this growth factor has not been demonstrated at the fracture site. Release of PDGF into the fracture site from platelet ct granules is likely to be similar in a fracture and in a soft-tissue wound (Skoog et al. 1990; Wahl et al. 1987; Assoian & Sporn 1986). The finding of diffuse immunostaining for PDGF in the hematoma probably indicates release from platelets. Some mononuclear cells in the hematoma also contained PDGF but did not appear to be actively synthesizing it. We have previously dem-
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Figure 9. PDGF demonstrated by immunohistochemistry in osteoclasts in day-21 humeral neck fracture. Hematoxylin counterstain; original magnification ×400.
onstrated that transforming growth factor-131 (TGF-I31) mRNA is not detectable in mononuclear ceils at this stage of fracture healing (Andrew et al. ]L993). This may indicate that mononuclear cells are not activated to produce growth factors at this stage of fracture healing, although they are migrating into the fracture site. The presence of PDGF in the wound may be important to promote chernotaxis (Deuel et al. 1991; Pierce et al. 1991). PDGF A gene was expressed by more cells in the granulation tissue phase of fracture healing than was PDGF B, and signal levels were considerably higher for PDGF A. The marked preponderance of cells expressing PDGF A make PDGF AA the most likely isoform to be: active in this situation, although further investigation would be required to confirm this. The round mononuclear cells that expressed both PDGF gene and protein may be macrophages. The relative expression of PDGF A and B chains is similar to tha'I observed in a study of carotid artery injury in a rodent model, in which PDGF A-chain expression was markedly elevated tMajesky et al. 1990). The cells studied were principally smooth-muscle cells, but arterial endothelium appeared to produce PI)GF A, as did endothelial cells in the current study. This is likely to cause paracrine effects only, as endothelium is thought not to respond to PDGF (Ross & Raines 1988; Weinstein & Weng 1986). PDGF is known to have marked effects on osteoblast function in vitro. Canalis et al. (1989) found that PDGF BB enhanced mitosis in cultured rat c~Llvariae, but also enhanced collagen degradation. The enhancement of collagen degradation was prevented by insulin-like growth factor (IGF). We have previously found evidence of both IGF II and I gene expression by osteoblasts at the stage of matrix formation in healing human fractures (Andrew et al. 1993). The role of different isoforms of PDGF may be important (Centrella et al. 1990), but this area is relatively unexplored, as is the possibility of species differences in both expression and effects of PDGF isoforms. Thus, Wergedal et al. (1990) failed to find enhanced [3H]thymidine uptake in human bone cell cultures treated with PDGF. The role of PDGF in cartilage is not clear. Under some circumstances, PDGF m~Ly inhibit chondrogenesis (Joyce et al. 1991; Chen et al. 1992). However, in culture PDGF enhances
J.G. Andrew et al. PDGF in human fracture callus
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both proliferation and differentiation of chondrocytes (Fukuo et al. 1989). Our finding of PDGF A gene expression in early and hypertrophic chondrocytes is accordingly difficult to interpret. The finding that PDGF protein is present in cartilage matrix around hypertrophic chondrocytes may be important in understanding coupling of bone formation during endochondral bone formation in fracture healing. PDGF A-chain gene and PDGF protein were expressed by osteoclasts. Although PDGF has been shown to stimulate collagen degradation in fetal rat calvariae (Canalis et al. 1989), much of this may be caused by osteoblastic matrix degradation. PDGF also has effects on tissue inhibitor of metalloproteinases production by osteoblasts (Meikle et al. 1991). The role of this growth factor in control of osteoclasts is not clear. In view of the clear evidence of PDGF expression, further investigation of PDGF in osteoclasts and in conditions of unbalanced bone resorption would be of interest. It is possible that some or all of the PDGF expression seen during fracture healing may be induced as a secondary phenomenon by other growth factors. Thus, TGF-13 has been shown to cause release of PDGF AA in culture from smooth-muscle cells (Battegay et al. 1990); it was also shown that the bimodal effect of TGF-13 on cellular proliferation was mediated by effects on PDGF and PDGF receptor expression. Because chondrocytes and osteoblasts have a similar bimodal response to TGF-I3, it is possible that this is important in cartilage and bone cells as well. Recently, TGF-I3 has been shown to increase PDGF A and PDGF B transcript levels in osteoblast-like cells (Takaishi et al. 1994). TGF-13 has been shown to be expressed locally in both rodent (Joyce et al. 1990, 1991) and human fracture healing (Andrew et al. 1993). PDGF has received a great deal of attention in wound healing research, but less is known about its role and effects in fracture healing. In view of the extensive expression, especially of PDGF A, found in normally healing human fractures, further investigations of the expression and role of this growth factor in bone repair may be rewarding. Investigation of PDGF receptor expression would also be useful.
Acknowledgments: This work was supported by the North Western Regional Health Authority. J.G.A. was the Sir Harry Platt research fellow, Department of Orthopedics, University of Manchester. We thank Mrs. Pauline Baird for technical assistance. The PDGF A and B cDNA were generous gifts from Dr. C.-H. Heldin, Uppsala, Sweden.
References Andrew, J. G., Hoyland, J. H., Andrew, S. M., Freemont, A. J., and Marsh, D. R. Demonstration of TGFI3 mRNA in human fracture callus by in situ hybridisation. CalcifTissue Int 52:74-78; 1993. Andrew, J. G., Hoyland, J. H., Freemont, A. J., and Marsh, D. R. Insulinlike growth factor gene expression in human fracture callus. Calcif Tissue Int 53:97-102; 1993. Assoian, R. K. and Spore, M. B. Type 13transforming growth factor in human platelets during platelet degranulation and action on vascular smooth muscle cells. J Cell Biol 102:1217-1223; 1986. Battegay, E. J., Raines, E. W., Seifert, R. A., Bowen-Pope,D. F., and Ross, R. TGF-betainducesbimodal proliferationof connectivetissue cells via complex control of an autocrine PDGF loop. Cell 63:515-524; 1990. Canalis, E., McCarthy,T. L., and Centrella, M. Effectsof platelet-derivedgrowth factor on bone formation in vitro. J Cell Physiol 140:530--537; 1989. Centrella, M., McCarthy, T. L., Ladd, C., and Canalis, E. Expressionof PDGF and regulation of PDGF binding are isoform specific in osteoblast enriched cultures from fetal rat bone. J Bone Miner Res 5:$86; 1990. Chen, P., Carrington, J. L., Paralkar, V. M., Pierce, G. F., and Reddi, A. H.
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Chick limb bud mesodermal cell chondrogenesis: inhibition by isoforms of platelet-derived growth factor and reversal by recombinant bone morphogenetic protein. Exp Cell Res 200:110-117: 1992. Deuel, T. F., Kawahara, R. S., Mustoe, T. A., and Pierce, A. F. Growth factors and wound healing: platelet-derived growth factor as a model cytokine. Annu Rev Med 42:567 584; 1991. Fukuo, K., Morimoto, S., Kaji, K., Koh, E., Hironaka, T., Morita, R., Kim, S., and Onishi, T. Association of increased intracellular free Ca2 + by plateletderived growth factor with mitogenesis but not with proteoglycan synthesis in chondrocytes~ffect of suramin. Cell Calcium 10:2%35; 1989. Graves, D. T., Valentin-Opran, A., Delgado, R., Valente, A. J., Mundy, G., and Piche, J. The potential role of platelet-derived growth factor as an autocrine or paracrine factor for human bone cells. Connect Tissue Res 23:20%218; 1989. Howes, R., Bowness, J. M., Grotendorst, G. R., Martin, G. R., and Reddi, A. H. Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation. Calcif Tissue Int 42:34-38; 1988. Hoyland, J. A., Thomas, J. T., Donn, R., Marriott, A., Ayad, S., Boot-Handford, R. P., Grant, M. E., and Freemont, A. J. Distribution of type X collagen mRNA in normal and osteoarthritic human cartilage. Bone Min 15:151-164; 1991. Joyce, M. E., Jingushi, S., and Bolander, M. E. Transforming growth factor-beta in the regulation of fracture repair. Orthop Clin North Am 21:199-209; 1990. Joyce, M. E., Jingushi, S., Scully, S. P., and Bolander, M. E. Role of growth factors in fracture healing. Prog Clin Biol Res 365:391-416; 1991. Lynch, S. E., Nixon, J. C., Colvin, R. B., and Antoniades, H. N. Role of platelet-derived growth factor in wound healing: synergistic effects with other growth factors. Proc Natl Acad Sci USA 84:7696-7700; 1987. Majesky, M. W., Reidy, M. A., Bowen-Pope, D. F., Hart, C. E., Wilcox, J. N., and Schwartz, S. M. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol 111:214%2158; 1990. Marles, P. J., Hoyland, J. A., Parkinson, R., and Freemont, A. J. Demonstration of variation in chondrocyte activity in different zones of articular cartilage: an assessment of the value of in situ hybridization. Int J Exp Pathol 72:171-182; 1991. Meikle, M. C., McGarrity, A. M., Thomson, B. M., and Reynolds, J. J. Bonederived growth factors modulate collagenase and TIMP (tissue inhibitor of metalloproteinases) activity and type I collagen degradation by mouse calvarial osteoblasts. Bone Min 12:41-55; 1991. Nash, T., Howlett, C. R., Martin, C., Steele, J., Johnson, K., and Hicklin, D. J. The effect of PDGF on tibial osteotomies in rabbits. Calcif Tissue lnt 50:A 11; 1992. Pfeilschifter, J., Oechsner, M., Naumann, A., Gronwald, R. G. K., Minne, H. W., and Ziegler, R. Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor I, platelet-
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derived growth factor, and transforming growth factor beta. Endocrinology 127:69-75; 1990. Pierce, G. F., Brown, D., and Mustoe, T. A. Quantitative analysis of inflammatory cell influx, procollagen type 1 synthesis, and collagen cross-linking in incisional wounds: influence of PDGF-BB and TGF-beta 1 therapy. J Lab Clin Med 117:373 382; 1991. Ross, R. and Raines, E. Platelet derived growth factor--its role in health and disease. Adv Exp Med Biol 234:9-21; 1988. Shimokado, K., Raines, E. W., Madtes, D. K., Barrett, T., Benditt, E. P., and Ross, R. A significant part of macrophage derived growth factor consists of at least two forms of PDGF. Cell 43:277-286; 1985. Skoog, V., Widenfalk, B., Ohlsen, L., and Wasteson, A. The effect of growth factors and synovial fluid on chondrogenesis in perichondrium. Scand J Plast Reconstr Surg Hand Surg 24:89 95; 1990. Smits, A., Funa, K., Vassbotn, F. S., Beausang-Linder, M., af-Ekenstam, F., Heldin, C. H.. Westermark, B., and Nister, M. Expression of platelet derived growth factor and its receptors in proliferative disorders of fibroblastic origin. Am J Pathol 140:639-648; 1992. Takaishi, Y., Matsui, T., Tsukamoto, T., Ito, M., Taniguchi, T., Fukase, M., and Chihara, K. TGF-[3 induced macrophage colony stimulating factor gene expression in various mesenchymal cell lines. Am J Physiol 267:25-31; 1994. Tsukamoto, T., Matsui, T., Fukase, M., and Fujita, T. Platelet-derived growth factor B chain homodimer enhances chemotaxis and DNA synthesis in normal osteoblast-like cells (MC3T3-EI). Biochem Biophys Res Commun 175:745 751; 1991. Wahl, S. M., Hunt, D. A., Wakefield, I. M.. et al. Transforming growth factor beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci USA 84:5788-5792; 1987. Weinstein, R. and Weng, K. Growth factor responses of human arterial endothelial cells in vitro. In Vitro Cell Dev Biol 22:549-556; 1986. Wergedal, J. E., Mohan, S., Lundy, M., and Baylink, D. J. Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells. J Bone Miner Res 5:179186; 1990. Whitby, D. J. and Ferguson, M. W. Immunohistochemical localization of growth factors in fetal wound healing. Dev Biol 147:20~215; 1991. Zhang, L., Leeman, E., Cames, D. C., and Graves, D. T. Human osteoblasts synthesize and respond to platelet-derived growth factor. Am J Physiol 261:C348 C354; 1991.
Date Received: July 20, 1994 Date Revised: December 14, 1994 Date Accepted: December 14, 1994
ELSEVIER
Platelet-Derived Growth Factor Expression in Normally Healing Human Fractures J. G. A N D R E W , 1 J. A. H O Y L A N D , 1 A. J. F R E E M O N T , 2 and D. R. M A R S H 1 i Department of Orthopedics, Hope Hospital, Salford, United Kingdom z Department of Pathological Sciences, Manchester University Medical School, Manchester, United Kingdom
PDGF B-chain mRNA by Northern analysis in rodent fracture callus; PDGF A-chain expression was not examined. Thus, the demonstrated in vitro effects of PDGF on the cell types involved in fracture healing have not been demonstrated to be relevant in normal fracture-healing models, and we have been unable to find previous reports on PDGF in human fracture material. This paper reports an immunohistochemical and in situ hybridization study of PDGF expression in normally healing fractures.
Platelet-derived growllh factor (PDGF) has been shown to have effects on bone and cartilage cells in vitro, but its role in vivo in bone repair is not clear. We studied biopsy material from 16 normally healing fractures at a variety of times after injury, using immun¢~histochemistry for PDGF and in situ hybridization for PDGF A and B chains. PDGF A-chain gene was found to be expressed by many cell types over a prolonged period during fracture healing. These cells included endothelial and mese~hchymai cells in granulation tissue and osteoblasts, chondroc,ctes, and osteoclasts later during fracture healing. PDGF B-chain gene expression was more restricted, being detected principally in osteoblasts at the stage of bone formation. PDGF was detected using immunohistochemistry in the cell types expressing PDGF A. These findings indicate that PDGF is expressed during normal human fracture repair, and the in vitro data also suggest that PDGF is likely to be an important local regulator in this process.
Materials and Methods Callus specimens were obtained from normally healing fractures at operation for malunion between 1 and 6 weeks after fracture. Ethical committee approval and informed consent were obtained. Specimens were examined from 16 fractures. All of the fractures had united normally at follow-up 1 year after operation, except in one patient who died of unrelated causes shortly after operation. Tissue was fixed in neutral buffered formalin for 24 h and then decalcified in 20% ethylenediamine tetraacetic acid, pH 7.2, until radiologically decalcified (10-14 days). Tissue was then processed routinely into paraffin wax, and 7-1~m sections were cut and mounted onto slides coated with vectorbond (Vector Laboratories) for in situ hybridization. Human cDNA probes were used. The PDGF A-chain cDNA (1.3 kb) and the PDGF B-chain cDNA (2.0 kb) were obtained from Dr. C. H. Heldin (Uppsala, Sweden). Specificity of the cDNA probes for the individual chains has been shown previously (Smits et al. 1992). All probes were random prime-labeled to specific activities of approximately 1 × 108 cprn/ixg using [35S]otdCTP. For control purposes, we used similarly sized fragments of bacteriophage DNA labeled to the same specific activity. The in situ hybridization method used in this study was similar to that detailed in previous reports (Marles et al. 1991; Hoyland et al. 1991; Andrew et al. 1993). After hybridization and stringency washing, slides were dehydrated in 99% ethanol, air dried, and subjected to autoradiography, performed with K5 emulsion (Ilford) melted at 40°C and diluted 1:1 in distilled water. Slides were exposed at 4°C for 7-14 days and developed in Kodak D-19 developer for 5 min, rinsed, fixed for 5 min, and counterstained with hematoxylin and eosin (H&E).
(Bone 16:455-460; 1995) Key Words: Platelet-derived growth factor; Fracture healing; Bone; Cartilage; Immunohistochemistry; In situ hybridization. Introduction Platelet-derived growth factor (PDGF) has potent effects on connective tissue cells, including osteoblasts and chondrocytes. Accordingly, it has been suggested that PDGF may be important in soft-tissue and bone repair. PDGF may be released into fractures as a result of passive release from platelet a granules or released from macrophages (Shimokado et al. 1985) and cells of osteoblast lineage (Zhang et al. 1991). PDGF may also be produced by endothelial cells in response to injury (Majesky et al. 1990). PDGF has effects in vitro on several cell lines that may be important in fracture healing. These include osteoblasts (Pfeilschifter et al. 1990; Graves et al. 1989; Tsukamoto et al. 1991) and chondrocytes and their precursors (Skoog et al. 1990). The role of PDGF in wound healing has been investigated in some detail (Deuel et al. 1991; Whitby & Ferguson 1991; Lynch et al. 1987), but there is relatively little information on its function in fracture healing (Joyce et al. 1991; Nash et al. 1992). We have found only one previous study of PDGF expression in fracture healing (Joyce et al. 1991). This failed to demonstrate
Immunohistochemistry
Sections were initially dewaxed, and then endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in ethanol. Following this, sections were treated with chymotrypsin (0.01% with 0.1% CaCI 2 in TRIS-buffered saline [TBS]) at 37°C
Address for correspondence and reprints: Dr. J. A. Hoyland, Department of Orthopedics, c/o Department of Rheurnatology, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT UK. © 1995 by Elsevier Science Inc.
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for 20 min. Sections were then washed in TBS, blocked with 10% normal swine serum (15 min), and incubated for 1 h with polyclonal antiserum to human platelet PDGF (British Biotechnology). After extensive washing, secondary antibody (swine anti-rabbit immunoglobulin G, conjugated with biotin) was applied for 30 min. The sections were washed again and avidinbiotin-peroxidase complex was applied for 30 min. After washing, the sections were immersed in 0.2 mg/ml 3,3'diaminobenzidine tetrahydrochloride containing 0.05% (v/v) H202 for 15 rain. Nuclei were stained with hematoxylin. Characterization and specificity of the antibody have been published and are summarized in the product data sheet. It has been shown that prior incubation of the diluted antibody with 10 ng of human PDGF completely abolished staining (Whitby & Ferguson 1991). It should be noted that this antibody detects both the A and B chains of PDGF.
Results Human fracture callus presents a heterogeneous appearance at histological examination, with several of the elements of normal fracture healing being present in close proximity in any one section. These elements include blood clot, fibrous tissue, woven and compact bone, and both calcified and uncalcified cartilage. Because of this heterogeneous appearance, we graded callus specimens according to the predominant appearance of the callus, and related cellular events to the histological grade. The grades considered were as follows: 1) fracture hematoma and granulation tissue; 2) stage of definitive matrix formation: cartilage and/or bone formation, without remodeling; and 3) stage of matrix remodeling. In all cases, the results were consistent regardless of the time between fracture and biopsy.
Figure 1. PDGF A gene expression in day-8 fibular fracture demonstrated by in situ hybridization. Most cells, including endothelial (e) and mesenchymal cells (arrowhead), are positive. A = test; B = RNase control. H&E counterstain; original magnification × 150.
Grade 1
PDGF was detected by immunohistochemistry at the early stages of fracture healing. It was seen diffusely in the fracture hematoma. Some mononuclear cells in the fracture hematoma contained the protein, especially those that were close to areas of invading mesenchymal tissue. Gene expression was not detected for either PDGF A or B in these cells. In granulation tissue, gene expression for PDGF A was seen in endothelial cells (Figure 1), and PDGF was detected immunohistochemically in these cells (Figure 2). PDGF A gene expression was also seen in other cells at this stage; these were both round cells and the predominantly spindle-shaped "mesenchymal" cells between capillaries in the granulation tissue (Figure 1). Again, PDGF was seen in these cells on immunohistochemistry (Figure 3), although staining was more marked over the round than the spindle cells. PDGF B gene expression was also seen in the same cell types as for PDGF A expression (Figure 3), but in fewer cells and with lower signal levels over the cells. Grade 2
At the stage of matrix formation, PDGF A mRNA was detected in hypertrophic chondrocytes (Figure 4). PDGF B gene expression was not seen in these cells. PDGF was detected using immunohistochemistry in the cytoplasm of both early and hypertrophic chondrocytes and in the matrix surrounding hypertrophic chondrocytes (Figure 5). PDGF B mRNA was detected only in osteoblasts, which were of cuboidal morphology and several layers thick, adjacent to,
and apparently synthesizing, woven bone. However, levels of expression were low. PDGF A mRNA expression also was found in these cells, in flat surface osteoblasts on bone trabeculae and in osteoblasts trapped within trabeculae of woven bone (Figure 6). These osteoblasts all stained for PDGF using immunohistochemistry, although the staining of flat surface osteoblasts was variable (Figure 7). PDGF staining was also seen in osteoid as a rim around woven bone trabeculae, but not in the center of trabeculae. At this stage, the connective tissue between trabeculae was also stained for PDGF, with the areas adjacent to osteoblasts and woven bone being more strongly positive than those at some distance from evidence of bone formation. Endothelium in small vessels in areas of granulation tissue was positive for PDGF A gene expression and for PDGF on immunohistochemistry. Endothelium and perivascular cells of larger vessels were variably positive, with no obvious morphological difference between those that were positive and negative. PDGF B mRNA was not found in endothelial or perivascular cells at this stage. Grade 3
At the stage of matrix remodeling, both chondrocytes and osteoblasts expressed PDGF A gene in a way similar to that at the stage of matrix formation. PDGF A expression was thus seen in osteoblasts and chondrocytes, but areas of multiple layers of osteoblasts laying down woven bone were rare, and few osteoblasts expressed PDGF B mRNA at this stage. PDGF was seen
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Figure 2. Immunohistocheraical localization of PDGF in day-8 fibular fracture. Endothelial cells (arrowhead) and round cells (possibly monocytes) (arrow) are positive. Hematoxylin counterstain, x 400. in some osteoblasts and in cartilage matrix using immunohistochemistry. Osteoclasts were nearly all positive for PDGF A mRNA (Figure 8), but very few were positive for PDGF B. Osteoclasts were positive for PDGF on immunohistochemistry (Figure 9). The amount of detectable PDGF in the bone matrix
Figure 4. PDGF A-chain gene expression in chondrocytes (arrowhead) in day-17 femoral fracture. A = test; B = RNase control. H&E counterstain; original magnification x200. was less, possibly because of remodeling or crosslinking of the collagen.- It was striking that many round cells in the spaces between trabeculae of bone were positive for PDGF on immunohistochemistry and for PDGF A- and B-chain gene expression. These cells were single and their nature was not certain, but they
Figure 3. PDGF B-chain gene expression in day-8 fibular fracture. A few cells, principally endothelial cells of capillaries (arrowhead), are positive. Contrast this with PDGF A gene expression in Figure 1. A = test; B = RNase control. H&E counterstain; original magnification × 150.
Figure 5. PDGF demonstrated in cartilage matrix in day-17 femoral fracture. Chondrocytes were also positive for PDGF on immunohistochemistry. Hematoxylin counterstain; original magnification x200.
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Figure 6. PDGF A-chain gene expression in osteoblasts (ob). The cells with strongest signal were characteristically those several layers deep against woven bone; these cells were also often positive for PDGF B expression, although signal intensity was much lower. A = test; B = RNase control. H&E counterstain; original magnification ×250. appeared to be mononuclear cells. The connective tissue between bone trabeculae was either negative or very weakly positive at this stage. Endothelium was positive for PDGF protein and for PDGF A and B expression.
Figure 8. PDGF A-chain gene expression demonstrated by in situ hybridization in an osteoclast (oc) in day-21 humeral neck fracture. These cells were nearly all negative for PDGF B-chain expression. A = test; B = RNase control. H&E counterstain; original magnification x250.
Discussion
Figure 7. Immunohistochemical localization of PDGF in osteoblasts and osteoid in day- 11 radial fracture. The central part of bone trabeculae was usually negative. Hematoxylin counterstain; original magnification ×300.
There is surprisingly little information available about the role of PDGF in fracture healing. This is particularly striking in vmw of the large number of studies of the role of this growth factor in wound healing. PDGF B gene expression was not detected by Northern blotting in a rodent fracture model at any stage (Joyce et al. 1991). However, injection of suramin, which inhibits PDGF action, into the fracture site appeared to reduce markedly intramembranous bone formation. Conversely, subperiosteal injection of PDGF BB in rodents resulted in marked enhancement of intramembranous bone formation. Similar enhancement of intramembranous bone formation was found in tibial osteotomies in rabbits (Nash et al. 1992). One can conclude from these studies that although PDGF appears rate limiting at some stages of fracture repair, local production of this growth factor has not been demonstrated at the fracture site. Release of PDGF into the fracture site from platelet ct granules is likely to be similar in a fracture and in a soft-tissue wound (Skoog et al. 1990; Wahl et al. 1987; Assoian & Sporn 1986). The finding of diffuse immunostaining for PDGF in the hematoma probably indicates release from platelets. Some mononuclear cells in the hematoma also contained PDGF but did not appear to be actively synthesizing it. We have previously dem-
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Figure 9. PDGF demonstrated by immunohistochemistry in osteoclasts in day-21 humeral neck fracture. Hematoxylin counterstain; original magnification ×400.
onstrated that transforming growth factor-131 (TGF-I31) mRNA is not detectable in mononuclear ceils at this stage of fracture healing (Andrew et al. ]L993). This may indicate that mononuclear cells are not activated to produce growth factors at this stage of fracture healing, although they are migrating into the fracture site. The presence of PDGF in the wound may be important to promote chernotaxis (Deuel et al. 1991; Pierce et al. 1991). PDGF A gene was expressed by more cells in the granulation tissue phase of fracture healing than was PDGF B, and signal levels were considerably higher for PDGF A. The marked preponderance of cells expressing PDGF A make PDGF AA the most likely isoform to be: active in this situation, although further investigation would be required to confirm this. The round mononuclear cells that expressed both PDGF gene and protein may be macrophages. The relative expression of PDGF A and B chains is similar to tha'I observed in a study of carotid artery injury in a rodent model, in which PDGF A-chain expression was markedly elevated tMajesky et al. 1990). The cells studied were principally smooth-muscle cells, but arterial endothelium appeared to produce PI)GF A, as did endothelial cells in the current study. This is likely to cause paracrine effects only, as endothelium is thought not to respond to PDGF (Ross & Raines 1988; Weinstein & Weng 1986). PDGF is known to have marked effects on osteoblast function in vitro. Canalis et al. (1989) found that PDGF BB enhanced mitosis in cultured rat c~Llvariae, but also enhanced collagen degradation. The enhancement of collagen degradation was prevented by insulin-like growth factor (IGF). We have previously found evidence of both IGF II and I gene expression by osteoblasts at the stage of matrix formation in healing human fractures (Andrew et al. 1993). The role of different isoforms of PDGF may be important (Centrella et al. 1990), but this area is relatively unexplored, as is the possibility of species differences in both expression and effects of PDGF isoforms. Thus, Wergedal et al. (1990) failed to find enhanced [3H]thymidine uptake in human bone cell cultures treated with PDGF. The role of PDGF in cartilage is not clear. Under some circumstances, PDGF m~Ly inhibit chondrogenesis (Joyce et al. 1991; Chen et al. 1992). However, in culture PDGF enhances
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both proliferation and differentiation of chondrocytes (Fukuo et al. 1989). Our finding of PDGF A gene expression in early and hypertrophic chondrocytes is accordingly difficult to interpret. The finding that PDGF protein is present in cartilage matrix around hypertrophic chondrocytes may be important in understanding coupling of bone formation during endochondral bone formation in fracture healing. PDGF A-chain gene and PDGF protein were expressed by osteoclasts. Although PDGF has been shown to stimulate collagen degradation in fetal rat calvariae (Canalis et al. 1989), much of this may be caused by osteoblastic matrix degradation. PDGF also has effects on tissue inhibitor of metalloproteinases production by osteoblasts (Meikle et al. 1991). The role of this growth factor in control of osteoclasts is not clear. In view of the clear evidence of PDGF expression, further investigation of PDGF in osteoclasts and in conditions of unbalanced bone resorption would be of interest. It is possible that some or all of the PDGF expression seen during fracture healing may be induced as a secondary phenomenon by other growth factors. Thus, TGF-13 has been shown to cause release of PDGF AA in culture from smooth-muscle cells (Battegay et al. 1990); it was also shown that the bimodal effect of TGF-13 on cellular proliferation was mediated by effects on PDGF and PDGF receptor expression. Because chondrocytes and osteoblasts have a similar bimodal response to TGF-I3, it is possible that this is important in cartilage and bone cells as well. Recently, TGF-I3 has been shown to increase PDGF A and PDGF B transcript levels in osteoblast-like cells (Takaishi et al. 1994). TGF-13 has been shown to be expressed locally in both rodent (Joyce et al. 1990, 1991) and human fracture healing (Andrew et al. 1993). PDGF has received a great deal of attention in wound healing research, but less is known about its role and effects in fracture healing. In view of the extensive expression, especially of PDGF A, found in normally healing human fractures, further investigations of the expression and role of this growth factor in bone repair may be rewarding. Investigation of PDGF receptor expression would also be useful.
Acknowledgments: This work was supported by the North Western Regional Health Authority. J.G.A. was the Sir Harry Platt research fellow, Department of Orthopedics, University of Manchester. We thank Mrs. Pauline Baird for technical assistance. The PDGF A and B cDNA were generous gifts from Dr. C.-H. Heldin, Uppsala, Sweden.
References Andrew, J. G., Hoyland, J. H., Andrew, S. M., Freemont, A. J., and Marsh, D. R. Demonstration of TGFI3 mRNA in human fracture callus by in situ hybridisation. CalcifTissue Int 52:74-78; 1993. Andrew, J. G., Hoyland, J. H., Freemont, A. J., and Marsh, D. R. Insulinlike growth factor gene expression in human fracture callus. Calcif Tissue Int 53:97-102; 1993. Assoian, R. K. and Spore, M. B. Type 13transforming growth factor in human platelets during platelet degranulation and action on vascular smooth muscle cells. J Cell Biol 102:1217-1223; 1986. Battegay, E. J., Raines, E. W., Seifert, R. A., Bowen-Pope,D. F., and Ross, R. TGF-betainducesbimodal proliferationof connectivetissue cells via complex control of an autocrine PDGF loop. Cell 63:515-524; 1990. Canalis, E., McCarthy,T. L., and Centrella, M. Effectsof platelet-derivedgrowth factor on bone formation in vitro. J Cell Physiol 140:530--537; 1989. Centrella, M., McCarthy, T. L., Ladd, C., and Canalis, E. Expressionof PDGF and regulation of PDGF binding are isoform specific in osteoblast enriched cultures from fetal rat bone. J Bone Miner Res 5:$86; 1990. Chen, P., Carrington, J. L., Paralkar, V. M., Pierce, G. F., and Reddi, A. H.
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Chick limb bud mesodermal cell chondrogenesis: inhibition by isoforms of platelet-derived growth factor and reversal by recombinant bone morphogenetic protein. Exp Cell Res 200:110-117: 1992. Deuel, T. F., Kawahara, R. S., Mustoe, T. A., and Pierce, A. F. Growth factors and wound healing: platelet-derived growth factor as a model cytokine. Annu Rev Med 42:567 584; 1991. Fukuo, K., Morimoto, S., Kaji, K., Koh, E., Hironaka, T., Morita, R., Kim, S., and Onishi, T. Association of increased intracellular free Ca2 + by plateletderived growth factor with mitogenesis but not with proteoglycan synthesis in chondrocytes~ffect of suramin. Cell Calcium 10:2%35; 1989. Graves, D. T., Valentin-Opran, A., Delgado, R., Valente, A. J., Mundy, G., and Piche, J. The potential role of platelet-derived growth factor as an autocrine or paracrine factor for human bone cells. Connect Tissue Res 23:20%218; 1989. Howes, R., Bowness, J. M., Grotendorst, G. R., Martin, G. R., and Reddi, A. H. Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation. Calcif Tissue Int 42:34-38; 1988. Hoyland, J. A., Thomas, J. T., Donn, R., Marriott, A., Ayad, S., Boot-Handford, R. P., Grant, M. E., and Freemont, A. J. Distribution of type X collagen mRNA in normal and osteoarthritic human cartilage. Bone Min 15:151-164; 1991. Joyce, M. E., Jingushi, S., and Bolander, M. E. Transforming growth factor-beta in the regulation of fracture repair. Orthop Clin North Am 21:199-209; 1990. Joyce, M. E., Jingushi, S., Scully, S. P., and Bolander, M. E. Role of growth factors in fracture healing. Prog Clin Biol Res 365:391-416; 1991. Lynch, S. E., Nixon, J. C., Colvin, R. B., and Antoniades, H. N. Role of platelet-derived growth factor in wound healing: synergistic effects with other growth factors. Proc Natl Acad Sci USA 84:7696-7700; 1987. Majesky, M. W., Reidy, M. A., Bowen-Pope, D. F., Hart, C. E., Wilcox, J. N., and Schwartz, S. M. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol 111:214%2158; 1990. Marles, P. J., Hoyland, J. A., Parkinson, R., and Freemont, A. J. Demonstration of variation in chondrocyte activity in different zones of articular cartilage: an assessment of the value of in situ hybridization. Int J Exp Pathol 72:171-182; 1991. Meikle, M. C., McGarrity, A. M., Thomson, B. M., and Reynolds, J. J. Bonederived growth factors modulate collagenase and TIMP (tissue inhibitor of metalloproteinases) activity and type I collagen degradation by mouse calvarial osteoblasts. Bone Min 12:41-55; 1991. Nash, T., Howlett, C. R., Martin, C., Steele, J., Johnson, K., and Hicklin, D. J. The effect of PDGF on tibial osteotomies in rabbits. Calcif Tissue lnt 50:A 11; 1992. Pfeilschifter, J., Oechsner, M., Naumann, A., Gronwald, R. G. K., Minne, H. W., and Ziegler, R. Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor I, platelet-
Bone Vol. 16, No. 4 April 1 9 9 5 : 4 5 5 - 4 6 0
derived growth factor, and transforming growth factor beta. Endocrinology 127:69-75; 1990. Pierce, G. F., Brown, D., and Mustoe, T. A. Quantitative analysis of inflammatory cell influx, procollagen type 1 synthesis, and collagen cross-linking in incisional wounds: influence of PDGF-BB and TGF-beta 1 therapy. J Lab Clin Med 117:373 382; 1991. Ross, R. and Raines, E. Platelet derived growth factor--its role in health and disease. Adv Exp Med Biol 234:9-21; 1988. Shimokado, K., Raines, E. W., Madtes, D. K., Barrett, T., Benditt, E. P., and Ross, R. A significant part of macrophage derived growth factor consists of at least two forms of PDGF. Cell 43:277-286; 1985. Skoog, V., Widenfalk, B., Ohlsen, L., and Wasteson, A. The effect of growth factors and synovial fluid on chondrogenesis in perichondrium. Scand J Plast Reconstr Surg Hand Surg 24:89 95; 1990. Smits, A., Funa, K., Vassbotn, F. S., Beausang-Linder, M., af-Ekenstam, F., Heldin, C. H.. Westermark, B., and Nister, M. Expression of platelet derived growth factor and its receptors in proliferative disorders of fibroblastic origin. Am J Pathol 140:639-648; 1992. Takaishi, Y., Matsui, T., Tsukamoto, T., Ito, M., Taniguchi, T., Fukase, M., and Chihara, K. TGF-[3 induced macrophage colony stimulating factor gene expression in various mesenchymal cell lines. Am J Physiol 267:25-31; 1994. Tsukamoto, T., Matsui, T., Fukase, M., and Fujita, T. Platelet-derived growth factor B chain homodimer enhances chemotaxis and DNA synthesis in normal osteoblast-like cells (MC3T3-EI). Biochem Biophys Res Commun 175:745 751; 1991. Wahl, S. M., Hunt, D. A., Wakefield, I. M.. et al. Transforming growth factor beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci USA 84:5788-5792; 1987. Weinstein, R. and Weng, K. Growth factor responses of human arterial endothelial cells in vitro. In Vitro Cell Dev Biol 22:549-556; 1986. Wergedal, J. E., Mohan, S., Lundy, M., and Baylink, D. J. Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells. J Bone Miner Res 5:179186; 1990. Whitby, D. J. and Ferguson, M. W. Immunohistochemical localization of growth factors in fetal wound healing. Dev Biol 147:20~215; 1991. Zhang, L., Leeman, E., Cames, D. C., and Graves, D. T. Human osteoblasts synthesize and respond to platelet-derived growth factor. Am J Physiol 261:C348 C354; 1991.
Date Received: July 20, 1994 Date Revised: December 14, 1994 Date Accepted: December 14, 1994